Zhou

Dr Jean-Pierre Renou

Zhou N N

Hibrand Saint-Oyant

Zhou Nn

Tang Kx

Lopez Arias

Hibrand-Saint Oyant

Prickle number on four internodes of two types of stems for three years in OW progeny . . provided new insights

Keywords: Je, soussignée Ning-ning Glandular and non-glandular structure, Trichome, Prickle anatomy, QTL, RNA sequencing, Transcriptomics v en génétique évolutive, en génétique quantitative, en sélection, en biologie cellulaire

par tous les moyens raisonnables, sans toutefois suggérer que l'offrant vous soutient ou soutient la facon dont vous avez utilisé son oeuvre.

Acknowledgments

Time flies so quickly. The adventure of this thesis was not done alone and here I would like to thank all of them who have been part of this journey and making this thesis possible.

Firstly, I would like to express my appreciation to Chinese Government Scholarships (China Scholarship Council), for supporting me to do my Ph.D project in France. Thank the National Natural Science Foundation of China for supporting the research funding of this project.

Further, I express my warmest gratitude to my supervisors Dr. Fabrice FOUCHER and Dr. Laurence HIBRAND-SAINT OYANT for their continued support, patience, motivation throughout this project. Their immense knowledge and guidance helped me in all the time of research and writing of this thesis. Besides work, I would also like to thank them for their responsive help in my daily life. I also wish to thanks the UMR IRHS (Institute for Research in Horticulture and Seeds, INRAE, Agrocampus west, University of Angers) and all its member's staff for providing a great platform that let me perform my research in the best condition.

Many thanks to all the members of the GDO team for the welcome to me. Thank in particular Julien Jeauffre for the guidance of molecular biology experiments and qPCR data analysis. Thank Tatiana Thouroude for her contribution to manage my materials in the greenhouse and for the good times spent together. A great thank Jordan Marie Magdelaine for helping me to pass my Python course on MOOC and ignited my interest in bioinformatics. Thank Sophie Paillard for her serval key tips to solve the bug I met during the transcriptome data analyses, and also for her enthusiasm and humor brings to me much happiness. Thank Jérome Chameau for sampling with me. He was so meticulous which left a deep impression on me. Thank Laurent Ogé for vii his helping in the lab and for his kindness and warm smiles. Thanks Alix, Jérémy, Agnès, Valéry, Latifa, Gilles. . . for all your kindness. Many thanks also to the Loubert Rose Gardens (Rosiers sur Loire, France) for providing the experimental materials. Thank madam Loubert and her family for preparing the annual rose pruning activity and for the great lunch we spend together ( all the members of GDO).

Special thanks to the leadership of Yunnan Academy of Agricultural Sciences for encouraging young researchers to pursue doctoral degrees while retaining their positions. Particularly, I'm deeply indebted to my master director Dr. Kai-Xue TANG who directly influenced my academic and work journey. I would also like to thank Prof. Hao ZHANG and Dr. Ji-Hua WANG of Yunnan Academy of Agricultural Sciences (YAAS) for accepting to be the recommenders for applying for the project funding. I would like to thank my colleagues in FRI for their help in all aspects. Especially, I am grateful to thank Dr. Hong-Ying JIAN for her contribution to revising the manuscript of the funding project and doing the oral presentations of the project acceptance defense for me, and for her responsive help for many kinds of stuff. Thank Prof. Qi-Gang WANG for sharing his experience in the rose breeding program and guide me to participate in corporate services in the rose company. Thank Dr. Xian-Qin QIU and Prof. Hui-Jun YAN for sharing their experience in performing the research projects. Thank all the term members of Woody ornamental plants R & D, FRI for the happiness or even conflict moments we spend together in years.

Thank the IMAC technical platforms (Fabienne Simonneau, Aurelia Rolland) of SFR Quasav for supervising the histological experiment and the good times spent together. Thank Muriel Bahut of the PTM ANAN of the SFR Quasav for designing the program and guide me to use the Pipetting Robot for qPCR. Thank Sandrine Balzergue of VALEMA team for participates in the discussion of experimental design and the RNA data analysis. Thank the experimental unit (UE Horti) for their technical assistance in plant management and the ImHorPhen team (Dominique Besnard, Rémi Gardet) of IRHS for taking care of the plant cuttings in the greenhouse.

Thank Eric Montaudon, Sylvain Gaillard of the BIDEFI term for helping fix several problems in install the bioinformatics tools and thanks to the bioinformatics platform (Angers, IRHS) for providing computing and storage resources. Thank the IRHS doctoral students Ming (graduated), Diana, Kevin (graduated), Aliex (graduated), and postdoctorate Deepika for the support that we have given to each other during our respective journeys. Thank Xue-Wu Dou (doctoral student of Angers university) for helping me to reproduce the structure figures which were presented in Chapter 1.

Finally, my deepest and sincere gratitude to my parents and young brother for their support and unconditional love throughout my life. I am grateful to my cousins for taking care of my parents when I and my brother were not there. Thanks also to my sister-in-law who brought a babe to our life. I also would like to thank my little nephew for bringing much happiness to us as well as for saving me from mom's attention about my aging without a child. And now that I will have time to tent to relationships, I want to thank my two unborn

Glossaries list

Bark

(botany) It refers to all tissues exterior to the vascular cambium, including a number of tissue types that are periderm (composed of the cork, cork cambium, and the phelloderm), cortex (comprised of ground tissues), and phloem, and epidermis

Biseriate

(botany) Arranged in two whorls, cycles, rows, or series Chimera (genetics) A single organism composed of cells with more than one distinct genotype Downy plant (botany) The plants that organ(s) covered with fine, soft hair Emergences (botany) Any of various superficial outgrowths of plant tissue usually formed from both epidermis and immediately underlying tissues Epidermis (botany) A single layer of cells that covers the organs of plants.

Genetic redundancy

(genetics) Two or more genes are performing the same function and inactivation of one of these genes has little or no effect on the phenotype

Ground meristem

(botany) The primary meristematic tissue from where the ground tissues (i.e.

non-dermal or non-vascular tissues) develop from. It directly come from the apical meristem.

Meristem

(botany) A meristem (meristematic tissue) is a plant tissue that is made up of undifferentiated, actively dividing cells. Its fundamental function is growth. Some of the cells continue to give rise to new cells by cell division while others develop into differentiated cells comprising a particular permanent tissue.

Multiseriate

(botany) Arranged in rows or composed of more than one cell layer

Prickles

(botany) Sharp appendages of plant that originate from ground meristem (just under protoderm), they do not have vascular bundles and generally easy to be removed at mature stage due to the formation of abscission layer structure-like.

Prickle meristem (botany) Derive from the ground meristem, associate with the growth of prickle

Distinctions and definitions based on anatomical studies

The basic terminology describe appendages on plants (trichomes, prickles, thorns and spines) is frequently inaccurately cited in scientific reports, making it even more difficult for most researchers to understand how to distinguish between the different terms. For example, commonly confused words include trichomes, emergences and prickles, since their definitions are not consistent in the literature. Some authors have described emergences as prickles, e.g., prickles on the stems or leaves of plants such as Solatium torvium, Aiphanes acanthophylla, roses, etc. [START_REF] Bell | Plant form: an illustrated guide to flowering plant morphology[END_REF], and and some have referred to trichomes as emergences, e.g. grape emergences (Ma et al., 2016b). Another common confusion is between prickles, thorns and spines.

Many plants described as having thorns or spines [START_REF] Mcpheeters | Histogenic layer manipulation in chimeral thornless evergreen trailing blackberry[END_REF][START_REF] Hall | The inheritance of thornlessness from tissue culture-derived 'thornless evergreen'blackberry[END_REF][START_REF] Canli | A review on thornless roses[END_REF][START_REF] Coyner | Thornlessness in blackberries[END_REF][START_REF] Castro | Genetic map of the primocane-fruiting and thornless traits of tetraploid blackberry[END_REF][START_REF] Kariyat | Plant spines deter herbivory by restricting caterpillar movement[END_REF], actually have prickles. For instance, Rosa and Rubus have the most representative prickles. Thus, before conducting any molecular research on this subject, an essential step is to clearly describe their anatomy and to understand their origin. In this paper, we have reviewed the anatomical structure and development of those tissues in order to guide subsequent research by more effectively understanding the difference and connection between them. In this Chapter, the sharp appendages (prickles, thorns and spines) are collectively referred to as 'spinescences'.

Generally, depending on the presence or absence of vascular bundles, we can divide these structures into two categories: (i) trichomes (Figure 1a andb) and prickles (Figure 1c andd) ), which are not vascularized and are generally easy to remove; and (ii) thorns (Figure 1e andf) and spines (Figure 1g) ), which have vascular bundles and cannot be easily separated from the organs that have vascular tissues (spines, usually from leaves, and thorns from stems or shoots) (Figure 1h). [START_REF] Esau | Plant anatomy[END_REF] has defined trichomes as "epidermal appendages of diverse form, structure and functions ...... represented by protective, supporting, and glandular hair, by scales, by various papillae, and by absorbing hairs of roots." The major feature that indicates that these structures are trichomes is that trichomes are epidermal appendages that originate in the protoderm (or epidermis) only. Thus, they are usually distinguished from emergences, which are formed from both epidermal cells and sub-epidermal tissues [START_REF] Werker | The essential oils and glandular hairs in different chemotypes of Origanum vulgare l[END_REF]. However, the boundary between trichomes and emergences is not always clear due to some intermediate forms. In some cases, no sub-epidermal cells take part in the development of a trichome but they are locally differentiated below the trichome [START_REF] Werker | Trichome diversity and development[END_REF]. Coupled with a lack of understanding of anatomy, some authors use "trichome" as a general term for both trichomes and emergences. For instance, [START_REF] Payne | A glossary of plant hair terminology[END_REF] created hundreds of terminology glossaries and described their morphological characteristics in order to distinguish different types of trichomes. This extraordinary undertaking was expected to serve as a reference for researchers concerning the description of trichomes. However, a precise definition of a type of terminology is impossible without ontogenetic studies. Many definitions on his list should be revised according to their anatomical structure. Thus, researchers need to be careful to cite these terms in their reports in order to prevent inappropriate references from creating more confusion. A developmental study is necessary to determine whether some outgrowths are solely epidermal in origin or both epidermal and sub-epidermal in origin. However, such studies are often ignored before assigning a classification.

Trichome diversity

Trichomes vary widely in their final forms and structures, locations, functions, etc. Although no classification method is totally satisfactory, some categories are remarkably uniform in a given taxon and have been used for a long time [START_REF] Metcalfe | Anatomy of The Dicotyledons Vol I[END_REF][START_REF] Netolitzky | Plant Hairs[END_REF][START_REF] Leelavathi | Structure, distribution and classification of plant trichomes in relation to taxonomy iii[END_REF][START_REF] Wagner | Secreting glandular trichomes: more than just hairs[END_REF][START_REF] Werker | Trichome diversity and development[END_REF][START_REF] Evert | Esau's plant anatomy: meristems, cells, and tissues of the plant body: their structure, function, and development[END_REF][START_REF] Osman | Trichome micromorphology of Egyptian Ballota (Lamiaceae) with emphasis on its systematic implication[END_REF].

Depending on their morphology and secretion ability, trichomes are mainly divided into two general categories: "non-glandular" and "glandular". Non-glandular trichomes (NGT) are diverse in their morphology, anatomy and microstructure. They may be unicellular (Figure 2 a and b) or multicellular (Figure 2 c, d ande), and both types can be branched or unbranched. Unbranched multicellular trichomes and the stalk of branched multicellular trichomes can be uniseriate (Figure 2 c), biseriate or multiseriate (Figure 2 d). Within these categories, they may differ in size, shape, length, and may be jagged or smooth. They may also be soft, sinuous or stiff because of variations in cell wall thickness and substances in the cell. Branched multicellular trichomes may have unicellular or multicellular branches, constituting a variety of tufted appearances. Glandular trichomes (GTs) are defined by the presence of cells that have the ability to secrete or store large quantities of secretions [START_REF] Fahn | Secretory tissues in plants[END_REF][START_REF] Fahn | Glandular trichomes of Fagonia l.(Zygophyllaceae) species: structure, development and secreted materials[END_REF][START_REF] Huchelmann | Plant glandular trichomes: natural cell factories of high biotechnological interest[END_REF]. They also can be unicellular (rarely, Figure 2 f) or multicellular (commonly, Figure 2 g, h, i, j and k), branched or unbranched, and have various shapes. Depending on the location of the gland cell or "collecting cell" and the morphology properties, some GTs have been classified as capitate (Figure 2 i and j) and peltate (Figure 2 h) [START_REF] Ascensão | Glandular trichomes on vegetative and reproductive organs of Leonotis leonurus (Lamiaceae)[END_REF][START_REF] Corsi | Glandular hairs of salvia officinalis: new data on morphology, localization and histochemistry in relation to function[END_REF][START_REF] Turner | Distribution of peltate glandular trichomes on developing leaves of Peppermint[END_REF], or as stinging hairs [START_REF] Macfarlane | The stinging properties of Laportea[END_REF][START_REF] Thurston | The morphology and toxicology of plant stinging hairs[END_REF] (Figure 2 k ). Some exceptional cases cannot be simply classified in GT or NGT. For example, some trichomes have both non-glandular and glandular branches (Figure 2 m).

Origin and development of the different trichomes

Trichome development starts at a very early stage of leaf development, often prior to stomatal development, and sometimes even before the leaf primordium can be distinguished. [START_REF] Werker | Trichome diversity and development[END_REF] extensively studied and distinguished the seven different types of trichome development: "(1) One protoderm cell that gives rise to a unicellular (Figure 3 (3) "Twin hairs" that were classified by [START_REF] Metcalfe | Anatomy of the Dicotyledons: Volume II: Wood Structure and Conclusion of the General Introduction[END_REF] as two armed hairs; (4) More than one epidermal cell that gives rise to a multiseriate trichome; (5) Hair development by splitting of cells;

(6) Subepidermal cells, in which one to all the local tissues subtending the epidermis may take part in the formation of a trichome/emergence; (7)"False hairs" that develop by partial disintegration of cell walls."

1)

2)

3)

Figure 3: The origin and development of different types of trichomes. (a1) non-glandular trichome (unicellular and branched) development in wild type of Arabidopsis [START_REF] Hülskamp | Plant trichomes: a model for cell differentiation[END_REF]). (a2) non-glandular unbranched and branched; (a3) trichome development in Origanum vulgare [START_REF] Kintzios | Oregano: the Genera Origanum and Lippia[END_REF]. Glandular trichome development on leaves in (b) Origanum dictamnus L [START_REF] Bosabalidis | Glandular scale development and essential oil secretion in Origanum Dictamnus l[END_REF]. and (c) Sideritis syriaca [START_REF] Karousou | Sideritis syriaca ssp. syriaca: Glandular trichome structure and development in relation to systematics[END_REF]. Reproduced by NN ZHOU and XW DOU.

1.1 Trichomes, prickles, thorns and spines

Prickles

In a glossary of plant hair terminology list, [START_REF] Payne | A glossary of plant hair terminology[END_REF] defined a prickle as "a sharp, rigid, epidermal outgrowth, often relatively massive, as for Rosa; frequently and erroneously termed spine.". In blackberries and raspberries, prickles were described as a stiffer structure, morphologically similar to glandular trichomes and originnating from multiple cellular divisions of the epidermis and a lack of internal vascular material that later became cutinized as hard sharp appendages [START_REF] Peitersen | Blackberries of New England-genetic status of the plants[END_REF][START_REF] Esau | Anatomy of seed plants[END_REF][START_REF] Coyner | Thornlessness in blackberries[END_REF], whereas there was no evidence to support the fact that the origin of prickles is from epidermal cells. Later, a few reports described the prickle's anatomical structure [START_REF] Asano | Growth, structure and lignin localization in rose prickle[END_REF][START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF][START_REF] Li | Studies on anatomical structure and chemical composition in prickles of Rosa hybrida[END_REF][START_REF] Angyalossy | Iawa list of microscopic bark features[END_REF] and suggested that prickles may develop from sub-epidermal or/and epidermal cell layers (regularly confused with trichomes). The conclusion based on microscopic pictures of young prickles is regularly subject to controversy and confusion.

Concerning rose, [START_REF] Asano | Growth, structure and lignin localization in rose prickle[END_REF] described prickles as sharply pointed spines, lacking vascular tissue and growig from tissue under the outer layer of the plant. By analyzing the sections of a tender-soft prickle and a mature-hard one, he observed a cell layer in the young and mature prickles but not in the very early stages of development. This cell layer resembles the abscission layer of deciduous leaves. Considering that the mature prickles are easy to peel off with the fingers, and based on previous observations, the authors suggested that the rose prickles were spines or modified leaves (see section of spines and thorns). This conclusion is not supported by strong evidence. Later, [START_REF] Li | Studies on anatomical structure and chemical composition in prickles of Rosa hybrida[END_REF] suggested that cells in the prickle abscission region were different from cells in the petiole abscission zone by studying the anatomical structure and chemical composition of tender prickles. Then, based on examination of the longitudinal sections of immature prickles and microscopic observations of prickle development, [START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF] defined rose and raspberry prickles as "epidermal tissue, and modified from glandular trichomes by continuing to grow and hardening into the prickle structure". This conclusion is based on the observation of the glandular head on the tip of a prickle, that falls off during prickle development. They added: "if these glandular structures were absent or reduced in size, the resulting cultivars were prickless or almost prickless, respectively".

Based on this, they further hypothesized that secondary metabolites produced in the glandular trichome may play a role in prickle development. Although this argument was supported by other researchers (Ma et al., 2016b;[START_REF] Pandey | Transcriptome analysis provides insight into prickle development and its link to defense and secondary metabolism in Solanum viarum dunal[END_REF]Khadgi and Weber, 2020a), the questions remain: firstly, do prickles originate from epidermal tissues like trichomes? Secondly, why is the origin of prickles is closer to that of glandular trichomes but not non-glandular trichomes? Later, [START_REF] Angyalossy | Iawa list of microscopic bark features[END_REF] defined prickles as "sharp outgrowths from the bark, without vascular tissue", based on examination of longitudinal sections of the young prickles of Polyscias mollis, Piptadenia gonoacantha and Oplopanax horridus. However, there is still no anatomical evidence to support the prickle origin. Moreover, "bark" is an imprecise term that refers to all tissues exterior to the vascular cambium including a number of tissue types that are periderm (composed of the cork, cork cambium, and the phelloderm), cortex (comprised of ground tissues), phloem and epidermis [START_REF] Dickison | Integrative plant anatomy[END_REF][START_REF] Evert | Esau's plant anatomy: meristems, cells, and tissues of the plant body: their structure, function, and development[END_REF]. These tissues are formed at a relatively later stage of stem development, while prickle formation normally takes place earlier (almost simultaneously with leaf primordia) [START_REF] Asano | Growth, structure and lignin localization in rose prickle[END_REF]. Therefore, the definition of prickles needs to be defined in relation

Chapter 1 Introduction and literature review to their origin.

Do prickles originate from which tissue(s) or cell layer(s)? Are epidermal cells involved in the differentiation of parts? To answer these questions, we need to provide anatomical evidence of prickle development, including initiation and later developmental stages, which are still absent.

Thorns and spines

Thorns and spines are usually distinguished based on the modified organ from which they come. Thorns are defined as a sharp-pointed stems or shoots [START_REF] Delbrouck | Die Pflanzen-Stacheln[END_REF][START_REF] Blaser | Morphology of the determinate thorn-shoots of gleditsia[END_REF][START_REF] Michael | Plant Systematics[END_REF]. They can sometimes bear leaves (Figure 1 e, Prunus spinosa, Crataegus laevigata L.), flowers (Ulex europaeus), or be bare (Figure 1 f), and can be unbranched (Crataegus monogyna, Ccrataegus crus-galli) or branched (Gleditsia triacanthos). By comparing the non-thorny and thorny branches of Crataegus laevigata L., Prunus spinosa L. and Pyrus communis L, Pyrus Matus L, Mespilus germanica L, [START_REF] Aubertot | Sur l'anatomie comparée des rameaux polymorphes chez quelques arbustes épineux de la famille des Rosacées[END_REF] described the thorny branches as being shorter than the ordinary ones, growing in all directions (Prunus spinosa), with smaller leaves(Pyrus, Prunus), and more indented lobes (Crataegus). The stipules were often reduced in

Crataegus. Based on anatomical study, one remarkable trait of thorny was the woody conductive tissue [START_REF] Aubertot | Sur l'anatomie comparée des rameaux polymorphes chez quelques arbustes épineux de la famille des Rosacées[END_REF], which consist of a considerable reduction in the vascular network, a reduction that may affect the number of vessels only (Pynus) or their diameter ( which decreased from an average of 50% in Crataegus,).

Delbrouck (1875) classified numerous spinescences and noted that thorns may be produced from normal axillary buds, from supernumerary buds, or from terminal buds. In Gleditsia, thorns are usually branched and bare, represented by the typical three-parted thorn that arises from one axillary meristem. This meristem gives a shoot that will become a thorn. This shoot also bears two additional axillary meristems that can grow later and give rise to two new thorns, leading to the branched thorn [START_REF] Blaser | Morphology of the determinate thorn-shoots of gleditsia[END_REF].

Spines are variously described as leaves or leaflet spines (as in Cactaceae, Ulex europaeus, Phoenix), stipule spines (as in Vachellia xanthophloea), petiolar spines (as in Fouquieria), or parts of leaves (as in Ilex aquifolium) [START_REF] Michael | Plant Systematics[END_REF]. Some authors prefer not to distinguish between thorns and spines because, unlike prickles, they are both usually vascularized [START_REF] Bell | Plant form: an illustrated guide to flowering plant morphology[END_REF]. For instance, Cacti have evolved succulent stems (in order to store water) with specialized buds called areoles. Areoles produce a cluster of spines where the spines are vestigial leaves, sometimes with no vascular tissue visible. Most cacti spines are diverse in terms of form: 'long or short', 'hairlike, papery, hooked, corrugated, or flattened', 'heavy or thin', 'barbed, smooth, or glandular'. Even though some have none as adults (e.g. Ariocarpus, Lophophora, Rhipsalis), they usually have them at the juvenile stage [START_REF] Boke | Developmental morphology and anatomy in Cactaceae[END_REF]. In Bartschella or Opuntia, spine primordia arose in the same manner as leaf primordia, but they both lack lamina and vascular tissue. The spine appears in the spine-leaf transition forms that usually occur on expanding areoles [START_REF] Boke | Histogenesis of the leaf and areole in Opuntia Cylindrica[END_REF][START_REF] Boke | Developmental anatomy and the validity of the genus Bartschella[END_REF].

Apical maturation of spine primordia is both precocious and covers the entire structure. Further growth is restricted to a well-defined basal intercalary meristem in which continuation of growth activity and planes of cell division determine the length, breadth and cross-sectional configuration of the mature structure (Boke, 1.1 Trichomes, prickles, thorns and spines 1980). In non-cacti plants, spines are mostly described at the macroscopic level, and anatomical evidences is rarely. [START_REF] Gallenmüller | Rose prickles and asparagus spines-different hook structures as attachment devices in climbing plants[END_REF] examined the cross and longitudinal sections of young spines in Asparagus setaceus and Asparagus falcatus. They are both composed of different types of tissues, including leaf, stem, axillary bud or lateral shoot. The scale of the different tissues differs between species, with a higher contribution of leaf and lateral shoot tissue in A. setaceus, where a cork layer is formed between leaf and stem tissues of the spines. The cork layer was not observed in spines of A. falcatus.

Many structures cannot clearly be classified in these categories (thorns, prickles or spines). The most important point for biologists is to understand the initiation and development of this structure (origin). The same or similar developmental patterns of organs and tissues may be controlled by similar genetics pathways.

Good knowledge of its structure and development patterns will be an assist for reverse genetics research.

An efficient adaptive strategy to response to biotic and abiotic stress

Superficial tissues (epidermis) and appendages structures (trichomes, spinescences) of plant organs are the first line of plant defense. They play a crucial role against multiple abiotic and biotic stresses. Since they comprise the outermost boundary between the plant and its environment, they mediate a plethora of plant-environment interactions, plant-pathogen interactions and plant-herbivorous interactions, as well as plant-plant interactions. In this section, I will review the protective and defensive role of these appendage structures in plant-environment and plant-herbivorous interactions.

Adaptation to extreme environments

A wide range of hypotheses concerning trichome functions in plant-environment interactions have been reported, but have not always been experimentally tested. Hundreds of different forms of trichomes are found on different organs in different plants and may have a great variety of functions, whereas a number of trichome functions have only been either hypothesized or are totally unknown [START_REF] Werker | Trichome diversity and development[END_REF].

Investigation of the morphological characteristics of plants in extreme environments shows how trichomes play an important role in protecting plants against increasing solar radiation, extreme temperatures, drought and windy environments. The most typical representative examples are alpine plants [START_REF] Körner | Alpine plant life: functional plant ecology of high mountain ecosystems[END_REF] and cacti [START_REF] Nobel | Environmental Biology of Agaves and Cacti[END_REF][START_REF] Nobel | Cacti: Biology and Uses[END_REF], whose younger organs are always covered with high-density trichomes or spinescences, which may remain over the lifetime of the plant.

Plant adaptation to the light environment

In plants, radiation absorptance is a major driver of both carbon and energy balance, two important aspects of plant survival and reproduction in any ecosystem. Regulating the absorptance and reflection of radiation is important for plants living in a variety of light environments [START_REF] Shull | A spectrophotometric study of reflection of light from leaf surfaces[END_REF][START_REF] Billings | Reflection of visible and infrared radiation from leaves of different ecological groups[END_REF][START_REF] Ehleringer | Leaf absorptances of mohave and sonoran desert plants[END_REF][START_REF] Körner | Alpine plant life: functional plant ecology of high mountain ecosystems[END_REF]. Apine plants, in particular, are under heightened selective pressure to

Chapter 1 Introduction and literature review structurally and physiologically adapt to their unusual light environment. Trichomes are one of the essential adaptation structures to control absorptance and reflection radiation [START_REF] Johnson | Plant pubescence: an ecological perspective[END_REF]. In desert and subalpine plants, [START_REF] Billings | Reflection of visible and infrared radiation from leaves of different ecological groups[END_REF] observed consistently higher reflectance in the pubescent leaf exposed to visible spectrum radiation, whereas in soybean lines (varying from glabrous to pubescent), leaf trichomes increase their absorptance and decrease their reflectance through diffuse radiation, and entrap incoming near-IR light [START_REF] Gausman | Light reflectance by leaflets of pubescent, normal, and glabrous Soybean Lines[END_REF]. The optical properties of trichomes in plants vary greatly in response to visible spectrum radiation. For dynamic lights adaptation, the variation in trichomes response is explained by trichome density [START_REF] Ehleringer | Leaf pubescence: effects on absorptance and photosynthesis in a desert shrub[END_REF][START_REF] Ehleringer | Leaf absorptances of mohave and sonoran desert plants[END_REF], and as well as their forms and structures [START_REF] Pierce | The jeweled armor of tillandsia -multifaceted or elongated trichomes provide photoprotection[END_REF][START_REF] Mershon | Linkage between trichome morphology and leaf optical properties in New Zealand alpine Pachycladon (Brassicaceae)[END_REF]. Ultraviolet (UV)-mediated induction of trichome density has been demonstrated using genetic approaches. In A. thaliana, using wild type and several trichome-related mutants (gl1, gis, gis2, zfp8, try82, and gl3), and overexpressing trichome positive regulator lines (35S::GIS and 35S::GIS2), [START_REF] Yan | The responses of trichome mutants to enhanced ultraviolet-B radiation in Arabidopsis Thaliana[END_REF] demonstrated that trichome density significantly increased under UV-B enhanced radiation conditions, suggesting a clear induction of trichome formation by UV-B.

Considering that trichomes absorb UV-B radiation, some authors have suggested that trichomes covering different organs play a protective role against damage from UV radiation [START_REF] Karabourniotis | The dense indumentum with its polyphenol content may replace the protective role of the epidermis in some young xeromorphic leaves[END_REF][START_REF] Karabourniotis | Protective and defensive roles of non-glandular trichomes against multiple stresses: Structure-function coordination[END_REF][START_REF] Skaltsa | UV-B protective potential and flavonoid content of leaf hairs of Quercus ilex[END_REF][START_REF] Ntefidou | Optical properties of hairs during the early stages of leaf development in Platanus orientalis[END_REF][START_REF] Liakoura | Trichome density and its UV-B protective potential are affected by shading and leaf position on the canopy[END_REF][START_REF] Agati | Flavonoids as antioxidants in plants: location and functional significance[END_REF], although no direct evidence has shown that genotypes that lack trichome protection suffer from increased injury by UV-B. Plants have very effective systems of UV-protection and repair because plants in the natural environment rarely show any signs of UV damage [START_REF] Jenkins | Signal transduction in responses to UV-B radiation[END_REF]. The benefits of UV-B to plants may far outweigh the damage. UV-B makes up just a small fraction of the total solar radiation, but it represents a crucial signal that initiates several responses in plants that affect metabolism, development, and viability [START_REF] Jenkins | Signal transduction in responses to UV-B radiation[END_REF]. It has been shown that UV-B-mediated induced morphological and physiological changes in plants as a reinforcement of plant defense in some cases (reviewed by [START_REF] Robson | Re-interpreting plant morphological responses to UV-B radiation[END_REF] and [START_REF] Escobar-Bravo | Interactive effects of UV-B light with abiotic factors on plant growth and chemistry, and their consequences for defense against arthropod herbivores[END_REF]). For example, in an experiment, where no pesticides were applied, [START_REF] Mazza | Perception of solar UV-B radiation by phytophagous insects: behavioral responses and ecosystem implications[END_REF][START_REF] Mazza | Beneficial effects of solar UV-B radiation on soybean yield mediated by reduced insect herbivory under field conditionss[END_REF] found that solar UV-B significantly indirectly reduced insect herbivory. This indirect benefit of UV-absorbtance by plants is mainly due to increased phenolic compounds and trichome density [START_REF] Mewis | UV-B irradiation changes specifically the secondary metabolite profile in broccoli sprouts: induced signaling overlaps with defense response to biotic stressors[END_REF][START_REF] Ðinh | UV-B radiation and 17-hydroxygeranyllinalool diterpene glycosides provide durable resistance against mirid (Tupiocoris notatus) attack in field-grown Nicotiana attenuata plants[END_REF][START_REF] Jeschke | Metabolism of glucosinolates and their hydrolysis products in insect herbivores[END_REF][START_REF] Zavala | Soybean resistance to stink bugs (Nezara Viridula and Piezodorus Guildinii) increases with exposure to solar UV-B radiation and correlates with isoflavonoid content in pods under field conditions[END_REF][START_REF] Escobar-Bravo | Interactive effects of UV-B light with abiotic factors on plant growth and chemistry, and their consequences for defense against arthropod herbivores[END_REF]. Use of the UV-B light component of solar radiation to enhance crop defense against pests and pathogens, as well as crop production, has aroused increasing interest [START_REF] Wargent | From ozone depletion to agriculture: understanding the role of UV radiation in sustainable crop production[END_REF].

Trichome may protect plants from extreme temperatures

Alpine plants that grow in the high alpine zone at high altitudes, always have a high density of trichomes.

This may indicate that these hairs play a certain role in adaptation to extreme conditions and particularly low temperatures. For example, Himalayan snowball plants (Figure 4) are considered to be an extreme form of downy plants. [START_REF] Tsukaya | Thermal insulation and accumulation of heat in the downy inflorescences of Saussurea Medusa (Asteraceae) at high elevation in yunnan, china[END_REF] examined the downy inflorescences of Saussurea medusa and analyzed the temperature within inflorescences after the absorbance of light energy. They found that the downy bracts of S. medusa have two major functions thermal insulation to protect the inside of flowers against cold and the accumulation of heat on the upper surfaces of the inflorescence.

Some trichomes and spinescences may be adapted to drought conditions

Plants growing in arid habitats had higher-density trichomes than similar plants in mesic habitats [START_REF] Ehleringer | Leaf pubescence: effects on absorptance and photosynthesis in a desert shrub[END_REF][START_REF] Ehleringer | Leaf absorptances of mohave and sonoran desert plants[END_REF][START_REF] Fahn | Structural and functional properties of trichomes of Xeromorphic leaves[END_REF][START_REF] Nobel | Environmental Biology of Agaves and Cacti[END_REF][START_REF] Nobel | Cacti: Biology and Uses[END_REF]. [START_REF] Evert | Esau's plant anatomy: meristems, cells, and tissues of the plant body: their structure, function, and development[END_REF] reviewed the studies on plants growing in aird regions and concluded that "an increase in leaf pubescence (hairiness) reduces the transpiration rate by [START_REF] Van Der | ). In AT, DOT5 is required for normal shoot and root development (Petricka et al., 2008). Both rose genes have a specific high expression in the early stage of glabrous stems, suggested that they may function as prickle initiation repressors (33b). RC3G0419900 (RcAS2) encodes ASYMMETRIC LEAVES 2 (AS2) like protein and belongs to LATE-RAL ORGAN BOUNDARIES (LOB) domain family (Supplementary Figure B.6 c). AS2 controls proximaldistal patterning in Arabidopsis petals[END_REF] increasing the reflection of solar radiation, which lowers leaf temperatures; and (2) increasing the boundary layer (the layer of still air through which water vapor must diffuse).". In addition, naturally arid conditions always corresponded to a higher frequency of spinescent plants distribution, indicating that spinescence may be adapted to arid conditions [START_REF] Shmida | Mediterranean vegetation in California and Israel: Similarities and differences[END_REF][START_REF] Nobel | Environmental Biology of Agaves and Cacti[END_REF].

Adaptive defense responses to herbivores

The defense system against herbivores is a large and changeable network and may involve the cooperation among of multiple strategies. To ward off an attack by different types of organisms, plants possess a surveillance system to perceive who attacked them and to thus introduce specific defenses [START_REF] Crawley | Herbivory: The dynamics of animal-plant interactions[END_REF]Van loon et al., 2009;[START_REF] Futuyma | Macroevolution and the biological diversity of plants and herbivores[END_REF][START_REF] Kant | Mechanisms and ecological consequences of plant defence induction and suppression in herbivore communities[END_REF][START_REF] Lev-Yadun | Color changes in old aposematic thorns, spines, and prickles. In Defensive (anti-herbivory) coloration in land plants[END_REF]. For instance, wounding can be chemically identified (Van loon et al., 2009;[START_REF] Karban | Plant sensing and communication[END_REF], plants can then recognize

Chapter 1 Introduction and literature review herbivore attacks for a long time [START_REF] Gagliano | Experience teaches plants to learn faster and forget slower in environments where it matters[END_REF][START_REF] Karban | Plant sensing and communication[END_REF]. Plants can employ bodyguards such as ants [START_REF] Jolivet | Interrelationship between insects and plants[END_REF] and use volatiles to attract the natural enemies of invertebrates to attack herbivores [START_REF] Kessler | Defensive function of herbivore-induced plant volatile emissions in nature[END_REF][START_REF] Lopresti | Chewing sandpaper: Grit, plant apparency, and plant defense in sand-entrapping plants[END_REF]. Different strategies include the production of large and strong fruits, reducing damage to other vital organs of herbivores [START_REF] Janzen | Why bamboos wait so long to flower[END_REF], visual or chemical intimidation of herbivores [START_REF] Lev-Yadun | Aposematic warning coloration in plants[END_REF], use of colors to pretend to be inedible [START_REF] Givnish | Leaf mottling: relation to growth form and leaf phenology and possible role as camouflage[END_REF][START_REF] Lev-Yadun | Aposematic warning coloration in plants[END_REF], ect. Plants have many types of mechanical defenses [START_REF] Lucas | Mechanical defences to herbivory[END_REF], even anisotropic structural defense arrangements, which may cause invertebrate herbivores to leave the plant [START_REF] Vermeij | Plants that lead: Do some surface features direct enemy traffic on leaves and stems?[END_REF]. Spinous plants can inject pathogens into the herbivores, causing injury or death [START_REF] Halpern | Plant biological warfare: thorns inject pathogenic bacteria into herbivores[END_REF][START_REF] Halpern | Chapter 4 -biological warfare of the spiny plant: introducing pathogenic microorganisms into herbivore's tissues[END_REF]. Of course, no single species can possess all of these defenses at the same time. However, the plant kingdom as a whole has a large weapon arsenal. In the following section, I briefly review the role of trichomes and spinescence as a defense against herbivores, including insects and large mammals.

Trichomes

From an evolutionary perspective, trichomes may have appeared much earlier than herbivores. This hypothesis is supported by evolutionary genealogy studies. Thus, non-glandular trichomes (NGTs) are normally thought to have evolved primarily as physiological barriers against extreme environmental conditions, as we discussed in a previous section. However, many types of trichomes may also have co-evolved with herbivores (especially insects), and perhaps the most widespread function of plant trichomes (especially GTs) today is to protect plants from herbivores [START_REF] Levin | The role of trichomes in plant defense[END_REF][START_REF] Johnson | Plant pubescence: an ecological perspective[END_REF][START_REF] Fahn | Secretory tissues in plants[END_REF][START_REF] Howe | Ecological relationships of plants and animals[END_REF]. The earliest evidence for the occurrence of modified trichomes comes from fossils of the late Carboniferous (Stephanian stage, ∼ 290 Mya) and these modified multicellular hairs were glandular trichomes (GTs) [START_REF] Krings | Trichomes of the seed fern blanzyopteris praedentata: implications for plant-insect interactions in the late carboniferous[END_REF][START_REF] Lange | The evolution of plant secretory structures and emergence of terpenoid chemical diversity[END_REF]. [START_REF] Krings | Trichomes of the seed fern blanzyopteris praedentata: implications for plant-insect interactions in the late carboniferous[END_REF] proposed that these GTs possessed a touch-sensitive mechanism that opened the secretory cell when touched. Compared to the plant trichomes of today, they further support the implications of GTs in plant-insect interactions in the Late Carboniferous. Indeed, many insect herbivores, including leaf beetles, leafhoppers, and caterpillars, have been shown to be physically-deterred or incapacitated by trichomes [START_REF] Levin | The role of trichomes in plant defense[END_REF][START_REF] Johnson | Plant pubescence: an ecological perspective[END_REF][START_REF] Ribeiro | Herbivory by chewing and sucking insects on Tabebuia ochracea[END_REF][START_REF] Webster | Leaf pubescence effects in wheat on yellow sugarcane aphids and greenbugs (homoptera: Aphididae)[END_REF][START_REF] Eisner | When defense backfires: detrimental effect of a plant's protective trichomes on an insect beneficial to the plant[END_REF][START_REF] Smith | Plant resistance to insects. Biological and biotechnological control of insects[END_REF][START_REF] Andres | The community-wide and guild-specific effects of pubescence on the folivorous insects of manzanitas Arctostaphylos spp[END_REF]. NGTs may form physical barriers that can reduce or prevent insects from moving on the plant or from feeding, whereas GTs may release various forms of chemical repellents or traps. Numerous studies have reported the negative relationship between trichome density and the rates of herbivore damage [START_REF] Pullin | The stinging nettle, Urtica Dioica, increases trichome density after herbivore and mechanical damage[END_REF][START_REF] Valverde | Defensive role of leaf trichomes in resistance to herbivorous insects in Datura Stramonium[END_REF][START_REF] Handley | Variation in trichome density and resistance against a specialist insect herbivore in natural populations of Arabidopsis thaliana[END_REF], and an induced resistance has been widely observed in plants following damage by insect herbivores [START_REF] Gibson | Glandular hairs providing resistance to aphids in certain wild potato species[END_REF][START_REF] Harvell | The ecology and evolution of inducible defenses[END_REF][START_REF] Karban | Induced responses to herbivory[END_REF][START_REF] Traw | Differential induction of trichomes by three herbivores of black mustard[END_REF][START_REF] Tian | Role of trichomes in defense against herbivores: Comparison of herbivore response to woolly and hairless trichome mutants in tomato (Solanum Lycopersicum)[END_REF]. In Brassica nigra (L.), for instance, leaf trichomes are induced differently in response to different herbivores attacks [START_REF] Traw | Differential induction of trichomes by three herbivores of black mustard[END_REF]. Leaves of plants damaged by Pieris rapae had 76% more trichomes per unit area than control plants, and leaves of plants damaged by Trichoplusia ni had 113% more trichomes. It has been suggested that specialized defense mechanisms in response to herbivores usually involve glandular trichomes [START_REF] Tingey | Potato glandular trichomes[END_REF], and some GTs secret antibiotic compounds that directly intoxicate arthropod 1.1 Trichomes, prickles, thorns and spines herbivores [START_REF] Wang | Transgenic Nicotiana tabacumL. with enhanced trichome exudate cembratrieneols has reduced aphid infestation in the field[END_REF][START_REF] Ranger | Glandular trichome extracts from Medicago sativa deter settling by the potato leafhopper empoasca fabae[END_REF][START_REF] Hare | Biological activity of acyl glucose esters from Datura wrightii glandular trichomes against three native insect herbivores[END_REF]. Molecular engineering of GT biochemistry has been successfully targeted as a measure to promote pest resistance [START_REF] Haudenschild | Molecular engineering of monoterpene production[END_REF][START_REF] Wang | Transgenic Nicotiana tabacumL. with enhanced trichome exudate cembratrieneols has reduced aphid infestation in the field[END_REF][START_REF] Calo | Leaf hairs influence phytopathogenic fungus infection and confer an increased resistance when expressing a Trichoderma α-1,3-glucanase[END_REF], and some plant genes responsible for the antibiotic compounds have been used to engineer microbes that produce specific compounds [START_REF] Ro | Production of the antimalarial drug precursor artemisinic acid in engineered yeast[END_REF]. In some cases, very small insects such as spider mites and aphids use trichomes to protect themselves against their natural enemies like coccinellid beetles [START_REF] Eisner | When defense backfires: detrimental effect of a plant's protective trichomes on an insect beneficial to the plant[END_REF], which means that some insects may be specifically adapted to glandular trichomes [START_REF] Dam | Differences in distribution and performance of two sap-sucking herbivores on glandular and non-glandular Datura wrightii[END_REF][START_REF] Hare | Variable impact of diverse insect herbivores on dimorphic Datura Wrightii[END_REF]. Some insects may utilize specific terpenes to locate their host [START_REF] Roda | Allometric analysis of the induced flavonols on the leaf surface of wild tobacco (Nicotiana attenuata)[END_REF]. Based on this hypothesis, there has been increasing interest in incorporating trichome-based resistance in plant breeding programs [START_REF] Simmons | Entrapment of helicoverpa armigera (hübner) (lepidoptera: Noctuidae) on glandular trichomes of Lycopersicon species[END_REF](Simmons et al., , 2006;;[START_REF] Glas | Plant glandular trichomes as targets for breeding or engineering of resistance to herbivores[END_REF]Simmons and Gurr, 2006). Except for their well-recognized roles in entrapping or impeding small insects, [START_REF] Peiffer | Plants on early alert: Glandular trichomes as sensors for insect herbivores[END_REF] supposed that GTs also function as an early detection system against herbivores.

Following insect movement, GTs release plant defense signals that quickly activate the expression of defense genes' in response to insect movement. Other studies have reported that herbivore feeding induces local and systemic changes in gene expression. For example, van de [START_REF] Van De Wilhelmina | Local and systemic changes in squash gene expression in response to silverleaf whitefly feeding[END_REF] identified squash genes (SLW1 and SLW3) that were systemically induced after silverleaf whitefly feeding. [START_REF] Yoshida | Jasmonic acid control of GLABRA3 links inducible defense and trichome patterning in Arabidopsis[END_REF] suggested GL3 is a key transcription factor of wound-induced trichome formation acting downstream of JA signaling in Arabidopsis.

Spinescence

Spinescence has been proposed as a defense against herbivory. It has been the working assumption of scientists who study ecological interactions between plants and animals [START_REF] Cooper | Effects of plant spinescence on large mammalian herbivores[END_REF][START_REF] Belovsky | Effects of spines and thorns on Australian arid zone herbivores of different body masses[END_REF][START_REF] Burns | Are there general patterns in plant defence against megaherbivores?[END_REF][START_REF] Wilcox | The thorny truth about spine evolution[END_REF]. A study of paleontological fossils offered evidence that spinescent structures may have appeared in the late Silurian (∼ 400 million years ago), which is before the advent of large herbivores [START_REF] Chaloner | The rise of the first land plants[END_REF]. This evidence assumed that the evolution of prickles may be a response to the pressure of herbivorous insects. [START_REF] Kariyat | Plant spines deter herbivory by restricting caterpillar movement[END_REF] later reported that prickles may play a role in deterring insects by restricting caterpillar movement. In the process of co-evolution with herbivores (especially mammals), plants have evolved an impressive diversity of defenses. Many types of spinescence have been produced (or modified) from the shoots, leaves, fruits, pedicels and even roots.

They specifically react against vertebrates rather than against invertebrates or insects, especially thorn and spines (?). After secondary compounds, spinescence has been proposed as the most successful defense strategy against herbivory [START_REF] Belovsky | Effects of spines and thorns on Australian arid zone herbivores of different body masses[END_REF]. Those spinescent structures frequently team up with predatory pathogenic bacteria, fungi, toxic chemicals, volatiles, and coloration, to enhance their attack or defense ability [START_REF] Lev-Yadun | Weapon thorn automimicry and mimicry of aposematic colorful thorns in plants[END_REF][START_REF] Halpern | Plant biological warfare: thorns inject pathogenic bacteria into herbivores[END_REF][START_REF] Hartmann | The lost origin of chemical ecology in the late 19th century[END_REF][START_REF] Halpern | Chapter 4 -biological warfare of the spiny plant: introducing pathogenic microorganisms into herbivore's tissues[END_REF]. A number of diseases caused by injecting pathogenic bacteria and fungi into predators have been reported. [START_REF] Halpern | Plant biological warfare: thorns inject pathogenic bacteria into herbivores[END_REF] identified 58 bacterial isolates from the thorns of Phoenix dactylifera and Crataegus aronia, belonging to 22 different bacterial species, 13 of them known to be pathogenic for animals or humans, such as Bacillus anthracis, Clostridium perfringens and Clostridium tetani. Bacillus anthracis is the causative agent of anthrax, primarily a disease in mammals, including humans [START_REF] Jensen | The hidden lifestyles of Bacillus cereus and relatives[END_REF]. Clostridium perfringens is a ubiquitous pathogen that produces many toxins and hydrolytic enzymes [START_REF] Petit | Clostridium perfringens: Toxinotype and genotype[END_REF].

Clostridium tetani is the causative agent of tetanus, and injuries caused by spinescences have been reported

to cause tetanus in humans in several countries [START_REF] Hodes | Tetanus in ethiopia: analysis of 55 cases from addis ababa[END_REF][START_REF] Pascual | Tetanus surveillance-united states, 1998-2000[END_REF][START_REF] Ergonul | Analysis of the case fatality rate of tetanus among adults in a tertiary hospital in Turkey[END_REF][START_REF] Campbell | Microbiologic characterization and antimicrobial susceptibility of clostridium tetani isolated from wounds of patients with clinically diagnosed tetanus[END_REF][START_REF] Tadele | Clinical profile and outcome of pediatrics tetanus: The experience of a tertiary hospital in Ethiopia[END_REF]. Mycetoma caused by Eumycetoma (fungi) or Actinomycetoma (filamentous bacteria), and sporotrichosis diseases caused by Sporothrix schenckii (fungi) are also referred to as "plant thorn synovitis" and "rose-thorn or rose-gardeners' disease" , respectively. The most common route of infection is the introduction of spores to the subcutaneous cellular tissue through a skin wound [START_REF] Fahal | Mycetoma: a thorn in the flesh[END_REF][START_REF] Barros | Sporothrix Schenckii and Sporotrichosis[END_REF][START_REF] Vásquez-Del-Mercado | Sporotrichosis[END_REF][START_REF] Mahajan | Sporotrichosis: an overview and therapeutic options[END_REF][START_REF] Kieselova | Rose thorn injury[END_REF].

Dermatophytes that cause subcutaneous mycoses are unable to penetrate the skin and must be introduced into the subcutaneous tissue by a puncture wound [START_REF] Willey | Prescott, Harley, and Klein's microbiology[END_REF]. Thus, the physical defense provided by thorns, spines and prickles against herbivores might be only the tip of the iceberg in a much more complicated story [START_REF] Halpern | Plant biological warfare: thorns inject pathogenic bacteria into herbivores[END_REF][START_REF] Halpern | Chapter 4 -biological warfare of the spiny plant: introducing pathogenic microorganisms into herbivore's tissues[END_REF]. These sharp plant structures inject bacteria into herbivores by wounding them, enabling the microorganisms to pass the animal's first line of defense (the skin), and in so doing, may cause severe infections that are much more dangerous and painful than the mechanical wounding itself [START_REF] Lev-Yadun | Color changes in old aposematic thorns, spines, and prickles. In Defensive (anti-herbivory) coloration in land plants[END_REF].

Understanding the evolutionary process of these defense strategies and their potential joint relationship will help biological scientists to better understand the genetic collaboration network hidden behind them.

The genetic and molecular bases of trichome and prickle initiation and development

Concerning the genetic basis of trichome and prickle development, the main results were obtained on trichomes in model plants such as Arabidopsis. In this section, I will review what is known about molecular networks that control trichome initiation and development.

Trichomes have been one of the models for studying plant differentiation and cell factories

As mentioned in the first section, trichomes are mainly divided into two general categories, glandular (GT) and non-glandular (NGT), depending on their morphology and secretion ability. Among all the appendages structures, the studies of NGT are the most systematic and comprehensive, and are mainly done on the unicellular branched trichome of A. thaliana, which has been considered as a model system to study the structure initiation and development at the single-cell level [START_REF] Hülskamp | Plant trichomes: a model for cell differentiation[END_REF]. Over 30 genes involved in the initiation and development of NGTs have been identified and have been used to generate a developmental framework, whereas GTs have been chiefly studied to reveal the biochemical pathways of the compounds they secrete and to advance our understanding of secondary metabolism in plants (Champagne and Boutry,1.2 The genetic and molecular bases of trichome and prickle initiation and development 2013; [START_REF] Lange | Terpenoid biosynthesis in trichomes -current status and future opportunities[END_REF][START_REF] Huchelmann | Plant glandular trichomes: natural cell factories of high biotechnological interest[END_REF][START_REF] Hao | Light-induced artemisinin biosynthesis is regulated by the bZIP transcription factor AaHY5 in Artemisia annua[END_REF][START_REF] Pradhan | Plant glandular trichomes: the natural pesticide factories[END_REF].

Non-glandular trichomes

Depending on the cell's position, each cell perceives different signaling, responds to signaling pathways, and adopts a specific cell fate [START_REF] Larkin | How do cells know what they want to be when they grow up? lessons from epidermal patterning in Arabidopsis[END_REF][START_REF] Schiefelbein | Cell-fate specification in the epidermis: A common patterning mechanism in the root and shoot[END_REF]. The subsequent differentiation of the cell often involves complex changes, e.g., cells may exit the mitotic cycle or enter an endoreplication cycle.

Cellular architecture is modified to meet the functional requirements of the respective cell type, and cell metabolism changes according to its function [START_REF] Hülskamp | Plant trichomes: a model for cell differentiation[END_REF]. A branched unicellular NGT originates from one cell of the protodermal epideris [START_REF] Hülskamp | Genetic dissection of trichome cell development in Arabidopsis[END_REF][START_REF] Werker | Trichome diversity and development[END_REF][START_REF] Hülskamp | Plant trichomes: a model for cell differentiation[END_REF]. This cell only initiates endoreduplication cycles but no mitotic cell division, which causes the cell to increase in size and to branch to form the mature structure [START_REF] Melaragno | Relationship between endopolyploidy and cell size in epidermal tissue of Arabidopsis[END_REF][START_REF] Hülskamp | Genetic dissection of trichome cell development in Arabidopsis[END_REF][START_REF] Folkers | Cell morphogenesis of trichomes in Arabidopsis: differential control of primary and secondary branching by branch initiation regulators and cell growth[END_REF]. In A. thaliana, leaf trichome nuclei have elevated ploidy levels, reaching 4C, 8C, 16C, 32C, and 64C (1C is the nuclear DNA content of the unreplicated haploid genome) [START_REF] Melaragno | Relationship between endopolyploidy and cell size in epidermal tissue of Arabidopsis[END_REF]. The molecular network was deciphered using mutants impaired in NGT initiation and development [START_REF] Marks | Molecular genetic analysis of trichome development in Arabidopsis[END_REF][START_REF] Hülskamp | Pattern formation and cell differentiation: trichomes in arabidopsis as a genetic model system[END_REF]. The genetic of NGT in A. thaliana is well understood as this time and numerous genes have been identified. [START_REF] Hülskamp | Plant trichomes: a model for cell differentiation[END_REF] assumed that only very few genes are trichome-specific and that most genes are related to many cell types and are involved in more general cellular processes.

Gene network controlling unicellular NGT initiation and differentiation

A development framework for unicellular NGT formation has been extensively studied [START_REF] Folkers | Cell morphogenesis of trichomes in Arabidopsis: differential control of primary and secondary branching by branch initiation regulators and cell growth[END_REF][START_REF] Hülskamp | Plant trichomes: a model for cell differentiation[END_REF][START_REF] Balkunde | Chapter ten -trichome patterning in Arabidopsis thaliana: from genetic to molecular models[END_REF][START_REF] Yang | Trichomes as models for studying plant cell differentiation[END_REF][START_REF] Pattanaik | An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis[END_REF]. We will recall here the main transcription factors that control unicellular NGT initiation in A. thaliana, and show how they work together (Figure 5). They generally belong to MYB, bHLH, WD40, WRKY and C2H2 zinc finger proteins families.

A trimeric activator complex (MBW) consisting of MYB (GLABRA1, GL1)-bHLH (GLABROUS3/ ENHANCER OF GL3, GL3/EGL3)-WDR (TRANSPARENT TESTA GL1, TTG1) plays a key role in trichome initiation [START_REF] Schiefelbein | Cell-fate specification in the epidermis: A common patterning mechanism in the root and shoot[END_REF][START_REF] Zhang | A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis[END_REF][START_REF] Hülskamp | Plant trichomes: a model for cell differentiation[END_REF][START_REF] Kirik | Functional diversification of MYB23 and GL1 genes in trichome morphogenesis and initiation[END_REF][START_REF] Pattanaik | An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis[END_REF]. Mutations in both the GL1 and TTG1 genes both lead to the complete absence of trichomes [START_REF] Koornneeff | The complex syndrome of ttg mutants[END_REF][START_REF] Koornneeff | EMS-and relation-induced mutation frequencies at individual loci in Arabidopsis thaliana l. heynh[END_REF], while the gl3 mutant still exhibits fewer trichomes compared to wild-type plants, probably caused by a close homologue, EGL3, that may be able to rescue the failed function of GL3.

In fact, gl3 egl3 double mutants are devoid of trichomes [START_REF] Zhang | A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis[END_REF]. Downstream, the MBW trimeric complex finely regulates the temporal and spatial expression of GLABRA2 (GL2) and TRANSPARENT TESTA GL2 (TTG2), determining the fate and pattern of trichome precursor cells [START_REF] Rerie | The GLABRA2 gene encodes a homeo domain protein required for normal trichome development in Arabidopsis[END_REF][START_REF] Ishida | Arabidopsis TRANSPARENT TESTA GLABRA2 is directly regulated by R2R3 MYB transcription factors and is involved in regulation of GLABRA2 transcription in epidermal differentiation[END_REF][START_REF] Ishida | A genetic regulatory network in the development of trichomes and root hairs[END_REF]. The MBW complex is a hub that integrates different signals that controls trichome initiation. The R3MYB subfamily genes, TRY, CPC, TCL1, ECT1, ETC2 and ETC3, act as negative regulators by competing with GL1 for binding to GL3 [START_REF] Schnittger | Generation of a spacing pattern: The role of TRIPTYCHON in trichome patterning in Arabidopsis[END_REF][START_REF] Scheres | Plant patterning: TRY to inhibit your neighbors[END_REF][START_REF] Esch | A contradictory GLABRA3 allele helps define gene interactions controlling trichome development in Arabidopsis[END_REF]; Wang et al., 2008;[START_REF] Wester | Functional diversity of R3 single-repeat genes in trichome development[END_REF][START_REF] Wang | Regulation of cell fate determination by single-repeat R3 MYB transcription factors in Arabidopsis[END_REF]. try mutants produce trichome clusters whereas all other inhibitors are involved in trichome density regulation [START_REF] Pesch | Role of TRIPTYCHON in trichome patterning in Arabidopsis[END_REF]. The active TTG1 complex, in interaction with TTG2, regulates the expression of the R3 MYB inhibitors that move to the neighboring cells where they repress trichome initiation [START_REF] Pesch | Creating a two-dimensional pattern de Novo during Arabidopsis trichome and root hair initiation[END_REF][START_REF] Hülskamp | Plant trichomes: a model for cell differentiation[END_REF].

GL1

Giberellins

Cytokinins

Upstream, plant growth regulators, such as gibberellin (GA), iasmonic acid (JA) and cytokinin (CK) regulate trichome initiation by regulating transcription upstream of the MBW complex. Different C2H2 zinc finger proteins such as GLABROUS INFLORESCENCE STEM (GIS), GIS2, GIS3, ZINC FINGER PROTEIN5, 6 and 8 [START_REF] Gan | GLABROUS INFLORESCENCE STEMS modulates the regulation by gibberellins of epidermal differentiation and shoot maturation in Arabidopsis[END_REF][START_REF] Gan | Genetic and molecular regulation by DELLA proteins of trichome development in Arabidopsis[END_REF] include GA and cytokinin signaling pathways [START_REF] Zhou | ZINC FINGER PROTEIN 6 (ZFP6) regulates trichome initiation by integrating gibberellin and cytokinin signaling in Arabidopsis thaliana[END_REF]. The novel transcription factor TRP interacts with ZFP5 and negatively regulates trichome initiation through the gibberellic acid pathway [START_REF] Kim | The novel transcription factor TRP interacts with ZFP5, a trichome initiation-related transcription factor, and negatively regulates trichome initiation through gibberellic acid signaling[END_REF]. In addition, Ultraviolet-B-mediated induction can increase trichome density and GL3 expression in the zfp8 and gis mutants [START_REF] Yan | The responses of trichome mutants to enhanced ultraviolet-B radiation in Arabidopsis Thaliana[END_REF]. JASMONATE ZIM DOMAIN1 (JAZ1) protein, an important repressor in the JA signalling pathway is degraded by JA. This degradation releases MBW activity and promotes trichome initiation [START_REF] Qi | The Jasmonate-ZIM-domain proteins interact with the WD-Repeat/bHLH/MYB complexes to regulate Jasmonate-mediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana[END_REF].

Cell-cycle control during unicellular NGT development

The cell cycle is the indispensable process required for organ or tissue development. It consists of four phases: Gap 1 (G1), DNA synthesis (S) and Gap 2 (G2) and Mitosis (M) (Figure 6). Endoreplication (or endoreduplication), is a common cell cycle variant in which cells increase their genomic DNA content without dividing [START_REF] Edgar | Endoreplication cell cycles: more for less[END_REF]. A survey of Arabidopsis revealed polyploidy in unicellular NGT (uNGT) [START_REF] Melaragno | Relationship between endopolyploidy and cell size in epidermal tissue of Arabidopsis[END_REF], and the number of endoreplication cycles determines the ploidy level 1.2 The genetic and molecular bases of trichome and prickle initiation and development and trichome growth; mutants with reduced endoreplication cycles result in smaller trichomes with fewer branches, while increased endoreplication cycles have larger trichomes with more branches [START_REF] Hülskamp | Genetic dissection of trichome cell development in Arabidopsis[END_REF]. As in other eukaryotic organisms, cyclin-dependent kinases (CDKs) control progression of the plant cell cycles [START_REF] Morgan | CYCLIN-DEPENDENT KINASES: Engines, clocks, and microprocessors[END_REF]. Different CDK-cyclin complexes phosphorylate a plethora of substrates at the key 'G1 to S' and 'G2 to M' transitions, triggering the onset of DNA replication and mitosis, respectively.

All eukaryotic organisms studied to date possess at least one CDK with the PSTAIRE hallmark in their cyclin-binding domain. In plants as well, a bona fide PSTAIRE CDK, designated CDKA plays a pivotal role at both the 'G1 to S' and 'G2 to M' transitions. Plants possess a unique class of CDKs, the so-called B-type CDKs (CDKB) that have not been described for any other organism [START_REF] Joubès | Cdk-related protein kinases in plants[END_REF][START_REF] Boudolf | Identification of novel cyclin-dependent kinases interacting with the CKS1 protein of Arabidopsis[END_REF]. Arabidopsis harbors two CDKB1 (CDKB1;1 and CDKB1;2) and two CDKB2 (CDKB2;1 and CDKB2;2) family members. CDKB1 transcripts are accumulatef during the S, G2 and M phases, whereas CDKB2 expression is specific to the G2 and M phases [START_REF] Segers | The Arabidopsis cyclin-dependent kinase gene cdc2bAt is preferentially expressed during S and G2 phases of the cell cycle[END_REF][START_REF] Porceddu | A plant-specific cyclin-dependent kinase is involved in the control of G2/M progression in plants[END_REF][START_REF] Breyne | Transcriptome analysis during cell division in plants[END_REF][START_REF] Corellou | Atypical regulation of a green lineage-specific B-type cyclin-dependent kinase[END_REF]. CDK activity is regulated by phosphorylation and associated with their binding activators and inhibitors [START_REF] Inzé | Cell cycle regulation in plant development[END_REF]. Cyclins, as the activators of CDKs, exist extensively in plants and are classified into many distinct types, regulating the transition between different phases of the cell cycle. B-type cyclins mainly control the transition of G2 to M (reviewed by [START_REF] Ishida | A genetic regulatory network in the development of trichomes and root hairs[END_REF]; [START_REF] Yang | Trichomes as models for studying plant cell differentiation[END_REF]). Ectopic expression of B-Type Cyclin CYCB1;2 and D-Type Cyclin CYCD3:1 in the uNGT genotype induces normal cell divisions resulting in multicellular trichomes (Schnittger et al., 2002a,b), and the latter also increase the DNA content in trichomes. Interestingly, CYCB1;2 is not expressed during wild-type unicellular trichome development whereas it was detected in pGL2::CYCD3;1 and sim mutant trichomes (multicellular type) [START_REF] Schnittger | Ectopic B-Type cyclin expression induces mitotic cycles in endoreduplicating Arabidopsis trichomes[END_REF], suggesting that the expression of CYCB1;2 is inhibited by SIM. The SIAMESE (SIM) gene encodes a putative CDK inhibitor with a key function in the switch from mitosis cycle to endoreplication cycles [START_REF] Churchman | Siamese, a plant-specific cell cycle regulator, controls endoreplication onset in Arabidopsis thaliana[END_REF]. The CCS52A1 gene, which encodes a CDH1/FZR-like protein, is a genetic modifier of the multicellular trichome phenotype of sim mutants. Overexpression of CCS52A1 completely suppresses the sim mutant phenotype, while the ccs52A1 mutation enhances the multicellularity of sim mutant trichomes, supporting the hypothesis that CCS52A1

and SIM cooperate in repressing the accumulation of B-type cyclins to switch the trichome cell from mitotic to endoreplication (no M phase) [START_REF] Kasili | SIAMESE cooperates with the CDH1-like protein CCS52A1 to establish endoreplication in Arabidopsis thaliana trichomes[END_REF].

Two of the cell fate genes described above, GL3 and TRY, also act as positive and negative regulators of endoreplication cycles. Different gl3 mutant alleles showed an astonishingly contrasting function in endoreplication, one that reduced DNA content (smaller NGTs with fewer branches) [START_REF] Koornneeff | EMS-and relation-induced mutation frequencies at individual loci in Arabidopsis thaliana l. heynh[END_REF][START_REF] Hülskamp | Genetic dissection of trichome cell development in Arabidopsis[END_REF] and another one that increased it (oddly shaped NGT with a striking nuclear morphology) [START_REF] Esch | A contradictory GLABRA3 allele helps define gene interactions controlling trichome development in Arabidopsis[END_REF]. The try mutants have NGTs with more branches and increased DNA contents (64C). These pleiotropic effects raise the hypothesis that trichome cell-fate choice is functionally linked to cell-cycle regulation [START_REF] Hülskamp | Plant trichomes: a model for cell differentiation[END_REF]. Cell-cycle control is usually thought to act downstream of the regulatory complex of trichome cell fate. Strikingly, a reduction of endoreplication leads to fewer trichomes, whereas promoting endoreplication in glabrous patterning mutants could restore the initiation of trichomes. Therefore, the endoreplication cycle plays a role in trichome cell fate [START_REF] Bramsiepe | Endoreplication controls cell fate maintenance[END_REF]. [START_REF] Evert | Esau's plant anatomy: meristems, cells, and tissues of the plant body: their structure, function, and development[END_REF]. G1 phase refers to cell size increases and cellular contents duplicated, S phase to DNA and chromosomes replication, G2 refers to cell enlargement and organelles development and proteins synthesis in preparation for cell division. M phase refers to mitosis followed by cytokinesis (cell separation). Reproduced by NN ZHOU.

SIM is most probably a direct target for GL3 for controlling endoreplication during the maintenance of trichome cell fate [START_REF] Morohashi | A systems approach reveals regulatory circuitry for Arabidopsis trichome initiation by the gl3 and gl1 selectors[END_REF].

In addition, gibberellin (GA) signaling may also control endoreplication cycles by regulation of GL1

or its homolog [START_REF] Ishida | A genetic regulatory network in the development of trichomes and root hairs[END_REF]. In spindly (spy) mutants, which exhibit a constitutive GA response, trichomes have eight branches and a high level of endoreplication (64C). In kaktus (kak), rastafari (rfi), polychome (poc) and hirsute (hir) mutants, trichomes all show a very similar phenotype and ploidy level, like in the spy mutant (reviewed by [START_REF] Hülskamp | Plant trichomes: a model for cell differentiation[END_REF]). The KAK gene encodes a member of the HECT ubiquitin-protein ligase family, suggesting that ubiquitin-regulated protein degradation negatively controls the progression of endoreplication and thereby reduces trichome branching [START_REF] Downes | The hect ubiquitin-protein ligase (upl) family in Arabidopsis: Upl3 has a specific role in trichome development[END_REF][START_REF] El Refy | The Arabidopsis KAKTUS gene encodes a HECT protein and controls the number of endoreduplication cycles[END_REF].

A regulatory link exists between the progression of endoreplication cycles (or cell proliferation), programed cell death, and resistance to pathogens. The cpr5 mutant was reported to be constitutively resistant to virulent pathogens. [START_REF] Bowling | The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance[END_REF]. In cpr5 mutant trichomes, endoreplication cycles stop after two rounds instead of four, and the trichomes then die, the nucleus disintegrates, and the cell collapses.

It is therefore suggested that CPR5 is also involved in programmed cell death control and endoreplication cycles [START_REF] Kirik | Cpr5 is involved in cell proliferation and cell death control and encodes a novel transmembrane protein[END_REF]. A similar relationship between cell death and endoreplication cycles is found in transgenic plants that misexpress CDK inhibitor (ICK1/KRP1). ICK1/KRP1 expression in uNGTs reduces endoreplications and induces cell death [START_REF] Schnittger | Misexpression of the cyclin-dependent kinase inhibitor ick1/krp1 in single-celled Arabidopsis trichomes reduces endoreduplication and cell size and induces cell death[END_REF].

1.2 The genetic and molecular bases of trichome and prickle initiation and development

Glandular trichomes

A key and unique characteristic of GTs is their ability to synthesize and secrete large compounds, mainly terpenoids, but also phenylpropanoids, flavonoids, methylketones and acyl sugars [START_REF] Huchelmann | Plant glandular trichomes: natural cell factories of high biotechnological interest[END_REF].

Since no GTs are found in A. thaliana, research on GT has been carried out on various other plant species, but most researchers are committed to elucidating the biosynthetic pathway of the compounds, and the molecular genetic aspects of GTs development are still unclear (see [START_REF] Huchelmann | Plant glandular trichomes: natural cell factories of high biotechnological interest[END_REF] and [START_REF] Chalvin | Genetic control of glandular trichome development[END_REF]). In Nicotiana tabacum, GL1 homologue overexpression did not alter the glandular trichome phenotype of the tobacco plants [START_REF] Payne | Heterologous myb genes distinct from GL1 enhance trichome production when overexpressed in Nicotiana tabacum[END_REF], perhaps indicating a different pathway between GT and NGT. Several studies have recently reported genes and protein complexes that regulate glandular trichome development in A. annua [START_REF] Yan | HOMEODOMAIN PROTEIN 1 is required for jasmonate-mediated glandular trichome initiation in Artemisia Annua[END_REF][START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF][START_REF] Shi | The roles of aa mixta 1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF][START_REF] Hao | Light-induced artemisinin biosynthesis is regulated by the bZIP transcription factor AaHY5 in Artemisia annua[END_REF] and tomato [START_REF] Yang | A regulatory gene induces trichome formation and embryo lethality in tomato[END_REF][START_REF] Nadakuduti | Pleiotropic phenotypes of the sticky peel mutant provide new insight into the tole of CUTIN DEFICIENT2 in epidermal cell function in tomato[END_REF][START_REF] Ewas | Manipulation of SlMXl for enhanced carotenoids accumulation and drought resistance in tomato[END_REF][START_REF] Vendemiatti | Loss of Type-IV glandular trichomes is a heterochronic trait in tomato and can be reverted by promoting juvenility[END_REF][START_REF] Gao | The tomato B-type cyclin gene, SlCycB2, plays key roles in reproductive organ development, trichome initiation, terpenoids biosynthesis and prodenia litura defense[END_REF][START_REF] Chang | Hair, encoding a single c2h2 zinc-finger protein, regulates multicellular trichome formation in tomato[END_REF][START_REF] Xu | SlMYC1 regulates type VI glandular trichome formation and terpene biosynthesis in tomato glandular cells[END_REF]. The majority of regulators belong to the R2R3-MYB and HD-ZIP IV transcription factor subfamilies, and may play essential roles in glandular trichome initiation [START_REF] Chalvin | Genetic control of glandular trichome development[END_REF]. In A.

annua, for example, two members of the R2R3-MYB factors have been characterized as positive regulators of glandular trichome initiation: AaMYB1 (Matías-Hernández et al., 2017) and AaMIXTA1 [START_REF] Shi | The roles of aa mixta 1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua[END_REF].

A homeodomain-leucine zipper (HD-ZIP) IV transcription factor, AaHD8, interactes with a MIXTA-like protein AaMIXTA1 forming a regulatory complex. This complex activates AaHD1 to induce glandular trichome initiation [START_REF] Yan | A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua[END_REF].

Genetic and molecular studies in prickles

A few studies on the quantitative trait locus (QTL) of prickles have been reported, mainly in the genus Rosa and a few in Rubus (Table 1). In the diploid rose population (with a predominance of the Rosa multiflora genotype), the presence of prickles on the stem was assumed to be controlled by a single dominant gene [START_REF] Debener | Genetic analysis of horticulturally important morphological and physiological characters in diploid roses[END_REF][START_REF] Shupert | Inheritance of flower traits, leaflet number and prickles in roses[END_REF]) located on linkage group 3 (LG3) [START_REF] Linde | Powdery mildew resistance in roses: QTL mapping in different environments using selective genotyping[END_REF], whereas two QTLs were detected on LG3 with the scoring of prickle density [START_REF] Crespel | Mapping of qualitative and quantitative phenotypic traits in Rosa using AFLP markers[END_REF] using an F1 population from a cross with a hybrid of Rosa wichurana as the paternal parent. Using two F1 progenies (including a genetic background of Rosa wichurana), we also identified a large QTL (or two neighboring QTLs) on

LG3 (between positions 31 Mb and 46.5 Mb, corresponding to the end of chromosome 3) which was further supported by a GWAS analysis on a diversity panel showing a significant association in positions 31 and 32.4 Mb (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF]. In a tetraploid F1 roses population (referred to as K5), three QTLs were identified in relation to the number of prickles on the stem: two located on LG2 and one on

LG3 [START_REF] Koning-Boucoiran | Specific mapping of disease resistance genes in tetraploid cut roses[END_REF]. Using the same K5 population with the same phenotype data but a new SNP dense genetic map, different QTLs were detected on LG3, 4 and 6 and on LG2 (one year) (Bourke et al., 2018a). In Rubus idaeus, two QTLs were detected on LG4 and 6 (Molina-Bravo et al., 2014).

In roses, the gene network of prickle initiation and development are still largely unknown. No gene has been identified to control prickle formation. Only a WRKY transcription factor (RcTTG2), homologous to Arabidopsis TTG2, was found to be located close to the major QTL of the prickle trait in rose and LG3@K7826_576,

LG4@K5629_995, LG6

Bourke et al. (2018a)

Rubus idaeus NC497 2n \ Scored visually on a scale of 0-5, where 0 is no prickle and 5 is densely covered in prickles

LG4@Rub116a,

LG6@Rub103a [START_REF] Molina-Bravo | Quantitative trait locus analysis of tolerance to temperature fluctuations in winter, fruit characteristics, flower color, and prickle-free canes in raspberry[END_REF] the gene transcripts are differentially accumulated between roses with prickles and roses without prickles [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF].

Rose as a model to study the genetic mechanism of prickles 1.3.1 Rose: a complex genus

Wild roses belong to the genus Rosa in the family Rosaceae, a medium-sized family of flowering plants, including 2825 ∼ 4900 species in 95 ∼ 125 genera, although these numbers should be seen as estimates since much taxonomic work remains to be done (Yu, 1974;Gu et al., 2003;Royal Botanic Gardens and Kew and Missouri Botanic Garden, 2013;[START_REF] Christenhusz | The number of known plants species in the world and its annual increase[END_REF]. The name Rosaceae is derived from the genus type Rosa. Up to 90 species of the Rosaceae family are of economic importance and include the following fruit trees (e.g. apple, pear, plum, cherry, peach, apricot, hawthorn, strawberry, raspberryand blackberry) and ornamental plants (e.g., rose) [START_REF] Longhi | Molecular genetics and genomics of the Rosoideae: State of the art and future perspectives[END_REF].

Several fossils of wild roses have been found in the North America [START_REF] Becker | The fossil record of the genus Rosa[END_REF], Europe (Edelman, 1975) and China [START_REF] Su | A miocene leaf fossil record of Rosa (R. Fortuita n. sp.) from its modern diversity center in SW China[END_REF]. Using time calibration based on fossil records, Fougère-Danezan et al.

(2015) estimated that an early lineage of wild roses evolved during the Eocene-Oligocene 24 MY ago (54 Mya -30 Mya). Today, the genus Rosa is composed of ∼ 200 species (or variants), widely distributed from cold temperate to subtropical regions [START_REF] Rehder | Manual of cultivated trees and shrubs hardy in North America. Manual of cultivated trees and shrubs hardy in North America[END_REF]. A total of 95 species (65 endemic) are distributed in China (Gu and Robertson, 2003).

Rose characteristics and classification

As a member of the genus Rosa, roses have their own characteristics that distinguish them from other genera of the family Rosaceae. In particular, carpels are usually numerous (Figure 7 (Yu, 1974;Gu and Robertson, 2003).

Based on the phenotypic variations, several attempts to classify wild roses have been reported, and

Chapter 1 Introduction and literature review

Rose species as woody perennial shrubs can be climbing, erect or diffuse (Yu, 1974;Gu and Robertson, 2003) (Figure 8). They are distinguished from trees by their multiple stems without clear trunks and their shorter height, usually less than 10 m tall [START_REF] Allaby | A dictionary of plant sciences[END_REF]. Roses grow in all different shapes and sizes, from tiny miniatures only 30 cm high to climbers that can sprawl over a house. A few climbing species can exceed 10 m if they find a support nearby. For example, Rosa banksiae 'Albo Plena' (Lady Banksia Rose) is a native species in China. Unlike most roses, it is prickless or almost prickless. This species can climb to the top of the tree and occupy the entire canopy.

Stems and stem prickles

Rose stems are terete and usually green or red during the first years of growth, and then turn gray/brown when older. Many young stems secrete a thin waxy film that disappears on older stems. Stems are mostly prickly, glabrous or puberulous, and the prickles and bark in some cases peel off from old stems, which can be frequently observed in the platyrhodon subgenus, represented by R. roxburghii and R. minutifolia. Here, we selected some representative genotypes in four subgenera and a few of the sections under subgenus Rosa to show the prickle diversity on stems (Figure 9). Their distribution can be paired at leaf bases (Figure 9 p, q, s and t), unpaired but regularly distributed (Figure 9 g, h and i), randomly scattered (Figure 9 e, l ando ), or densely bristly (Figure 9 b,[START_REF]MM Closest molecular marker (MM) associated. d bp Location in base pair (bp) on the Rosa chinensis Genome v1.0[END_REF]d,j and m). Their shapes present a large diversity; they can be straight, slightly curved or curved, thin-soft or thick-hard, acicular (soft or hard), gradually tapering off to an elliptic base or abruptly flaring into a broad base, glabrous or hairy. These prickle shapes are also varied in size and color. Young prickle colors are normally green, sometimes slight red, bright red, ruby red, and some may even be white (Figure 9 i and j), and the colors usually change to rust, gray, or white when the prickles mature and dry. Some species can be reliably recognized by their prickles' characteristics.

Leaves

The alternating leaves are generally odd pinnate (Figure 9 p, s and t), 5 ∼ 15cm long (except for extreme cases), and the stipules are adnate or inserted at the petiole (Figure 7 c), except for the leaves of R. persica that are simple, sessile and lack stipules (Figure 9 r). They are composed of 5 ∼ 9 leaflets on average with a range that varies from 3 ∼ 17 between species, which is considered as an important character for rose classification. A variation of serrated patterns can often be observed on the leaflet and stipule margins.

Leaflets can be glabrous, or bear pubescence on both surfaces or only on the abaxial surface, and some have glandular emergences. Glandular emergences can often be observed on both sides of the rachis, while prickles are normally only present underneath. usually have five petals and five sepals, are imbricate, except for R. mairei, R. morrisonensis, R. omeiensis, R. sericea, R. sikangensis and R. taronensis, that only has four (Yu, 1974). Some wild variants have multiple petals, such as R. praeluceus and R. banksiae. Petal colors may be white, pink, red, yellow, and rarely green (eg. R. chinensis f.viridiflora, Figure 10 l), and the frequently observed gradient varies from white to pink or white to yellow, depending on the species. Sepals are arranged into two outer, two inner, and one middle sepal, with a the margin that is entirely or variously pinnately lobed, and they sepals usually bear high-density hairs and glandular trichomes or emergences. The understructure that carries petals and sepals is called the hypanthium (Figure 7 a and b), and may be globose, urceolate, or cupular and constricted at the neck. A disc is inserted at mouth of the hypanthium, and there are numerous stamens in several whorls inserted in the disc (Figure 7 a). The numerous carpels (there are rarely few) are free (i.e., not fused together in a single ovary), inserted at the margin or base of the hypanthium. Styles are terminal or lateral, exserted or not, free or connate in the upper part (Figure 7 a) (Yu, 1974;Gu and Robertson, 2003).

Inflorescences and flowers

The history of rose breeding and application in human society

Roses have always been greatly appreciated at different periods of history and in many different civilizations. Rose domestication is usually related to the most prosperous periods of history. As of this time, more than 33,000 roses have been created [START_REF] Young | Modern roses-12: The comprehensive list of roses in cultivation or of historical or botanical importance[END_REF] and roses have been the most economic important ornamental plant in the world.

Ancient rose domestication and human selection

Little archaeological evidence of rose cultivation has been found. Rose seeds along with other fruits and nuts, including Corylus, Pyrus, Crataegus, and Rubus were found at a formally inhabited site of the early Neolithic (5000 B.P.) near Swifterbant in the Netherlands [START_REF] Zeist | Seeds and fruits from the swifterbant s3 site[END_REF]. Humans may have intentionally gathered rose fruit for food. Possible rose seeds have also been described at sites in Switzerland (3500 B.P) and in Britain [START_REF] Renfrew | The prehistoric food plants of the near east and europe[END_REF][START_REF] Widrlechner | History and utilization of Rosa damascena[END_REF].

The origin and domestication of rose are always topics of debate for scientists [START_REF] Hurst | Notes on the origin and evolution of our garden roses[END_REF][START_REF] Widrlechner | History and utilization of Rosa damascena[END_REF][START_REF] Tucker | Identification of the rose, sage, iris, and lily in the "blue bird fresco" from knossos, crete (ca. 1450 bce)[END_REF][START_REF] Wang | A study on the history of chinese roses from ancient works and images[END_REF][START_REF] Chwalkowski | Symbols in arts, religion and culture: The soul of nature[END_REF][START_REF] Bombarely | Roses for darwin[END_REF]Debray, 2020). Although it is reported that the oldest known historical evidence of rose dates back to the Minoan civilization [START_REF] Hurst | Notes on the origin and evolution of our garden roses[END_REF][START_REF] Widrlechner | History and utilization of Rosa damascena[END_REF][START_REF] Tucker | Identification of the rose, sage, iris, and lily in the "blue bird fresco" from knossos, crete (ca. 1450 bce)[END_REF]Ministry of Culture and Sports, 2007;Debray, 2020), this assumption is extremely weak if we carefully analyze the evidence, the restoration of "The Bluebird Fresco" (∼ 1450 B.C.)

by Émile Gilliéron (Figure 11). In the photo, the flower, described as a rose, has six petals, whereas roses have usually five and rarely four petals. Even if there is a debate concerning the number of petals since [START_REF] Tucker | Identification of the rose, sage, iris, and lily in the "blue bird fresco" from knossos, crete (ca. 1450 bce)[END_REF] mentioned that the 6th petal was added during the restoration, we can still deny that hypothesis on the basis of the characteristics of the bud or fruit (not clear from the photo), the plant posture and the leaves. As we described in the previous section, rose flower buds have a hypanthium structure that holds the petals and sepals during the flowering period and from which the fruit develops at the end (Figure 7), but it is clear that the flower in the photo does not have a hypanthium like roses. The number of leaflets of the compound rose leaf is rarely three and the leaves are usually alternating (Figure 9), not opposite (Figure 11). Taking a step back, five-petals is not a specific characteristic for the genus Rosa, since many other plants have five petals, including roses. [START_REF] Hurst | Notes on the origin and evolution of our garden roses[END_REF] suggested that the painted flower is a natural R. × richardii hybrid between R. phoenicia Boiss and R. gallica L., whereas these species are much too different compared to the painted flowers. [START_REF] Tucker | Identification of the rose, sage, iris, and lily in the "blue bird fresco" from knossos, crete (ca. 1450 bce)[END_REF] argues that it is more similar to R. pulverulenta (syn. R. glutinosa) based on the 3-leaflets leaves on flowering shoots and the distribution of its current cultivation, but this species also has five-leaflets and major differences with the flower in the photo. In conclusion, the nature of this painted flower is still a source of debate.

In China, roses were bred for ornamental purposes, with a history that goes back more than 1500 years.

According to the records, there are three names for roses in Chinese, 'Qiángwēi', 'Méigui', and 'Yuèjì', and these names are still used today. 'Qiángwēi' generally refers to wild roses; except for R. rugosa, rugosa varieties and hybrids, which referred to as 'Méigui'. 'Yuèjì' refers to the roses that can bloom every month.

Many poems have highly praised the roses because of their scent, different colors, beautiful posture, and blooming in four seasons. A rose with bright-red petals and a light fragrance was described in the poem 'Yǒng Qiángwēi shī' by Xiè Tiǎo (464 -499). He described the gradient of colors from purple-red to bright red from bud and bloom, with new flowers that bloom at the same time as the old ones (may fruit) on the . A number of poems about roses were written during the Tang Dynasty (618 -907) and Song Dynasty (960 -1279) and were passed on to later generations. To capture the fragrance, we know that people in the Song Dynasty made sachets from roses and carried to cover up body odors, based on the description in the poem 'Yù Méigui', by the poet Sòng Qí (998 -1061). In the poem 'Yǐng zhōu dào zhōng xiàn cì Méigui huā', Xiàng An-shí (1129 -1208) described an fragrance that belonged to a prickly rose that accompanied him passing the Yǐng-zhōu road on a snowy day in early spring. The Yǐngzhōu road may be in the Yǐngzhōu county of the Song Dynasty since the Xiàng An-shi lived nearby, and Yǐngzhōu is currently located in Zhongxiang county in Hubei province. The above poems were obtained by searching for the keyword 'Qiángwēi', 'Méigui', or 'Yuèjì' in the ancient Chinese poems included in the online "Han Dian" (http://sc.zdic.net/) and "Gu shi wen wang" (https://so.gushiwen.org/) sites. There are still many records describing roses in ancient Chinese books. The 'earliest records' and 'impact of human participation on the rose domestication' questions need to be studied with the participation of historians, linguists and botanists. The Ming (1368Ming ( -1644) ) and Qing (1636Qing ( -1912) ) dynasties was a heyday for porcelain development, and many flowers were painted on brightly colored porcelain, including roses (Figure 12). The paintings will help botanists, as a source for classification and tracing of the origin of rose traits based on ancient records. These records are useful information to speculate about the genetic background of ancient Chinese roses which are one of the most important ancestors of modern roses. From the late Qing Dynasty (1840) to Chairman Mao Zedong proclaiming the foundation of the People's Republic of China (1949), invasion and civil war never ceased in China, and rose breeding entered a period of stagnation. It was in this period that rose breeding in Europe was entering in the golden age [START_REF] Liorzou | Nineteenth century french rose (Rosa sp.) germplasm shows a shift over time from a european to an asian genetic background[END_REF]. In Europe, roses were once integrated into the religious culture, and the prosperity and decline of rose planting are always associated with different religious cultures in different periods [START_REF] Joret | La rose dans l'antiquité et au Moyen âge: histoire, légendes et symbolisme[END_REF][START_REF] Touw | Roses in the middle ages[END_REF]Debray, 2020). A recent review of the history of rose culture in Europe was recently published by Debray (2020).

Modern rose selection

In the 18th century, the introduction of Chinese cultivars into Europe was a major landmark in rose breeding history. It changed the history of ancient European roses that can only bloom once or occasionally twice [START_REF] Hurst | Notes on the origin and evolution of our garden roses[END_REF][START_REF] Joyaux | Nouvelle encyclopédie des roses anciennes[END_REF]. According to [START_REF] Joyaux | Nouvelle encyclopédie des roses anciennes[END_REF], R. chinensis may be the first Chinese rose brought into Europe. The evidence is based on a specimen under the name "Chineeshe Eglantier Roosen", which was grown by the Dutch botanist Gronovius in 1733, and probably brought back to Europe through the Dutch East India Company. This specimen looks like the R. chinensis var. spontanea (Rehd. Wils.) Yu Ku.

Another Chinese cultivar probably originating from a nursery in Guangdong (Canton), was introduced into England in the late 18th century. This cultivar is called 'Yuè yuè fěn' in Chinese (which means 'blooming monthly with pink petals'), renamed R. chinensis 'Old Blush' or 'Parson's Pink China' in 1793 by the Colville Nursery. Concurrently, a red cultivar, called 'Yuè yuè hóng', meaning 'blooming monthly with red petals' in Chinese, was planted in Calcutta in 1789, and renamed Rosa chinensis var. semperflorens Koehne in Europe [START_REF] Joyaux | Nouvelle encyclopédie des roses anciennes[END_REF].

Rose as a model to study the genetic mechanism of prickles

At the beginning of the 19th century, these Chinese roses were crossed with old European cultivated roses, and led to the creation of thousands of hybrids (1st generation of hybrids like the Bourbon or Noisette roses and 2nd generation like the recurrent blooming hybrid). In 1867, Jean-Baptiste André Guillot created the first hybrid tea rose, named Rosa 'La France', by crossing Chinese tea-type roses with a hybrid of unknown parentage, but 'Madame Falcot' is considered as a possible parent [START_REF] Beales | Klassische rosen classic roses in German[END_REF]. The 'La France' ushered in a new era of modern roses. From that year on, several thousand of rose cultivars were created and the industry flourished worldwide [START_REF] Joyaux | Nouvelle encyclopédie des roses anciennes[END_REF]. Genetic analysis reveals that during this important breeding period in Europe, the genetic background of cultivated roses has shifted from an old European genetic background to a Chinese genetic background [START_REF] Liorzou | Nineteenth century french rose (Rosa sp.) germplasm shows a shift over time from a european to an asian genetic background[END_REF].

Rose breeding objectives depending on the markets

Currently, roses are major ornamental plants with an important economic value, a wide diversity and diverse application forms, including cut roses, garden roses, roses for urban beautification, house roses in small pots, roses for essential oil, rose teas and roses for culinary purposes. The breeding target of roses is different depending on their application form, and breeding requires different environments for varietal testing.

Cut roses

The global market for cut flowers was estimated at 29.2 billion USD in the year 2020, and is projected to reach a revised size of 41.1 billion USD by 2027, growing at a CAGR of 5% over the analysis period 2020-2027. The rose industry is projected to reach 14.3 billion USD by the end of the analysis period (5.8% growth annually) (ReportLinker, 2020). The cut rose industry involves multiple processes, including rose breeding, variety field testing, propagation, promotion, efficient cultivation, post-harvest processing, transportation logistics, wholesale and retail marketing, etc. For breeding, the main companies are historically located in European countries, especially in France and the Netherlands, and still occupy an important share of the market today. From planting to sales, the Netherlands currently has the most complete production system and the world's largest action market. However, due to costly labor and energy requirements in Europe, European companies are gradually shifting their production bases to areas with low-cost labor and favorable climates (temperature and photoperiod) such as Africa (Kenya), South America (Ecuador, Colombia) and Asia (China, India) [START_REF] Leus | Ornamental crops, Handbook of plant breeding[END_REF]. Production centers are far away from the trading centers and the products earmarked for the consumer require more transfer and packaging, which greatly promotes the development of cold chain transportation. The tolerance of cut roses to storage and transportation is one of the most important factors to assess the market recognition of cut rose cultivars.

Tolerance of rose to transportation helps the product maintain freshness and avoid premature withering during the marketing process. Therefore, vase life has become an important trait to assess the quality of cultivars and for breeding, and many factors affect vase life, such as genetic factors, pre-harvest and post-harvest conditions, storage and transportation, etc. [START_REF] Mortensen | Effects of air humidity, lighting period and lamp type on growth and vase life of roses[END_REF][START_REF] Marissen | Effects of pre-harvest light intensity and temperature on carbohydrate levels and vase life of cut roses[END_REF][START_REF] Dahal | Post harvest handling of cut-flower rose[END_REF]. The number of prickles will undoubtedly largely affect transportation capacity and vase life since Chapter 1 Introduction and literature review removing prickles is an essential step before packaging. This process will inevitably cause branch injuries, with an impact on vase life. Moreover, the injured branches will decrease the ornamental value. Therefore, even if the market requires roses with exceptional ornamental characteristics (form, color, scent), and producers require roses with disease resistance, good yield and long vase life, the presence of too many prickles is not acceptable for cut roses (personal communication Arnaud Delbard, "Société nouvelle des Pépinières Georges Delbard").

Changes in the production area also promote the development of local breeding. For instance, the rapid development of cut flower production in Yunnan province (China) in recent years is remarkable.

Benefiting from favorable climatic conditions and cheap labor, China's Yunnan is now one of the largest production areas for cut roses, and has developed the world's second largest flower auction market (Kunming International Flower Auction Exchange Center) [START_REF] Xu | China's Kunming remains one of world's top flower markets[END_REF]. With the improvement of the laws protecting Plant Variety Rights (PVR), more and more companies and applied research institutes have begun to focus on cultivating new varieties of independent intellectual property rights and strive to carry out market promotion. The promotion and planting of new cultivars in Yunnan are mainly through the "company + production base + farmers" model. Using the radiation effect of the business management model, it was possible to convince growers in the region to adjust the product structure, thereby improving agricultural production efficiency and ensuring increased income for rose farmers. The new cultivars rapidly reach the market and provide substantial economic benefits for enterprises and farmers. The opinion of growers concerning the planting new cultivars has changed from passive acceptance to active demand. The gradual regulation of the payment of PVR fees has attracted international breeders to contribute new outstanding roses to China's market. The production of cut roses in Yunnan has developed from traditional, classic cultivars (single-head and cup-shaped flower buds) to more diverse ones (single or multiple-head buds, rich colors and diversity shaped flower buds). For rose breeders, the market is full of challenges and opportunities and constitutes new area for rose breeding in China (personal communication with Prof. Qi-Gang Wang (FRI, Yunnan Academy of Agricultural Sciences, Kunming, China)).

Garden roses Garden roses have the most abundant application form, but their market is much smaller than that of cut roses. The garden rose market still has much room in which to expand. Floribundas and flowers (color, type, perfume) are important selection traits. However, disease resistance and the absence of prickles are definitely the most important traits that require improvement. Black spot disease (BSD), caused by the hemibiotrophic fungus, D. rosae, is responsible for the presence of round dark spots on leaves, followed by chlorosis and premature defoliation of the host, thus reducing the aesthetics of the host [START_REF] Blechert | Distribution of rose black spot (Diplocarpon rosae) genetic diversity in eastern north america using amplified fragment length polymorphism and implications for resistance screening[END_REF][START_REF] Gachomo | Microscopic evidence for the hemibiotrophic nature of Diplocarpon Rosae, cause of black spot disease of rose[END_REF]. Prickles and other spinescent structures make the plants difficult to plant in gardens or urban greening areas because of their safety risks. Indeed, prickles may cause diseases by infection resulting from stabbing injuries, [START_REF] Vincent | Enterobacter agglomerans osteomyelitis of the hand from a rose thorn: A case report[END_REF][START_REF] Fahal | Mycetoma: a thorn in the flesh[END_REF][START_REF] Halpern | Plant biological warfare: thorns inject pathogenic bacteria into herbivores[END_REF][START_REF] Cruz | Pantoea agglomerans, a plant pathogen causing human disease[END_REF][START_REF] Barros | Sporothrix Schenckii and Sporotrichosis[END_REF][START_REF] Vásquez-Del-Mercado | Sporotrichosis[END_REF][START_REF] Kieselova | Rose thorn injury[END_REF][START_REF] Frothingham | Rose thorns: a risk for bacterial and fungal infection[END_REF]. Thus, for cultivars with the same ornamental value, those with no or only very few prickles may be more popular for using in public urban greening areas. Garden roses with no prickles will provide growers 1.3 Rose as a model to study the genetic mechanism of prickles with more choices.

Indoor roses Roses used as houseplants are mainly miniature type of roses. Miniature roses have become more and more popular in recent years especially in cities, since people have progressively less room (or even room at all) to cultivate plants. Thus, the first breeding target of indoor roses is to change them into smaller than usual species by selecting miniature plants or plants that respond to regularly used plant growth retardants (such as paclobutrazol and chlorocholine chloride). The producers require rose varieties with many flowering branches and a good level of resistance to diseases. Due to insufficient sunlight and ventilation in the room, and because the root system is trapped in the flowerpot, the maintenance of indoor roses is much more difficult than that of garden roses. Spider mites and powdery mildew are always the biggest problems for growers. Benefiting from the high productivity of automated modern greenhouses, a new form of miniature rose was created for consumption in recent years. As for cut flowers, these miniature roses are produced quickly, 4 ∼ 6 cuttings that are assembled into a plastic flowerpot and sold after buds growth. These products are usually eliminated by the consumer after their first blooming.

Oils roses

The cultivation of essential oil roses has a long history with applications that include cultural rituals, aromatherapy, perfumery, body care, ayurveda, flavoring and spiritual traditions [START_REF] Apothecary | Cultural & historical impact of rose oils[END_REF].

Rose oil is extracted from petals. It is an expensive, labor-intensive procedure that takes more than 4000 kg (around 1,600,000 rose blossoms) of rose petals to produce 1kg of 100% pure distilled rose essential oil [START_REF] Lubbe | Cultivation of medicinal and aromatic plants for specialty industrial materials[END_REF]. Consequently, rose oil is one of the most expensive essential oils, and a kilo of good quality oil will fetch around 5000-7000 USD. Only a few species (mainly R.damascena and R. centifolia) are used to produce rose oil and rose absolute. R. rugosa, R. alba, R. bourbonia and R. moschata are also used to produce rose otto and rose absolute, but in limited quantities. The aroma of R. damascena is very rich, deep, sweet-floral, slightly spicy and honey-like. These roses are mainly grown in Bulgaria's Rose Valley and in Turkey. On the other hand, R. centifolia has an aroma that is rich, sweet, deeply floral, spicy, slightly honey, intensely rosy and somewhat woody, and is mainly grown in Morocco. The chemical compounds present in rose oils are different [START_REF] Apothecary | Cultural & historical impact of rose oils[END_REF]. The key fragrance compounds that contribute to the distinctive scent of rose oil, are beta-damascenone, beta-damascone, beta-ionone, and rose oxide, and the presence of a proportion of beta-damascenone is an important criterion for the quality of rose oil [START_REF] Leffingwell | Aroma from carotenoids -rose[END_REF][START_REF] Khan | Leung's encyclopedia of common natural ingredients: used in food, drugs and cosmetics[END_REF]. Even though these compounds make up less than 1% of rose oil, they contribute to more than 90% of the odor due to their low odor detection thresholds [START_REF] Leffingwell | Aroma from carotenoids -rose[END_REF]. What should be mentioned here is that beta-damascenone does not naturally exists in roses. It is formed during distillation from precursors present in the petals. Therefore, the breeding targets for the rose oil industry are the scent components and the increase in plant productivity (number of petals, rose blossoms per plant, compound content, ability to emit scents etc.).

The high quality of rose genome sequences supports the study of roses at the omics level 1.3.3.1 Roses have a complex genome with different levels of ploidy and high heterozygosity

The genus Rosa (Rosaceae) has a base chromosome number of 7 (x = 7) [START_REF] Täckholm | On the Cytology of the Genus Rosa[END_REF][START_REF] Hurst | Chromosomes and characters in Rosa and their significance in the origin of species[END_REF][START_REF] Roberts | DNA amounts of roses (Rosa l.) and their use in attributing ploidy levels[END_REF]Jian et al., 2010b). The ploidy level of wild rose species ranges from diploid (2n =2x = 14) to decaploid (2n = 10x = 70) with almost all levels of even and odd ploidy with around half of the species being diploid. The highest level of ploidy (decaploid) were only found in R. praelucens , a critically endangered alpine rose with an extremely narrow geographic distribution in Northwestern Yunnan, China (Jian et al., 2010a[START_REF] Jian | High genetic diversity and differentiation of an extremely narrowly distributed and critically endangered decaploid rose (Rosa praelucens): implications for its conservation[END_REF]. Its high level of ploidy may be due to inter-specific hybridization, but not directly to auto polyploidization (Jian et al., 2010a). Several intersectional hybridizations reveal the need to consider the genus Rosa as a hybrid system (Debray, 2020). For instance, the hybrid origin of R. spinosissima (syn. R. pimpinellifolia) is derived from a cross between R. sect. Rosa and R. sect. Pimpinellifoliae. Some individuals of the Caninae section are derived from R. sect. Synstylae and R. sect. Rosa. R. marginata is an intersectional hybrid between R. sect. Rosa and R.sect. Caninae (Debray, 2020).

Genetic resources: development of genetic maps from F1 crosses

Molecular markers are useful tools for assessing genetic diversity and for mapping studies. In roses, several genetic maps have been built with microsatellites or simple sequence repeats (SSR), amplified fragment length polymorphism (AFLP) markers, restriction fragment length polymorphism (RFLP) markers, sequence specific amplified region (SCAR) markers and nucleotide binding site (NBS) markers, mostly in diploid crosses [START_REF] Debener | Construction of a genetic linkage map for roses using RAPD and AFLP markers[END_REF][START_REF] Crespel | Mapping of qualitative and quantitative phenotypic traits in Rosa using AFLP markers[END_REF][START_REF] Yan | Construction of an integrated map of rose with AFLP, SSR, PK, RGA, RFLP, SCAR and morphological markers[END_REF][START_REF] Dugo | Genetic mapping of QTLs controlling horticultural traits in diploid roses[END_REF][START_REF] Linde | Powdery mildew resistance in roses: QTL mapping in different environments using selective genotyping[END_REF][START_REF] Oyant | Genetic linkage maps of rose constructed with new microsatellite markers and locating QTL controlling flowering traits[END_REF][START_REF] Terefe | An SSR from the leucine-rich repeat region of the rose Rdr1 gene family is a useful resistance gene analogue marker for roses and other Rosaceae[END_REF]. Genetic maps from four different populations [START_REF] Yan | Construction of an integrated map of rose with AFLP, SSR, PK, RGA, RFLP, SCAR and morphological markers[END_REF][START_REF] Linde | Powdery mildew resistance in roses: QTL mapping in different environments using selective genotyping[END_REF][START_REF] Shupert | Inheritance of flower traits, leaflet number and prickles in roses[END_REF][START_REF] Remay | A survey of flowering genes reveals the role of gibberellins in floral control in rose[END_REF] were firstly integrated into a consensus map by [START_REF] Spiller | Towards a unified genetic map for diploid roses[END_REF], based on four diploid populations and more than 1000 initial markers. This integrated consensus map comprises 597 markers that are distributed over a length of 530 cM on seven linkage groups (LGs). Then later, high-density SNP (single-nucleotide polymorphism) maps were developed and used for anchoring and ordering the rose genome sequences [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF]. These genetic maps were used for genetic mapping and QTL studies in rose stem prickles [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF] and black spot disease [START_REF] Lopez-Arias | Characterization of black spot resistance in diploid roses with QTL detection, meta-analysis and candidate-gene identification[END_REF].

Next-generation sequencing facilitated the creation of transcriptomes and large numbers of SNP markers.

Using the 68K WagRhSNP array, ultra-dense genetic maps have been produced in diploid (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF] and tetraploid rose [START_REF] Vukosavljev | High-density SNP-based genetic maps for the parents of an outcrossed and a selfed tetraploid garden rose cross, inferred from admixed progeny using the 68k rose SNP array[END_REF][START_REF] Bourke | Partial preferential chromosome pairing is genotype dependent in tetraploid rose[END_REF] populations. In diploid rose, two genetic maps, female and male, were built using 151 hybrids obtained from a cross between R. chinensis 'Old Blush' and a hybrid of R. wichurana (OW). There were 5635 and 2331 SNPs for seven LGs of the female and male genetic maps, respectively, which contained 556 and 427 unique loci with a map density of 0.87 and 0.9 markers/cM, respectively (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF]. In tetraploids, SNP array data 1.3 Rose as a model to study the genetic mechanism of prickles currently provides the clearest information on the allele dose of markers. Using the dosage of SNP markers, tetraploid SNP genotypes can be used for linkage analysis with dedicated polyploid mapping software such as TetraploidSNPMap [START_REF] Hackett | TetraploidSNPMap: software for linkage analysis and QTL mapping in autotetraploid populations using SNP dosage data[END_REF] or polyMapR (Bourke et al., 2018c,b). One genetic map was developed using 151 hybrids of the K5 rose population (a cross between the cut roses cultivars 'P540' and 'P867') [START_REF] Bourke | polymapr-linkage analysis and genetic map construction from f1 populations of outcrossing polyploids[END_REF], including 25,695 SNP markers (not unique positions). Another map was built using 177 hybrids obtained from a cross between garden rose cultivars 'Red New Dawn' and 'Morden Centennial' [START_REF] Hackett | TetraploidSNPMap: software for linkage analysis and QTL mapping in autotetraploid populations using SNP dosage data[END_REF]. In addition, 96 diverse tetraploid garden rose genotypes were using for the genome-wide association study (GWAS) of certain traits in roses, such as of the anthocyanin and carotenoid contents of rose petals [START_REF] Schulz | Genome-wide association analysis of the anthocyanin and carotenoid contents of rose petals[END_REF], the number of rose petals and prickle density (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF], root formation in vivo and in vitro (Nguyen et al., 2020a) and callus formation (Nguyen et al., 2020b).

Rose has now entered the area of genomics studies

The first rose genome was released from wild and heterozygous Rosa multiflora [START_REF] Nakamura | Genome structure of Rosa multiflora, a wild ancestor of cultivated roses[END_REF].

However, the quality of the genome was low, with 83,189 scaffolds. Soon afterward, two high-quality genome sequences were published both by sequencing a doubled haploid line from R. chinensis 'Old Blush' [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF][START_REF] Raymond | The Rosa genome provides new insights into the domestication of modern roses[END_REF] .

Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF] used PacBio long-read sequencing, and obtained a total genome length of 512 Mb haploids. These sequences were corrected with Illumine short-read sequencing technologies and anchoring to a high-density genetic female and male maps, which was developed from a diploid OW population, as mentioned in the previous sub-section. Finally, the doubled haploid rose genome assembly was anchored to seven pseudo-chromosomes containing 512 Mb with N50 of 3.4 Mb and 551 contigs (the N50 is defined as the sequence length of the shortest contig at 50% of the total genome length). A total of 95% of the sequence is contained in only 196 contigs. The length of 512 Mb represents 90.1 ∼ 96.1% of the estimated haploid genome size of rose. The genome includes 39,669 protein-coding genes and 4,812 non-coding genes. Evidence of transcription was found for 87.8% of a total 44,481 predicted genes. The predicted non-coding genes included 99 microRNA, 170 small nuclear RNA, 186 ribosomal RNA, 384 small nuclear RNA, 751 transfer RNA, and 3,222 unclassified genes (annotated as non-coding RNA) with evidence of transcription but no consistent coding sequence. [START_REF] Raymond | The Rosa genome provides new insights into the domestication of modern roses[END_REF] also used PacBio long-read sequencing and assembled a total length of 515 Mb with 82 contigs for an N50 of 24 Mb. The seven pseudo chromosomes were built by integrating 86.4% of the 25,695 markers (not all unique positions) of a tetraploid K5 high-density SNP genetic map [START_REF] Bourke | Partial preferential chromosome pairing is genotype dependent in tetraploid rose[END_REF], which was developed from a cross between the cut roses cultivars [START_REF] Koning-Boucoiran | The mode of inheritance in tetraploid cut roses[END_REF]. From an evolutionary point of view, rose is a very interesting model species as it includes species at several ploidy levels as well as having a rich phenotype and a broad genetic diversity. However, owing to the highly heterozygous character, the assembly of a rose genome is always a challenging task, which has greatly limited the progress of related research in rose. The completion of these two high-quality genomes is a starting point that has propelled rose into the area of genomic [START_REF] Smulders | In the name of the rose: A roadmap for rose research in the genome era[END_REF]. Both are publicly available at the GDR [START_REF] Jung | 15 years of GDR: new data and functionality in the genome database for Rosaceae[END_REF] website.

Rationale/issues and thesis objectives

Economic issues

In the agricultural production process, prickles are an undesirable trait, not only in rose but in most crops in general. Roses are among the most popular and economically important horticultural crops, especially cut rose cultivars. Cut roses with prickles are more difficult to handle, harvest and transport, and also constitute safety hazards for consumers and workers. In addition, the tolerance of rose cultivars to storage and transportation is one of the most important factors to assess the market recognition of cut rose cultivars since they are important factors that affect vase life. Prickles on the stem will undoubtedly considerably affect transportation capacity and vase life since removing them is an essential step before packaging. This process will inevitably cause branch injuries, with an impact on vase life and a decrease in ornamental value.

Thus, cultivars with no or very few prickles are in high demand from producers and breeders. Further studies are necessary to develop markers for breeding selection and to identify the molecular bases. In the General Discussion, I will elaborate on how the availability of markers can be used in rose breeding in general and to obtain dedicated roses without prickles.

Scientific issues

Prickles are sharp appendages of plants that was thought to be a defense against insect and mammalian herbivores. Many studies have been carried out concerning on their ecological, evolutionary and biogeographic implications, but the histological origin, genetic and molecular mechanisms underlying prickle initiation and development remain largely unknown. A wide diversity of prickles is present in roses. With the recent development of genetic and genomic resources, rose can be a good model to study the molecular and genetic bases of prickle initiation and development. Our objectives are to decipher the genetic and molecular control of prickle initiation by studying the morphology, genetic determinism, and gene network (at the transcriptomic level) of stem prickles in roses. Through this project, we expect to build a genetic model system for studying prickles and to open new research areas in the plant sciences. We have developed histological, genetic and genomic approaches.

In Chapter 2, we investigated prickle types in wild species and 'Old Blush' × R. × wichurana F1 progeny, and carried out a comprehensive anatomical study for two representative types of prickles. We hope to answer the following questions: What types of prickles exist in roses? Where do the prickles originate from? How do they develop? Which stage leads to prickle absence in glabrous plants?

Rationale/issues and thesis objectives

In Chapter 3, we performed QTL analysis and anchored the SNP markers on the reference genome to determine the credible interval of prickle loci that control the presence and density of prickle on the 'Old Blush' genome. We studied the interaction of the QTL alleles to further discover the genetic background of prickles in the OW population. We used a candidate-gene approach to characterize rose gene homologues known in Arabidopsis, involved in trichome initiation to test the relationship between prickles and trichomes.

Through those studies, we expect to address the following issues: Which loci control the absence/presence of prickles on the rose stem? Which loci impact the prickle density? Do rose prickles share a similar gene network with trichomes?

In Chapter 4, we used a transcriptomic approach to decipher the gene network that controls prickle initiation and later development. We compare the transcriptome of glabrous and prickle shoots at different stages (from initiation and late developmental stages). We explore potential good candidate-genes that are involved in cell fate, cell proliferation, cell differentiation, cell division and cell cycle that are highly associated with prickle initiation. We highlight the best potential regulators of prickle formation by combining the transcriptomic results with genetic studies (Chapter 3). Through these studies, we hope to answer the following questions: Which genes are involved in prickle initiation? and which genes are involved in prickle development?

Experimental design and Results

Chapter 1 Introduction and literature review

The materials, methods, experimental design, and results will be displayed in details in each article of this thesis. The results part is divided in 3 Chapters corresponding to 3 articles. The second one has been accepted for publication in Theoritical and Applied Genetics.

Introduction

Prickles are common in plants [START_REF] Bagella | Thorn, spine and prickle patterns in the Italian flora[END_REF], with mainly knowledge of their function in defense against insects and large mammalian herbivores. The morphogenetic and molecular mechanisms underlying prickle formation and development remain still largely unknown. Although a few reports have described prickle's anatomical structure [START_REF] Asano | Growth, structure and lignin localization in rose prickle[END_REF][START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF][START_REF] Li | Studies on anatomical structure and chemical composition in prickles of Rosa hybrida[END_REF][START_REF] Angyalossy | Iawa list of microscopic bark features[END_REF], their conclusions based on microscopic analysis of prickles in late developmental stages are regularly controversial and confused. The most common assumption is that prickles are originated from multiple cellular division of the epidermis [START_REF] Peitersen | Blackberries of New England-genetic status of the plants[END_REF][START_REF] Esau | Anatomy of seed plants[END_REF][START_REF] Canli | Separation of thornless rose chimeras into their (Rosa sp.) consistent genotypes in vitro[END_REF]. Prickles were considered as modified glandular trichomes, with lignification leading to a hard-sharp appendage [START_REF] Coyner | Thornlessness in blackberries[END_REF][START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF]Ma et al., 2016b;Khadgi and Weber, 2020a), because glandular and non-glandular prickles were considered as different developmental stages of the same prickle. Based on this hypothesis, in rose F1 genotypes with no-, low-and high-density of prickles, (Hibrand-Saint Oyant 2. 1 Introduction et al., 2018) identified and studied the expression of candidate-genes presenting homology with genes from Regulatory Genes Networks, described for the initiation and development of trichomes in Arabidopsis.

In addition, in Rubus, using a transcriptomic approach, a genetic mechanisms of prickles initiation was proposed with similarity with the one described for trichome initiation in Arabidopsis [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF].

However, the origin tissues of prickle are not clearly identified in these studies, no anatomical evidence was provided to support the 'epidermis' origin. In a rose F1 progeny, glandular and non-glandular prickles were demonstrated to have their own developmental processes [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF]. Therefore, in the absence of strong evidence concerning the relationship between prickles and trichomes, cautions should be taken regarding the conclusions.

Another hypothesis was that the rose prickles are spines, defined as modified leaves and lack internal vascular tissues [START_REF] Asano | Growth, structure and lignin localization in rose prickle[END_REF]. The authors proposed that the abscission cell layer of prickle resembles the abscission layer of deciduous leaves, with mature prickles easy to peel off with fingers. This conclusion was not supported by strong evidences. [START_REF] Li | Studies on anatomical structure and chemical composition in prickles of Rosa hybrida[END_REF] suggested that cells in the prickle abscission region were different from cells of the petiole abscission zone by studying the anatomical structure and chemical composition of tender prickles.

Later, [START_REF] Angyalossy | Iawa list of microscopic bark features[END_REF] defined prickles as "sharp outgrowths from the bark, without vascular tissue", based on longitudinal sections through the developed prickle of Polyscias mollis, Piptadenia gonoacantha and Oplopanax horridus. However, the "bark" term is unprecise as it refers to all tissues exterior to the vascular cambium, including a number of tissue types like periderm (composed of the cork, cork cambium, and the phelloderm), cortex (comprised of ground tissues), phloem and epidermis [START_REF] Dickison | Integrative plant anatomy[END_REF][START_REF] Evert | Esau's plant anatomy: meristems, cells, and tissues of the plant body: their structure, function, and development[END_REF]. In vascular plants, the apical meristem give rise to the protoderm, the ground meristem, and the procambium. The protoderm differentiates into the epidermis. Beneath it, the ground meristem and procambium differentiates into the ground tissue (the pith and cortex) and the vascular tissues (the xylem, phloem, and vascular cambium), respectively [START_REF] Evert | Esau's plant anatomy: meristems, cells, and tissues of the plant body: their structure, function, and development[END_REF]. Therefore, the origin of prickles requires further investigations.

Wild roses belong to the genus Rosa in the family Rosaceae. The genus Rosa is composed of ∼ 200 species, widely distributed in cold temperate to subtropical regions [START_REF] Rehder | Manual of cultivated trees and shrubs hardy in North America. Manual of cultivated trees and shrubs hardy in North America[END_REF]. Roses were always popular at different periods and in many civilizations. Today, rose is one of the most economically important ornamental plant in the world. Most roses have prickles on their stems. For cut roses production, removing prickle is an essential step before packaging. This process causes wounding on the stem, largely affect transportation tolerance and vase life, and also reduces the ornamental value. Rose cultivars with a lot of prickles are not accepted for cut roses even if they have other outstanding ornamental traits.

Roses will become a good model plant to study the molecular and genetic bases of prickle initiation and development. Prickles in rose are very diverse with different types, shapes, sizes, density, and colors.

Several high-density SNP-based genetic maps from rose F1 populations [START_REF] Vukosavljev | High-density SNP-based genetic maps for the parents of an outcrossed and a selfed tetraploid garden rose cross, inferred from admixed progeny using the 68k rose SNP array[END_REF][START_REF] Bourke | Partial preferential chromosome pairing is genotype dependent in tetraploid rose[END_REF][START_REF] Lopez-Arias | Characterization of black spot resistance in diploid roses with QTL detection, meta-analysis and candidate-gene identification[END_REF][START_REF] Zhou | Genetic determinism of prickles in rose[END_REF] and GWAS panels [START_REF] Schulz | Genome-wide association analysis of the anthocyanin and carotenoid contents of rose petals[END_REF]Nguyen et al., 2017;[START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF] unpublished data from GDO team) have been used for genetic studies. The recent production of two high-quality genome sequences (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF][START_REF] Raymond | The Rosa genome provides new insights into the domestication of modern roses[END_REF] allows genomics approaches.

For precise genetic and genomic studies, histological approaches are necessary to clearly differentiate the rose shoot structures and identify the tissue of origin of the rose prickle. In this study, our main objectives were to characterize histologically in detail the initiation and development of prickles in roses and to investigate their diversity in terms of form. The major questions are: (i) which types of prickles do exist in roses? (ii) which tissue do prickles originate from? and (iii) how do the prickles develop? A thorough understanding of prickle morphology and anatomical histology in roses will help us to well organize the experiment design for subsequent genetic and genomic studies, and will help to complete the limited data on prickles in roses.

Materials and methods

Plant materials

A diploid OW population, obtained from a cross between the female Rosa chinensis 'Old Blush' (OB) and the male Rosa × wichurana (RW), was grown in a field and managed by the Horticulture Experimental Unit (INRAE, Angers, France). We have selected three once-flowering individuals, OW9068, OW9137 and OW9106, to have vegetative branches. Those genotypes were cut and managed in IRHS greenhouses in November 2017.

Rosa resources were planted in Loubert Rose Gardens (Rosiers sur Loire, France), INRAE (Angers, France) and Flower Research Institute (FRI, Kunming, China). We have selected twelve representative genotypes to perform details analyses of the type and developmental stages of prickles: Rosa ecae, Rosa laxa, Rosa sherardi, Rosa moschata, Rosa omeiensis, Rosa damascena, Rosa rugosa scabrosa, Rosa iwara, 'Grootendorst Supreme', Rosa rubella, 'General Kleber' and 'Parkzauber'. For 110 genotypes (Table 2), we scored the type of prickles on the stems.

Macroscopy and microscopy

The experiments were performed at Plateform IMAC (SFR QuaSav, Angers). Fresh rose stems were photographed with STEREO-MICROSCOPE M205FA-LEICA.

Histology study

Sample dissection was done under microscope to remove the leaves. Various steps were performed:

• Fixation at 4°C: The samples were immersed in the 4% (v/v) glutaraldehyde solution mixed with 0.2 mol/L phosphate buffer at pH 7.2. The volume of the solution must be equal to 50 times the volume of the

Results

sample. The sample were put under vacuum to remove air. The vacuum was broken every 4 minutes. After 2h of vacuum, the glutaraldehyde solution (4% v/v) is changed, the tubes are left 12h at 4 °C, then rinsed twice with phosphate buffer pH 7.2 and stored at 4 °C.

• Dehydration at room temperature: Samples are rinsed 3 times with distilled water, immersed in 50% (v/v) alcohol 10min, 70% (v/v) alcohol 10min, 90% (v/v) alcohol 10min, and 100% alcohol 15min, successively.

• Pre-infiltration: Sample are transferred in pre-infiltration solution (100°alcohol /Technovit® 7100 resin ((Heraeus Kulzer, Wehrhrim, Germany) (v/v)) at 4°C and under vacuum for 2h, then samples are stored for 12h at 4 °C.

• Infiltration: Samples are transferred to infiltration solution (dissolve 1 sachet of hardener I in 100 mL (Heraeus Kulzer, Wehrhrim, Germany) of Technovit ® 7100 resin) under vacuum for 20 minutes minimum at 4 °C, and the tubes are placed for 12h at 4 °C.

• Inclusion: The samples are included using an inclusion solution (1ml of hardener II ® (Heraeus Kulzer, Wehrhrim, Germany) and 15 ml of infiltration solution) and stored at 37 °C. The sections can be made after a week at 37 °C.

The samples were cut in 3 µm sections for anatomical observation using LEICA RM2165 Rotary Microtome. After staining with toluidine blue 1% [START_REF] O'brien | Polychromatic staining of plant cell walls by toluidine blue o[END_REF], they are observed and photographed using an ergonomic system microscope LEICA DM1000.

Score the type of prickle on the 110 roses

Taxonomical nomenclature followed the one described in Yu (1974), Gu and Robertson (2003) and [START_REF] Masure | Guide Des Rosiers Sauvages: 500 Espèces, Variétés et Hybrides Du Monde[END_REF]. Each species or hybrids was associated with the types of prickles that were previously determinate on the OW population and on the twelve representative roses To characterize the prickle types on each species were collected based on photographs which are mainly taken from Loubert Rose Gardens (Rosiers sur Loire, France) and Flower Research Institute (FRI, Kunming, China). For some species, the conclusion is based on professional knowledge and experience, and the photographs online. All the roses and their origin are presented in Table 2.

Results

For the anatomical study, first, we performed a detailed analysis on individuals of the OW progeny (photography and histological studies) as the OW progeny will be studied in details in subsequent genetic and transcriptomic studies. Then, based on these observations, we performed a survey of prickle diversity on the genus Rosa, with a more precise observation on twelve representative genotypes.

Prickles types determination and anatomical study in OW population

Prickle types determination

In the OW population, both parents present prickles on their stems. A very clear separation of prickle traits (type and density) on stem was observed in the F1 hybrids. Based on the macroscopic analysis, we have previously determined three types of prickle on the stems of the OW progeny: (i) the "prickless" referred to the glabrous stems, (ii) the "non-glandular prickle (NGP)" without glands and (iii) the "glandular prickle (GP)" referred to the prickles with glandular head (Figure 13).

We selected three representative genotypes of each type of prickle for the morphological and anatomical studies (Figure 13): * OW9068 is a genotype with glabrous stems. * OW9137 has the most common type of prickles which are non-glandular, unbranched, slightly curved, gradually tapering to a broad base. * OW9106 bears a mixture of prickles, NGPs and GPs.

Based on the macroscopic analysis, we determined three types of prickle on stem for the OW progeny:

(i) the "prickless" referred to the glabrous stems, (ii) the "non-glandular prickle (NGP)" with non-glandular head and (iii) the "glandular prickle (GP)" referred to the prickles with glandular head (Figure 13).

Prickles development and anatomy

In OW9137 and OW9106, we observed that the prickle development is associated with the stem development, it means that the prickle appears early during stem development and prickles develop in parallel with the stem. No new prickle appears on developed stems. According to the specific morphogenetic events during its development, we defined different stages for the development of NGP and GP on rose stem (Figure 13).

Stage I is defined as the prickle initiation and the first outgrowth. The initiation appeares at the very early stage of internode development (probably simultaneously with the first internode, under the petiole (Figure 14 a, white dotted frame)). It appears later than the formation of leaf primordium. The first visible sign of NGP is the cell proliferation of multiple cells of the ground meristem (Figure 14 d). The rapid division of those cells causes an oblique rise to triangular (100∼ 500 µm) which can be observed by macroscope (Figure 13a -stage PI and Figure 14 a). This process was absent in stems of the prickless genotype OW9068: no appendage was observed (Figure 13 c and Figure 15). For GP, 2∼4 cells located at the first (and/or second) layers of the ground meristem firstly appeared to differentiate and to divide (Figure 14 m), they gave rise to a cylindrical bump (around 50 µm) (Figure 14 n; Figure 13 b -stage PI). Subsequently, on the upper part of this new structure, multiple cells of the protoderm (meristem that will give the epidermis) gave

Results

the precursor gland cells (Figure 14 o). This is the main feature to distinguish GP from NGP: in GP, the protoderm (or maybe the epidermis) differentiated into glands cells; this differentiation is absent in NGP, where the protoderm (or the epidermis) only continues to grow by cell division.

In Stage II, both NGP and GP are continuously growing, coloring, and shaping, but the tissue remains juvenile (Figure 13 a and 13 b -Stage PII). The difference between GP and NGP is that the precursor gland cells of GP formed a new structure -glandular head, whereas no such a structure was observed at the tip of the NGP. We have divided stage II into three sub-stages:

For NGP, epidermal cells maintain normal cell proliferation during prickle development (Figure 14 g-l). In stage IIa, prickles are keeping upwards growth (Figure 13 a) due to covered under the unopened leaves. The anatomical analysis showed that the upper part cells (from up to down) of the prickle begin to enlarge (elongation process), suggesting that the cells gradually lose their division ability, while the cells of the lower part may still keep division (small cells) (Figure 14 The NGP and GP enter stage III when they begin to lignify and gradually hardening (Figure 13 a and 13 b -stage PIII). An abscission layer structure-like is also formed (Figure 14 q). Thus, they are easy to be separate from the stem. At the end of this stage, the cells are full-enlarged and lignified. Stage IV is defined as death stage, the NGP and GP completely hardened, lose moisture and cell might die gradually (Figure 13 a -Stage PIV).

Discover different types of prickles in rose resources

To identify and describe the different types of prickles present in the Rosa genus, we conducted a statistical survey of prickle types in 110 wild rose species, varieties or ancient hybrids (Table 2, Figure 16). Twelve representative individuals (highlighted in pink in Table 2), which represent different sections of Rosa classification, were selected for morphological studies. According to the macroscopic observations, we classified the prickles into two general categories: glandular prickles (GP) and non-glandular prickles (NGP), as we previously did for OW individuals. The majority of roses present NGPs (98 out of 110), and all these NGPs are unbranched (Figure 16). Few unbranched NGPs are covering with hairs ("hairy") whereas the majority (91 out of 98) do not have ("naked"). Among the 98 wild roses with NGPs, 17 present unbranched GPs simultaneously which includes 13 naked and 4 hairy. 5 genotypes present only GPs which are branched and naked. 7 roses are glabrous but can sometimes be described with rare NGP (as we mentioned above in NGP categories). Now, I will describe the developmental process of different types of prickles through examples in rose resources.

Unbranched NGPs

For all the 98 wild roses that present NGPs, all these NGPs are unbranched.

Naked

No other appendage grows on the surface of the unbranched NGPs. They are the most common type which were observed in 91 roses out of 110. In the five representative genotypes (R. ecae, R. sherardii, R.

moschata synstylae, and R. omeiensis, Figure 17, 18 and 19), the mature prickles have a large diversity in shapes, colors, sizes, and densities. We found that the early stage of development of the prickles in those species (except the bristles prickle, Figure 19 i-n) are similar to the one previous described for NGP (Figure NGP ( 98)

Unbranched ( 98)

Naked ( 91) Hair ( 7)

Stem prickles in roses GP ( 22)

Branched ( 5) Naked ( 5)

Unbranched ( 17) Naked ( 13) Hair ( 4)

Unbranched GPs

22 of 110 roses present GPs. 17 have unbranched with 13 "naked" and 4 "hairy" GPs. Five roses have branched and "naked" GPs (Figure 16).

Naked

No other appendage grows outside of the unbranched GPs surface. The developmental process and the origin of these prickles have been described in the previous section. We found that these prickles are generally not present alone in roses but always appear with NGPs (Table 2), as in the following species or

Hairy

Unbranched GPs are covering with hairs (or trichomes). Very rare species (4) have this type of prickles.

They are R. bracteata, R. rugosa 'scabrosa', 'Marie Bugnet' and 'Grootendorst Supreme' (two hybrids of R. rugosa) (Figure 20 t and 20 u).

Branched GPs

Branched GPs were only found only in five roses, R. centifolia 'chou', R. centifolia 'muscosa', R.

× damascena 'Quatre Saisons Blanc Mousseux', and two hybrids 'Général Kléber' (Figure 22 a-f) and 'Parkzauber' (Figure 22 g-l). The prickles are naked, no hairy type was found in this sub-category. Interestingly these roses are belonging to a particular type of roses, the moss roses (see discussion).

The development of branched GPs is more complicated than unbranched ones at stage I. Thus, we divided three sub-stages for the stage I. In stage Ia, multiple divisions give rise to a near round protuberance (Figure 22 a). Appearing branch bumps is a sign of entering the stage Ib (Figure 22 a). In stage Ic, the bumps continue to grow and to differentiate into glands and stalk (Figure 22b). The following stages are similar to the unbranched GPs (Figure 22 c-f).

Others

Other cases that are not easy to be classified in the previous categories: One or several small GP(s) can develop on a large NGP (Figure 22 All types of prickle formation will go through the process of initiation, development, and senescence.

Most of prickle do not fall off from stem, but few does (Figure 18 g).

Discussion

New insight in the type of prickles and their origin

The major objectives of this project were to understand prickle morphology, anatomy, and define the developmental stages to serve as the foundation for genetic and molecular studies.

We have found several types of prickles in roses, they can be glandular or non-glandular, branched or unbranched, naked or covering with hairs. We suggested to classify them into two general categories: glandular prickles (GPs) and non-glandular prickles (NGPs), depending on their morphology that presence and absence of glandular structures, respectively. The GPs which are observed in roses, have a glandular head or several glands positioned along the prickle that accompanies them throughout their lifetime, in contrast, NGP have no glandular structures at all. This conclusion is different from previous studies in raspberry and roses: prickles were extensions or modifications of glandular trichomes since the cell mass of glandular fall off the stalk when they reached a certain height in Rubus and Rosa habrid [START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF], and in other species (Ma et al., 2016b).

We also provide anatomical evidence for the very early stages. We revealed that the NGPs and GP are initiated from the ground meristem below the protoderm. For GPs, the main structure (stalk) of prickle originates from the ground meristem, the glandular head originates the protoderm (or epidermis) cells. These conclusions are also different from previous studies, which reported that prickles were origin from epidermal cells [START_REF] Peitersen | Blackberries of New England-genetic status of the plants[END_REF][START_REF] Esau | Anatomy of seed plants[END_REF][START_REF] Coyner | Thornlessness in blackberries[END_REF] and modified from glandular trichomes [START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF]Khadgi and Weber, 2020a), or including from glandular trichomes [START_REF] Pandey | Transcriptome analysis provides insight into prickle development and its link to defense and secondary metabolism in Solanum viarum dunal[END_REF], or from bark tissue [START_REF] Angyalossy | Iawa list of microscopic bark features[END_REF].

Furthermore, the glandular head was not produced at the prickle initiation but during the prickle's development (Figure 14 m-r), which may suggest that GPs might be modified from NGPs. A similar situation is present in the trichome: the earliest glandular trichomes are modified from the non-glandular trichome.

The earliest evidence for the occurrence of modified trichomes (glandular) comes from fossils of the late Carboniferous (Stephanian stage, ∼ 290 Mya) [START_REF] Krings | Trichomes of the seed fern blanzyopteris praedentata: implications for plant-insect interactions in the late carboniferous[END_REF][START_REF] Lange | The evolution of plant secretory structures and emergence of terpenoid chemical diversity[END_REF]. GTs possessed a touch-sensitive mechanism that opened the secretory cell when touched. This mechanism of GT may be important for plant-insect interactions in the late Carboniferous [START_REF] Krings | Trichomes of the seed fern blanzyopteris praedentata: implications for plant-insect interactions in the late carboniferous[END_REF]. The glands of prickle may also play an important role for plant-insect interactions. The GTs on the stem are relatively fewer in rose and found in different species presented large divergence time suggesting that GTs on stem appear several times during rose speciation (data not shown).

Suggestions for the genetic and genomic studies in rose prickles

The difference between GPs and NGPs is also related to their segregation in the OW population, demonstrating that different genetic determinisms are involved [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF]. Therefore, we suggested that they should be studied separately in genetic and molecular studies. Here we focused only on prickle present on the stem. Prickle on petiole, pedicel, and fruit may have different patterns, especially at the initiation stage (see discussion for moss roses).

For reverse genetics and RNAseq approach

According to the specific morphogenetic events during its maturity, we divided NGP and GP development into four stages, and three sub-stages for the stage II. These stages of NGPs have been used for reverse genetics [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF] and RNAseq approach (Chapter 4) to discovery the genes involved in prickle initiation and development.

For forward genetics approach

Quantitative trait locus (QTL) analysis and genome-wide association study (GWAS) are the most popular methods used to reveal the genetic base of quantitative traits. Both methods are achieved by looking for correlation between the phenotype and the genotype. Therefore, how to evaluate the phenotype is an important question for starting these two forward genetic approaches. Here we proposed a rapid way to phenotype the different prickles in rose according to few characteristics (Figure 16). First the presence of glandular head (GP vs NGP), then the presence of branched (unbranched vs branched trichomes) and the presence of hairs on the trichome ('naked' vs 'hairy').

In OW population, both parents only have NGPs, but a few individuals present NGPs and GPs. Coupled with the segregation of GP and NGP showed in different genetic determinisms, suggesting that they should be studied separately (see Zhou et al., 2020, Chapter 3).

Wild rose species or varieties have more diversity of phenotypes than the F1 hybrids such as OW population. This made it more complicated for this trait phenotyping in genome-wide association study (GWAS). Presently, we do not know whether the different types of prickles have the same genetic pathway for initiation or not, or perhaps they are sharing part of the pathway. Thus, the methods of phenotyping must be used by paying attention to the different types of prickles and their anatomy.

Prickless may be more adapted from human selection

We notice that glabrous roses are rare, in our 110 samples, we found only 7 glabrous roses (no GP or NGP). Among them, four were sometimes observed with few prickles, one (R. fraxinifolia Lindl) was described with few prickles occasionally (even by our scoring, no prickle was observed) [START_REF] Masure | Guide Des Rosiers Sauvages: 500 Espèces, Variétés et Hybrides Du Monde[END_REF].

61

Discussion

The majority of glabrous roses are in fact cultivars selected from wild species: R. multiflora 'inermis', R.

wichurana 'Bayses' Thornless', R. pimpinellifolia 'lutea', R. banksiae 'alba plena' and R. banksiae 'lutea'.

Only two wild species (R. banksiae var. normalis, R. fraxinifolia Lindl) were glabrous. It suggests that the glabrous is not adapted for roses in wild, and again highlight that prickle is a beneficial and important trait in rose domestication. Perhaps the absence of prickle is due to a mutation like the selection for the recurrent blooming. So, the hypothesis is that the mutant has been selected and rescue by humans. It could explain the rare genotypes without prickles found in the wild. Those glabrous roses are interesting material for genetic and genomic studies.

Branched GPs in moss roses

The branched GPs are also rare, only presented in 5 roses among the 110: R. centifolia (Rosa Chou) (Inconnu, < 1595), R. centifolia 'muscosa' (< 1700), R. × damascena 'Quatre Saisons Blanc Mousseux', 'Général Kléber' and 'Parkzauber' (1956) [START_REF] Nédelec | Roses grandeur nature: la collection des roses loubert[END_REF]. Interestingly these roses are all moss roses.

Moss roses are covered with a mossy growth on flower pedicel and calyx. They are old garden roses belonging to the subgenus Rosa sect. Caninae DC [START_REF] Masure | Guide Des Rosiers Sauvages: 500 Espèces, Variétés et Hybrides Du Monde[END_REF]. R. × centifolia 'muscosa' may be obtained by bud-mutation. R. × damascena 'Quatre Saisons Blanc Mousseux' may be a sport or bud-mutation of R. × damascena 'bifera' which is a repeat-blooming hybrid of R. × damascene [START_REF] Caissard | Chemical and histochemical analysis of 'quatre saisons blanc mousseux', a moss rose of the Rosa× damascena group[END_REF]. R. × damascena 'Quatre Saisons Blanc Mousseux' was the first repeat-blooming cultivar in moss roses [START_REF] Caissard | Chemical and histochemical analysis of 'quatre saisons blanc mousseux', a moss rose of the Rosa× damascena group[END_REF]. The exact genetic relationship between R. × damascena and R. centifolia are still unclear. The origin of the moss roses is also unknown. R. × damascena (< 1245) only presented unbranched GPs and NGPs on stem and leaves, suggesting that the branched GPs on the stem may obtain from R. × centifolia but not R. × damascena. +F means the genotype have this type of prickle, but only few.

- - + - + FRI R. beggeriana + - - + - + FRI R. bella + - - + - + FRI R. caudata + - - + - + FRI R. chengkouensis + - - + - + FRI R. corymbulosa + - - + - + FRI R. davidii + - - + - + FRI R. davurica + - - + - + FRI R. fedtschenkoana + - - + - + FRI R. forrestiana + - - + - + FRI R. fraxinifolia lindl - - - - - - LRG R. giraldii + - - + - + LRG R. laxa retzius + - - + - + LRG R. macrophylla + - - + - + FRI R. majalis plena + - - + - + LRG R. marretii + - - + - + LRG R. moyesii + - - + - + LRG R. multibracteata + - - + - + FRI R. murielae + - - + - + FRI R. oxyacantha + - - + - + FRI R. pendulina +F - - +F - +F LRG R. pinetorum + - - + - + WS2 R. prattii + + - + - + FOC R. pseudobanksiae + - - + - + FRI R. rubella + - - + - + LRG R. rugosa 'scabrosa' + + - + + - LRG R. sertata + - - + - + FRI R. sweginzowii + - - + - + LRG Continued......

63

Discussion

Pink shows the twelve representative species we used in the macroscopy study.

Green shows the glabours or almost glabours genotypes.

LRG Loubert Rose Gardens (Rosiers sur Loire, France).

INRAE Institut de Recherche en Horticulture Et Semences, Angers, France.

FRI Flower Research Institute, Yunnan Academy of Agricultural Sciences, Kunming, China.

FOC Flora of China (http://www.iplant.cn/foc).

WS1 web sourceshttps://fr.wikipedia.org/wiki/Rosa_minutifolia.

WS2 web sourceshttp://www.elkhornsloughctp.org/factsheet/factsheet.php?SPECIES_ID=97.

WS3 web sourceshttps://www.wikiwand.com/en/Rosa_pimpinellifolia.

Introduction

credible interval of the detected QTLs. Transcript accumulation analysis weakly supports the involvement of trichome homologous genes, in the molecular control of prickle initiation. Our studies provide strong evidence for a complex genetic determinism of stem prickle and could help to establish guidelines for glabrous rose breeding. New insights into the relationship between prickles and trichomes constitute valuable information for reverse genetic research on prickles.

Keywords Trichome, QTL, ZFP5, GIS2, MYB61, MYC1

Introduction

Rose is the major ornamental plant worldwide with a wide diversity, diverse application forms and an extensive cultivated area. Roses are sold as cut flowers, garden plants, in pots, for essential oil, flower tea and culinary purposes. In past centuries, with the continuous efforts of breeders, more than 33,000 varieties of roses were created [START_REF] Young | Modern roses-12: The comprehensive list of roses in cultivation or of historical or botanical importance[END_REF]. However, most of these varieties have persistent prickles on the stem. Prickles can protect against herbivores by deterring them from eating the stem [START_REF] Ronel | The spiny, thorny and prickly plants in the flora of Israel[END_REF][START_REF] Burns | Are there general patterns in plant defence against megaherbivores?[END_REF]. Furthermore, prickles can be desirable in roses when they are used in hedges to protect properties (as was the case in Reunion Island during the 19th century). However, garden roses without prickles are often desirable. Cut roses with prickles are more difficult to handle, harvest and transport and also constitute safety hazards for consumers and workers. Retailers commonly remove prickles from stems prior to sale. Removing the prickles increases labor costs and causes mechanical damage to the stems, which affects vase life and ornamental value. Although a strong market demand to develop roses without prickles exists [START_REF] Nobbs | American rose annual[END_REF][START_REF] Debener | Genetic analysis of horticulturally important morphological and physiological characters in diploid roses[END_REF][START_REF] Canli | A review on thornless roses[END_REF][START_REF] Canli | A review on thornless roses[END_REF]Skirvin, 2003, 2008), relatively little is known about the genetic and molecular bases of prickle initiation and development.

In plants, prickles are described as outgrowths of the epidermis and subjacent layers that lack vasculature, and mainly consist of lignin, suberin, cellulose and hemicellulose [START_REF] Asano | Growth, structure and lignin localization in rose prickle[END_REF][START_REF] Li | Studies on anatomical structure and chemical composition in prickles of Rosa hybrida[END_REF]. In rose and raspberry, it was thought that prickles were modified glandular trichomes that differentiate at the time of lignification into their final prickle morphologies [START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF].

Until recently, only a few studies had been published about the molecular regulation of prickle development, but great progress has been made in trichome initiation and development, especially in Arabidopsis. A trimeric activator complex consisting of MYB (GLABRA1) -bHLH (GLABROUS3/ENHANCER OF GL3) -WDR (TRANSPARENT TESTA GL1) plays a key role in trichome development [START_REF] Zhang | A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis[END_REF][START_REF] Kirik | Functional diversification of MYB23 and GL1 genes in trichome morphogenesis and initiation[END_REF][START_REF] Patra | Ubiquitin protein ligase 3 mediates the proteasomal degradation of GLABROUS 3 and ENHANCER OF GLABROUS 3, regulators of trichome development and flavonoid biosynthesis in Arabidopsis[END_REF]. This trimeric complex finely regulates the temporal and spatial expression of GLABRA2 (GL2) and TRANSPARENT TESTA GL2 (TTG2), determining the fate and pattern of trichome precursor cells [START_REF] Rerie | The GLABRA2 gene encodes a homeo domain protein required for normal trichome development in Arabidopsis[END_REF][START_REF] Ishida | A genetic regulatory network in the development of trichomes and root hairs[END_REF]. The bHLH family genes, MYC1 and TT8, belong to the same clade as GL3. AtMYC1 acts as a positive regulator of trichome initiation [START_REF] Symonds | Natural allelic variation defines a role for ATMYC1: Trichome cell fate determination[END_REF][START_REF] Zhao | A single amino acid substitution in IIIf subfamily of basic Helix-Loop-Helix transcription factor AtMYC1 leads to trichome and root hair patterning defects by abolishing its interaction with partner proteins in Arabidopsis[END_REF], and AtTT8 controls trichome development on leaf margins [START_REF] Maes | Functional specialization of the TRANSPARENT TESTA GLABRA1 network allows differential hormonal control of laminal and marginal trichome initiation in Arabidopsis rosette leaves[END_REF]. AaMYB1 and its orthologue AtMYB61, belonging to the R2R3MYB subfamily, were thought to affect terpene metabolism and trichome development in A. annua and A. thaliana, respectively (Matías-Hernández et al., 2017).

The active TTG1 trimeric complex can be repressed by R3 MYB subfamily genes: TRY/CPC/TCL1 act as negative regulars by competing with GL1 for binding to GL3 [START_REF] Wang | Terpene biosynthesis in glandular trichomes of hop[END_REF][START_REF] Wester | Functional diversity of R3 single-repeat genes in trichome development[END_REF][START_REF] Wang | Regulation of cell fate determination by single-repeat R3 MYB transcription factors in Arabidopsis[END_REF]. The active TTG1 complex, in interaction with TTG2, regulates the expression of the R3 MYB inhibitors that move to the neighboring cells where they repress trichome initiation [START_REF] Pesch | Creating a two-dimensional pattern de Novo during Arabidopsis trichome and root hair initiation[END_REF][START_REF] Pesch | Arabidopsis TTG2 regulates TRY expression through enhancement of activator complex-triggered activation[END_REF].

Different growth regulators positively affect trichome initiation, such as GA3, cytokinin and jasmonic acid [START_REF] Traw | Interactive effects of jasmonic acid, salicylic acid, and gibberellin on induction of trichomes in Arabidopsis[END_REF], through the activation of GL1 [START_REF] Gan | GLABROUS INFLORESCENCE STEMS modulates the regulation by gibberellins of epidermal differentiation and shoot maturation in Arabidopsis[END_REF]. Different C2H2 zinc-finger proteins such as GLABROUS INFLORESCENCE STEM (GIS), GIS2, GIS3, ZINC FINGER PROTEIN5, 6 and 8 [START_REF] Gan | GLABROUS INFLORESCENCE STEMS modulates the regulation by gibberellins of epidermal differentiation and shoot maturation in Arabidopsis[END_REF][START_REF] Gan | Genetic and molecular regulation by DELLA proteins of trichome development in Arabidopsis[END_REF] include GA and cytokinin signaling pathways [START_REF] Zhou | ZINC FINGER PROTEIN 6 (ZFP6) regulates trichome initiation by integrating gibberellin and cytokinin signaling in Arabidopsis thaliana[END_REF]. The novel transcription factor TRP interacts with ZFP5 and negatively regulates trichome initiation through the gibberellic acid pathway [START_REF] Kim | The novel transcription factor TRP interacts with ZFP5, a trichome initiation-related transcription factor, and negatively regulates trichome initiation through gibberellic acid signaling[END_REF].

In diploid rose, the presence of prickles on the stem was assumed to be controlled by a single dominant gene [START_REF] Debener | Genetic analysis of horticulturally important morphological and physiological characters in diploid roses[END_REF][START_REF] Shupert | Inheritance of flower traits, leaflet number and prickles in roses[END_REF]) located on linkage group 3, LG3 [START_REF] Linde | Powdery mildew resistance in roses: QTL mapping in different environments using selective genotyping[END_REF]. Furthermore, two QTLs were detected on LG3 with the scoring of prickle density [START_REF] Crespel | Mapping of qualitative and quantitative phenotypic traits in Rosa using AFLP markers[END_REF]. Using two F1 progenies,Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF] also identified a large QTL (or two neighboring QTLs) on

LG3 (between position 31 Mb -46.5 MB corresponding to the end of the chromosome 3) and a significant association between position 31 and 32.4 Mb using a GWAS approach. In tetraploid roses, three QTLs were identified in relation to the number of prickles on the stem: two located on LG2 and one on LG3 [START_REF] Koning-Boucoiran | Specific mapping of disease resistance genes in tetraploid cut roses[END_REF]. Using the same K5 population with the same phenotype data but a new genetic map, different QTLs were detected on LG3, 4 and 6 and on LG2 (one year) (Bourke et al., 2018a).

Recently, a WRKY transcription factor, homologous to Arabidopsis TTG2, was located close to a QTL controlling prickle density, and the gene transcripts are differentially accumulated between prickle and prickless roses (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF].

In this study, our objectives were to decipher the genetic determinism of stem prickles in rose and to characterize candidate genes involved in prickle initiation and development. First, we defined the different types of prickles on the stem and studied them separately. Using an F1 progeny, we detected QTLs and their position in the rose genome sequence. We further analyzed how the alleles of the major QTLs affect the presence of prickles. We identified putative candidate genes (homologues of genes involved in trichome initiation and development inArabidopsis) and studied their transcript accumulation. That study suggested that prickles and trichomes may carry two different genetic pathways, providing new insights into the relationship between prickles and trichomes.

Materials and methods

Materials and methods

Plant materials

A progeny of 151 diploid F1 hybrids obtained from a cross between the female Rosa chinensis 'Old Blush' (OB) × the male R. × wichurana (RW) was used for map construction (described in (Hibrand-Saint

Phenotypic data collection and analyses

To score prickle density, we selected three independent floral and main stems for each F1 progeny and the two parents. The prickle numbers were counted for each selected stem on four internodes (located in the middle of the stem) for three years (2016, 2017 and 2018).

Statistical analysis and visualization were performed using R version 3.2.3. We visualized the frequency distribution and Q-Q plot using the 'hist', 'legend', 'qqnorm' and 'qqline' functions. We performed mixed--factorial ANOVA analysis with 'aov'. A 'shapiro.test' was used to test the normality of the original data and the ANOVA residuals. When the null hypothesis was negated, 'kruskal.test' was used to test if there was any significant difference between the replicate shoots, years and the type of stem variance. 'pairwise.wilcox.test' with 'p.adjust.method =BH' was used to calculate pairwise comparisons between group levels with corrections for multiple testing. We displayed the distribution of prickle density with a boxplot to compare the difference between the variance using the ggplot2 and ggpubr packages.

Variance components were estimated with the restricted maximum likelihood (REML) method using the sommer package. Phenotype variance components of prickle density were obtained using the following model:

P ijlr = µ + G i + S l + Y (l)j + GS il + GY ij + ε ijlr (3.1)
Where P ijlr is the phenotypic value of a trait counted on a triplicate stem r of the stem type l of the individual i in the year j, µ is the overall mean, G i is the random effect of genotype i, S I is the random effect of stem type l, Y (l)j is the random effect of year j nested in stem type l, GS il is the random interaction between genotype i and stem type l, GY ij is the random interaction between genotype i and year j, and ε ijlr is the random residual error.

The phenotypic variance (σ 2 P ) of stem prickles was divided into the variance of genotypic effect (σ 2 G ) genotype × year interaction (σ 2 GY ), genotype × stem type interaction (σ 2 GS ), and the residual error variance (σ 2 E ).

Narrow-sense heritability (h 2 ) was calculated as follows:

h 2 = σ 2 G /(σ 2 G + σ 2 GY /y + σ 2 GS /s + σ 2 E /ysr) (3.2)
Where y is the number of years, r is the number of replication shoots per individual, and s is the number of stem types (PF and PM).

Genotypic data

The genetic determinism was conducted using the genetic map previously obtained by Hibrand-Saint

QTL Analysis

In this study, we performed QTL detection for prickles on the floral (PF) and main (PM) stems from data scored in 2016, 2017, 2018 (referred to as PF2016, PF2017, PF2018, PM2016, PM2017 and PM2018, respectively). QTL analyses were carried out using the R/qtl in R version 3.2.3. Based on the non-normal phenotype distribution data, single QTL analysis and LOD scores were calculated using the 'scanone'

function with non-parametric model (model='np', ties.random = FALSE, method = 'em') and the two-part model (model='2part', upper = FALSE) [START_REF] Boyartchuk | Multigenic control of listeria monocytogenes susceptibility in rice[END_REF].

In the non-parametric model, the genome-wide and chromosome-wide significance thresholds of LOD scores were estimated by permutations tests (n.perm = 1000, n.cluster = 20). The Bayesian credible interval was computed with 0.95 and 0.99 coverage probabilities. When QTLs for different traits had overlapping 0.95 credible intervals, they were declared to be a potentially "common QTL (cQTL)" [START_REF] Kawamura | Quantitative trait loci for flowering time and inflorescence architecture in rose[END_REF]. The percent of variance explained by each QTL was calculated by 'makeqtl' and 'fitqtl' with a 'normal' model.

In the two-part model, the phenotype was separated into two parts: first, the trait value was considered as without (0) or with [START_REF] Van Der | ). In AT, DOT5 is required for normal shoot and root development (Petricka et al., 2008). Both rose genes have a specific high expression in the early stage of glabrous stems, suggested that they may function as prickle initiation repressors (33b). RC3G0419900 (RcAS2) encodes ASYMMETRIC LEAVES 2 (AS2) like protein and belongs to LATE-RAL ORGAN BOUNDARIES (LOB) domain family (Supplementary Figure B.6 c). AS2 controls proximaldistal patterning in Arabidopsis petals[END_REF] prickles; if it had prickles, the trait value above zero was assumed to be normally distributed. Three LOD scores for each genomic position were calculated: LOD(p) and LOD(µ) were calculated for binary traits (0 or 1) and non-zero phenotype quantitative traits (> 0), respectively; LOD(p,µ)

is simply the sum of the LOD scores from the two separate analyses [START_REF] Broman | Mapping quantitative trait loci in the case of a spike in the phenotype distribution[END_REF]. The genome-wide significance thresholds of three LOD hypotheses were also estimated by 1000 permutation tests and summarized by a 0.05 alpha threshold. The percent of variance explained was calculated by 'makeqtl' and 'fitqtl'

with 'binary' and 'normal' models for binary(p) and quantitative(µ) traits.

Selection of rose candidate genes involved in prickle density

Proteins involved in trichome initiation and development were selected in A. thaliana from the TAIR database (https://www.arabidopsis.org) with searching terms GL1, MYB82, MYB61, CPC, TRY, GL3, TT8, MYC1, TTG1, TTG2, ZFP5, ZFP1, GIS2, GIS3, GL2. Rose homologues were searched using BLASTp in the Rosa chinensis Genome v1.0 (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF]. In addition, we also searched the transcription factors (TF) belonging to the bHLH, WD40, R2R3MYB, C2H2 and WRKY families in rose and which were located on the major cQTL interval of LG3. Using Geneious 9.1.7, 'Multiple Align' was performed for the family gene sequences. Conserved domains were used to build phylogenetic trees using the 'Geneious Tree Builder' tool with the Jukes-Cantor genetic distance model and the UPGMA tree build method. The rose candidate genes were named according to the following nomenclature corresponding to Rc (for Rosa chinensis) added to the corresponding gene name in Arabidopsis, e.g., RcTTG2 for the rose TTG2 homologue.

Gene expression analysis

Primers were designed using Primer Premier 5.0 software. To ensure the specificity of the primers, forward and reverse primers were designed in the last exon and in the beginning (first 100 bp) of the 3'UTR.

Primer length was between 18 and 25 bp, product length was between 70 and 200 bp, GC content was between 40% and 60%, and the annealing temperature was 58 ∼ 65 ℃. Primers are listed in Supplementary Table A.1. For the qPCR experimental design, we selected four contrasting once-flowering individuals from the OW progeny for prickle density: two with no prickle (OW9067 and OW9068) and two with prickles (OW9137 and OW9071 with means of 2.5 and 4 prickles per internode on the main stem, respectively). The materials were sampled in April 2018 in a greenhouse (three biological replicates). Stems were harvested at different stages of prickle development for roses with prickles, and stems at the same stages for roses without prickles (Chapter 2, Figure 13). Total RNA was extracted using the NucleoSpin RNA Plus-XS kit for early stages (I and IIa) and using the NucleoSpin RNA Plus-kit for later stages (IIb, IIc and III) according to the manufacturer's instructions, with minus modifications (2% PVP40 in lysis buffet). The purity of the RNA was checked on 1% agarose gel, and the concentration was measured by an UV spectrophotometer. cDNA was obtained from 500 ng of total RNA using iScriptTM Reverse Transcription Supermix for RT-qPCR (Bio-Rad, Hercules) accordant to the manufacturer's instructions. The purity and quality of the cDNA were checked by performing PCR amplification with a blank and RW's DNA sample control, and the concentration was measured with a UV spectrophotometer. RT-qPCR reactions were performed using the SsoAdvanced TM Universal SYBR ® Green Supermix (Bio-Rad) on the CFX Connect Real-time PCR system (Bio-Rad). The gene efficiency was evaluated with a serial dilution of the thirty cDNAs pooling (1: 10, 25, 50, 100, 250, 500, 1000). A 1: 25 dilution of each cDNA was used to analyse the expression pattern of ten candidate genes and two reference genes UBC and TCTP [START_REF] Randoux | Gibberellins regulate the transcription of the continuous flowering regulator, RoKSN, a rose TFL1 homologue[END_REF]. Data collection was performed using the Bio-Rad CFX Maestro1.1. Amplification efficiency of the ten genes ranged from 90.5 ∼ 104.1%. The reference genes UBC and TCTP presented high expression stability in all the samples.

For the technical replicates, potential outliers were excluded from the analysis when the standard deviation (SD) of samples is higher than 0.5.

Results

Type, distribution and genetic variability of stem prickles in OW progeny

Both parents of the F1 progeny ('Old Blush' and R. x wichurana) present prickles on their stems (Figure ) showed that the NGP densities on stems in the F 1 population were not normally distributed. We tried to transform data (log10, SQRT, box-cox) to make them normal but without success. The Kruskal-Wallis test reveals a genotype effect, a year effect and a stem effect (Table 3). The high heritability (h 2 0.97) demonstrated that the genetic analyses of stem prickle of this population were reliable (Table 3).

Results

OB(NGPs) X RW(NGPs)

Glabrous( 14) Prickles( 137)

NGPs and GPs ( 27) NGPs( 110) Prickless( 14

σ 2 G SE G σ 2 GY SE GY σ 2 GS SE GS σ 2 E SE E h 2 (%)

QTL analysis

Non-parametric QTL analysis

For the female and male maps, strong QTLs were detected on LG3 for the two types of stems and for the three years (Figure 24 and Table 4). The LOD scores are higher for the male map (between 8 and 11.5) and relatively lower for the female map (between 2.3 and 6.2). These QTLs explained between 6.65 to 37.4% of the phenotypic variance. The locations of these QTLs are very close. Indeed, on the female map, the marker at the peak of the QTLs is the same for both types of stems (Rh12GR_16570_782, 51.1 cM, located on the chr3 at 44,459,262 bp according to the Rosa chinensis Genome v1.0 (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF]), except for PM2018 (Rh12GR_34665_95, 45.7 cM, located on chr3 at 41,401,120 bp). On the male map, for the two types of stems and for the three years, the marker with the highest LOD for the QTLs detected on LG3 is the same, Rh12GR_52506_1218 (42.6 cM on the LG3, 42,317,122 bp on Chr3), which is the terminal marker on the genetic map but not on the physical map.

Furthermore, if we consider the common 0.95 Bayesian credible interval of these QTLs on LG3 on the female and male maps, all intervals are overlapping (Table 4 and26). For the female map, the interval on

LG3 was 40.38 ∼ 53.75 cM, which corresponds to the interval 36,517,224 ∼ 46,440,369 bp on the physical map of chr3 (Figure 26 a), and for the male map, the interval on LG3 was 37.69 ∼ 42.55 cM, corresponding to the interval 41,648,024 ∼ 42,317,122 bp on the physical map (Table 4, Figure 26 b).

On LG4, QTLs were only detected on the female map for the main stem for the three years (Figure 24, Table 4). The peak marker Rh12GR_60129_183 located at 30.6 cM, which is located on chr4 at 52,239,028 bp, explained 10.35 to 13.18% of the observed variance depending on the year of the phenotypic variance in the single QTL model. The common 0.95 credible interval on LG4 was 20.53 ∼ 48.59 cM (Figure 26 a), which covered from 46,189,407 ∼ 56,107,784 bp on the physical map (Table 4).

On LG6, QTLs were only detected on the male map for three years for PM and for two years (2017 and 2018) for PF (Figure 24 andTable 4). For PM (2016, 2017 and 2018) and PF (2017), the peak marker is the same, Rh12GR_56601_1304 (29.7 cM, located on chr6 at 31,814,891 bp). For PF2018, the peak marker is Rh88_37299_454 (11.5 cM, located on chr6 at 5,410,244 bp). These QTLs explained between 5.28 and 8.45% of the observed variance. The common 0.95 credible interval was from 15.59 to 42.49 cM, which covered from 8,578,645 to 44,264,630 bp on the physical map (Figure 26 b,Table 4).

On LG1, QTLs were only detected on the male map for PF for two years (2016 and 2018), and explained 6.52 and 6.99% of phenotypic variance, respectively. The common 0.95 credible interval was at 12.78 ∼ 44.11 cM, which covered from 20,231,658-62,553,371 bp on the physical map (Figure 26 b,Table 4).

We checked the interaction between OB3@Rh12GR_16570_782 and OB4@Rh12GR_ 60129_183, and between RW3@Rh12GR_52506_1218 and RW6@Rh12GR_56601_1304, and no significant interaction was detected. LOD( 2016) LOD( 2017) LOD( 2018)

Chapter 3 Genetic determinism of prickles in rose

Threshold( 2016) Threshold( 2017) Threshold( 2018) a.

Linkage group b.

Two-part QTL analysis

In order to extend the analysis even further, we performed a two-part QTL analysis to test the penetrance (presence/absence of prickles, LOD(p) were calculated with binary traits) and the severity (density of prickles on stems with prickles, LOD(µ) were calculated with non-zero quantitative phenotype) of these QTLs.

For the hypothesis LOD(p) on the female and male maps, we obtained a significant LOD(p) on the LG3

for the two types of stem (PF and PM) and the three years (Figure 25, Supplementary Table A.2). The marker with the highest LOD score on the OB map is the same: Rw35C24 (SSR marker) located at 44.4cM (Chr03: 40,215,502 bp). This QTL explained 13.38% to 16.72% of the variation. The peak marker on the RW map is also the same for PF and PM for the three years: Rh12GR_52506_1218 located at 42.6 cM (42,317,122 bp). This QTL explained 20.69 to 33.21% of the variation. These data suggested that the QTL detected on

LG3 mainly controls the presence/absence of prickles. Moreover, the LOD(p) on LG2 and LG6 for the male map were only significant in PF2016 and PM2016, respectively (Figure 25), and they showed a weak effect with an explanation of 1.80% and 2.70% of the variance, respectively (Supplementary Table A.2).

For the LOD(µ) hypothesis, we detected a significant QTL on the female map on LG4 for PM (2016 and 2017) and PF (2016) (Figure 25, Supplementary Table A.2). The QTLs explained 9.02% to 9.88% of the observed phenotypic variances. Therefore, this QTL might be involved in the control of prickle density. On

LG3, a significant QTL was detected on the male map for PM (2016,2017,2018) and PF ( 2016), suggesting that a QTL on LG3 might also control prickle density. This QTL is in the same region of the QTL detected for penetrance (Figure 25, Supplementary Table A.2). On LG1, the LOD(µ) peaks in OB (PM2018) and in RW (PF2018) were higher than the genome-wild threshold (µ); these QTLs explained 6.66% and 7.80%, respectively, of prickle density variation.

The interaction of the LG3-QTL allele between OB and RW

Based on non-parametric and two-part methods, we identified QTLs for the presence of prickles on LG3

for the OB and RW maps in the same region. To further investigate how the alleles on these QTLs affect the presence of prickles, we visualized the number of prickles for each genotype in the hybrid population depending of the Mendelian distribution of the SNP markers at the LOD peak (Figure 27). The female and male alleles are referred to as a,b and c,d, respectively. The separation ratio ac: ad: bc: cd in offspring is 33: 54: 16: 48, and was significantly different from the expected segregation of 1: 1: 1: 1 (37.5 for each) with a p-value = 0.004 estimated by a chi-squared test (Figure 27).

For PF and PM in all three years, we clearly see that the bd allele combination in hybrids is correlated with no-prickle individuals or individuals with only a few prickles (less than two on four internodes), whereas ac, ad and bc genotypes present prickles (Figure 27). These results suggest a dominant/recessive model for this QTL with the b and d alleles linked to the null or recessive alleles (prickless mutant) and the a and c alleles linked to the dominant alleles (prickles). For PM, a co-dominant effect can be detected since the phenotype for ac is significantly different from the one for ad and bc (ac > ad and ac > bc, p-value < 0.05,

OB4

a.

Continue to the next page.... Figure 26: Common QTLs (cQTLs) and candidate genes in (a) the female linkage groups 3 and 4, and (b) the male linkage groups 1, 3 and 6. Areas highlighted in pink, blue and yellow on the linkage groups represent the 0.95 Bayesian interval of cQTL for specific PF, PM and both, respectively. Bars and lines on the right of each chromosome represent 0.95 and 0.99 Bayesian intervals of the QTL with a different color for NGPs on the floral stem and the main stem (pink and blue, respectively). The red markers are the peak of the QTL. Brown markers are SSR markers and black markers are SNP markers.

Results

3670420-64122872

a PT Genome-wild LOD significance threshold was defined by a permutation test. b LG@positon Chromosomal linkage group, using the separate map (OB and RW) numbering of (Hibrand-Saint Oyant et al., 2018) @ peak location in cM. c r(%) Percentage of explanation. d MM Closest molecular marker (MM) associated. e bp Location in base pair (bp) on the Rosa chinesis Genome v1.0 (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF]. f cM CentiMorgan position of QTL peak.

Genotype

PrickleDensity_PM2018 ad( 54) bc( 16) bd( 48) ac( 33 The interaction of the different alleles of the LG3 QTL between OB and RW. Genotype: ac, ad, bc, bd (number of individuals), a/b and c/d alleles belong to females and males, respectively. For the phenotype, the mean values of prickle density for PF and PM for the three years are presented. Some individuals are highlighted with green dots (OW9106 and OW9107), blue dots (OW9062, OW9021, OW9052 and OW9109) and red dots (OW9067 and OW9068). The asterisk indicates that the difference is significant with a p-value of less than 0.05

Results

except for PM2016 between ac and ab; Figure 27), even if the effect is weak (no large difference between the mean for ac and ad/bc). For PF, no co-dominant effect was detected.

We also observed some odd phenotypes. For instance, OW9067 and OW9068 (red dots) had no prickle and were grouped in the ad genotype, perhaps due to recombination between the marker and the prickle locus (Figure 27). For individuals with the bd genotype, six individuals (blue and green dots) always have prickles: OW9062, OW9021, OW9052 and OW9109 (blue dots) look like the usual prickle genotypes and are probably caused by recombination, but the two extreme exceptions, OW9106 and OW9107 (green dots) with the highest prickle density are not that easy to clarify. Moreover, some individuals exist with both prickly and glabrous stems in the same plant.

Candidate genes in the QTL interval region and gene expression analysis

Candidate gene characterization and location in rose

Since it was proposed that prickles originate from a deformation of glandular trichomes in rose [START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF], we looked for rose homologues of transcription factors (TF) known to be involved in the molecular control of trichome initiation and development in Arabidopsis. The information from 15 TFs such as the bHLH (basic helix-loop-helix), C2H2 Zinc-Finger, MYB, WD40 repeat and WRKY families are presented in Supplementary Table A.3. For a more detailed annotation, we performed phylogenetic analyses on these protein families (Supplementary Figure A.2).

Concerning the bHLH family (Supplementary Figure A.2 a), RC7G0190300, RC1G0342400 and RC6G-0407800 showed strong similarity with GLABROUS3, MYC1 and TT8, respectively, where all of the proteins are in the same clade. They are referred to as RcGL3, RcMYC1 and RcTT8, respectively.

For the C2H2 family, RC3G0150000, RC4G0390900 and RC4G0476500 are closely related to GLABR-OUS INFLORESCENCE STEMS proteins (GIS, GIS2 and GIS3) and ZINC FINGER PROTEIN (ZFP5, 6 and 8). RC3G0150000 seems to be more closely related to GIS2, RC4G0390900 to GIS3 and RC4G0476500 to ZFP5. RC2G0415300 and RC6G0454700 are related to ZFP1 and ZFP3 and AT5G10970. They are referred to as RcZFP1-like1 and RcZFP1-like2, considering that they are closer to ZFP1 (Supplementary

R2R3 MYB and R3 MYB belong to the MYB family (Supplementary Figure A.2 c). In the R2R3 MYB

sub-family (blue sub-tree), RC7G0156100 is in the same clade as GLABROUS1, whereas RC2G0033100 and RC7G0261400 are more closely related to MYB82 and TT2, respectively. RC3G0322900 is in the same clade as MYB61, MYB50 and MYB86. In the R3 MYB sub-family (red sub-tree), RC2G0548400, RC1G0560100 and Chr1g0359121 [START_REF] Raymond | The Rosa genome provides new insights into the domestication of modern roses[END_REF] In the WD40 family, RC1G0586100 showed a strong similarity to TRANSPARENT TESTA GLABRA 1(TTG1), and RC3G0186600 and RC2G0693200 also belong to this clade. In the WRKY family, as previously shown by [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF], RC3G0244800 shows a strong similarity with AtTTG2 (TESTA-TRANSPARENT GLABRA2), whereas RC3G0309600 and RC3G0309700 seem to be more closely related to WRKY54 and WRKY70, RC3G0392200 to WRKY74, and RC3G0414600 appears to be related to WRKY34 and WRKY2. We then located these rose homologue genes on the rose genome and looked for co-location between these genes and the QTLs previously described (Figure 26 a andb). Concerning the QTLs on LG3 (male and female map, Figure 26 a andb), the most interesting TF among the detected genes was RcMYB61 (RC3G0322900, at Chr03: 39,896,892 -39,899,077bp) located in the cQTL interval for the female map (Figure 26 a). As previously described (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF]), a homologue of TTG2, a WRKY transcription factor (RC3G0244800), is also located in the credible interval. RcGIS2 (RC3G015000), a GIS2 homologue is also located on LG3 but not in the cQTL interval.

In addition to the candidate TFs, we also scanned the other TFs co-located in the cQTL interval on LG3 of the female map. There are four bHLH, two C2H2, three R2R3MYB and seven WD40 transcription factors (Supplementary Figure A.2, in blue) located under the cQTL.

Concerning the cQTL interval on LG4, RcGIS3 is positioned at Chr04: 50,315,805 -50,317,009 (1.21 Kb), and near the peak marker Rh12GR_55601_1304 (52,239,028 kb) on the female map (Figure 26 a). RC4G0476500, a ZFP5 homologue, is also located on the female LG4 but not in the QTL interval.

Concerning the QTL on the male LG1, RcMYC1, RcTRY and RcCPC, which are positioned at 44,468,298 -44,473,643 bp, 47,708,966-47,709,896 bp and 62,070,383 -62,072,848 bp, respectively, are located in the cQTL region (20.232Mb-62.553Mb) of PF (2016,2018) on the male LG1. The gene RC1G0586100 (RcTTG1) is also located on LG1 but outside this interval. For the male LG6, RC6G0407800, a homologue of TT8, is not located in the cQTL credible interval, and no studied gene was detected below this QTL.

Candidate gene expression in glabrous and prickle roses

Based on the positional approach, we identified ten interesting candidate genes, six within the QTL interval and the other four outside of QTL but near the credible interval (Figure 26). In order to obtain more information about these genes, we studied their transcript accumulation by RT-qPCR in tissues from prickle (P) and prickless (NP) stems at different developmental stages: I, IIa, IIb, IIc, III (Chapter 2, Figure 13).

The cluster analysis of gene expression clearly showed that all the samples can be divided into two main groups: PI, NPI, PIIa, NPIIa, PIIb, NPIIb were gathered into one group, and PIIc, NPIIc, PIII, NPIII into another group (Figure 28 a). At the sup-group level, PI and NPI, PIIa and NPIIa, PIIb and NPIIb, PIIc and NPIIc were clustered together, respectively. At the same stem developmental stage, prickle and glabrous samples (P and NP) behave similarly, suggesting no major difference of transcript accumulation between prickle and glabrous samples; the observed differences are more closely related to stem development.

To extend the analysis even further, we used NP as a control to compare the normalized expression of Using the 'non-parametric' QTL approach, we detected a stable QTL on LG3 in the three different years for both types of stems (PM and PF) on both the male and female genetic maps. We also demonstrated that this QTL mainly controls the presence/absence of prickles (Figure 24) using the 'two-part' QTL method.

Results

Interestingly, for PM in males, the QTL on LG3 may also be involved in regulating prickle density (severity in the two-part QTL analysis; Figure 24). A similar phenomenon was observed for the petal number with a locus on LG3 that controls the difference between simple and double petals, and a variance of the petal number that exists within the double petal flower is controlled by another locus [START_REF] Roman | Genetic analysis of the flowering date and number of petals in rose[END_REF][START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF].

We further enhanced the description of QTLs on LG3 that affect the presence/absence of prickles. A significantly distorted segregation was observed at the peak marker position. That unusual segregation ratio might be explained by the presence of a self-incompatibility locus (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF] near the peak marker for this QTL. On the basis of the phenotype-genotype relationship (Figure 27), we proposed that the PRICKLE alleles on this QTL are both heterozygous (np/P) in OB and RW, and that the presence of prickles is controlled by a dominant allele (np/P or P/P), and that the glabrous stem in the progeny is due to the combination of the two recessive alleles coming from both parents (np/np). These results are important for breeders who need to combine recessive alleles to obtain glabrous roses, an allelic combination that can be difficult in tetraploid roses. Development of specific molecular markers of the recessive allele may by useful for breeders. However, it should be noted that the actual markers used (peak of the QTL) are only closely linked to the PRICKLE locus and few recombinants are observed in the progeny. Furthermore, the phenotype of the individuals with the two recessive alleles (bd phenotype; Figure 27) are not stable and some of the hybrids were regularly seen to develop some prickles on parts of the stems. Indeed, this phenomenon is widespread in roses. Rose breeders have reported that glabrous mutants have either been unstable for the prickless trait [START_REF] Nobbs | American rose annual[END_REF][START_REF] Rosu | The development of putative adventitious shoots from a chimeral thornless rose (Rosa multiflora Thunb. ex J. Murr.) in vitro[END_REF], or reverted to the prickly character after a freezing winter or other environmental stresses [START_REF] Nobbs | American rose annual[END_REF][START_REF] Oliver | A pre ¸cis of thornless development. The rose[END_REF][START_REF] Druitt | Thornless roses[END_REF][START_REF] Canli | A review on thornless roses[END_REF]. Taken together, we assumed that a single major locus on LG3 controlled the absence/presence of stem prickles. Further investigations are necessary to more closely identify molecular markers (for molecular assisted breeding) and the mechanisms behind the instability.

In the ac, ad and bc genotypes, each genotype has a continuous quantitative trait, indicating that there are other loci responsible for prickle density variance. Other QTLs affecting quantitative traits were detected on

LG4 in OB and on LG1 and 3 in RW (Two-part QTL analysis; Figure 24). The LG4 QTL has a strong effect on PM but a weak effect on PF. For the QTL on LG1, it only had a weak effect on PF and on PM in 2018.

Those three loci are related to the density of prickles, indicating that there are multiple genes responsible for the density trait, and that those genes have a different effect on the different stems.

Detected QTLs are conserved in the Rosa genus and the Rosideae subfamily

Thanks to the link between genetic maps and reference genome sequences (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF], we were able to compare our results with previous genetic studies by associating genetic map markers.

A QTL was previously detected on LG3 in different diploid and tetraploid populations [START_REF] Crespel | Mapping of qualitative and quantitative phenotypic traits in Rosa using AFLP markers[END_REF][START_REF] Linde | Powdery mildew resistance in roses: QTL mapping in different environments using selective genotyping[END_REF][START_REF] Koning-Boucoiran | The mode of inheritance in tetraploid cut roses[END_REF][START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF]Bourke et al., 2018a), which is consistent with our results: a strong QTL on Chr3 with a high LOD value was detected in all of the environments (across and between years and types of stems). This demonstrated that Chr3 QTL is a robust QTL detected independently of ploidy and the environment, and is present in various genetic backgrounds.

Recently, three QTLs on LG3, 4 and 6 were detected in the tetraploid K5 population with a high density of SNPs genetic map (Bourke et al., 2018a). Interestingly, the QTLs identified from the diploid (OW) were almost identical to tetraploid (K5) populations (LG3, 4 and 6), with the slight difference that we also detected a weak QTL on LG1, which was only significant in males for two of the years. This slight difference might be due to the genetic background of the parents of the K5 and OW populations. In fact, in K5 populations, one parent is prickly and the other glabrous, whereas in OW populations, both parents have prickles. Bourke et al. (2018a) reported that two SNP markers, K7826_576 (located on the Chr3: 37,706,920 pb) and K5629_995

(located on the Chr4: 57,791,999 bp) are linked to the stem prickle trait. When compared with our results, K7826_576 is located within our Chr3 cQTL interval region (36,517,224 -46,440,369 bp; Figure 26), and K5629_995 is very close to our Chr4 QTL interval (46,189,107,784 bp). These results suggest that QTLs detected on LG3 and 4 could be similar between OW and K5 progenies.

In Rosaceae, the genetic determinism of prickle was studied in raspberry (Rubus idaeus), where two QTLs were detected on LG4 and 6 (Molina-Bravo et al., 2014). Using synteny viewer tools (https://www.

rosaceae.org/synview/search; Jung et al. ( 2014)), we checked the synteny. The region where the QTL is located on LG6 in R. occidentalis (position 6.028Mb) is syntenic with a region on rose chromosome 2 (position 42.330 Mb), where no QTL for prickle density was detected in our study. The region where the QTL 4 is located (position 0.101 Mb) is syntenic with the region on rose chromosome 4 (position 58.768

Mb), very close to the main QTL we detected on this chromosome (Table 4). These results could suggest that the two QTLs in rose and raspberry might be syntenic and share a common evolutionary history. In another publication, [START_REF] Graham | Mapping gene H controlling cane pubescence in raspberry and its association with resistance to cane botrytis and spur blight, rust and cane spot[END_REF] identified the gene H that controls cane pubescence. The locus is mapped on LG2, which is syntenic with the rose LG6 where one of the QTLs is located, detected in R. x wichurana. However, no precise location is available to allow us to assume a possible common origin.

Candidate gene below the QTL interval region

Prickles are assumed to originate from a 'trichome-like structure'. In order to find a putative candidate gene for the identified QTLs, we looked for homologue genes known to be involved in trichome initiation and development in Arabidopsis. We annotated 15 rose TFs that, based on similarity, can be involved in trichome development in rose: RcGL1, RcMYB82, RcMYB61, RcCPC, RcTRY, RcGL3, RcTT8, RcMYC1, RcTTG1, RcTTG2, RcZFP5, RcGIS3, RcGIS2, RcZFP1 and RcGL2 (Supplementary Table A.3). Among them, a few were below the detected QTLs: RcMYB61 and RcTTG2 below the QTL on LG3; RcGIS3 below the QTL on LG4; and RcCPC, RcTRY and RcMYC1 below the QTL on LG1. ZFP5 (Chr04: 57,125,905 bp) is out of the QTL interval on LG4 in OW, but close to the peak LOD marker K5629_995 of QTL in the K5 population (Chr04: 57,791,999 bp) (Bourke et al., 2018a). These genes are good candidates for the detected QTLs.

Candidate genes transcript expression in glabrous and prickle F 1 individuals

We quantified ten TF gene transcripts in glabrous and prickle F 1 individuals in different developmental stages using RT-qPCR. Surprisingly, minor differences were observed between glabrous and prickle samples, with the main differences occurring between developmental stages (as demonstrated by the heatmap analysis, Figure 28 a). Based on transcript accumulation, this suggests that these homologues, known to be involved in trichome initiation and development in Arabidopsis, are not implicated in prickle initiation in rose, leading to the hypothesis that the two processes (trichome initiation and prickle initiation) might involve different gene pathways. The candidate gene approach may not be appropriate and a non-a priori approach such as a transcriptomic analysis could be done between individuals with and without prickles. Nevertheless, some differences in transcript accumulation are observed between candidate genes. In the early stage (stage I), only RcMYB61 and RcGIS2 are slightly more highly accumulated in glabrous stems. However, GIS2 and MYB61 are positive regulators of trichome initiation [START_REF] Gan | GLABROUS INFLORESCENCE STEMS modulates the regulation by gibberellins of epidermal differentiation and shoot maturation in Arabidopsis[END_REF], which is difficult to reconcile with an increase in transcript accumulation in glabrous stems (Figure 28). Negative feedback regulation during prickle initiation can explain this point, as regularly observed in trichome initiation [START_REF] Pattanaik | An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis[END_REF] or, perhaps, differences are not at the transcriptional level. It could be interesting to sequence the genes in the two parents to see if a mutation can explain the phenotype.

RcZFP5 may also be an interesting candidate gene. This gene showed a different regulation between glabrous and prickly stems. At stage IIa, RcZFP5 shows a strong down-regulation in glabrous tissue, whereas this down-regulation is observed later at stage IIc in tissues with prickles (Figure 28 c). Furthermore, this gene is close to the QTL on LG4. Its early repression in glabrous stems might explain why no prickle developed. In A. thaliana, ZFP5 controls trichome initiation through GA signaling [START_REF] Zhou | ZINC FINGER PROTEIN 5 is required for the control of trichome initiation by acting upstream of ZINC FINGER PROTEIN 8 in Arabidopsis[END_REF].

These data (concerning ZFP5 and MYB61) might suggest an implication of GA in prickle development.

However, this hypothesis needs to be functionally validated in rose.

Conclusions

Prickle structure is an undesirable trait, not only in rose but in most crops in general. We identified a complex genetic determinism with a major locus on LG3 that controls the presence of prickles and a few QTLs that control prickle density. Further studies are necessary to develop markers for breeding selection and to identify the molecular bases. Using a candidate gene approach, we proposed different hypotheses concerning the gene involved in prickle initiation in rose. Approaches such as transcriptomics may help to identify new key regulators of prickle initiation and development in rose.

4

In-depth RNA sequencing analysis provides molecular mechanism insight into prickle initiation and development in rose

Zhouu NN 1,2 , Tang KX 2 , Jeauffreu J 1 , Thouroude T Corresponding author: ningning.zhou@aliyun.com or fabrice.foucher@inrae.fr

Introduction

Prickles are non-vascular sharp appendages and can be easily distinguished from thorns and spines which are modified stems and leaves respectively (and therefore they are usually vascularized). Many studies have been reported on their ecological, evolutionary, and biogeographic implications. From a functional perspective, spinescences (prickles, thorns, and spines) have been proposed as an effective defensive strategy against herbivores [START_REF] Janzen | Why bamboos wait so long to flower[END_REF][START_REF] Cooper | Effects of plant spinescence on large mammalian herbivores[END_REF][START_REF] Belovsky | Effects of spines and thorns on Australian arid zone herbivores of different body masses[END_REF][START_REF] Gowda | Spines of Acacia tortilis: what do they defend and how?[END_REF][START_REF] Burns | Are there general patterns in plant defence against megaherbivores?[END_REF][START_REF] Wilcox | The thorny truth about spine evolution[END_REF].

A piece of paleontological works offered evidence that spinescence structure may appear in the late Silurian (∼ 400 million years ago) which is before the mammal herbivores' appearance [START_REF] Chaloner | The rise of the first land plants[END_REF].

Thus, the evolution of spinescences may not be a response to the pressure of large herbivores (like mammals) but rather to other biotic or abiotic pressures (such as small herbivores like insects, reviewed by [START_REF] Wilcox | The thorny truth about spine evolution[END_REF]. While this is still a controversial topic since most researchers supported that spinescence evolved against mammalian herbivores [START_REF] Cooper | Effects of plant spinescence on large mammalian herbivores[END_REF][START_REF] Burns | Are there general patterns in plant defence against megaherbivores?[END_REF]. Nevertheless, in the process of co-evolution with herbivores, plants have evolved an impressive defense system where spinescences play a key role. Many types of spinescence have been produced (or modified) from the shoot, leaves, fruits, pedicels, and even roots. Besides providing mechanical protection, the spinescence structures are frequently cooperating with predatory pathogenic bacteria, fungi and toxic chemicals, enhancing their attack or defense

Introduction

ability [START_REF] Halpern | Plant biological warfare: thorns inject pathogenic bacteria into herbivores[END_REF][START_REF] Lev-Yadun | Color changes in old aposematic thorns, spines, and prickles. In Defensive (anti-herbivory) coloration in land plants[END_REF]. On thorns of Phoenix dactylifera and Crataegus aronia, fifty-eight bacterial isolates were found and they are belonging to 22 different bacterial species: 13 of them are known to be pathogenic for animals or humans, such as Clostridium tetani, a etiological agent of tetanus [START_REF] Halpern | Plant biological warfare: thorns inject pathogenic bacteria into herbivores[END_REF]. Spinescence injuries have been reported to cause tetanus on humans and others predators in several countries [START_REF] Hodes | Tetanus in ethiopia: analysis of 55 cases from addis ababa[END_REF][START_REF] Pascual | Tetanus surveillance-united states, 1998-2000[END_REF][START_REF] Ergonul | Analysis of the case fatality rate of tetanus among adults in a tertiary hospital in Turkey[END_REF][START_REF] Campbell | Microbiologic characterization and antimicrobial susceptibility of clostridium tetani isolated from wounds of patients with clinically diagnosed tetanus[END_REF][START_REF] Tadele | Clinical profile and outcome of pediatrics tetanus: The experience of a tertiary hospital in Ethiopia[END_REF]. Thus, rose bush prickles can cause puncture wounds that resulted in tetanus [START_REF] Pascual | Tetanus surveillance-united states, 1998-2000[END_REF]. Important human diseases are caused by prickle wounding: mycetoma caused by Eumycetoma (fungi) or Actinomycetoma (filamentous bacteria) and sporotrichosis diseases caused by Sporothrix schenckii (fungi), are called "plant thorn synovitis" and "rose-thorn or rose-gardeners' disease" respectively. The most common route of infection is the introduction of spores to the subcutaneous cellular tissues through a wound of the skin [START_REF] Fahal | Mycetoma: a thorn in the flesh[END_REF][START_REF] Barros | Sporothrix Schenckii and Sporotrichosis[END_REF][START_REF] Vásquez-Del-Mercado | Sporotrichosis[END_REF][START_REF] Mahajan | Sporotrichosis: an overview and therapeutic options[END_REF][START_REF] Kieselova | Rose thorn injury[END_REF]. Dermatophytes that cause subcutaneous mycoses are unable to penetrate the body and must be introduced into the subcutaneous tissue by a puncture wound [START_REF] Willey | Prescott, Harley, and Klein's microbiology[END_REF]. These spinescence structures inject bacteria into herbivores (or other mammals as human) by wounding, they may cause severe infections that are much more dangerous than the mechanical wounding itself [START_REF] Halpern | Plant biological warfare: thorns inject pathogenic bacteria into herbivores[END_REF][START_REF] Lev-Yadun | Color changes in old aposematic thorns, spines, and prickles. In Defensive (anti-herbivory) coloration in land plants[END_REF]. Some bacteria species probably are adapted to live and multiply on the spinescences (hence they are present in large numbers) and not just landed on the spinescence accidentally [START_REF] Halpern | Plant biological warfare: thorns inject pathogenic bacteria into herbivores[END_REF]. Spinescence color is also an important aspect of the protective property. The bright color prickles and other types of sharp appendages are frequently observed, especially in the juvenile phases of development. This special character confers a selective advantage as herbivores learn to associate conspicuous coloration with unpleasant qualities [START_REF] Cott | Adaptive coloration in animals[END_REF][START_REF] Edmunds | Defence in animals: a survey of anti-predator defences[END_REF][START_REF] Ruxton | Avoiding Attack: The Evolutionary Ecology of Crypsis, Aposematism, and Mimicry[END_REF][START_REF] Speed | Warning displays in spiny animals: One (more) evolutionary route to Aposematism[END_REF].

In addition, the role of spinescences against insects is often neglected nowadays. Some species (such as Acacia collinsii) developed large hollow spines as a habitat for ants, which protect the plants against herbivory insects [START_REF] Janzen | Why bamboos wait so long to flower[END_REF]. Prickles, such as those on roses, may play a role in reducing caterpillar feeding by restricting their movement [START_REF] Kariyat | Plant spines deter herbivory by restricting caterpillar movement[END_REF].

In the agricultural production process, prickle is an undesirable trait in many crops as roses because it can injure workers and also damage crops (especially for cutting roses). Rose is a typical representative of prickly plants with the most important economic value in ornamental plants. In roses, prickles are distributed wildly on stems, pedicel, peduncles, leaves, or fruits, and their distribution on the organs is a source of a large diversity within the Rosa genus. Two major categories of prickles on stem have been described in roses: 'non-glandular (NGP)' and 'glandular (GP)' prickles (see Zhou et al., 2020, Chapter 2).

NGPs are normally unbranded and can be naked on the surface or covering with hairs (hairy). GPs can be branched or unbranched, and the unbranched GPs also present the naked and hairy types (see Chapter 2, unpublished yet). Unbranched and naked NGP is the most common type of stem prickle found in wild (see Chapter 2). The genetic and molecular mechanisms underlying prickle initiation and development remain still largely unknown. Four loci on linkage group 1, 3, 4, 6 have been detected to control stem NGPs in OW population (Rosa chinensis 'Old Blush' × R. × wichurana) (see Zhou et al., 2020, Chapter 3). The major locus on LG3 was shown to control the absence/presence of NGPs. Another major locus on LG4

was associated with prickle density [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF]. NGP are described as modified glandular trichomes [START_REF] Peitersen | Blackberries of New England-genetic status of the plants[END_REF][START_REF] Coyner | Thornlessness in blackberries[END_REF][START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF]Ma et al., 2016b;Khadgi and Weber, 2020a).

However, this hypothesis was not supported by histological study and molecular data, as prickle does not come from protoderm (or epidermis) but from ground meristem under protoderm (see Chapter 2) and the rose homologue of genes involved in Arabidopsis trichome initiation and development might not be controlling prickle initiation and development in rose (see Zhou et al., 2020, Chapter 3). Further studies are necessary to identify new key regulators of prickle initiation and development in rose.

Here our objectives are to decipher the gene network that controls prickle initiation and development in rose using a transcriptomic approach. Using four F1 individuals of OW population, we present the first profile of the transcriptomic changes during prickle initiation and development by comparing the transcriptome of rose stems with and without prickles and a detailed time-course transcriptomic analysis of prickle development. This detailed analysis allows us to detect important regulators of prickle initiation and development. By combine with previous genetic data [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF], we spotlight the best potential regulators of prickle initiation. We provided pieces of evidence to support our new insight of non-glandular prickles and trichomes have different gene pathways.

Materials and methods

Plant materials

A diploid OW population obtained from the female Rosa chinensis 'Old Blush' (OB) × the male Rosa

x wichurana (RW), were grown in a field and managed by the Horticulture Experimental Unit (INRAE, Angers, France). We selected four once-flowering individuals: two presented non-glandular prickles (NGP) (OW9071 and OW9137) and two are glabrous (OW9067 and OW9068). Those genotypes were vegetatively propagated and grown in a greenhouse in November 2017. Harvesting was done in the morning from March 27th to April 5th, 2018. We mixed OW9067 and OW9068 materials as prickless samples, and OW9071 and OW9137 as prickle samples. We randomly divided 18 cuttings per genotype into three groups as three biological replicates. Under a microscope, unexpanded leaves and buds were removed, and we took samples of stages I, IIa, IIb, IIc and III strictly according to previously described stages of prickle (P) and glabrous stem (NP, stems at the same stages for P) (see Zhou et al., 2020, Chapter 2).

RNA isolation and experimental design

RNA was isolated according to [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF]. Total RNA concentration, RIN value, 28S/18S and fragment size were measured using Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit). Purity of RNA was determined by agarose gel electrophoresis and ultraviolet spectrophotometer NanoDropTM.

Results

have selected nine trichomes-candidate genes whose expression has been studied by RT-qPCR on the same samples at the same stages in a previous study to identify if the trichomes genes are controlling prickle initiation [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF]. Here, we have compared the nine genes' normalized expression (TMP) by RNA sequencing and relative transcript accumulation obtained by RT-qPCR. We found similar expression pattern, indicating those two data were reliable and can support subsequent results (Figure 29 b). Since the biological triplicates are highly reproducible, we used the sum of transcripts per million (TPM) values of the triplicates as the expression level of the gene in the corresponding sample. The normalized expression value (TPM) of all genes in all the samples are presented in the Supplementary Datasets 1 / sheet 2. As previously described (Chapter 2, Figure 13), prickles develop in parallel with stem: stage I correspond to early developmental stages of the stem after bud-outgrowth (prickle initiate just below the shoot apical meristem) and stage III corresponds to a stage where internode are fully developed (Chapter 2, Figure 13).

Which genes may be involved in

For glabrous individuals, we have selected stages corresponding to the same stage of stem development as stages I and IIa for prickle samples (Chapter 2, Figure 13). In order to obtain more useful information, we investigated the SDE genes that were shared or not shared with prickle and stem development. The 363 unique genes of PIIavsPI were supposed to be specifically involved in the early stage of prickle Figure 30: Caption on next page...... ome, cytosolic part, ribosome, nucleolus, supramolecular complex, phragmoplast, polysome, cytoskeleton, DNA packaging complex, protein-DNA complex, pre-ribosome, small nucleolar ribonucleoprotein complex, transferase complex, anchored component of membrane (Figure 30 d). Those processes are all important and highly associated with the anatomical features of prickle development from stage I to stage IIa (14d-f).

Results

Cluster 3 genes mainly expressed in stages IIa and IIb. Cluster 4 genes are mainly expressed in stages IIa, IIb and IIc (with highest expression in stage IIb) (Figure 30 c). No SSE-GO biological process was found in these two patterns, but we can observe some genes of cluster 3 that are involved in plant epidermis and embryo development (not shown in Figure 30 d since they are not significant), and several genes of cluster 4 that are involved in cell wall modification and different types of metabolic processes (not shown in Figure 30 d). The SSE-GO cellular component located in cytosolic part, ribosome, anchored component of membrane and plant-type cell wall.

Cluster 5 genes mainly expressed in stage IIc (Figure 30 c). In this cluster, we observed a large change of the SSE-GO terms (Figure 30 d). This cluster gathers mainly genes involved in response to different stimuli (nitrogen compound, wounding, ethylene, jasmonic acid, auxin, drug catabolic process, organic cyclic compound), defense response to insect, and phosphorelay signal transduction system. GO Cellular Component process mainly located in anchored component of membrane, plant-type cell wall and plasma membrane part.

Clusters 6 and 7 genes mainly expressed in stages IIc and III with different patterns. The transcript accumulation in stage IIc of cluster 6 is obviously higher than in stage III, but relatively higher in stage III of cluster 7 (Figure 30 c). Enriched genes are responding to bacterium were still observed in these clusters.

In Cluster 6 genes, new SSE-GO processes were observed that involved in the cell wall (modification, organization, biogenesis), protein autophosphorylation and polysaccharide metabolic processes. The cell wall (biogenesis, organization) and protein autophosphorylation were also enriched in cluster 7. Except that, we observed new SSE-GO biological processes involved in cell wall macromolecule metabolic, and cellular component macromolecule biosynthetic process. GO cellular component process mainly located in the anchored component of membrane and plant-type cell wall also observed in cluster 5 (Figure 30 d).

Clusters 8 and 9 genes mainly expressed in stage III. A slight difference between the two clusters is that the genes of cluster 8 are also relatively low expressed in stage IIc and cluster 9 genes are not (Figure 30 c). For the biological process, we still can observe cell wall (biogenesis, organization), defense genes response to bacterium and protein autophosphorylation processes in cluster 8 pattern. New and specific enriched processes appear in cluster 8 including cell surface receptor signaling pathway, cation transmembrane transport, and response to oomycetes and virus. 'cell recognition and inorganic ion transmembrane transport' as the new processes are both enriched in cluster 8 and 9. The 'anion transport, secondary metabolic process and cell death' processes were specifically enriched in cluster 9 (Figure 30 d). Interestingly, these kinds of genes are rarely expressed in prickle samples. We suggested those genes can be considered as a candidate gene pool which are negative regulators for prickle initiation or positive regulators for glabrous phenotype. Another set of genes are mainly expressed in prickle samples, and rarely expressed in glabrous samples, they were considered as a candidate pool of positive regulators that control prickles initiation and development.

Which genes may be involved in prickle initiation

In addition, we found that numerous genes specifically highly expressed in PIIb, PIIc, or PIII, but with relative lowly expression in PI, are also generally low expression in NPI (Figure 31 b). The expression patterns of these genes are similar to the expression pattern of genes from clusters 4, 5, 6, 7, 8 and 9 (Figure 30 c). Coupled that we have revealed those clusters' genes are mainly involved in the later stage of the cell wall (modification, organization, biogenesis) and response to different stimuli, etc (see the previous section, Figure 30 d). Hence, to identify the genes specifically involved in the prickle initiation, we removed 465 SDE genes that already presented in cluster 4, 5, 6, 7, 8 and 9 to gain insights into the function of the remained SDE genes (Figure 31 c). On the remaining 2118 SDE genes Supplementary Datasets 4 / sheet 3, 1075 genes have functional annotations (based homology search (Supplementary Datasets 4 / sheet 4)). Used their homology's UniProt-SwissProt ID, we performed gene ontology (GO) and enrichment analyses (Figure 31 h). Interestingly, the top of enriched biological process (non-redundant) is involved in post-embryonic plant morphogenesis (FDR = 0.005), then is fatty acid metabolic process (FDR = 0.01) and cell fate commitment (FDR = 0.05). In addition, we found several SSE-GO biological processes with FDR value higher than 0.05 (1) Unique and common genes differentially expressed in stage I and IIa between prickle and prickless samples.

(2) Highlight the good candidates by combining transcriptomics and genetic approaches

PIvsNPI PIIavsNPIIa

Rosa 'Old Blush' 44,481 a.

c.

(

) 1
(2) b.

d.

e.

f. Figure 31: Data-mining workflow to identify the best candidate-genes for prickle initiation. Significant differential expression (SDE) genes identified from stages I and IIa between prickle and prickless samples using DEseq2 (step1). Low expressed SDE genes (corresponding to whose sum TPM was less than 1.5) were removed (a, d). The relative expression of the remaining SDE genes were visualized (b, e) in order to check their expression patterns in all the samples (step 2). The genes that are mainly expressed in the later stages were removed (c and f): for SDE genes of stage I and IIa, we first filtered the genes with a relatively low expression in stage PI (c) and PIIa (f). We then filtered the genes presented in clusters 4, 5, 6, 7, 8 and 9 (c) and 5, 6, 7, 8 and 9 (f), respectively (step 3). Two methods to narrow the range of the best candidate genes (promoters or inhibitors) for prickle initiation were used (step 4): [START_REF] Van Der | ). In AT, DOT5 is required for normal shoot and root development (Petricka et al., 2008). Both rose genes have a specific high expression in the early stage of glabrous stems, suggested that they may function as prickle initiation repressors (33b). RC3G0419900 (RcAS2) encodes ASYMMETRIC LEAVES 2 (AS2) like protein and belongs to LATE-RAL ORGAN BOUNDARIES (LOB) domain family (Supplementary Figure B.6 c). AS2 controls proximaldistal patterning in Arabidopsis petals[END_REF] The two groups of SDE genes (g) are compared to each other to retrieve the specific and common SDE genes involved in stages I and IIa;

(2) The genetic and transcriptomic approaches were combined to reveal the SDE genes (PIvsNPI) that are located under the confidence interval of the prickle loci on chromosome 3 (OB3) and 4 (OB4) (i), '29' and '14' refer to the number of SDE genes under the intervals. Venn diagram (g) of the number of SDE genes between the different conditions. Total: total number of SDE genes; Up: up-regulated genes; Down: down-regulated genes; DR: differentially-regulated genes between the two comparison groups. Comparison group named as 'Treat'vs'Control'. Gene Ontogeny enrichment function analyses were used to check the function of the SDE genes in each step. (h) A graph showing the GO terms enriched (FDR < 0.1) for the unique, common, and total SDE genes of stages I and IIa.

but less than 0.1 are also highly associated with the morphological characters. Such as, 'cell death', 'cellular component morphogenesis', 'multi-multicellular organism process', 'ASFIM' are all important for the organ formation. 'Protein phosphorylation' is the most basic, common and important mechanism for regulating and controlling protein vitality and function. Thus, we suggested this relatively narrow range of SDE genes (2118) can be used as the candidate genes pool for further step analysis. The co-expression pattern of the genes involved in those SSE-GO biological processes were visualized in Supplementary Figure B.5 and the supporting databases presented in Supplementary Datasets 4 / sheet 5.

Narrow the range of good candidate genes for prickle initiation

Stage I of the prickle sample included initiation and the beginning of development (grow out). Considering the development of prickle in stage I is a result of the prickle cell division (or cell proliferation) process, and this process is also observed in stage IIa (see Chapter 2, Figure 14). Therefore, we investigated the 'unique' and 'common' SDE genes for stage I and IIa between the comparisons of PIvsNPI and PIIavsNPIIa (Figure 31 g) to mine the more interesting genes which specially involved in prickle initiation. We expected to concentrate on the genes who mainly involved in initiation. First, we discovered 2369 genes (987 upand 1382 down-regulars, Supplementary Datasets 4 / sheet 6) were significantly differential expression in PIIavsNPIIa (Supplementary Figure B.3 PIIavsNPIIa, Figure 31). Using the similar filtering methods as previously done for PIvsNPI, we first removed 324 low expression genes (Figure 31 d) and then filtered out the genes (300) that are present in clusters 5, 6, 7, 8, 9 (Figure 30 c), and 1745 SDE genes passed the filter (Figure 31 f). The normalized expression (TPM) and the functional annotation of these SED genes are presented in Supplementary Datasets 4 / sheet 7. We then compared these 1745 genes with the 2118 SDE genes of PIvsNPI (Figure 31 g, Supplementary Datasets 4 / sheet 8-15), and found that a large number of genes ( 1458) are in common between the two-stages comparison: 536 are up-and 921 are down-regulated and only 1 present an opposite pattern of expression (Figure 31 g). However, for genes of that large group, we only found 'cell recognition' enriched biological process (Figure 31 h), but we noticed that among the The network is clustered to three clusters using kmeans clustering method, and same color of node refers to one cluster. Filled and empty nodes indicate some 3D structure is known (or predicted) and unknown respectively. Edges represent protein-protein associations (associations are meant to be specific and meaningful, i.e. proteins jointly contribute to a shared function; this does not necessarily mean they are physically binding each other.). Different colors of line refer to the different types of data support, the detail have been showed on the Figure 32 c.

other enriched GO terms involved in both 'total' of PIvsNPI and PIIavsNPIIa, some genes belong to the 'common', some to the 'unique'. 660 SDE genes are unique in stage I with 308 up-and 352 down-regulated genes (Figure 31 g). Interestingly, we observed many unique SDE genes were especially involved in seven enriched (FDR>0.1) biological process which contents 'post-embryonic plant morphogenesis (PEPM)', 'fatty acid metabolic process (FAMP)', 'cell fate commitment (CFC)', 'monocarboxylic acid biosynthetic process (MABP)', 'ASFIM', 'secondary metabolic process (SMP)' and 'pattern specification process (PSP)'

(Figure 31 h). 287 SDE genes are unique in stages IIa with 137 up-and 160 down-regulated genes (Figure 31 g), and no enriched biological process was identified in this group (Figure 31 h). Those results of function prediction are highly consistent with the morphological characteristics: PI stage corresponds to initiation and first development processes (see Chapter 2), and the 2118 SDE genes of PIvsNPI contain the genes special involved in prickle initiation, development or in both processes.

The 660 unique SDE genes of PIvsNPI contents the most interesting of the SSE-GO biological processes associated with prickle initiation, we suggest that they can be as the priority candidate genes for studying prickle initiation. We visualized a Gene Ontology Tree using the weighted set cover of the WebGestalt tool to intuitively display the relationship network of SSE-GO processes of these 660 unique SDE genes (Figure 32 a, Supplementary Datasets 5 / sheets 1). The FMP, MABP and SMP processes are the sub-processes of metabolic process; the PEPM, CFC, PSP and ASFIM are the sub-processes of developmental and cellular process. A heatmap (Figure 32 b) showed the co-expression pattern of the genes which belong to the seven SSE-GO terms display in Figure 32 a. Most SDE genes are highly co-expressed in prickless samples, and only a few genes in prickle samples. Different GO terms sometimes shared common genes, such as the homology of AFO, JAG, ROXY1, DOT5, YBA5 (Supplementary Figure B.5). Some genes may be duplicated genes or genes from multifamily (same functional annotation). This is the case for the homologues of MYB106 (Four homologue genes are co-expressed in NPI), AFO and FAD5 (two homologues genes are co-expressed in NPI) (Figure 32 b). We also studied the protein-protein network between these genes (Figure 32 c, Supplementary Datasets 5 / sheet 2-3). A protein interaction networks between 25 (out of 37 in total) protein was detected with setting a minimum required interaction score (0.4 confidence). This network is clustered to three clusters using kmeans clustering method, and same color of node refers to one cluster. The green groups are associated with metabolic process. The blue and red groups are associated with

Results

developmental and cellular process and these genes have been reported to be involved in the different organs or tissues development, the best known were: petal, floral or inflorescence development process, represented by JAG, AFO, ROXY1, BOP2, SDD1; leaf development process, represented by AS2, JAG, KNAT1, BOP2, WOX1 and WOX3; embryonic shoot apical meristem development, represented by AFO, YAB5, YAB2, TCP4, KNAT1; the initial decision of protodermal cells, represented by ERL1, ERL2; the ovary transmitting tract cells and pollen tube growth, represented by NTT ; cutin or cuticular wax formation, represented by CER2, CER4, KSC6, ABCG11, LACS2. Our studies supported that those gene homologue in rose (Figure 32 b)

probably have function involved in promoting or inhibiting prickle initiation.

Highlight the best candidates by combining transcriptomics and genetic approaches

Using a genetic approach, we have identified two major QTLs on Linkage group (LG) 3 and 4, controlling the presence and the density of prickles on stem [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF]. By linked SNP markers with the reference Rosa chinensis Genome v1.0 (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF], we obtained 95% overlapping confidence interval of cQTLs (cQTLoci): LG3 (Chr03: 36.52Mb ∼ 46.44Mb), LG4 (46.18Mb ∼ 56.11Mb) (see Zhou et al., 2020, Chapter 3). Here, we combined the genetic and transcriptomic data: among the 2118 candidate genes discovered from PIvsNPI SDE genes, 29 and 14 SDE genes were located within cQTLoci of Chr03 and Chr04 respectively (Supplementary Table 4, Figure 31 i).

With TPM normalized expression value, we studied the co-expression patterns of those 43 SDE genes in all the samples. Two main expression patterns are observed with genes mainly co-expressed in prickle and prickless samples respectively at stage I (Figure 33 a). To gain information on the potential function of those 43 genes, we summarized the functional annotation based on their homologs in A. thaliana (AT) (Supplementary Table B.4). 13 out of 43 genes have no similarity (E-value < 1 e-6 ) in AT, our transcriptomic study supported that they might be involved in prickle initiation (and development). For others, we presented the nomlized expression (TPM) in all the samples (Figure 33 example, in shoot meristem of Petunia and Antirrhinum majus [START_REF] Stuurman | Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating cells[END_REF][START_REF] Kieffer | Analysis of the transcription factor wuschel and its functional homologue in Antirrhinum reveals a potential mechanism for their roles in meristem maintenance[END_REF], in root apical meristem of A. thaliana [START_REF] Sarkar | Conserved factors regulate signalling in Arabidopsis thaliana shoot and root stem cell organizers[END_REF], in ovule development of Arabidopsis [START_REF] Park | The structure of nucleosome assembly protein 1[END_REF], in petal and carpel fusion of Petunia and Arabidopsis [START_REF] Vandenbussche | Differential recruitment of WOX transcription factors for lateral development and organ fusion in petunia and Arabidopsis[END_REF], in adaxial/abaxial patterning of leaves [START_REF] Nakata | Roles of the middle domain-specific wuschel-related homeobox genes in early development of leaves in Arabidopsis[END_REF] in Arabidopsis, in lateral root development and root hair formation in rice [START_REF] Yoo | Rice WUSCHEL-related homeobox 3a (OsWOX3A) modulates auxin-transport gene expression in lateral root and root hair development[END_REF][START_REF] Sun | Hairy leaf 6, an AP2/ERF transcription factor, interacts with OsWOX3B and regulates trichome formation in rice[END_REF], in conferring glabrousness of rice leaves and glumes [START_REF] Asano | A WUSCHEL-related homeobox 3b gene, depilous (dep), confers glabrousness of rice leaves and glumes[END_REF]. In rose, WOX genes is a large family, we found 209/381 WUSCHEL-like annotated genes in the two Rosa chinensis 'Old Blush' Genomes (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF][START_REF] Raymond | The Rosa genome provides new insights into the domestication of modern roses[END_REF]. Interestingly by phylogenetic analysis, RC3G0389900 and RC4G0448500 belong to a clade A heatmap shows the co-expression pattern of the candidate-genes (forty-three SDE genes between PI and NPI stages) that located under the confidence intervals of prickle loci on LG 3 and 4. (b) The normalized expression (TPM) of candidate genes in the different stages of prickle (P) and glabrous (NP) samples. The best ten candidate-genes based on their potential function in prickle initiation were highlighted in the red.

Material and sampling limitations and improvement solution

In this project, one of the challenges was the material limitations for RT-qPCR and RNA-seq: to our knowledge, no single line of prickless mutant in roses resources was found. Thus, we selected sisters in the OW population and used a mixing pool for prickless and prickles samples to reduces the variance of genetic background. Since there are only two individuals completely prickless and once-time flowering (to obtained vegetative branch), we chose the individuals for the prickle pool following the principle of being as close as possible to the prickless progeny on the other traits. Although we have tried to limit the differences as much as possible, it is impossible to completely avoid that a few genes' differential expression in prickle and prickless may cause by the slight difference of genetic background.

Another challenge is the technical limitation for sampling: the prickle initiation happened at a very early stage of stem development (just below the shoot apical meristem) (Figure 14), and it is impossible to isolate the prickle from the stem tip during sampling (a perspective can be to used microdissection).

Therefore, in order to be consistent, we decide not to separate prickle from stem (leaves and buds were removed) as the strategy for the sampling of all the stages. This led to the stem development genes were mixed in the candidates of prickle development which brings more challenges for data analysis. We displayed a model method to obtain the 'specific' SDE genes involved in prickles development by comparing group NPIIavsNPI and PIIavsPI. We proposed that the specific SDE genes of PIIavsPI are more interesting for prickle development and the common SDE genes are more associated with stem development. It still needs to be reminded that the development of prickles and stems are probably sharing some genes. That means the 'common' SDE genes may also contain a number of genes that function in prickle development.

Due to the technical limitations for sampling the early stage of prickle initiation, we were unable to have the earliest stage (as initiation). Stage I samples included prickles initiation and development (grow out by cell proliferation) (see Chapter 2, Figure 14). In order to remove potential noise and obtain the most interesting candidates, I designed two unconventional processes in the analysis: 1) We removed the SDE genes of stage I that have been studied in the clusters 4, 5, 6, 7 and 8 (in the time-course study (Figure 30 c)). The reason is that these genes mainly expressed in the later stages (IIb, IIc, III (Figure 13 The best candidates for prickle initiation were selected by combining transcriptome with the genetic approaches. Since some genes may be simultaneously involved in initiation and development, we used the total 2118 candidate genes discovered between PI and NPI in this analysis step.

Mechanisms underlying prickle development

For the first time, we present a detailed analysis of processes that may control prickle development in rose. By clustering the different expressed genes (DE) in different developmental stages (I, IIa, IIb, IIc, III) of prickle sample (Figure 30), we revealed these DE genes' expression pattern in development stages. Using GO enrichment for the genes of each cluster, we observed many interesting biological processes that are highly associated with the different stages of prickle morphogenesis. (Figure 30 d).

For the genes mainly expressed in early stages I or/and IIa (Figure 30 c, clusters 1 and 2) of prickle samples, we discovered genes that are enriched in biological processes such as cell fate commitment, differentiation, proliferation and cell division. Those genes were described to be involved in organ or tissue development (floral, shoot, leaf, meristem, post-embryonic plant morphogenesis). These early steps correspond to the prickle initiation with acquisition of a new fate for some cells from the ground meristem (below the protoderm, see Chapter 2). Then, through cell proliferation, the prickle develops. The DE genes are therefore good candidate to be involved in prickle initiation and first steps of prickle development.

For the genes mainly expressed in later stages: [START_REF] Van Der | ). In AT, DOT5 is required for normal shoot and root development (Petricka et al., 2008). Both rose genes have a specific high expression in the early stage of glabrous stems, suggested that they may function as prickle initiation repressors (33b). RC3G0419900 (RcAS2) encodes ASYMMETRIC LEAVES 2 (AS2) like protein and belongs to LATE-RAL ORGAN BOUNDARIES (LOB) domain family (Supplementary Figure B.6 c). AS2 controls proximaldistal patterning in Arabidopsis petals[END_REF] in cluster 4 (mainly in PIIa, PIIb, PIIc) and 5 (mainly in IIc), we discovered genes have potential function involved in cell wall modification. These genes associated with cell wall modifications might be required for cell elongation that are observed in the stage IIa ,IIb and IIc of prickle development (Chapter 2, Figure 14); (2) in clusters 6 (mainly in PIIc), 7 (mainly in PIIc and PIII), and 8 (mainly in PIII), we observed genes have potential function involved in cell wall organization, biogenesis and macromolecule metabolic processes. These processes might be related to cell wall thickening and lignification in developing and maturing of prickle. In addition, among the genes specifically highly expressed in stage IIc (cluster 5), we also observed genes involved in ethylene, jasmonic acid and auxin response. It may be related to the prickle (or/and stem) growth. 'Cell death', 'secondary metabolic' and 'anion transport' processes were also enriched among the genes that are a specifically highly expression in stage III, that may be associated with the prickles maturing.

Future more, in stages IIc and III (corresponding mainly to cluster 5, 6, 7 and 8), we found many genes that present GO enrichment in responding to different stimuli (wounding, insect, bacterium, virus, oomycetes), organic cyclic compound and drug catabolic process. These results are quite interesting from the perspective of defense function. Prickles are usually covered with a lot of bacteria, viruses or fungus (microorganisms). Some microorganisms species may randomly scatter on prickles. Some species probably are inhabitants that multiply on the spinescences (hence they are present in large numbers) and not just landed on the spinescence accidentally [START_REF] Halpern | Plant biological warfare: thorns inject pathogenic bacteria into herbivores[END_REF]. Prickle as a defense structure plays an important role against insects and mammal herbivores and the aggregated bacteria strengthen the defense of its physical structure (as we introduced in Chapter 1). Our results showed that the microorganisms may start

Discussion

to aggregating in stage IIc of prickle development. These results are highly consistent with the environment of prickles growth, because the prickles completely expose to the external environment when they are get in stage IIc.

Mechanisms underlying prickle initiation

We investigated in detail the genetic network that is controlling prickle initiation by comparing the transcriptome between prickle (P) and sample glabrous (NP) samples at the early stages of prickle initiation and development (or the corresponding stages in NP tissues). We provide a candidate gene pool contains 2118 genes (inhibitors and promoters) for prickle initiation and we recommended 660 priority genes by comparing the SDE gene in stage I and IIa. We explored these 660 specific genes of stage I included the most interesting GO enrichment processes for organ initiation, such as 'cell fate commitment', 'post-embryonic plant morphogenesis', 'anatomical structure formation involved in morphogenesis'. Among the genes involved in these seven SSE-GO terms, a large interaction network between these genes (25 out of 37 Arabidopsis protein) were detected. While most of these genes are highly co-expressed in stage I of prickless samples, and only few genes in prickle samples. This majority of genes are therefore putative inhibitors of prickle initiation that control cell fate, anatomical structure or post-embryonic plant morphogenesis (26 a). Moreover, many SDE genes expressed in PI (but not in NP) also highly expressed in other stages of P (Figure 31 b,d). That means some promotor genes may both involved in prickle initiation and development, and these genes are mostly presented in the 'common' SDE genes list of stage I and IIa (Figure 31 g). Therefore, there are probably a few interesting activators that exist in the common list.

Our final objective was to obtain the best candidate genes (inhibitors or promoters) for prickle initiation.

We combined the transcriptomics and genetic approach with a functional prediction. It allows us to obtain more confidence and a limited number of candidate genes (Figure 27). We exported the SDE genes located under the confidence interval of the major prickle loci (QTL on LG3 and LG4, (see [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF] Chapter 3)), and only 43 (out of 2118) genes meet these conditions.

Among them, we explored seven (RcAS2, RcJAG, RcWOX-g1, RcWOX-g2, RcDOT5, RcAKR1) that may positively regulate glabrous stem development (or inhibitor for prickle initiation). AS2 is a later organ boundaries protein as known to control proximal-distal patterning in Arabidopsis petals [START_REF] Chalfun-Junior | ASYMMETRIC LEAVES2-LIKE1 gene, a member of the AS2/LOB family, controls proximal-distal patterning in Arabidopsis petals[END_REF] and leaves [START_REF] Ueno | Histone deacetylases and ASYMMETRIC LEAVES2 are involved in the establishment of polarity in leaves of Arabidopsis[END_REF], adaxial/abaxial axis specification of leaves [START_REF] Wu | KANADI1 regulates adaxial-abaxial polarity in Arabidopsis by directly repressing the transcription of ASYMMETRIC LEAVES2[END_REF].

as2 mutant presents leaves with defect phenotype (Bumpy). scal-l as2 double mutant develops spines on the edge of the leaf [START_REF] Mateo-Bonmatí | Plastid control of abaxial-adaxial patterning[END_REF].

JAG is a C2H2 and C2HC zinc fingers superfamily protein, controls the morphogenesis of lateral organs.

Functions with AS1 and AS2 in the sepal and petal primordia to repress boundary-specifying genes, thereby, define these organs from their boundaries [START_REF] Xu | Arabidopsis genes as1, as2, and jag negatively regulate boundary-specifying genes to promote sepal and petal development[END_REF]. Loss JAG function causes organs to have serrated margins [START_REF] Dinneny | The role of JAGGED in shaping lateral organs[END_REF][START_REF] Ohno | The Arabidopsis jagged gene encodes a zinc finger protein that promotes leaf tissue development[END_REF].

WUSCHEL-related homeobox (WOX) family genes are known to be involved in different organ development.

WOX1 and WOX3 (or PRS) are important for lateral-specific blade outgrowth and margin-specific cell fate in Arabidopsis,nakata2012. A WUSCHEL-WOX 3B gene, dep, conditions the glabrous phenotype of rice leaves and glumes [START_REF] Asano | A WUSCHEL-related homeobox 3b gene, depilous (dep), confers glabrousness of rice leaves and glumes[END_REF]. WUSHEL is known to maintain the identity of the undifferentiated cells in the shoot apical meristem [START_REF] Laux | The WUSCHEl gene is required for shoot and floral meristem integrity in Arabidopsis[END_REF][START_REF] Ma | WUSCHEL acts as an auxin response rheostat to maintain apical stem cells in Arabidopsis[END_REF]. In roses, RcWOX-g1

and RcWOX-g2 are co-expressed with AS2 and JAG that may function in deciding the stem boundaries.

Interestingly, RcJAG, RcAS2, RcWOX-g1 and RcWOX-g2 were detected to be central in the protein--protein network (26c). They are co-regulating glabrous stem phenotype in roses with other important genes (as AFO, YAB2, YAB5, KNAT1, TCP4, NTT, ROXY1 homologue), indicating that lost function of these genes may cause out control of stem boundary, thereby, caused prickle initiation. Therefore, they are considered inhibitors for prickle formation. The expressed dose of these inhibitors may regulate the prickle density. That means more inhibitors lost function may cause more ground meristem cells to get the ability to out of stem boundary fate and driven into the first step of prickle (grow out).

We also explored two genes (RcMMD1, RcNAP1-like1) may be involved in the cell differentiation and division for prickle initiation and development, and an auxin gene (RcAMI1) may regulate prickle initiation and development.

RcMMD1 encodes a PHD (plant homeodomain) zinc finger protein. PHD fingers are Zn-coordinating domains that generally recognize unmodified or methylated lysines [START_REF] Jain | Characterization of the plant homeodomain (PHD) reader family for their histone tail interactions[END_REF]. They are central "readers" of histone post-translational modifications (PTMs) [START_REF] Jain | Characterization of the plant homeodomain (PHD) reader family for their histone tail interactions[END_REF], and can modify chromatin as well as mediate molecular interactions in gene transcription [START_REF] Sanchez | The PHD finger: a versatile epigenome reader[END_REF]. 14 times expression of RcMMD1 in PI more than NPI, suggesting that RcMMD1 play an important role in prickle initiation.

RcAMI1 encodes an amidase signature homolog protein. In AT, AMI1 involves in auxin and indoleacetic acid biosynthetic process [START_REF] Pollmann | Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone, indole-3-acetic acid[END_REF]. Auxin signaling regulates lateral root (LR) initiation and subsequent LR primordium formation in Arabidopsis [START_REF] Laskowski | Formation of lateral root meristems is a two-stage process[END_REF]; mutants defective in auxin biosynthesis, homeostasis, transport and signaling caused increased or decreased numbers of LRs [START_REF] Casimiro | Dissecting arabidopsis lateral root development[END_REF][START_REF] Fukaki | Auxin-mediated lateral root formation in higher plants[END_REF]. Interestingly, RcPKL encodes a CHD homolog protein, and its ortholog AtPKL negatively regulates auxin-mediated LR formation by chromatin remodeling [START_REF] Fukaki | PICKLE is required for SOLITARY-ROOT/IAA14-mediated repression of ARF7 and ARF19 activity during Arabidopsis lateral root initiation[END_REF].

In roses, RcAMI1 relatively highly expressed in all the stages of prickle (P) samples and lowly expressed in glabrous (NP) samples, while RcPKLis highly expressed in NP and lowly in P. These may suggest that:

prickle formation is also regulated by auxin signaling; RcAMI1 is a positive regulator for prickle formation and RcPKL is a repressor.

Nucleosome assembly protein 1 (NAP-1) shuttles histones into the nucleus, assembles nucleosomes, and promotes chromatin fluidity, thereby affecting the transcription of many genes [START_REF] Park | The structure of nucleosome assembly protein 1[END_REF].

AtNAP1 is involved in cell differentiation and cell population proliferation [START_REF] Galichet | Developmentally controlled farnesylation modulates AtNAP1; 1 function in cell proliferation and cell expansion during Arabidopsis leaf development[END_REF].

RcNAP1-like1 is highly expressed in both prickles and non-prickle samples, and more than two times higher in stage I of the prickle sample than in the glabrous sample. This supported RcNAP-1 may play an important role in cell proliferation both in the meristem tissue of prickle.

This thesis focused on filling the gaps in prickle knowledge using morphological, genetics, and genomic approaches. Our objective was to decipher the molecular and genetic control of the initiation and development of prickles on rose stems. Since the basic botanical terminologies (trichomes, prickles, thorns and spines)

were frequently inaccurately cited in scientific reports, and these words were confusing for most researchers, we first described and compared them in the Introduction in Chapter 1. One of the most confusing points is the relationship between trichomes and prickles, which is important for the experimental design in our study. The current mainstream hypothesis is that prickles originate from the epidermis [START_REF] Peitersen | Blackberries of New England-genetic status of the plants[END_REF][START_REF] Canli | A review on thornless roses[END_REF] and are considered to be modified glandular trichomes. In the late developmental stage, the prickles become cutinized as a hard-sharp appendage, like in roses [START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF], Rubus [START_REF] Peitersen | Blackberries of New England-genetic status of the plants[END_REF][START_REF] Coyner | Thornlessness in blackberries[END_REF][START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF]Khadgi and Weber, 2020a) and grapes (Ma et al., 2016b). In fact, the tissues from which the prickles originated are not clearly defined, since no anatomical evidence was ever provided to support the 'epidermis' hypothesis. To well understand the origin of prickles, their types, and their development, we investigated the prickle types in rose wild species, in parents and progeny of a F1 population (OW). We carried out a comprehensive anatomical study for two representative types of prickles (Chapter 2). In the OW progeny, we classified rose prickles into two major categories: non-glandular (NGPs)

and glandular (GPs) prickles. We also discovered other types of prickles in wild roses and classified them into the sub-categories of NGPs and GPs. We found that NGPs and the major structure (stalk) of GPs come from the ground meristem (the underlayers of protoderm). GP and NGP initiation take place at an early stage of shoot development (just below the shoot apical meristem). The first layers of gland cells of GP originate from epidermal cells (or protoderm) of the bump (the first stage of GPs). These new insights constitute valuable information for further study. We adjusted the experimental design in the middle of the project and decided to study the NGPs and GPs separately.

For the genetic approach (Chapter 3), we performed QTL analysis for these two types of prickles.

However, for the GPs, we were unable to detect any significant QTLs since the individuals presenting GPs

were not numerous enough in the OW population. Thus, we focused on exploring genetic determinism and the gene network for the NGPs, the most common type of prickles on rose stems. We detected a major locus on LG3 that controls the absence/presence of prickles on rose stems and several other loci controlling prickle density. In addition, to identify the relationship between prickles and trichomes at the molecular level, we 5.1 Two types of prickles in roses, glandular and non-glandular, and two different gene networks used a candidate-gene approach to characterize rose gene homologues known in Arabidopsis to be involved in trichome initiation. Almost no difference of transcript accumulation for these candidate genes between prickle and prickless roses were detected, suggesting that prickle and trichome initiation is controlled by two different genetic pathways. This conclusion was also supported by results from the transcriptomic approach (Chapter 4).

In Chapter 4, by transcriptomic approach (RNA-Seq), using a mixed pool of F1 individuals with or without NGPs, respectively, we discovered many genes involved in prickle initiation and development. We developed unconventional methods to optimize the candidate gene discovery. By combining a genetic approach and gene function prediction, we mined the ten best candidate genes for prickle initiation. By combining the results obtained by the anatomical/histological (Chapter 2), genetic (Chapter 3) and transcriptomic (Chapter 4) approaches, we proposed a genetic and molecular mechanism for prickle initiation and development (see Discussion below).

During this project, we provided a general framework to study the genetics and genomics of prickles in woody plants. Our study significantly improved the knowledge of the genetic determinism and molecular mechanisms underlying prickle formation in rose. Here, we discuss the implication of such approaches for rose breeding.

Two types of prickles in roses, glandular and non-glandular, and two different gene networks

In Chapters 2 and 3, by studying the morphology and anatomy of prickles, from their initiation to their complete development, and their distribution in the OW population, we proposed that non-glandular prickles (NGPs) and glandular prickles (GPs) are different types of prickles with different genetic determinism.

The most common prickles observed in the OW population are the NGPs. They have no glandular structure at any point in their development. Another type of prickles appears in some F1 individuals (17.9%)

but not in parents: these prickles have a gland head and were denominated by glandular prickles (GPs). So our results did not support the previous hypothesis that 'GP and NGP are the early and later stages of same prickle [START_REF] Coyner | Thornlessness in blackberries[END_REF][START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF]Ma et al., 2016b;Khadgi and Weber, 2020a).

For the first time, NGP and GP initiation and development were histologically characterized in-depth from the early to later stages. NGPs are initiated from the ground meristem below the protoderm.At the initiation stage, no essential difference was observed between GPs and NGPs. Both are initiated from the ground meristem below the protoderm. In GPs, the gland at the head of the prickle originates from the protoderm (or epidermis) cells of the newly formed prickle at the early stages of development. So our results did not support the previous hypothesis that 'Prickles originate from the epidermis' [START_REF] Peitersen | Blackberries of New England-genetic status of the plants[END_REF][START_REF] Esau | Anatomy of seed plants[END_REF][START_REF] Canli | Separation of thornless rose chimeras into their (Rosa sp.) consistent genotypes in vitro[END_REF][START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF].

In Chapter 2, at the morphological level, we reported more different types of prickles in the Rosa genus.

There are 'naked' and 'hairy' forms in NGPs and GPs. GPs can be unbranched and branched. Some GPs are present with a gland head, whereas some have several glands randomly distributed on their surface.

As in OW population, the most common type found in wild rose species or cultivars are the 'unbranched and naked' NGPs. The unbranched (naked or hairy) GPs are generally not present alone but always associated with NGPs. Moreover, the gland (a specific structure of GPs) was not produced at the prickle initiation but during the prickle's development (Chapter 2, Figure 14 m-r). Therefore, we supposed that GPs may be modified from NGPs.

A similar hypothesis was proposed for the trichomes: in an evolutionary perspective, the earliest glandular trichomes (GTs) are modified from the non-glandular trichomes (NGTs) [START_REF] Krings | Trichomes of the seed fern blanzyopteris praedentata: implications for plant-insect interactions in the late carboniferous[END_REF][START_REF] Lange | The evolution of plant secretory structures and emergence of terpenoid chemical diversity[END_REF].

The NGTs and GTs evolve from protoderm (or epidermis) only, and different gene networks (Chapter 1) have been reported to control the initiation of NGTs [START_REF] Folkers | Cell morphogenesis of trichomes in Arabidopsis: differential control of primary and secondary branching by branch initiation regulators and cell growth[END_REF][START_REF] Hülskamp | Plant trichomes: a model for cell differentiation[END_REF][START_REF] Balkunde | Chapter ten -trichome patterning in Arabidopsis thaliana: from genetic to molecular models[END_REF] and GTs [START_REF] Payne | Heterologous myb genes distinct from GL1 enhance trichome production when overexpressed in Nicotiana tabacum[END_REF][START_REF] Huchelmann | Plant glandular trichomes: natural cell factories of high biotechnological interest[END_REF][START_REF] Chalvin | Genetic control of glandular trichome development[END_REF]. This difference can be explained by morphogenetic evidences: mother protoderm cell(s) produced different structures, NGTs and GTs, which are probably controlled by different regulators. However, we do not know if the developmental program is different at the first mother cell initiation (cell fate) or later in the developmental process. In Chapter 1, I gave examples to show the developmental stages of GTs and NGTs (Figure 3). They look similar in the beginning:

one mother protoderm cell starts to enlarge. This enlarged cell continues to grow without cell cycle division and begins endoreplication (unicellular NGT, Figure 3 a1) [START_REF] Hülskamp | Plant trichomes: a model for cell differentiation[END_REF], or divides into two cells. The two cells then continue normal division (multicellular NGT, Figure 3 a2 and a3) [START_REF] Kintzios | Oregano: the Genera Origanum and Lippia[END_REF], or one of the two cells becomes a clear gland cell (multicellular GT, Figure 3 b andc) [START_REF] Bosabalidis | Glandular scale development and essential oil secretion in Origanum Dictamnus l[END_REF][START_REF] Karousou | Sideritis syriaca ssp. syriaca: Glandular trichome structure and development in relation to systematics[END_REF]. In Arabidopsis, it has been shown that interruption of endoreplication can modify unicellular NGTs into multicellular NGTs (Schnittger et al., 2002b,a). Since the molecular mechanism of GT initiation is still unclear, we do not know how the genetic pathways interact between NGTs and GTs.

For glandular and non-glandular prickles on rose stems, the major difference is the presence/absence of a gland. This difference appears the early developmental stage of GPs and NGPs. In GP initiation, ground meristem cells first rise and become a bump. At this stage, GP initiation is similar to that of NGP. Then, gland cells evolve from the protoderm (or epidermis) cells of the newly initiated prickle (Chapter 2). We can hypothesize that two genetic pathways control the GP and gland initiation. One controls the cell fate of the ground meristem cells (a process that may be shared with NGPs), and another pathway controls the cell fate of the protoderm (or epidermis) cells (only in glandular trichomes and may be similar to the gland initiation in glandular trichomes). Another possibility is that GPs and NGPs have different genetic pathways that control the cell fate of the first mother cell. Further studies are necessary to clarify these hypotheses. However, as demonstrated by the genetic approach (Chapter 3), GP and NGP genetic determinism is different, suggesting that the genetic network controlling GP and NGP development is different in rose. Since GPs were rare in the OW progeny and we were unable to study their genetic determinism in OW progeny, our further analyses focus on NGPs, the most common prickles in rose.

123

Genetic determinism and molecular mechanisms underlying NGP formation

These conclusions are different from the current mainstream hypothesis: prickles evolve from multiple cellular divisions of the epidermis [START_REF] Peitersen | Blackberries of New England-genetic status of the plants[END_REF][START_REF] Canli | A review on thornless roses[END_REF] and prickles are modified glandular trichomes that later become cutinized as a hard-sharp appendage [START_REF] Peitersen | Blackberries of New England-genetic status of the plants[END_REF][START_REF] Coyner | Thornlessness in blackberries[END_REF][START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF]Ma et al., 2016b;Khadgi and Weber, 2020a), or induced by glandular trichome [START_REF] Pandey | Transcriptome analysis provides insight into prickle development and its link to defense and secondary metabolism in Solanum viarum dunal[END_REF].

In the next section, we will compare the studies in Rosa and Rubus in detail.

Genetic determinism and molecular mechanisms underlying NGP formation

Prickle formation from initiation to late stages

For the first time, we present a detailed transcriptomic analysis of processes that may control prickle formation from initiation to late developmental stages, in rose (Chapter 4). We have combined anatomical and transcriptomic approaches to propose molecular mechanisms underlying prickle formation (Figure 34).

Just like other organs, prickle formation involves many cellular processes, including cell fate, cell differentiation, cell-to-cell communication, cell proliferation, cell cycle division, cell polarity, etc. For the genes mainly expressed in stages I or/and IIa (cluster 1 and 2), we discovered genes that are enriched in these processes. Stage I corresponds to prickle initiation with several cells from the ground meristem (below the protoderm) that acquire a new fate. These cells then grow to give the first stage of prickles (Figure 34, Chapter 2, Figure 14 a). Cluster 1 genes are specifically highly expressed in stage I, with genes described to be involved in organ or tissue development (floral, shoot, leaf, meristem, post-embryonic plant morphogenesis (Chapter 4, Figure 30)). This suggests that many genes in this cluster may be related to the cell fat and differentiation required for prickle initiation. Then, through cell proliferation, the prickle develops. Genes from cluster 2 are highly expressed in stages I and IIa, and their expression decreases as the prickle and stem develop. Numerous genes in this cluster are involved in cell proliferation and in the processes of cell cycle division. These results are highly consistent with the development of the prickle, i.e., prickle meristem cells gradually lose their meristematic activity as the prickle develops (Chapter 2, Figure 14 d-l ). We therefore propose that cluster 2 genes may be highly associated with cell proliferation required for prickle and/or stem growth at the early stage. In stage II, some cells still maintain their cell division ability (IIa an IIb), some (from top to bottom) lose this ability and start to elongate and to gradually enlarge Furthermore, in stages IIc and III (corresponding mainly to clusters 5, 6, 7 and 8), we found many genes that present GO enrichment in response to different stimuli (wounding, insects, bac-teria, viruses, oomycetes), organic cyclic compounds and drug catabolic processes. These results are quite interesting in terms of defense functions. Prickles are usually covered with many bacteria, viruses or fungi [START_REF] Halpern | Plant biological warfare: thorns inject pathogenic bacteria into herbivores[END_REF][START_REF] Halpern | Chapter 4 -biological warfare of the spiny plant: introducing pathogenic microorganisms into herbivore's tissues[END_REF][START_REF] Lev-Yadun | Color changes in old aposematic thorns, spines, and prickles. In Defensive (anti-herbivory) coloration in land plants[END_REF]. Some microorganisms (bacteria, viruses or fungi) may randomly scatter on prickles, such as Sporothrix, a fungus taht causes sporotrichosis (also known as 'rose gardener's disease'). This fungus lives throughout the world in soil and on plant matter. The infection occurs when the fungus enters the skin through a small injury. The 'organic cyclic compound' and 'drug catabolic' processes may also be related to the defense function against certain microorganisms. Some species are probably to spinescences (hence, they are present and can multiply to reach large numbers) and not just present accidentally [START_REF] Halpern | Plant biological warfare: thorns inject pathogenic bacteria into herbivores[END_REF]. Prickle as a defense structure plays an important role against insects and mammal herbivores, and the aggregated microorganisms strengthen the defense of its physical structure (as reported in Chapter 1). Our results suggest that the microorganisms may start to aggregate in stage IIc of prickle development. These results are highly consistent with the prickles environment at this stage, since the prickle is completely exposed to the external environment (no longer protected by leaves).

Based on our knowledge, the genomic studies on prickle formation are rare. In Solanum vivarium, a prickle (wild type, WT) and prickless (mutant) individuals were used in a transcriptomic study. [START_REF] Pandey | Transcriptome analysis provides insight into prickle development and its link to defense and secondary metabolism in Solanum viarum dunal[END_REF] sequenced the total RNA of the 'skin' that they peeled off from the later stage of stem development.

Although the samples were described as epidermis in the article (the epidermis is the first cell layer of the organ), the 'skin' might be more complex and may correspond to the term 'bark' in woody plants. 2018)). Thus, the wild type and mutant of S. vivarium are good materials to study glandular prickles. However, the sample (stem skin) was harvested when the stem and prickle were well developed. By comparing them with rose prickles, we consider that these samples may correspond to stages IIc and III (Chapter 2, Figure 14) in our study. To study prickle initiation, it is necessary to do so at the time of their initiation. Furthermore, the presence of trichomes and prickles at the same time (mix of structures) may complexify the interpretation of the results. By comparing the prickle and prickless samples, they found a higher percentage of DE genes that were enriched to responses to stimulus (both biotic and abiotic), responses to stress and responses to defense. If we consider that their material corresponds to stages IIc and III of the GPs, these processes were also found in stages IIc to III of the NGPs in our study. This may suggest that NGPs and GPs in the later stages both aggregate many microorganisms.

Zoon in prickle initiation

Through an anatomical study, we confirmed that prickle initiation does not happen in prickless individuals (Chapter 2, Figure 13 c and 15). That means: (1) prickless phenotype is caused by the absence of prickle initiation and not by an interruption during prickle development;

(2) prickle density is related to prickle initiation that takes place in the ground meristem. Those facts allowed us to combine the genetic and transcriptomic approaches to raise the question of prickle initiation. We thus combined these two approaches to be discussed and, furthermore, proposed a detailed hypothesis for the genetic determinism and molecular mechanisms underlying prickle initiation (Figure 35).

A complex genetic determinism for prickles in rose

In Chapter 3, we revealed a complex genetic determinism of NGPs in rose stems with a major locus on

LG3 that controls the absence/presence of prickles and several other loci controlling prickle density. We detected three QTLs on LG4, 3 and 1 which are involved in prickle density in the OW population.

We extended the description of the QTLs on LG3 that affect the presence/absence of prickles. We designated this locus as PRICKLE. On the basis of the phenotype-genotype relationship, we proposed that at this locus, the female OB and the male RW parents are both heterozygous (np/P), and that the presence of prickles is controlled by a dominant allele (P), and that the glabrous stem in the progeny is due to the combination of the two recessive alleles from both parents (np/np). Based on the genetic data, the presence of prickles seems to be controlled by activator(s) that is (are) present in prickle genotypes (P) and absent in glabrous genotypes (np/np). This np allele might be explained by a loss of function of the activator P (Figure 35 a). In addition, the PRICKLE locus is in the vicinity of other important loci for ornamental traits (continuous flowering, double flower and self-incompatibility (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF]).

These results are important for breeders who need to combine recessive alleles to obtain glabrous roses.

However, since modern roses are tetraploid, a recessive allelic combination could be difficult to abtain in a tetraploid background. Furthermore, we do not know how the np and P alleles would behave in a tetraploid background. Development of specific molecular markers of the recessive allele may by useful for breeders.

However, it should be mentioned that the two markers actually used (peak of the QTL) are not closely linked with the PRICKLE locus, and few recombinants are observed in the progeny.

Moreover, the phenotype of the individuals with the two recessive alleles is not stable and some of the hybrids regularly developed some prickles on parts of the stems, suggesting a leaky mutation. Indeed, this phenomenon is widespread in roses. Rose breeders have reported that glabrous mutants have either been unstable for the prickless trait [START_REF] Nobbs | American rose annual[END_REF][START_REF] Rosu | The development of putative adventitious shoots from a chimeral thornless rose (Rosa multiflora Thunb. ex J. Murr.) in vitro[END_REF], or have reverted to the prickly character after a freezing winter or other environmental stresses [START_REF] Nobbs | American rose annual[END_REF][START_REF] Oliver | A pre ¸cis of thornless development. The rose[END_REF][START_REF] Druitt | Thornless roses[END_REF][START_REF] Canli | A review on thornless roses[END_REF]. These reversions may be explained by the chimeric nature of the prickless mutation in roses [START_REF] Rosu | The development of putative adventitious shoots from a chimeral thornless rose (Rosa multiflora Thunb. ex J. Murr.) in vitro[END_REF][START_REF] Canli | A review on thornless roses[END_REF]. In our case, this hypothesis is unlikely as the mutation (np) is heritable. Another explanation is that the 'prickle state reversion' ability in rose could possibly be caused by unstable mutations involving transposable elements [START_REF] Canli | In vitro separation of a rose chimera[END_REF]. Such an unstable and reverted phenotype has been already observed in rose for the continuous-flowering phenotype. This phenotype is due to the insertion of a retrotransposon in a floral repressor [START_REF] Iwata | The TFL1 homologue KSN is a regulator of continuous flowering in rose and strawberry[END_REF]. Recombination of the retrotransposon can occur and can restore a hypomorphic allele. Another explanation could be genetic redundancy or a phenotypic compensation mechanism. Previous studies have reported that many engineered mutants do not exhibit an obvious phenotype, and several studies have provided evidence on how the compensatory network is activated to buffer against deleterious mutations, such as in Caenorhabditis [START_REF] Burga | Predicting mutation outcome from early stochastic variation in genetic interaction partners[END_REF], zebrafish [START_REF] Rossi | Genetic compensation induced by deleterious mutations but not gene knockdowns[END_REF] and mouse [START_REF] El-Brolosy | Genetic compensation triggered by mutant mRNA degradation[END_REF]. This evidence supports the hypothesis that phenotypic outcome does not depend on the intrinsic properties of a mutation but on a range of compensatory mechanisms within the individual [START_REF] Casci | Genetic support network to the rescue[END_REF] instead and, of course, on the plant environment. Prickles, as a beneficial structure, have gone through a long evolutionary history to protect plants against herbivores. Consequently, prickles in roses may carry a second genetic compensation to pathway to maintain their genetic robustness, which can be triggered when the major function gene of prickle initiation is mutated and loses its function.

This importance of prickle in rose co-evolution is also highlighted by the rareness of wild glabrous roses (see Chapter 2), suggesting an important role of prickles in rose adaptation.

The mechanisms underlying prickle initiation

Our final objective was to reveal genetic determinism of prickle and to obtain the best candidate genes (inhibitors or promoters) for prickle initiation. In the Chapter 3, and as discussed in the previous section, we detected a major locus on LG3 (PRICKLE) that controls the presence / absence of prickles [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF]. We have proposed that PRICKLE(P) is an activator of prickle initiation. We also detected three QTLs Concerning the PRICKLE locus on LG3, the best candidates are potential activators. We detected three activators (RcMMD1, RcAMI1, RcNAP1-like1), whicha re located in the confident interval of the main QTL on LG3(Chapter 3). Among them, the most interesting gene is RcMMD1, which encodes a PHD (plant homeodomain) zinc finger protein. PHD fingers are Zn-coordinating domains that generally recognize unmodified or methylated lysines [START_REF] Jain | Characterization of the plant homeodomain (PHD) reader family for their histone tail interactions[END_REF]. They are central "readers" of histone post-translational modifications (PTMs) and can modify chromatin and mediate molecular interactions in gene transcription [START_REF] Sanchez | The PHD finger: a versatile epigenome reader[END_REF]. RcMMD1 transcripts are accumulated fourteen times more in stage I of P than in NP tissues, supporting the hypothesis that RcMMD1 plays an important role in prickle initiation.

Interestingly, protein-protein network analysis showed that MMD1 was linked to an inhibitor protein network by interacting with AS2, JAG and WOX (Figure 35 b). RcMMD1 and RcJAG are both zinc finger genes with an opposite pattern of expression in stage I of P and NP tissues (Figure 35 c). They both interact with AS2 in the protein network (Figure 35 b). RcMMD1 might inhibit the expression of repressors such as RcJAG, RcAS2, RcWOX1-g1, or RcWOX-g2 (see below). Thus, we propose that RcMMD1 may activate prickle initiation by repressing the activity of the 'JAG-AS2-WOX' gene pathway.

RcAMI1 encodes an amidase signature homolog protein. In A. thaliana, AMI1 is involved in auxin and biosynthesis [START_REF] Pollmann | Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone, indole-3-acetic acid[END_REF]. Auxin signaling regulates lateral root (LR) initiation and subsequent LR primordium formation in Arabidopsis [START_REF] Laskowski | Formation of lateral root meristems is a two-stage process[END_REF]. Mutants, defective in auxin biosynthesis, homeostasis, transport or signaling, caused an increase or decrease in the numbers of LRs [START_REF] Casimiro | Dissecting arabidopsis lateral root development[END_REF][START_REF] Fukaki | Auxin-mediated lateral root formation in higher plants[END_REF]. Interestingly, RcPKL encodes a CHD homolog protein, and its ortholog ATPKL negatively regulates auxin-mediated LR formation by chromatin remodeling [START_REF] Fukaki | PICKLE is required for SOLITARY-ROOT/IAA14-mediated repression of ARF7 and ARF19 activity during Arabidopsis lateral root initiation[END_REF]. In roses, RcAMI1 is relatively highly expressed in all the stages of prickle (P) samples and weakly expressed in glabrous (NP) samples, while RcPKL is highly expressed in NPs and poorly in P samples. We can hypothesize that: prickle formation might also be regulated by auxin signaling with; RcAMI1 as a positive regulator of prickle initiation and RcPKL as a repressor.

AtNAP1 is involved in cell differentiation and cell population proliferation [START_REF] Galichet | Developmentally controlled farnesylation modulates AtNAP1; 1 function in cell proliferation and cell expansion during Arabidopsis leaf development[END_REF]. RcNAP1-like1 is highly expressed in both prickle and non-prickle samples, and its expression is twice as high in stage I of the prickle samples than in the glabrous ones. We can imagine that the speed of cell proliferation of the prickle precursor is faster than that of the surrounding meristem cells, and the mechanical force of the precursor cell proliferation pushes the formation of the early prickles. This supported the hypothesis that RcNAP-1 may play an important role in cell proliferation both in the meristem tissue of prickles and stems, and the dose of this gene expression may impact the speed or ability of the cell proliferation.

Surprisingly, we detected many SDE genes highly expressed in the glabrous samples, suggesting the importance of inhibitors that may block prickle initiation (Figure 31 • NGPs and GPs are different types of prickle and have their own developing process.

• Origin of prickles NGP: Ground meristem under protoderm. GP: multiple cells of the ground meristem first give rise to a bump, and then the protoderm (or epidermis) of the bump is differentiated into gland cells.

QTL (see Chapter 3)

The genetic determinism of prickle in rose is complex, with a major locus on LG3 that controls the absence/presence of prickles on the rose stem and several loci that control the prickle density. One major QTL on LG4 and two minor QTLs on LG1 and 3 were detected in the OW population [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF].

Limitation: Genetical study was conducted in a restricted genetic materials (a F1 population)

RNA sequencing (see Chapter 4):

Materials: Individuals of the F1 OW progenies (rose). Sampling: Samples of stages I, IIa, IIb, IIc and III strictly according to the described stages of prickle (P) and glabrous stem (NP, stems at the same stages as P) (see Chapter 2; [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF]. Workflow:

Results from previous studies

Prickle morphology:

• NGP and GP are the same type but present different development stages.

• Origin of prickles Materials: Prickly 'Caroline', Prickless 'Joan J.' and their offspring. (Rubus ideaus). Morphology of three prickly and three prickless offspring are not clear. Sampling: Sampled 'epidermal' tissues (the sampling method was not described). Basic hypothesis: NGPs, GTs or/and GPs on the stem of prickly 'Caroline' were considered as different stages of prickle. They were mixed in the sampling.

Workflow:

The limits of conclusions: 1) Used later stages of prickle development to answer the question of prickle initiation.

2) Based on an incorrect 'basic hypothesis' to propose the gene pathway is controversial.

3) The materials correspond to different stages of initiation and development of several structures (trichomes and prickles) making the results are difficult and distorted conclusions.

Detect SDE genes

Selected 75 genes belongs to 15 TF families as the key genes

Later stages of 'NGP+GT or/and GP + NGT' vs NGT Materials and technical limitations : 1) Some differential expressed genes can be due to the different genetic background, even if we try to limit the differences by using individuals from the F1 progeny (brother/sister).

2) Due to technical limitations, the early stages of prickle initiation and development could not be separated from the stem. We, thus, took the prickle and stem together for all the stages (leaves and buds were removed). We could also not obtain the prickle initiation stage only; stage I represents initiation and the first growth of the prickle. More details and Improvement solutions are presented in Chapter 4

NGP GP

Raspberry (A) and blackberry (B) [START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF].

Results from our study QTL

In raspberry (Rubus idaeus), a major QTL on LG4 and a minor QTL on

LG6 were detected [START_REF] Molina-Bravo | Quantitative trait locus analysis of tolerance to temperature fluctuations in winter, fruit characteristics, flower color, and prickle-free canes in raspberry[END_REF]). The QTL region on LG4 (position 0.101 Mb) is syntenic with the region on rose Chr4 (position 58,768 Mb), very close to the major QTL we detected on this chromosome. In another publication, [START_REF] Graham | Mapping gene H controlling cane pubescence in raspberry and its association with resistance to cane botrytis and spur blight, rust and cane spot[END_REF] detected a major QTL on LG2 (locus H) that controls cane pubescence. The locus is mapped on LG2, which is syntenic with the rose LG6 where one of the QTLs is detected in R. x wichurana.

Limitation: 1) Genetical study was conducted in a restricted genetic background.

2) Low density of genetic maps with SSR and AFLP makers 3) Prickle types was not considered as a factor in these studies.

1)

2)

Developing prickles in Rosa hybrid [START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF].

Limitation: Lack of morphological evidence from early stage. (46,189,407 -56,107,784 bp). Thus we proposed that the QTLs on LG3 and 4 were detected in diploid OW [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF] and tetraploid K5 populations (Bourke et al., 2018) are probably the same QTL. A minor QTL was detected on LG1 in OW but not in K5. This is perhaps due to the different genetic background of the parents. In fact, one parent of K5 is prickly and the other one is glabrous, whereas both parents of OW have prickles.

In Rosaceae, a major QTL on LG4 and a minor QTL on LG6 were detected in raspberry (Rubus idaeus) [START_REF] Molina-Bravo | Quantitative trait locus analysis of tolerance to temperature fluctuations in winter, fruit characteristics, flower color, and prickle-free canes in raspberry[END_REF]. We checked the synteny using synteny viewer tools (https://www.rosaceae.

RNA sequencing

Only a few pieces of transcriptomic studies have been published. Unfortunately, as previously mentioned, these studies are based on potentially incorrect assumptions: GP and NGP are the different developmental stages of same type pf prickle, and prickles either develop directly from GTs (e.g., raspberry) or the signal coming from GTs induces the prickle development [START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF][START_REF] Pandey | Transcriptome analysis provides insight into prickle development and its link to defense and secondary metabolism in Solanum viarum dunal[END_REF]Khadgi and Weber, 2020a). [START_REF] Khadgi | RNA-Seq analysis of prickled and prickle-free epidermis provides insight into the genetics of prickle development in red raspberry (Rubus ideaus l.)[END_REF] used two cultivars of red raspberry (Rubus idaeus L) to identify differentially expressed genes in the 'skin' tissue of 'Caroline' (a cultivar with prickle) and 'Joan J.' (a glabrous cultivar), and three prickly and prickless offspring. They sampled the 'epidermis' (the sampling method was not described) [START_REF] Khadgi | RNA-Seq analysis of prickled and prickle-free epidermis provides insight into the genetics of prickle development in red raspberry (Rubus ideaus l.)[END_REF], as prickles previously described as having originated from the epidermis (Khadgi and Weber, 2020a). As in [START_REF] Pandey | Transcriptome analysis provides insight into prickle development and its link to defense and secondary metabolism in Solanum viarum dunal[END_REF], an imprecise term, epidermis, was used to describe the tissues. A more accurate description of the materials is needed. Another issue in this study the sampling of later stages of prickle development to decipher prickle initiation. Indeed, GP or GT were considered as the early stage of prickle development. It should also be mentioned that the prickly cultivar 'Caroline' presents NGPs, GPs or/and GTs and NGTs, while the prickless cutivar 'Joan J.' presented only

NGTs. This means that in this study, the experimental design does not consider all the variable factors (NGP, GP, or/and GT). The six offspring were only described as prickly and prickless. Do they have NGPs, GPs, GTs or NGTs? This point is not clear. Furthermore, we do not know if the genetic background of the two cultivars is close or not. All these elements make it very difficult to interpret the results and to compare them with our results.

especially when the morphological characteristics of trichomes and prickles are different, and when almost every chromosome has one or more candidate genes. Therefore, it is particularly important to gain further information by studying the transcript accumulation of these candidate-genes during prickle initiation and development. Consequently, we quantified the transcripts accumulation of 10 TF genes in glabrous and prickle F1 individuals at different developmental stages by RT-qPCR. Unfortunately, only minor differences were observed between glabrous and prickle samples. The main differences are between developmental stages but not between the type of stems (as demonstrated by the heatmap analysis). In Chapter 4, we compared the nine genes' whose expression was normalized by RNA sequencing and obtained quite similar expression patterns to those previously found by the RT-qPCR in Chapter 3. In addition, through Gene

Ontology enrichment studies, we observed that some candidates for prickle initiation and development might be involved in organ development, including flowers, leaves, shoot, but not trichomes.

In conclusion, we suggest that NGPs and NGTs have different genetic pathways that control their initiation and development. This conclusion is different from the previous hypothesis: rose prickles and trichomes share a common genetic pathway [START_REF] Khadgi | RNA-Seq analysis of prickled and prickle-free epidermis provides insight into the genetics of prickle development in red raspberry (Rubus ideaus l.)[END_REF].

Beyond this study, what we can do next?

The genus Rosa is a very interesting model as it includes species at different ploidy levels, as well as rich phenotypes and a large genetic diversity. However, due to the highly heterozygosity character, the assembly of a rose genome is always a challenging task, which greatly limits the progress of related research in rose.

The completion of two high-quality genomes is a starting point of genomic research in rose (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF][START_REF] Raymond | The Rosa genome provides new insights into the domestication of modern roses[END_REF]. For genetic studies on prickles, we compared different genetic studies thanks to the positioning of the genetic markers on the sequence of the reference genomes. We easily combined genetics, candidate gene approaches and transcriptomic approaches. This integrative analysis has greatly enhanced our understanding of the genetic mechanism of prickle initiation and development.

In this study, we provide a framework for studying the genetics and genomics of quantitative traits in a heterozygous woody species. Further studies that will combine GWAS, genomics (DNA sequencing) and functional validation can be done to go deeper into the analysis of the molecular mechanisms underlying the complexity of prickles in rose and to develop markers for breeding prickless roses.

Using genetic diversity to decipher prickle regulation and to develop markers

for assisted breeding selection of prickless roses

GWAS study of different types of prickles (as a complex trait)

As a complement to QTLs analyses in F1 progeny, a genome-wide association study (GWAS) could be used on a rose core collection to study genetic determinism. QTL mapping is an efficient approach to study the genetic determinism in a target population and for a given trait. It provides high statistical power for detecting QTLs. Despite the fact that QTL mapping has proved and remains a powerful method, it suffers from two fundamental limitations: the genetic basis is narrow in biparental approaches and the number of recombinations present in the F1 individuals limits the mapping resolution (especially if the population size is low). Consequently, we need larger populations to increase the resolution [START_REF] Korte | The advantages and limitations of trait analysis with GWAS: a review[END_REF].

GWAS overcomes the limitations of QTL mapping but introduces other issues [START_REF] Korte | The advantages and limitations of trait analysis with GWAS: a review[END_REF].

One of the main advantages of GWAS is to work on a larger genetic diversity, while the disadvantage is that the method is sensitive to the population structure that may lead to many false positives. GWAS can offer a very fine resolution (based on the LD decay). However, the power of QTL detection will be determined by the allele frequency. For instance, effects of rare alleles are difficult to detect. In GWAS, the genetic interaction between loci (epistasis), or the interaction between loci and the environment are not considered.

Therefore, GWAS is often complementary to QTL mapping and, when performed together, they alleviate each other's limitations [START_REF] Zhao | An Arabidopsis example of association mapping in structured samples[END_REF][START_REF] Brachi | Linkage and association mapping of Arabidopsis thaliana flowering time in nature[END_REF].

Since QTL mapping and GWAS are both based on the detection of correlations between phenotypes and molecular markers, the evaluation of the phenotype is an important issue when initiating genetic approaches.

For example, as we discussed in the first section, we suggested that GPs and NGPs should be studied separately in F1 population ( [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF], Chapter 3). Wild rose species or cultivated varieties have more diversified phenotypes than the F1 hybrids from the OW population. The scoring of the trait is then more complicated in GWAS. In Chapter 2, we proposed a rapid method to phenotype the different prickles in rose according to several characteristics. First, the presence of a glandular head (GPs vs. NGPs), then the presence of branched (unbranched vs. branched prickles) and, finally, the presence of hairs on the prickles ('naked' vs. 'hairy'). This method of phenotyping can be used by focusing on the different types of prickles and their anatomy. Different GWAS panels have recently been developed in roses [START_REF] Schulz | Genome-wide association analysis of the anthocyanin and carotenoid contents of rose petals[END_REF][START_REF] Nguyen | Genetic dissection of adventitious shoot regeneration in roses by employing genome-wide association studies[END_REF][START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF], and unpublished data from the GDO team) with a high-density genotyping using an AXIOM-ARRAY. These panels can be studied for different types of rose prickles (with the proposed method). It can help to identify new loci and to then validate other good candidates identified by the transcriptomic approach (Chapter 4).

Develpment of genetic markers for assisted breeding of prickless roses

Molecular markers remain essential for genomic research and for molecular breeding of any crop. The latest developments in rose genomics have led to the generation of a wide range of genomic tools and technologies for application in ornamental trait improvement. In Chapter 3, the peak markers of QTLs on LG3 are highly associated with the absence and present of prickles. However, few recombinants are observed in the progeny between the peak marker and the PRICKLE locus. Further studies are necessary to develop specific markers for assisted selection of prickleless roses. Combining QTL and GWAS results may help to develop markers that will be closer to the PRICKLE locus.

Single-nucleotide polymorphism (SNP) markers have now become the most popular markers due to advances in sequencing technologies. DNA sequencing (or whole genome sequencing) by Illumina technology is now becoming cheaper and easier and could be used for genotyping in GWAS (Genotyping by Sequencing).

Through mapping on the reference genome (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF][START_REF] Raymond | The Rosa genome provides new insights into the domestication of modern roses[END_REF], it was possible to detect SNPs and Indels everywhere on the genome. SNP and Indel effects can then be annotated as frameshift variants, new stop codons, splicing variants (splice donor or splice acceptor variant) or intron variants. Combined with the genetic knowledge from Chapter 3, a strategy can be developed to mine the SNP markers and associated SNP with prickless or prickle alleles. The preferred materials for DNA sequencing are the prickle and prickless genotype in another population, which was obtained in China from a cross between R. chinensis 'Old Blush' (OB) and R. wichuraiana 'Basyes' Thornless' (which may be a mutant or offspring of RW) [START_REF] Zhou | Hybrids identification and genetic analysis in diploid roses population (f1) using ssr markers[END_REF]. The genetic information for prickle trait in this population will facilitate the data analysis in subsequent studies, and the sister genotype can reduce the uncertain genetic background factors.

Detecting the genetic diversity of the candidate genes discover in Chapter 4

Through the whole genome sequencing we mentioned above, a large number of genes can be assembled and annotated based on the reference genome. Presently, around 20 roses (prickly) genomes (short reads)

have been released [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF][START_REF] Raymond | The Rosa genome provides new insights into the domestication of modern roses[END_REF]. Rose comparative genomic strategies in prickle and prickless samples can be used to detect the genetic diversity of the candidate genes discovered in Chapter 4. However, the genome of prickless genotypes should be re-sequenced. The seven glabrous genotypes we presented in Chapter 2 are a material of choice to be re-sequenced. They are R. multiflora 'inermis', R. wichurana 'Bayses' Thornless', R. pimpinellifolia 'lutea', R. banksiae 'alba plena' and R. banksiae 'lutea', R. banksiae var. normalis, R. fraxinifolia Lindl.

RT-pPCR to identify if the inhibitors can impact NGP density

In Chapter 4, we mentioned many good candidate genes related to prickle initiation. Most of them are mainly expressed in prickless samples, and are considered to be inhibitors of prickle formation. We proposed that the inhibitors may be involved in a complex regulatory network that could impact prickle density. To test this hypothesis, we can use a RT-qPCR method to test if the transcript accumulation of those genes can be correlated with the density of NGPs. Different F1 individuals from the OW progenies with different density can be used. Materials should be sampled at an early developmental stage such as Stage I.

We can expect to detect a correlation between the transcript level and prickle density. These inhibitors are RC3GO350900 (RcPKL), R3G0389900 (RcWOX-g1), RC4G0448500 (RcWOX-g2), RC3G0419900 (RcAS2), RC4G0393200 (RcJAG), RC4G0398800 (RcDOT5), RC3G0386900 (RcAKR1), RC1G0289800

(AFO), RC70233600 (AFO), RC4G0428600 (MYB16), RC1G0527600 (KCS6), RC7G0011600 (KNAT1), RC7G0049900 (MYB106), RC3G0071300 (MYB106), RC2G03132700 (BOP2), RC2G0036100 (YAB5), RC5G0428600 (YAB2) etc. The first seven can be studied in priority.

Functional validation

Through this project, we have explored ten good candidate genes for prickle initiation. Seven RcAS2,RcJAG,RcDOT5,RcAKR1) were proposed to be positive regulators of glabrous stem development (or repressors of prickle initiation). Three (RcMMD1, RcAMI1, RcNAP1-like1) were suggested to be activators of prickle initiation and development. The candidate genes described in this study are highly associated with prickle formation since we combined transcriptomic and genetic approaches. However, it is still too early to say that they are the key regulators. Further research is necessary and, especially, functional validation.

For the seven potential repressors, we have suggested that using a RT-qPCR method to identify their relationship with prickle density as in the previous section. Another hypothesis is that they are the important regulators for the boundaries of glabrous stem development. To validate this argument, we can knock-down or silence these genes in individuals with glabrous stems to check if it will give a prickle phenotype or a defect stem (compare with glabrous).

For the three potential promotors (RcMMD1, RcAMI1, RcNAP1-like1), two approaches can be proposed for a functional validation: (1) An ectopic expression of these genes in glabrous individuals to check if the transgenic roses will have prickles;

(2) A knock-down or silencing of the genes in individuals with prickles to check if transgenic roses will be prickless or will have a lower prickle density.

Since prickle and trichome development might not share a similar regulatory network (see previous discussion), functional validation in A. thaliana is not essential. Functional validation has to be done in rose where transient and stable transformation protocols exist [START_REF] Firoozabady | Regeneration of transgenic rose (hybrida) plants from embryogenic tissue[END_REF][START_REF] Randoux | Gibberellins regulate the transcription of the continuous flowering regulator, RoKSN, a rose TFL1 homologue[END_REF].

A recent review [START_REF] Smulders | In the name of the rose: A roadmap for rose research in the genome era[END_REF] summarized rose genetic transformation. It should be observed that some candidates RcAKR1,RcMMD1,RcNPI1) are duplicated and present paralogs. This may affect functional validation of single genes and may complicate the analysis. We should therefore pay more attention to this point for the experimental design and for the interpretation of the results. Abstract : Prickle is an undesirable trait in many crops as it makes crops difficult to handle, harvest, and can injure workers. Roses are among the most important ornamental plants, and most roses present prickles on their stems. There is a strong demand from producers and breeders for glabrous rose cultivars, particularly in cut roses. The genetic and molecular mechanisms underlying prickle initiation and development remain still largely unknown. Our objectives are to decipher the genetic and molecular control of prickle initiation and development in rose using anatomic, genetic and genomic approaches. By a survey of the different types of prickle within the genus Rosa, we classified them in two types: non-glandular (NGP) and glandular prickles (GP), with the NGP being the most common. We demonstrated that NGP are originated from a cell layer below the protoderm contrary to what was previously described. Using a F1 progeny, we detected four QTLs controlling the presence and density of stem prickle. We characterized rose gene homologues known in Arabidopsis that involved in trichome initiation. Minor different expression of the homologues in P and NP, suggesting different gene pathway between prickles and trichomes. Molecular bases of prickle initiation and development were explored using an RNA-Seq strategy by comparing the transcriptome (i) of glabrous and prickle shoots and (ii) during prickle development. We have identified key genes and regulatory networks controlling prickle initiation and development, with interesting genes below the QTLs. Through this project, we have built a genetic model system for studying prickles and open new research areas in the plant sciences.

I

  would like to extend my gratitude to Dr. Jean-Claude CAISSARD and Dr. Michel HERNOULT for being my individual monitoring committee. Their insightful comments and encouragement incented me to improve my research from various perspectives. I'm extremely grateful to the members of my thesis juries : Dr. Sylvie BAUDINO (Jean Monnet University, Saint-Etienne) and Dr. Patrick LAUFS (INRAE, Jean-Pierre Bourgin Institute, Versailles) for accepting to be the rapporteurs of my thesis and Dr. Beatrice DESNOYES-ROTHAN (INRAE, Fruit Biology and Pathogens, Bordeaux) and Dr. Nathalie LEDUC (University of Angers, Angers) to be the examiners.

Hair

  (botany) Hair like structure, they refers to trichomes Hypomorphic mutation A type of mutation wherein the change in gene leads to the partial loss of the normal (wild-type) gene function, such as by reduced expression of the RNA or reduced activity of the protein xv

  in early stageProtoderm(botany) The primary meristem from where the epidermis of the plant are derived Pubescent (botany) Covered with short soft hair Uniseriate (botany) Arranged in a single row, layer, or serie Thorns (botany) Thorns are modified from stem or shoot, have vascular bundles Trichomes (botany) Trichomes from the Greek τ ριχωµα (trichôma) meaning "hair", are epidermal appendages of diverse form, structures, and functions. They originate from the protoderm (or epidermis) only. Spines (botany) Spines are modified from leaves or part of leaves (leaflet, stipules, petiolars), have vascular bundles Spinescence (botany) A general designation for the sharp appendages, such as prickle, thorn and spine xvi List of Supplementary Figures A.1 Frequency distribution and Q-Q plot of non-glandular prickles on four internodes in the OW population for three years . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.2 Phylogenetic analysis of the transcription factor family involved in trichome initiation and development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.1 An overview of the pipeline for RNAseq Bioinformatics analysis . . . . . . . . . . . . . . B.2 Quality control of clean reads in each dataset . . . . . . . . . . . . . . . . . . . . . . . . B.3 MA plot visualized the differences between expression taken in two compared samples . . B.4 GO biological process tree for the specific and common SDE genes of each compared groups PIIavsPI and NPIIavsNPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.5 Heatmaps showed the 2118 SDE genes of PIvsNPI involved in the interesting GO enrichment and their co-expression pattern. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.6 A phylogenetic tree forWOX protein family . . . . . . . . . . . . . . . . . . . . . . . . . B.6 C2H2 zinc finger proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.6 LATERAL ORGAN BOUNDARIES (LOB) domain family proteins . . . . . . . . . . . . B.6 NAD(P)-linked oxidoreductase superfamily proteins . . . . . . . . . . . . . . . . . . . . . B.6 PHD finger (C4HC3-type zinc finger-like) proteins and Nucleosome assembly protein (NAP) family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.6 Chromodomain helicase DNA-binding (CHD) subfamily proteins and Amidase signature family proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Figure 1 :

 1 Figure 1: General methods to distinguish trichomes, prickles, thorns and spines. (a) trichomes on Arabidopsis leaves; (b) the anatomy of an Arabidopsis trichome; (c) prickles on a rose stem; (d) an anatomical illustration of prickles; (e) a shoot thorn of Pyrus cordata; (f) a citrus thorn; and (g) an anatomical illustration of thorns and spines. Produced by NN ZHOU and XW DOU.

Figure 2 :

 2 Figure 2: Diversity in trichomes. Non-glandular trichomes: unicellular (a) unbranched and (b) branched trichomes; multicellular (c) uniseriate unbranched, (d) multiseriate unbranched, and (e) uniseriate branched trichomes. Glandular trichomes: (f) unicellular unbranched; multicellular (g, h, i, and k) unbranched and (j) branched. (m) Trichome with non-glandular and glandular branches. Reproduced by NN ZHOU and XW DOU.

  a) or multicellular trichome (Figure 3 b and 3 c); (2) One protoderm cell that gives rise to a uniseriate or multiseriate trichome, but neighboring epidermal cells give rise to the pedestal of the trichome;

1. 1

 1 Figure 4: Himalayan snowball plants. (a) Saussurea medusa. (b) Saussurea gnaphalodes. (c) Saussurea aster. (d) Saussurea glacialis. Credits: CGIN nature film crew (a); iNaturalist (Kulbhushansingh Suryawanshi (b); Harry Jans (c); Ruslan (d)).

Figure 5 :

 5 Figure 5: Summary the genes network of non-glandular trichome initiation.

Figure 6 :

 6 Figure6: Regulation of the cell cycle during trichome development[START_REF] Evert | Esau's plant anatomy: meristems, cells, and tissues of the plant body: their structure, function, and development[END_REF]. G1 phase refers to cell size increases and cellular contents duplicated, S phase to DNA and chromosomes replication, G2 refers to cell enlargement and organelles development and proteins synthesis in preparation for cell division. M phase refers to mitosis followed by cytokinesis (cell separation). Reproduced by NN ZHOU.

1. 3 Figure 7 :

 37 Figure 7: Morphology and anatomy of wild rose. (a) Terms used for the position of the flower (cross-section of a flower), (b) bud, (c) leaves and fruit, (d) fruit and pedicel prickles. Produced by NN ZHOU and XW DOU.

  a), rarely few; the fruit is an achene, rarely drupaceous (Figure 7 b); sepals are persistent (Figure 7 c); leaves are alternate, odd-pinnate (Figure 7 d), and very rarely simple (except in R. persica)

Figure 8 :

 8 Figure 8: Rose growth habits: (a) Erect type of roses in a variety of crown widths and heights; (b) Semi-erect rose; (c) Trailing rose; (d) Climbing rose. Reproduced by NN ZHOU and XW DOU.

1. 3 Figure 9 :Figure 10 :

 3910 Figure 9: Continued on the next page

Flowers

  are generally solitary (Figure 10 a-f) or in a corymb (commonly consisting of flowers 2 ∼ 3 flowers (Figure 10 g-i and j), a few contain more than 10, e.g., R. banksiae 'Alba Piena' (Figure 10 j)), and a few in form a compound corymb (Figure 10 k-n) or panicle inflorescence (Figure 10 o). Wild rose flowers

1. 3

 3 Figure 11: The blue bird fresco (detail from a modern reproduction by Émile Gilliéron fils) from the House of Frescoes at Knossos, Late Bronze Age (∼ 1450 B.C.); Paintings on wet lime plaster, H. 60cm, Heraklion Museum of Crete, Greece.

Figure 12 :

 12 Figure 12: Continued on the next page

Figure 12 :

 12 Figure 12: Chinese old garden rose paintings throughout history. (a) 'Bái qiángwéi tú', Chinese Silk Painting; Painter: Mǎ Yuǎn ( 1160-1225 BC), Song Dynasty; Texture: Silk; Size: 26.2cm x 25.8cm; Collecting of the Palace Museum in Bejing, China. (b) Qiángwēi shān niǎo zhóu, Chinese Handscrolls; Painter: huá yán (1682-1756 BC), Qing Dynasty, Kangxi mark; Size: 127.1cm x 55.5cm; Texture: paper; Collection of the Palace Museum in Bejing, China. (c) Yùfú túzhóu and Shuāngzhì Túzhóu (d) Chinese Silk Painting; Painter: Lü Jì (1477-unknown), Ming Dynasty; Size: 153.4 cm x 98.3 cm (c); 128.4 cm x 84.9 cm (d); Texture: Silk; Collection of the Shanghai Museum. (e,f,g) Falangcai ('foreign color') vase, Chinese porcelain, Qing Dynasty, Qian long enamel mark and period (1736-1795 BC); Collecting in the Shanghai Museum. (h) Wucai shieryue huā huì wenbēi -Yuèjì, Chinese porcelain, Qing Dynasty Kangxi mark and period (662-1722 BC); Size: 4.9 cm x 6.7 cm; Collection of the Shanghai Museum. Description: Twelve flowers correspond to twelve months, and the Rose cup to November: 'Unlike a thousand other species that die out, this one alone blazes in red throughout the year.'

  g). These cell proliferation ability and cell division orientation may determine the prickle shape and the width of the prickle base in the later stages. In stage IIb, as the leaves open, the prickles are growing outwards (Figure 13 a -stage PIIb). The cells (from up to down) of the lower part of the prickle are gradually stopping proliferation and begin to enlarge and elongate, except the bottom part (Figure 14 h and i). In stage IIc, after the leaves are fully opened, prickles are almost fully developed and forming downward curved hooks (only for curved prickle) (Figure 13 astage PIIc). All the cells continue to elongate, and most cells gradually stop dividing at the end (Figure 14 j-l). For GPs, their developmental stages are similar to NGP, except the development of the gland head (Figure 13 b; Figure 14 p and 14 r). The gland is usually surrounding by one cell layer and occasionally two cell layers (Figure 13), and occasionally two cell layers (Figure 14 o and 14 p). The division stopped at early stage IIa and then the cells only enlarge, thereby forming a glandular head (Figure 14 p and 14 r). The size of the glandular head only slightly increases during GP development (100 ∼ 150 µm).

Figure 13 :

 13 Figure 13: Developmental stages of non-glandular and glandular prickles in OW individuals. (a) non-glandular prickles (NGP, OW9137), (b) glandular prickles (GP, OW9106), and (c) glabrous stems (NP, OW9068). For the glabrous stems, the developmental stages correspond to the same developmental stages for the stem with prickles. 49

Figure 14 :Figure 15 :

 1415 Figure 14: Anatomy of Non-glandular and glandular prickles in OW9106. (a-c) Macroscopic pictures of the different stages of GP and NGP on the stem; Anatomy of stage I (d-f), IIa (g), IIb (h and i) and IIc (j-l) of non-glandular prickle; Anatomy of stage I (m-o), IIa (p) and III (q and r) of glandular prickle. White dotted frame: the first internode; I: prickle initiation; Pro: protoderm; GM: ground meristem; PM: prickle meristem; PMCL: prickle meristematic cell like; E: epidermis; PG: precursor gland; G: gland; AL: abscission layer structure-like.

  13 a -stage I and IIa). The primordial cells give rise to an oblique triangular structure (100 ∼ 500 µm) that keeps upwards growth. Then a large difference in shapes appears at the later stages. The size of the triangular structure in stage I is one of the important factors that decides the size of mature prickle. Prickle density is determined by the number of prickle initiation at early stage of shoot development and normally no prickle initiates at later stages (stage IIb and later). But there are slightly differences for prickle initiation in different genotypes. In R. ecae (Figure17a-e), R. laxa (Figure17 f-i) and R. omeiensis (large winglike prickle, Figure19 a-h), prickle initiation only happened at shoot tip; therefore, all the prickles are at the same stage on the same location of the stem. Their developmental stages are quite similar to the one previously described for the prickle of OW9137 (previous section). While in R. sherardi and R. moschata, prickle initiation happened not only at the shoot tip, but also appears on stages PIIa or PIIb in different parts of stem (Figure18b and 18 i). Prickle initiation can take place during a longer period, and the prickles that initiate later are relatively smaller at maturity (Figure18a-f, 18 h-k). Thus, location of initiation is other important factors that impact the density and the size of prickles present on the mature stem. Prickless (NP) or almost (7) NP or almost (4) N P ( 3 )

Figure 16 :

 16 Figure16: Different types of prickles in rose resources. The number of roses is presented in the brackets. The '4' in red means that 4 genotypes were sometimes prickless or sometimes have a few NGPs (R. banksiae var.normalis , R. banksiae 'alba plena', R. banksiae 'lutea', R. pimpinellifolia 'lutea'). OW individuals (showed in blue) didn't count in the number.

  varieties: R. iwara (Figure 20 n and 20 o), R. stellate, R. caninae 'freya', R. horrida, R. rubella (Figure 21 a-f), R. damascena (Figure 21 g-j), R. gallica officinalis, R. prattii, R. willmottiae, R. tsinglingensis, R. marmorata, R. pimpinellifolia 'king of the scots', R. pimpinellifolia 'aïcha' and R. anemoniflora.

Figure 18 Figure 19 :Figure 21 :Figure 22 :

 18192122 Figure 17: Non-glandular prickle development process in (a-e) R. ecae and (f-i) R. laxa

  l); A prickle has a similar size of non-glandular and glandular branches 59 2.4 Discussion (Figure 20 s).

  Several transcription factors (TFs) such as MYB, bHLH, WD40, WRKY and C2H2 zinc finger families' proteins have been identified as being involved in trichome initiation and development (reviewed in Balkunde et al. (2010); Pattanaik et al. (2014); Ma et al. (2016a); Huchelmann et al. (2017); Chopra et al. (2019)).

  [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF] and QTL analysis. The plants were grown in a field and managed by the Horticulture Experimental Unit (INRAE, Angers, France). The plants were pruned each December. In the following spring, new stems developed from the axillary buds from the old pruned stems, and are referred to as "floral stems" since they develop flowers. Later, new stems arise from the base of the plant and are referred to as "main stems". They remain vegetative in once-flowering individuals and may become floral in continuous-flowering individuals.

  [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF] and modified by[START_REF] Lopez-Arias | Characterization of black spot resistance in diploid roses with QTL detection, meta-analysis and candidate-gene identification[END_REF].

  23 a) (a mean of around ten prickles on four internodes). In the F1 progeny, hybrids without prickle can be observed (14 out of 151; no prickle on the three stems scored over three years). These hybrids with glabrous stems (Figure23 b) are referred to as 'prickless' individuals (Figure23 c). Out of the 137 F1 individuals with prickles (Figure23 b), nine hybrids were nearly prickless (prickle number on four internodes < 1 for three scored years and two types of stems (Figure23 d)), and numerous stems were glabrous for some individuals, whereas other stems presented a few prickles (variable between the genotypes with unstable states between years and types of stems). Macroscopic analysis shows that parents that present prickles originated from a 'non-glandular' structure. These prickles are referred to as Non-Glandular Prickles (NGP). All the F1 prickly individuals (137 out of 151) have NGP. However, some individuals with NGP prickles also present another type of prickle (27 out of 137). These prickles present a 'glandular head' structure and are referred to as Glandular Prickles (GP) (Figure23b and 23 c, Supplementary Figure A.1). Since the presence of GP in the OW progeny is rare (27 and 12 out of 151 on flowers and main stems, respectively; Figure 23 d) and very irregular, we decided to consider only NGPs in this study. The Shapiro-Wilk normality test and the Q-Q plot of original data (W = 0.692 ∼ 0.936, p -value < 2.96 × 10 -8 ) (Supplementary Figure A.1) and variance residuals (W = 0.88591, p -value < 2.2e -16

Figure 23 :

 23 Figure 23: Different types of prickles on the OW progeny stem and their distribution. (a) Stem prickles in the female 'Old Blush' (OB) and the male R. x wichurana (RW); NGPs: non-glandular prickles. (b) Stem prickles in F1 progeny. Glabrous: no prickle whatsoever on the recorded stems in the three years. (c) Macroscopic photos of the terminal part of the stems with different types of prickles (number of offspring); GPs: glandular prickles. (d) The distribution and Q-Q plot of NGPs and GPs in the F1 progeny in 2018; PF: prickles on the floral stem; PM: prickles on the main stem.

  Q1) means 25% observations are below this quantity (approx) 3rd Qu third quartile(Q3) means 75% observations are below this quantity (approx) σ 2 G variance components of genotype, genotype x year interaction (σ 2 GY ), genotype x stem interaction (σ 2 GS ), and the residual error (σ 2

Figure 24 :

 24 Figure 24: LOD curves of the QTL scan for the NGPs on the floral (FM) and main (PM) stems in (a) female (OB) and (b) male map (RW) calculated with a non-parametric model for the three years (2016, 2017 and 2018, with red, blue and green lines, respectively). The LOD threshold value is represented by a dotted line in red, blue and green for 2016, 2017 and 2018, respectively.

Figure 25 :

 25 Figure 25: LOD curves of the QTL scan for the NGPs on the floral (FM) and main (PM) stems in (a) female (OB) and (b) male (RW) maps calculated using the two-part approach. The LOD (p) value (penetrance) is in red, the LOD (µ) value (severity) is in blue, and the LOD (p, µ) value is in black. The dotted line represents the LOD threshold. QTLs above threshold value are indicated by stars: red for penetrance, blue for severity.

  Figure27: The interaction of the different alleles of the LG3 QTL between OB and RW. Genotype: ac, ad, bc, bd (number of individuals), a/b and c/d alleles belong to females and males, respectively. For the phenotype, the mean values of prickle density for PF and PM for the three years are presented. Some individuals are highlighted with green dots (OW9106 and OW9107), blue dots (OW9062, OW9021, OW9052 and OW9109) and red dots (OW9067 and OW9068). The asterisk indicates that the difference is significant with a p-value of less than 0.05

Figure

  Figure A.2 b).

  are in the same clade of CPC, TRY, ETC1 and ETC3. RC1G0560100 and Chr1g0359121 are more closely related to TRY and CPC, and are referred to as RcTRY and RcCPC, respectively. (Supplementary Figure A.2 d).

Figure 28 :

 28 Figure 28: Transcript accumulation of candidate genes followed by qPCR during prickle development. (a) A heatmap of samples and genes. (b) The scatter plot of the candidate genes' normalized expression in prickle and glabrous individuals in different stages (as defined in Chapter 2, Figure13). The red and green lines represent a two-fold change in the accumulation with an increase or a decrease, respectively. Gene transcripts differentially accumulated (p-value < 0.05) are represented by red or green dots for upor down-accumulation, respectively.(c) Transcript accumulation in the different stages of prickle (P) and glabrous (NP) stems with NPI as a control.

Figure 29 :

 29 Figure 29: Verify the credibility of expression quantification of the RNAseq data. (a) a PCA Biplot showed the biological replication of all samples. (b) genes expression patterns of nine genes previously studied by RT-qPCR (line) (based on data published in Zhou et al. (2020) and by RNA-Seq (bar) (this study, using the sum of replicates' TPM)

Figure 30 :

 30 Figure30: DE gene expression patterns in the development stages and their function enrichment. Venn diagrams (a, b) show the number of SDE genes across compared conditions. Total: total number of SDE genes; Up: up-regulated genes; Down: down-regulated genes; DR: differently-regulated genes in two comparison groups. Comparison group referred to as'Treat'vs'Control'. (c) Time course sequencing (Tcseq) analysis showed the DE gene expression patterns in all the stages of prickle tissues. (d) A heatmap presented the enriched GO processes of genes in each pattern.

1

 1 Figure B.3 PIvsNPI, Supplementary Datasets 4 / sheet 1). Then 356 genes with low expression (corresponding to a gene whose sum TPM was below 1.5) both in PI and NPI were removed (Figure 31). Using a heatmap, we visualized the co-expression pattern of the 2583 SDE genes (z-score transformed TPM value) in all the samples (Figure 31 b, Supplementary Datasets 4 / sheet 2). A large set of genes are specifically highly expressed in NPI, or both in NPI and NPIIa (Figure 31 b). Interestingly, these kinds of genes are rarely

Figure 31 :

 31 Figure 31: Caption on next page......

Figure 32 :

 32 Figure32: Detailed analysis of the 660 specific SDE genes of stage I between P and NP samples. (a) GO tree view showing the enriched GO terms involved. The dark blue background with white font and the light blue background with blank font indicate that the GO terms are enriched at a significant level FDR < 0.05 and 0.05 < FDR < 0.1, respectively. (b) Heatmap showing the co-expression pattern of the SDE genes (involved in the seven enriched biological process Figure32 a) in all the samples. Arabidopsis protein inside brackets refers to the homologue of rose protein, rose proteins are obtained from automatic annotation.(c) Protein-protein network between the SDE genes which are presented in Figure32 b. Network nodes represent proteins. The network is clustered to three clusters using kmeans clustering method, and same color of node refers to one cluster. Filled and empty nodes indicate some 3D structure is known (or predicted) and unknown respectively. Edges represent protein-protein associations (associations are meant to be specific and meaningful, i.e. proteins jointly contribute to a shared function; this does not necessarily mean they are physically binding each other.). Different colors of line refer to the different types of data support, the detail have been showed on the Figure32 c.

  b), and we selected the ten most promising genes based on their putative function (from AT similarity) (Figure 33 b in red) and discussed in details: RC3G0389900 and RC4G0448500 belong to WUSCHEL-related homeobox (WOX) gene family (Supplementary Figure B.6 a). WOX members are known to be involved in different organ development, for

Figure 33 :

 33 Figure33: Best candidate-genes were selected by combining genetic and transcriptomic approaches. (a) A heatmap shows the co-expression pattern of the candidate-genes (forty-three SDE genes between PI and NPI stages) that located under the confidence intervals of prickle loci on LG 3 and 4. (b) The normalized expression (TPM) of candidate genes in the different stages of prickle (P) and glabrous (NP) samples. The best ten candidate-genes based on their potential function in prickle initiation were highlighted in the red.

  a)) of prickle development but expressed quite lowly in stage I. We have revealed the potential functions of these genes are not related to prickle initiation, and mainly involved in the cell wall modification, organization, biogenesis, macromolecule metabolic process, cell death and response to the different stimuli (Figure30 d);2) We compared the SDE genes involved in stage I and IIa between prickle and glabrous sample. This analysis is based on the histological study in Chapter 2, we proposed that the stages PI (Figure14 d-f) and PIIa (Figure14 g) are both including cell proliferation and cell division required for prickle growth. Fortunately, the 660 'specific' SDE genes of stage I included the most interesting GO enrichment processes for prickle initiation, thereby, they were considered as priority gene for further function prediction and the protein-protein network studies.

(Figure 34 :

 34 Figure 34: The molecular mechanisms of prickle formation from initiation to the developed stages

  on LG4, 3 and 1 (Y) that regulate prickle density in the OW population (Figure36 a). By combining the transcriptomic and genetic approaches with a functional prediction, we mined ten best candidate-genes for prickle initiation. Seven are negatively associated with prickle initiation and three are positively associated: they were proposed as inhibitors and activators, respectively.

Figure 35 :

 35 Figure 35: The molecular mechanisms of prickle initiation. (a) A general scheme for prickle initiation. P and np alleles were proposed to be an activator(s) and a mutant of activator(s) on LG3, respectively. Y means genes on QTL on LG4, 3, 1. (b) protein-protein interaction network between AS2, JAG, WOX1, WOX3, MMD1 in A. thaliana. (c) The best candidate gene expression pattern in stage I of prickle and glabrous samples.

  d, 32 b and 33 a). Among the seven best inhibitors (RcAS2, RcJAG, RcWOX-g1, RcWOX-g2, RcDOT5, RcAKR1 and RcPKL), AS2, JAG and WOX are known to be positive regulators of the organ boundary in Arabidopsis. Indeed, AS2 is the LATERAL ORGAN BOUNDARIES (LOB) protein that are positively regulates lateral organ boundaries within the Prickle morphology (see Chapter 2):

Figure 36 :

 36 Figure 36: Comparison of the different studies of prickles in Rosa and Rubus. NGP: non-glandular prickle, GP: glandular prickle, NGT: non-glandular trichome, GT: Glandular trichome, NP: no prickle

Titre:

  Génétique et génomique des aiguillons de la tige du rosier Mots clés : Structure glandulaire et non glandulaire, Trichome, Anatomie de l'aiguillon, QTL, Séquençage d'ARN, Transcriptomique Résumé : Les aiguillons sont un caractère indésirable. Leur présence rend difficile la culture et la récolte et provoque des blessures sur les producteurs. Le rosier est la plante ornementale la plus importante économiquement. Du fait de la présence d'aiguillons chez de nombreuses roses, la demande des obtenteurs et producteurs est forte pour des rosiers inermes. Les mécanismes génétiques et moléculaires de l'initiation et du développement des aiguillons sont peu connus. Les objectifs sont d'identifier les réseaux génétiques et moléculaires de l'initiation et du développement des aiguillons par des approches anatomique, génétique et génomique. Une analyse anatomique au sein du genre Rosa a mis en évidence 2 types d'aiguillons : glandulaires et non-glandulaires. Ces derniers sont les plus courants et ont une origine sous-épidermique. Par une approche génétique, nous avons identifié 4 QTLs responsables de la présence et de la densité des aiguillons sur la tige. L'analyse d'homologues, connus pour contrôler les trichomes chez Arabidopsis, n'a pas montré de différence d'expression entre rosiers épineux et glabres, suggérant peu de lien entre trichomes et aiguillons. Les bases moléculaires de l'initiation et du développement des aiguillons ont été étudiées par RNA-Seq en comparant le transcriptome de tiges (i) avec et sans aiguillons et (ii) à différents stades de développement des aiguillons. Nous avons identifié des réseaux de régulation contrôlant l'initiation et le développement des aiguillons, avec des gènes intéressants sous les QTLs. Par cette étude, nous avons construit un modèle génétique pour l'étude des aiguillons et ouvert des perspectives de recherche de ces structures. Title : Genetics and genomics of prickles on rose stem Keywords : Glandular and non-glandular structure, Trichome, Prickle anatomy, QTL, RNA sequencing, Transcriptomics

Table 1 :

 1 Overview of previous genetic studies for prickle in rose and Rubus

	Chapter 1 Introduction and literature review

Table 2 :

 2 Types of stem prickle in the list of rose resources.

	Stem prickles

Table 2 -

 2 Continued from previous page..

	Stem prickles

Table 2 -

 2 Continued from previous page..

	Stem prickles

Table 2 -

 2 Continued from previous page..

	Stem prickles

-/+ means the genotype is prickless or almost and if have few prickles, it's NGPs.

  Only seven technical replicates (seven out of 390) were excluded: CPC

	in PIIb (biological group A, C) and in NPIIc (group A), GIS2 in NPIIc (group C), NPIII (group B) and PIII
	(group B).
	Normalized expression (	Cq) was calculated using Bio-Rad Maestro1.1 software by applying the
	'gene study' tool. The cluster analysis for sample and target genes with the mean value of normalized
	expression was performed using R software with the 'pheatmap' package. NP samples were used as controls
	to compare the normalized expression of genes between P and NP samples in the different stages. | Fold
	change (FC) | > 2 and the Wilcoxon signed rank test (p-value < 0.05) as cut-off values in scatter plots were
	used to demonstrate the significant difference of normalized expression between P and NP samples. NPI
	was used as a control to visualize the relative normalized expression during stem development in prickle and
	glabrous stems.

Table 3 :

 3 Mean, median and range values for prickle number on 4 internodes, phenotypic variance components and the trait heritability

	Variance component in σ 2 P
		Stem type
	Kruskal-Wallis test	RepS Year
		Genotype
	OB RW F1	Trait Year Mean±SD Mean±SD Range 1st Qu Mean Median 3rd Qu

Table 4 :

 4 Summary of QTLs for NGP with non-parametric model in OW progeny

		PF2018 2.36	4 PF2016 2.42	PM2018 2.5	PM2017 2.41	PM2016 2.51	PF2018 2.36	3 PF2017 2.38	PM2018 2.51	PM2017 2.49	2 PM2016 2.31	PM2018 2.5	PM2017 2.41	PM2016 2.51	PF2018 2.36	PF2017 2.38	1b PF2016 2.42	PM2018 2.51	PM2017 2.49	PM2016 2.31	PF2018 2.43	PF2017 2.42	1a PF2016 2.45	QTL Phenotyping PT a
		3.04 RW1@34.2 6.99 RhK5_3678_875	3.08 RW1@34.2 6.52 RhK5_3678_875	2.58 RW6@29.7 5.28 Rh12GR_56601_1304	2.7 RW6@29.7 7.07 Rh12GR_56601_1304	4.16 RW6@29.7 8.45 Rh12GR_56601_1304	2.81 RW6@11.5 6.67 Rh88_37299_454	2.8 RW6@29.7 6.73 Rh12GR_56601_1304	3.3 OB4@30.6 10.35 Rh12GR_60129_183	3.15 OB4@30.6 13.18 Rh12GR_60129_183	2.89 OB4@30.6 11.64 Rh12GR_60129_183	11.48 RW3@42.6 37.40 Rh12GR_52506_1218	9.56 RW3@42.6 12.12 Rh12GR_52506_1218	5.88 RW3@42.6 12.76 Rh12GR_52506_1218	8.03 RW3@42.6 21.45 Rh12GR_52506_1218	8.31 RW3@42.6 12.94 Rh12GR_52506_1218	8 RW3@42.6 20.05 Rh12GR_52506_1218	4.48 OB3@45.7 11.78 Rh12GR_34665_95	3.58 OB3@51.1 6.65 Rh12GR_16570_782	2.31 OB3@51.1 7.29 Rh12GR_16570_782	6.22 OB3@51.1 14.84 Rh12GR_16570_782	4.33 OB3@51.1 6.92 Rh12GR_16570_782	5.03 OB3@51.1 11.74 Rh12GR_16570_782	LOD LG@positon b r(%) c MM d	QTL Characteristics
		59006755	59006755	31814891	31814891	31814891	5410244	31814891	52239028	52239028	52239028	42317122	42317122	42317122	42317122	42317122	42317122	41401120	44459262	44459262	44459262	44459262	44459262	bp e
	-62553371	6.74-44.11 7764439	12.78-44.11 20231658-62553371	4.21-48.53 3340353-48180089	15.59-45.19 8578645-45439915	0-38.43 1518964-48896977	6.74-44.11 7764439-62612495	8.11-42.49 4339433-44264630	11.81-48.59 36731337-56107784	11.81-48.59 36731337-56107784	20.53-48.59 46189407-56107784	28.3-42.55 36807925-42317122	37.69-42.55 41648024-42317122	16.8-42.55 32557591-42317122	37.69-42.55 41648024-42317122	37.69-42.55 41648024-42317122	37.69-42.55 41648024-42317122	36.99-53.75 35014990-46440369	20.14-53.75 24888779-46474274	27.59-53.75 27934327-46440369	39.71-53.75 36517224-46440369	30.31-53.75 33612066-46440369	40.38-53.75 36517224-46440369	cM f bp	0.95 bayes interval
		1.34-54.05	7764439-64122872 6.74-54.05	1518964-52689670 0-53.21	4339433-41715319 8.11-49.9	1518964-44264630 0-42.49	1461254-64122872 1.34-54.05	1518964-48810479 0-48.52	34803638-56107784 7.78-48.59	30431277-56107784 4.43-48.59	33319795-56107784 5.77-48.59	32557591-42317122 16.8-42.55	40854291-42317122 35.67-42.55	16767733-42317122 9.44-42.55	41648024-42317122 37.69-42.55	41648024-42317122 37.69-42.55	40854291-42317122 35.67-42.55	24888779-46440369 20.14-53.75	24000000-46440369 18.8-53.75	23585838-46440369 15.43-53.75	33612066-46440369 32.98-53.75	24888779-46440369 20.14-53.75	27934327-46440369 27.59-53.75	bp cM	0.98 bayes interval

prickle development? 4.3.3.1 Discover SDE genes from comparing prickle developmental stages

  To decipher the gene network controlling prickle development, we selected stages I, IIa, IIb, IIc, and III of prickle samples and stages I and IIa of glabrous samples. Condition design set as PIIavsPI, PIIbvsPIIa,

PIIcvsPIIb, PIIIvsPIIc (as 'Treat'vs'Control')

. 914

(650 up-and 264 down-regulation)

, 580 (509 up and 71 down), 3444 (2434 up and 1010 down), 3837 (1929 up and 1908 down) SDE genes (svalue < 0.05, |LF C| ≥1) were detected from those comparisons, respectively (Supplementary Figure B.3, Supplementary Datasets 2 / sheet 1-4). The SDE genes between early stages (PI and PIIa, PIIa and PIIb) are fewer in number comparing to those differentially expressed between later stages (PIIb and PIIc, PIIc and III). There are 329, 84, 1820 and 2259 unique SDE genes between PIIa and PI, PIIb and PIIa, PIIc and PIIb, PIII and PIIc respectively; only 38 are in common between the 4 comparisons (Figure 30 a, Supplementary Figure B.3, Supplementary Datasets 2 / sheets 6-17).

  Genetic determinism and molecular mechanisms underlying NGP formation enhance secondary metabolism. However, this hypothesis is not supported by strong evidence: the larger prickles in S. vivarium look like glandular prickles, with or without branches (see from the macroscope graph,Pandey et al. (

Both the WT and mutant (prickle and prickless) present many glandular trichomes. The larger prickles in the WT were considered to have induced from those glandular trichomes, but no prickle is induced in the mutant. Based on this hypothesis, they proposed a pathway that would govern prickle initiation from GTs, and that would 125 5.2

  Originated from epidermis(Peiterson 1921; Esau 1977;[START_REF] Coyner | Thornlessness in blackberries[END_REF]) and modified from GT[START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF] Ma et al. 2016; Khadgi, 2020a) or induced from GT[START_REF] Pandey | Transcriptome analysis provides insight into prickle development and its link to defense and secondary metabolism in Solanum viarum dunal[END_REF]).

		Prickles types in roses
		NGP		GP
		unbranched	unbranched	branched
		naked hairy	naked hairy	naked
	RNA sequencing (Khadgi, 2020b):
				NP vs NGP
	Question: prickle initiation	NPI	PI
	QTL interval	SDE genes
			NPIIa	PIIa
	Best candidate genes for prickle initiation		PIIb
		1) SDE genes in prickle development	PIIc
		2) Time cluster reveal genes' expression patterns in different stages of prickle samples	PIII
		Question: prickle development

Limitation: no anatomical studies on the origin stages.

NGP GP

Developing process of NGP and GP in OW9106 GM(ground meristem), P(protoderm), E(epidermis), PG( precursor gland), G(gland)

  org/synview/search;Jungetal.2014). The QTL region, located on LG6 in Rubus occidentalis (position 6,028 Mb), is syntenic with a region on rose Chr2 (position 42,330 Mb), where no QTL was detected in our study. The major QTL region on LG4 (position 0.101 Mb) is syntenic with the region on rose Chr4 (position 58,768 Mb), that is very close to the major QTLs we detected on this chromosome (Table2, Chapter 3). These results suggest that the two QTLs on LG4 in rose and raspberry might be syntenic and share a common evolutionary history. In another publication,[START_REF] Graham | Mapping gene H controlling cane pubescence in raspberry and its association with resistance to cane botrytis and spur blight, rust and cane spot[END_REF] identified the gene H that controls cane pubescence. The locus is mapped on LG2, which is syntenic with the rose LG6 where one QTL is detected in R. x wichurana. However, no precise location is available in Rubus, and no clear comparison can be made with rose, making it difficult to draw any conclusions concerning a possible common origin.

  Supplementary Table A.4: Prickle number on four internodes of two types of stems for three years in OW progeny. Continued...... Supplementary Table A.4-Continued from previous page.. Supplementary Table A.4-Continued from previous page.. Supplementary Table B.1: Quality control statistics per sample Supplementary Table B.4: Summary of the annotated information for 43 SDE genes located on QTL-LG3,4. Supplementary Table B.4 -Continued from previous page..

			Annexe B Supplementary tables and figures associated with Chapter 4
	Gene_ID	Indiciduals Indiciduals PF2016 Gene_Position	PF2017 PF2016 TAIR	PF2018 PF2017 UNIPROT PM2016 PF2018 PM2016 PM2017 PM2017 PM2018 PM2018 Annotation Annotation	Potential function
	Indiciduals Ow9057 0 Ow9058 NA Ow9122 Ow9123 RC3G0350900 42174997..42181428 AT2G25170 0 PF2016 0 0 9 11.67 AT Gene e-value PF2017 0 0 9.67 7.67 SwissProt ID PF2018 0 0 10.67 7.67 Q9S775 Gene_ID Gene_Position TAIR UNIPROT AT Gene e-value SwissProt ID Ow9001 0.67 5 2.67 Ow9003 0 0 0 Ow9004 2 3 0.33 Ow9005 10.67 12 11.67 Ow9006 3.67 4 9 Ow9007 3.67 4.67 8.33 Ow9008 6 0.67 0 Ow9009 7 4.33 2 Ow9010 11.67 11 7.67 Ow9011 11 11.67 9 Ow9012 11.67 14.67 11.67 Ow9013 1 2.67 4 Ow9014 2.33 10 0.67 Ow9016 NA NA NA Ow9017 15.67 17 14.33 Ow9018 9.67 8.67 11.33 Ow9019 0 0 0 Ow9021 13.67 15.33 14.67 Ow9022 9.67 10 7.67 Ow9023 15.33 15.67 13.67 Ow9024 16 13 11.33 Ow9025 14 13 11.67 Ow9027 13 8.67 8.33 Ow9029 10 10 7.67 Ow9030 10.67 10.67 11.67 Ow9031 12.33 12.33 8.33 Ow9032 9.33 6.67 3.33 Ow9033 12.33 11 9 Ow9034 15.67 17.33 13.33 Ow9035 14 15 16.67 Ow9036 0 0 0 Ow9037 0 0.67 2 Ow9038 0 0 0 Ow9039 10.33 13 10.33 Ow9040 11.67 11.67 9 Ow9041 5.67 6.67 8.67 Ow9042 11.67 9.67 13 Ow9044 0.67 1 0.67 Ow9045 3.33 4.33 4.33 Ow9046 0 0.33 4 Ow9047 4.33 2 6 Ow9049 1 0 4 Ow9050 14 15.33 16 Ow9051 0.67 0.33 0 Ow9052 13.67 14.33 12.33 Ow9054 0 0 0 Ow9055 6.67 6.67 9.33 Ow9056 NA 0 0 Ow9121 4.67 5.33 5.67 11.67 e-value PM2016 0 0 9 10 name PM2017 0 0 9.67 9 0 PKL CHD3-type chromatin-remodeling factor PICKLE 7.33 9 Annotation Annotation e-value name PM2018 10 12.67 10.33 14 12.33 8.33 3 3 0 10.67 12.33 11 3.67 4 0 10 4.67 5.67 6 2.67 1.33 7 5.33 12.33 11.67 15.67 12.67 11 11.67 11.67 15 17 14.33 2.67 2.67 4 9.67 10 2 8.33 NA NA 15.67 18.33 14.67 9.67 8.67 10 0 0 0 13.67 15.33 14.33 11.33 9.67 9.33 15.33 15.67 14.33 16 16.67 16.33 14 13 13.67 13 8.67 9 10 10 8.33 12 11 11.33 12.33 12.33 15.33 14.33 2 0.33 12.33 12 13.33 15.67 17.33 15 14 15 5 0 0 0 0.33 0.67 1 7.67 2.33 5.33 13.67 12 14.33 11.67 12 10.33 5.67 6.67 10.33 11.67 12.67 13.67 3.33 1 0 3.33 2.67 3.67 12.67 0.33 0.33 4.33 2 0 1 0 0 14 15.33 0.67 0.33 16.33 14.33 0 0 6.67 11 0 0 Continued...... Continued...... 0 10.33 9.67 Ow9190 11.67 16.33 10.67 11.67 13 13 11.67 Ow9120 8 10 9.33 8 10 7 Ow9186 0 NA 2.67 4.67 NA 2 0 Ow9119 0 0 0 0 0 0 Ow9185 12.67 11.33 11.67 12.67 11.33 11.67 12.33 Ow9117 13.33 14 15 15 16 14.33 Ow9182 7.67 NA 7.67 9.67 NA 3.67 0.33 Ow9116 12 12.67 12 12 12.67 11.33 Ow9181 0 0 0 0 0 0 13.33 Ow9059 0.33 1.33 0.33 0.33 1.33 0.67 Ow9060 1 1.33 2.67 1 1.33 1 Ow9061 12.67 12 9.33 11.33 12 12.33 Ow9062 12.67 9.67 7.67 14.67 14.33 14.67 Ow9065 2 1.67 0 3.67 8.67 4 Ow9066 2.33 6.33 3.67 10.67 10 11 Ow9067 0 0 0 0 0 0 Ow9068 0 0 0 0 0 0 Ow9069 14.33 14.67 11 14.33 14.67 13 Ow9071 14 21.33 12.67 18.67 16 13.67 Ow9072 16.67 16.67 13 16.67 16.67 12.33 Ow9074 11.67 11 10.33 11.67 11 5.67 Ow9075 11.67 14 9.33 11.67 14 12.67 Ow9076 2.67 3 1.67 11.67 3 0 Ow9077 10.67 9.67 10.33 10.67 15 14.33 Ow9078 0.67 0 0 0.67 0 0 Ow9079 11.67 8.67 7.67 11.67 12 12 Ow9080 12.67 10.33 9.67 13.33 16.33 12.33 Ow9081 13 13 12.33 13 15 15.33 Ow9082 9.33 12 11.33 9.33 12 11.33 Ow9083 12 9 9 12 9 12 Ow9084 14.33 14 13.67 15.67 20.33 17 Ow9085 12 3 10 12 12 13 Ow9087 14.33 15 12.67 14.33 15.33 14.67 Ow9088 10.33 11.33 10.33 12.33 11.33 11 Ow9089 13.33 13.67 13 13.33 15 15.33 Ow9091 0.67 1 0.67 0.67 1 0 Ow9092 8 7.33 8.33 15.67 12 6 Ow9095 11 11.33 7.33 11 11.33 12.33 Ow9096 5.67 10.67 9.33 9.67 10.67 11 Ow9098 2.67 10.67 9.33 9.33 11 11.33 Ow9099 13.33 12 14.67 13.33 12.67 9.33 Ow9100 6.67 8 8.33 6.67 8 9.33 Ow9101 15 16 11.33 15 15 17.33 Ow9103 15 8.67 11 15 15 14.33 Ow9104 10.67 12.33 9 13 11.67 13 Ow9105 12.67 12 8 12.67 12.33 12 Ow9106 43 45.67 38 48.67 48 30 Ow9107 38 52 35 32.33 42.33 29 Ow9109 12 10.67 9.67 12 10.67 11.33 Ow9111 6 2.67 0.33 16.33 2 10.33 Ow9113 15.67 14.33 13.33 15.67 18.33 13.33 Ow9115 6.67 0.33 0.33 6.67 6 1.33 Ow9124 5 10 9.67 12.67 12.67 RC3G0384500 44273324..44274796 AT5G45060 1E-36 Q9XGM3 2E-34 RPS4 Disease resistance protein RPS4 12.67 Ow9125 7.67 9 7 11.33 11.67 9.33 Ow9126 9.67 15 10 24 20.33 12.33 Ow9127 13.33 11.67 8.67 13.33 11.33 11 Ow9128 0 1.33 0 0 1.33 1.33 Ow9129 5 10.67 11.33 12 11 11.33 Ow9132 0 0 0 0 0 0 Ow9133 0.67 1.67 0 0.67 1.67 0 Ow9134 18 13.67 16.67 18 13.67 18.67 Ow9137 5 7.67 3.67 8.33 11 8 Ow9138 10.67 10 8 15 15.67 12.33 Ow9139 18.67 20 20 17.33 17.33 15 Ow9140 6.67 9 9 6.67 11.67 10.67 Ow9142 10.67 10.33 9.67 10.67 10.67 12.67 Ow9143 16.67 12 9.33 13 12.33 9.33 Ow9144 21.33 15 16 16 14.33 14.67 Ow9147 16.33 18 13.67 16.33 18 11.33 Ow9148 NA 16 16 NA 16 8 Ow9149 0 0 0 0 0 0 Ow9150 0 0 0 0 0 0 Ow9151 NA 12.33 12 NA 12.33 13.33 Ow9152 13.33 13.33 16.33 13.33 15 20 Ow9153 0 2.33 2.33 3.67 2.33 3 Ow9154 16.33 15.33 15.33 16.33 15 12.33 Ow9155 8 9 9 11.67 11.67 10.33 Ow9156 8.33 8 7.67 11.67 11.67 10.67 Ow9158 NA 9 3 NA 8.67 4 Ow9159 9.67 10 10.67 11 12.33 11.33 Ow9160 17 15.33 10 12 12.33 14.33 Ow9161 10.33 9.67 10 10.33 12 7.33 Ow9163 0.67 2.33 0 0.67 2.33 0.67 Ow9166 0 0 0 2 0 0 Ow9167 11 9.67 10 11 9.67 10 Ow9168 6.33 9 7 6.33 9 6 Ow9169 10.67 10.67 11 10.67 10.67 11.33 Ow9171 0 0.67 0 0 0.67 0 Ow9172 13.33 16.33 17 13.33 16.33 15.33 Ow9173 NA 16.67 16.67 NA 16.67 16.67 Ow9174 NA 8 8 13.33 8 5.33 Ow9175 11.67 9 8.33 13.33 14 12.33 Ow9178 0 0 0 0 0 0 Ow9179 12.67 13 12.33 12.67 12.67 16.33 Ow9180 11 10.33 10.67 11 11.33 12.33 Sample NPI1 6.703 NPI2 6.623 NPI3 6.687 NPIIa1 6.596 NPIIa2 6.581 NPIIa3 6.61 PI1 6.73 PI2 6.734 PI3 6.626 PIIa1 6.635 PIIa2 6.67 PIIa3 6.613 PIIb1 6.689 PIIb2 6.589 PIIb3 6.609 PIIc1 6.557 PIIc2 6.625 PIIc3 6.647 PIII1 6.697 PIII2 6.64 PIII3 6.693 Average 6.65 Unmapped reads Reads unmapped too short % 3.73 3.39 3.69 3.53 3.68 3.37 4.23 4.16 4.02 3.82 4.35 3.93 4.22 3.86 4.10 4.56 4.56 4.58 5.25 4.76 5.16 4.14 Total Raw Reads(G) Total Clean Bases(Gb) Clean Reads Ratio(%) Clean Reads Q20(%) Clean Reads Q30(%) 6.54 97.55 98.89 93.3 6.46 97.56 98.96 93.58 6.53 97.7 98.93 93.6 6.43 97.47 98.93 93.54 6.39 97.14 98.97 93.67 6.45 97.55 98.93 93.53 6.55 97.4 98.96 93.69 6.56 97.38 98.96 93.66 6.44 97.23 98.97 93.56 6.45 97.17 98.93 93.43 6.43 96.34 98.92 93.39 6.4 96.84 98.87 93.23 6.52 97.47 98.91 93.62 6.42 97.42 98.91 93.43 6.4 96.89 98.96 93.53 6.39 97.49 99.01 93.88 6.45 97.32 98.91 93.5 6.44 96.82 98.91 93.37 6.51 97.16 98.87 93.42 6.46 97.34 98.95 93.65 6.53 97.55 98.94 93.59 6.46 97.28 98.93 93.53 Samples Unique mapped reads Muti mapped reads Uniquely mapped read number Uniquely mapped read % Reads mapped to multiple loci % Reads mapped to too many loci % NPI1 30792634 94.01 2.21 0.04 NPI2 30562437 94.40 2.16 0.04 NPI3 30774402 94.01 2.24 0.04 NPIIa1 30387845 94.27 2.13 0.05 NPIIa2 30171264 94.06 2.15 0.07 NPIIa3 30515397 94.45 2.11 0.05 PI1 30700001 93.38 2.34 0.04 PI2 30692586 93.37 2.39 0.05 PI3 30237122 93.58 2.34 0.04 PIIa1 30355036 93.78 2.33 0.05 PIIa2 30180023 93.28 2.29 0.05 PIIa3 30112583 93.65 2.34 0.05 PIIb1 30435351 93.09 2.58 0.08 PIIb2 30092350 93.47 2.57 0.07 PIIb3 30020480 93.30 2.50 0.07 PIIc1 29676004 92.61 2.65 0.13 PIIc2 29927308 92.52 2.71 0.15 PIIc3 29953711 92.66 2.57 0.13 PIII1 29995551 91.91 2.69 0.13 PIII2 29948334 92.41 2.67 0.14 PIII3 30054770 91.89 2.74 0.19 RC3G0373700 43671059..43678116 AT1G29320 4E-41 Q6RFH5 1E-10 WDR74 WD repeat-containing protein 74 RC3G0398600 45359094..45359645 AT5G28850 2E-25 Q5QIT3 4E-24 BETA Serine/threonine protein phosphatase 2A regulatory RC3G0356400 42523016..42526787 AT3G47570 0 C0LGP4 0 AT3G47570 Probable LRR receptor-like serine/threonine-protein Average 30265961 93.34 2.41 0.08 subunit B"beta RC3G0408700 46043054..46044287 AT2G02230 2E-64 Q6NPT8 3E-63 PP2B1 F-box phloem protein PP2-B1 kinase RC3G0380200 44011688..44014786 AT3G14470 1E-111 Q9LRR4 2E-110 RPPL1 Putative disease resistance RPP13-like protein 1 RC3G0385400 44343641..44345479 AT4G19510 4E-40 Q9XGM3 4E-30 RPS4 Disease resistance protein RPS4 RC3G0397000 45215053..45216638 na na na na na na RC3G0322100 39819975..39821329 na na na na na na RC3G0402100 45588629..45590604 AT5G38260 1E-103 P93604 1E-137 LRK10 Rust resistance kinase Lr10 RC3G0321700 39748324..39749807 na na na na na na RC4G0343600 46347068..46347836 AT4G15900 5E-74 Q42384 9E-73 PRL1 pleiotropic regulatory locus 1 RC3G0307000 38567684..38568951 AT2G40610 1E-136 O22874 2E-135 EXPA8 Expansin-A8 RC4G0344200 46402314..46404541 na na na na na na RC4G0347100 46567791..46573060 na na na na na na RC3G0389900 44624768..44626218 AT3G18010 1E-29 Q6X7K0 2E-28 WOX1 WUSCHEL-related homeobox 1 RC4G0359000 47552702..47553360 na na na na na na RC3G0351100 42192172..42192742 AT1G35910 2E-15 Q67XC9 3E-14 TPPD Probable trehalose-phosphate phosphatase D RC4G0365800 48216896..48219183 AT1G67730 5E-105 Q8L9C4 1.064E-147 KCR1 BETA-KETOACYL REDUCTASE 1 RC3G0419900 46667066..46669290 AT1G65620 1E-79 O04479 2E-78 AS2 Lateral organ boundaries (LOB) domain family protein RC4G0366900 48308508..48309066 AT2G18980 2E-26 Q96518 4E-25 PER16 RC3G0285500 36789437..36792822 AT3G47570 0 C0LGP4 0 AT3G47570 Probable LRR receptor-like serine/threonine-protein kinase RC3G0307300 38580886..38581780 AT2G38530 1E-29 P83434 6E-33 AT2G38530 Non-specific lipid-transfer protein 1 RC3G0390600 44685108..44688663 na na na na na na RC3G0386900 44426300..44430239 AT1G10810 4E-127 C6TBN2 8E-173 AKR1 Probable aldo-keto reductase 1 RC3G0399800 45435579..45437570 AT5G62970 7E-18 Q9FM55 1E-16 AT5G62970 Putative F-box/FBD/LRR-repeat protein RC3G0385500 44348086..44348695 na na na na na na RC3G0359600 42723887..42724684 AT1G08980 2E-29 Q9FR37 4E-28 AMI1 Amidase 1 RC3G0353900 42370775..42371639 AT1G55020 4E-41 Q43191 2E-43 LOX1 Probable linoleate 9S-lipoxygenase 5 RC4G0393200 50528968..50532271 AT1G68480 6E-35 Q6S591 1.45187E-51JAG C2HC zinc fingers superfamily protein RC4G0398700 51013336..51015521 na na na na na na RC4G0398800 51048761..51050484 AT1G13290 4E-116 Q9FX68 4.6623E-126WIP6, DOT5 C2HC zinc fingers superfamily protein RC4G0400600 51242442..51246144 na na na na na na RC4G0401600 51299705..51303048 AT3G25670 2E-158 Q5PP26 0 PII-2 Leucine-rich repeat (LRR) family protein RC4G0418500 52778042..52781021 AT1G13740 3E-43 Q9LMX5 5E-42 AFP2 ABI five binding protein 2 Supplementary Table B.2: Mapped read statistics observed per sample Mismatch rate per base % 0.67 0.68 0.67 0.67 0.69 0.68 0.77 0.77 0.76 0.77 0.76 0.77 0.79 0.79 0.78 0.80 0.80 0.79 0.80 0.80 0.81 0.75 RC3G0407300 45947848..45949114 na na na na na na RC3G0394400 45041606..45044025 AT1G66170 2E-134 Q7X6Y7 3E-133 MMD1 PHD finger protein MALE MEIOCYTE DEATH 1 RC3G0416400 46439606..46443677 AT4G26110 7E-31 Q9SZI2 1E-29 NAP1 Nucleosome assembly protein 1;1 RC4G0428600 53515878..53518069 AT5G15310 5E-110 Q9LXF1 9E-109 MYB16 R2R3 factor gene family RC3G0379900 43996353..44002099 na na na na na na RC4G0448500 55069906..55071397 AT2G28610 7E-24 Q9SIB4 6.52049E-18WOX3 WUSCHEL-related homeobox 3 RC3G0297000 37583666..37587460 na na na na na na	Potential function cell division: response to "auxin, gibberellin, water deprivation" defense response: guarding the plant against cell fate: preimplantation development pathogens others: Required for organ development others: Required for organ development others: phloem protein defense response: guarding the plant against defense response: bacterial pathogens na defense response: bacterial na cell development: cell elongation and stess responses na cell division: na plant-type cell wall loosening, na plant-type cell wall organization na cell fate: organ development cell fate: embryo development defense response: abiotix stess tolerance cell fate: organ development others: Required for organ development cell development: cuticle-cell wall interface integrity na cell fate: organ development na na Auxin biosynthesis defense response: bacterial Continued...... cell fate:Controls the morphogenesis of lateral organs na cell fate: organ development na response to symbiotic fungus signal transduction: response to abscisic acid, water na deprivation cell division: mitosis others: cell morphogenesis, cuticle pattern formation cell differentiation and proliferation cell fate: Required to initiate organ founder cells in a na na lateral domain of shoot meristems
				163

Supplementary Figure B.6: Continued on the next page....

Acknowledgements

We gratefully thank the IMAC technical platforms of SFR Quasav for supporting the histological experiments, especially Aurelia Rolland for participating in the discussion during the histological experiments. We gratefully thank the Loubert Rose Gardens and especially Thérèse Loubert (Rosiers sur Loire, France) for providing the experimental materials. We would also like to thank Jian Hong-Ying and Shu-Fa Li (FRI, Kunming, China) for the wild roses collection and for identifying the species. We thank Latifa Hamama (IRHS, Agrocampus-ouest, Angers, France) for the discussion and advice on anatomical terms. We thank the PHENOTIC platform (IRHS, Angers, France) to manage the plants.

Acknowledgments

We are grateful to the experimental unit (UE Horti) for their technical assistance in plant management, and the ImHorPhen team (Besnard D, Gardet R) of IRHS for taking care of the plant cuttings in the greenhouse.

We would also like to thank the IMAC technical platforms (Simonneau F, Rolland A) of SFR Quasav for supervising the histological experiment, and the PTM ANAN (Bahut M) of the SFR Quasav for overseeing the RT-qPCR experiment. We acknowledge Chameau J of the GDO team for helping to obtain the different stages of the sample.

Acknowledgments

We thank the experimental unit (UE Horti) for their technical assistance in plant management, and the ImHorPhen team (D. Besnard, R. Gardet) of IRHS for taking care of the plant cuttings in the greenhouse.

We thank S. Balzergue of VALEMA team for participates in the discussion of experimental design and data analysis. We thank J. Chameau of the GDO team for helping to obtain the different stages of the sample.

Further, NN ZHOU thanks Prof. Hao Zhang and Dr. Ji-hua Wang of Yunnan Academy of Agricultural Sciences (YAAS) for accepting to be the recommenders for applying for the project funding. Thanks also HY Jian (FRI, YAAS) for revising the project manuscript.

Scholarship Council ([2017] 3109) et de la National Natural Science Foundation of China (31760585). Le projet a été mené au This project was initiated on October 1 st , 2017 for more than three years of research on the genetics and genomics of prickles on rose stem. The work was financially supported by the China Scholarship Council ([2017]3109) and the National Natural Science Foundation of China (31760585). The research was carried out within the UMR IRHS (Institute for Research in Horticulture and Seeds, INRAE, Institut Agro, University of Angers), a major player in plant research in France, based in Angers and directed by Dr. Jean-Pierre Renou (Research Director, INRAE). This project was supervised by Dr.

Funding

This work was supported by funding from the China Scholarship Council ([2017]3109) and the National Natural Science Foundation of China (31760585).

Funding

This work was supported by funding from the National Natural Science Foundation of China (31760585), the China Scholarship Council ([2017]3109) and the Natural Science Foundation of Yunnan (2016FB061).

Funding

This work was supported by funding from the National Natural Science Foundation of China (31760585), the China Scholarship Council ([2017]3109) and the Natural Science Foundation of Yunnan (2016FB061).

Results

OB3

RcTTG2(RC3G0244800)

Chapter 3 Genetic determinism of prickles in rose b.

RcGIS2(RC3G0150000)

Genotype PrickleDensity_PM2017 PrickleDensity_PM2016 PrickleDensity_PF2017 PrickleDensity_PF2018 ad( 54) bc( 16) bd( 48) ac( 33) ad( 54) bc( 16) bd( 48) ac( 33) ad( 54) bc( 16) bd( 48) ac( 33) ad( 54) bc( 16) bd( 48) ac( 33) ad( 54) bc( 16) bd( 48) ac (33)

Author's contributions

Zhou NN, Foucher F, Hibrand Saint-Oyant L conceived and designed the study. Zhou NN performed experiments, collected and analyzed data, and drafted the manuscript. Foucher F and Hibrand-Saint Oyant L were responsible for supervising the project and for revising the manuscript. Simonneau F provided full technical knowledge for performing the histological experiments. Thouroude T was contributed to the F1 cutting plant management in the greenhouse and to record planting information on wild rose resources.

Chapter 3 Genetic determinism of prickles in rose genes between P and NP samples in the different stages (Figure 28 b). In stage I, two genes are differentially expressed: RcMYB61 and RcGIS2 were down-regulated in prickly stems, with a significant p-value = 4.1 e-5 and 2.9 e-4 (Figure 28 b), respectively. In stage IIa, only RcZFP5 was significantly differentially expressed between P and NP, with a p-value = 0.0056 and FC = -5.7606 (Figure 28 b). A different pattern is observed in stage IIb where RcZFP5 expression was up-regulated with FC = 8.2240 and a p-value = 0.0025. In addition, the transcripts of RcMYC1, RcCPC and RcGIS2 were also significantly accumulated (p-value = 4.1 e-5 , 0.0048, 0.0012, respectively) in stage IIb. In stage IIc, no significant change in gene expression was detected. In stage III, the RcGIS2 transcript is differentially accumulated with FC = -4.908 and a p-value = 0.043. The same pattern is observed for RcMYB61 with a p-value = 4.9 e-4 .

We followed the transcript accumulation during stem development in prickly and glabrous stems (NPI as a control; Figure 28 c). All the studied genes are regulated between the different samples. For instance, RcMYB61 is up-regulated and RcMYC1 is down-regulated between the different stages. For RcZFP5, we observed a delay in the decrease of transcript accumulation, with a decrease in stage IIa for glabrous stems and in stage IIb for stems with prickles (Figure 28 c).

Discussion

Two types of prickles are present in the OW progeny, originating from different structures

A good understanding of prickle morphology is required to serve as the foundation for genetic and molecular studies. We identified two different types of prickles in our population: it appears that GP and NGP originate from glandular and non-glandular structures, respectively. This conclusion is different from previous studies in rose, which reported that prickles were extensions or modifications of glandular trichomes [START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF], and in other species (Ma et al., 2016b;[START_REF] Pandey | Transcriptome analysis provides insight into prickle development and its link to defense and secondary metabolism in Solanum viarum dunal[END_REF]. [START_REF] Asano | Growth, structure and lignin localization in rose prickle[END_REF] observed two types of prickles in the cultivated rose 'Laura', described as large size and small size prickles.

The large size prickles look similar to NGPs in our study. The small size prickles, referred to as acicles [START_REF] Asano | Growth, structure and lignin localization in rose prickle[END_REF], are more closely related to the glandular prickles (GP) we observed since they have a glandular head that accompanies them throughout their lifetime. The difference between these two types of prickles is also related to their segregation in the OW population (Figure 23 d), demonstrating that different genetic determinisms are involved. In this study, since only a few F 1 individuals had GPs, we cannot perform a genetic analysis on GPs, we concentrated our analysis on NGPs.

A complex genetic determinism for prickles in rose

Prickles on stems exhibited transgressive segregation in diploid OW, the same as for the tetraploid K5 population [START_REF] Koning-Boucoiran | The mode of inheritance in tetraploid cut roses[END_REF][START_REF] Gitonga | Genetic variation, heritability and genotype by environment interaction of morphological traits in a tetraploid rose population[END_REF]Bourke et al., 2018a), supporting the hypothesis that multiple loci may be responsible for this trait.

Author's contributions

Zhou NN designed the projects and obtained funding, planned and performed experiments, collected and analyzed data, and drafted the manuscript. Foucher F and Hibrand-Saint Oyant L were responsible for piloting and supervising the project, and for revising the manuscript. Tang KX contributed to designing the project and obtaining funding. Jeauffre J led the qPCR experiment and analyzed data. Thouroude T prepared the plant cuttings of F1 individuals in the greenhouse. Lopez Arias D modified the genetic map and helped to develop the R/qtl script.

Orcid-ID

Zhou NN: orcid.org/0000-0002-5208-6788

Tang KX: orcid.org/0000-0003-3807-784X Jeauffre J: orcid.org/0000-0001-6770-0552 Thouroude T: orcid.org/0000-0001-7908-7353 Lopez Arias D: orcid.org/0000-0001-8129-2786 Foucher F: orcid.org/0000-0002-3693-7183 Hibrand-Saint Oyant L: orcid.org/0000-0002-4451-8798

Materials and methods

Experiment design depending on the morphological studies of developing rose prickle (see Chapter 2).

We designed two experiments to study genes involved in prickle initiation and development. 1) For prickle development, stage I, IIa, IIb, IIc, III of P samples (refers to PI, PIIa, PIIb, PIIc, PIII respectively) and I and IIa of NP samples (refers to NPI and NPIIa) were selected to search for gene expression patterns throughout the prickle initiation and development. Compare the differentially expressed genes in PIIavsPI and NPIIavsNPI to understand the specific and common genes that expressed in glabrous and prickle samples' development.

We expected to answer what genes may be involved in prickle development. 2) Focus on prickle initiation, stages I and IIa of prickless (NP, control) and prickle (P, treatment) samples were selected to research genes' differential expressed in prickle and prickless. We expected to answer what genes may be involved in prickle initiation. In total, 21 samples were sent for RNA sequencing, they are NPI, NPIIa, PI, PIIa, PIIb, PIIc, PIII, each sample has three biological replications to verify the repeatability of the gene expression.

Generation of RNA-seq data

Library preparation and cDNA sequencing were performed at the Laboratory of the Beijing Genomics Institute (BGI), Hongkong, China. All the samples were sequenced by BGISEQ-500 platform in a Pair-End (PE)100 base pair run.

Bioinformatics analysis approach

We developed an RNA-seq analysis workflow that suitable for our data situation and for our project purpose (Supplementary Figure B.1). We provide details of the parameters used in each step as follows:

Quality control of data processing and genome alignment

FastQC version 0.11.2 [START_REF] Andrews | Fastqc: a quality control tool for high throughput sequence data[END_REF] was used to assess the quality of the raw and clean reads. RSeQC version 2.6.4 [START_REF] Wang | RSeQC: quality control of RNA-seq experiments[END_REF] was used to evaluate sequencing saturation, mapped reads distribution, coverage uniformity, strand specificity and transcript level RNA integrity. IGV [START_REF] Robinson | Integrative genomics viewer[END_REF][START_REF] Thorvaldsdóttir | Integrative genomics viewer (IGV): High-performance genomics data visualization and exploration[END_REF] tool was used to visualize the bam format file to check the distribution of reads alignment on the reference genome. MultiQC [START_REF] Ewels | MultiQC: summarize analysis results for multiple tools and samples in a single report[END_REF] was used to have an overview of the results from FastQC, RSeQC, and STAR mapping.

Raw data filtering

Trimmomatic [START_REF] Bolger | Trimmomatic: a flexible trimmer for illumina sequence data[END_REF] was used to trim low-quality bases and remove the shorter reads with the following parameters: LEADING:3,TRAILING:3, and MINLEN:50. We kept the paired files as clean data for the next step analysis.

Chapter 4 In-depth RNA sequencing analysis provides molecular mechanism insight into prickle initiation and development in rose

Reference genome alignment and expression analysis

Rose reference genome sequence (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF] and annotation files were downloaded from the Genome Database for Rosaceae (GDR, [START_REF] Jung | 15 years of GDR: new data and functionality in the genome database for Rosaceae[END_REF]). STAR source code [START_REF] Dobin | Star: ultrafast universal RNA-seq aligner[END_REF] and binaries were used to generate genome indexes and to run mapping jobs. Parameters 'sjdbOverhang 99' and 'genomeSAindexNbases 13' were used to build rose reference genome indexes. For reference genome alignment, non-default parameters were set as follows: 'outFilterType BySJout ', 'outFilterMultimapNmax 10', 'outFilterMismatchNmax 6', 'alignIntronMin 20', 'alignIntronMax 20000', 'alignMatesGapMax 20000', 'outFilterIntronMotifs RemoveNoncanonicalUnannotated', 'outSAMprimaryFlag AllBestScore', 'outFilter--MultimapScoreRange 0', 'outSAMtype BAM SortedByCoordinate', 'outSAM-unmapped Within'.

FeatureCount source code [START_REF] Liao | Webgestalt 2019: Gene set analysis toolkit with revamped uis and apis[END_REF] was used to count the number of sequence fragments that have been assigned to each gene and summary the assigned information of unique mapped reads. StringTie version 1.3.6 (Pertea et al., 2015) was used to calculate the normalized expression value, transcripts per million (TPM), of each gene.

Identification of differential expression genes

DESeq2 package [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]

Principal Component Analysis (PCA) analysis

PCA plot was carried out in R using the calibrate package. The input value (log transformation) is calculated by DEseq2 with DESeqDataSetFromMatrix and rlogTransformation function.

Clustering expression pattern in the prickle development stage

TCseq package was used to do clustering analysis and visualization of prickle development sequencing data [START_REF] Wu | KANADI1 regulates adaxial-abaxial polarity in Arabidopsis by directly repressing the transcription of ASYMMETRIC LEAVES2[END_REF]

Gene ontology and enrichment analysis

The similarity of the 44,481 annotated genes of Rosa chinensis 'Old Blush' was determined by pairwise sequence comparison using the blastx algorithm against various protein databases (GDR, https://www. rosaceae.org/species/rosa/chinensis/genome_v1.0) with an expectation value cutoff less than 1 e-9 for the NCBI nr (Release 2017-07) and 1 e-6 for the Arabidopsis proteins (TAIR10) (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF]. 28,760 genes that have matched uniport SwissProt ID which was set as a reference background for GO enrichment analysis. With A. thaliana as the organism database and Over-Representation Analysis (ORA) as the method of interest, gene ontology (Biological process nonredundant, cellular componentnonredundant, and molecular function nonredundant) functional database and Uniprot-SwissPort gene ID were used for performing functional enrichment analysis, on the webGestalt 2019 platform [START_REF] Liao | Webgestalt 2019: Gene set analysis toolkit with revamped uis and apis[END_REF]. For the SDE genes of PIvsNPI, we reported enrichment of biological process nonredundant when the false discovery rate (FDR) was ≤ 0.1. For the nine clusters of genes expressed in different patterns of the prickle developmental stages, we reported the enriched category with the FDR ≤ 0.05. With log10 of FDR, we visualized the enriched GO in the different patterns of genes. When FDR = 0, we convert to -log10 (0) as 10.

Protein-Protein Interaction Networks

We used STRING tool (https://string-db.org/) to search protein-protein interaction networks between the candidate genes by searching SwissProt ID in A. thaliana dataset. The network type is full network (the edges indicate both functional and physical protein associations). Active interaction sources including text mining, experiments, databases, co-expression, neighborhood, gene fusion, and co-occurrence.

Minimum required interaction score is medium confidence (0.4). Clustering method is kmeans clustering (number of clusters: 3).

Functional prediction for best candidate-genes

We first summarized the function information from the TAIR (https://www.arabidopsis.org/) by using the A. thaliana (AT) homologs name. Then we used the UNIPROT (https://www.uniprot.

org/) to replenish the function information by searching SwissProt ID. AT homologs and SwissProt ID were obtained from automatic annotation of 'Rosa chinensis Genome v1.0' (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF]. For the most interesting genes, we performed gene family phylogenetic trees to represent evolutionary relationships among family genes as previously described in [START_REF] Zhou | Genetic determinism of prickles in rose[END_REF]. Using Geneious 9.1.7, the family genes were searched in genome protein databases: A. thaliana (https://www.arabidopsis.

org), Rosa species (R. chinensis 'Old Blush' (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF][START_REF] Raymond | The Rosa genome provides new insights into the domestication of modern roses[END_REF], R.

multiflora [START_REF] Nakamura | Genome structure of Rosa multiflora, a wild ancestor of cultivated roses[END_REF] [START_REF] Jiang | The apricot (Prunus armeniaca l.) genome elucidates Rosaceae evolution and beta-carotenoid synthesis[END_REF], P. avium 'Tieton' [START_REF] Wang | A de novo assembly of the sweet cherry (Prunus avium cv. tieton) genome using linked-read sequencing technology[END_REF], P. persica genome [START_REF] Verde | The peach v2.0 release: High-resolution linkage mapping and deep resequencing improve chromosome-scale assembly and contiguity[END_REF]. The genome protein databases of Rosaceae recourses were downloaded from the GDR databases. Of the 'common' SDE genes, we noticed many genes involved in organ development, such as the top 5 enriched GO biological processes 'anatomical structure formation involved in morphogenesis (ASFIM)', 'post-embryonic plant morphogenesis', 'pattern specification process', 'floral whorl development' and 'shoot system development'. We also gained 'common' genes response to auxin, cytokinin, and nitrogen compound. We even discovered common genes that are involved in the phosphorelay signal transduction system and tropism (Supplementary involved in response to auxin, and then in 'monovalent inorganic cation transport', 'protein autophosphorylation', 'fatty acid derivative metabolic process' and 'secondary metabolic process'. These 933 SDE genes are supposed to be specifically involved in the early stages of glabrous stem development.

Results

DE genes expression pattern in developmental stages

To well understand the genes expression patterns in the prickle developmental stages, we performed time course sequencing (Tcseq) analysis for all stages of prickle samples. A total of 6958 differential expression (DE) genes were detected and clustered in nine groups, showing different expression patterns during the prickle and stem development (Figure 30 c). The gene list and annotation information for the genes of each cluster are presented in Supplementary Datasets 3 / sheets 1-9. We exported the DE genes that 'membership value' > 0.5 of each cluster, and obtained 523, 1018523, , 291, 195, 424, 458, 619, 1097523, , 1157 DE genes involved DE genes involved in clusters 1, 2, 3, 4, 5, 6, 7, 8 and 9 respectively. For those DE genes of each pattern, we performed the Gene Ontology (GO) and enrichment analyses to hunt their functional characteristics. We then checked the GO enrichment biological process and cellular component process. Statistically significance enriched (SSE) categories (FDR < 0.05 set as cut-off value) were presented in Figure 30 d. Cluster 2 genes mainly expressed in stages I and IIa, and their expression decreases as the prickle and stem develop (Figure 30 c). In this cluster, we clearly observed a large number of SSE-GO (FDR < 0.05) processes associated with cell proliferation, cell cycle, cell division, organelle fission/assembly, RNA

Results

Biological process

Discussion

In the gene pathway, AS2 was detected to be a center that linked with JAG, WOX1, WOX3 and MMD1.

RcJAG and RcMMD1 are both zinc fingers genes and their expression are totally opposite expression, indicating that RcMMD1 may inhibit RcJAG expression, and there by, repress RcAS2 expression as well as RcWOX1-g1 and RcWOX-g2 expression. Therefore, we propose that RcMMD1 may activate prickle initiation by repressing the activity of the 'JAG-AS2-WOX' gene pathway.

Author's contributions

Orcid-ID

Zhou NN: orcid.org/0000-0002-5208-6788

Tang KX: orcid.org/0000-0003-3807-784X

Jeauffre J: orcid.org/0000-0001-6770-0552

Thouroude T: orcid.org/0000-0001-7908-7353

Foucher F: orcid.org/0000-0002-3693-7183

Hibrand-Saint Oyant L: orcid.org/0000-0002-4451-8798

Genetic determinism and molecular mechanisms underlying NGP formation

shoot apex [START_REF] Xu | Lob domain proteins: beyond lateral organ boundaries[END_REF]. Interestingly, the accurate regulation of AS2 expression may involve epigenetic regulation [START_REF] Chen | Quantitative control of asymmetric leaves2 expression is critical for leaf axial patterning in Arabidopsis[END_REF]. As a putative repressor of AS2, RcMMD1 protein (see above) encodes a PHD zinc finger, which was proposed as a versatile epigenome reader [START_REF] Sanchez | The PHD finger: a versatile epigenome reader[END_REF]. In Arabidopsis, the as2 mutant presents leaves with a defective phenotype (Bumpy). The scal-l as2 double mutant develops spines on the edge of the leaf [START_REF] Mateo-Bonmatí | Plastid control of abaxial-adaxial patterning[END_REF]. JAG is a member of the C2H2 and C2HC zinc fingers superfamily protein, and controls the morphogenesis of lateral organs. In Arabidopsis, JAG, in interaction functions with AS1 and AS2, defines the boundaries between sepal and petal [START_REF] Xu | Arabidopsis genes as1, as2, and jag negatively regulate boundary-specifying genes to promote sepal and petal development[END_REF].

Loss of JAG function causes organs to have serrated margins [START_REF] Dinneny | The role of JAGGED in shaping lateral organs[END_REF][START_REF] Ohno | The Arabidopsis jagged gene encodes a zinc finger protein that promotes leaf tissue development[END_REF].

WUSCHEL-related homeobox (WOX) family genes are known to be involved in different organ development. WUS, as an integrator of multiple signals plays a central role in the maintenance of the cell population in the shoot apical meristem. This maintenance is intimately balanced with cell recruitment into differentiating tissues through intercellular communication [START_REF] Dodsworth | A diverse and intricate signalling network regulates stem cell fate in the shoot apical meristem[END_REF] Interestingly, RcJAG, RcAS2, RcWOX-g1 and RcWOX-g2 were detected to be central in the proteinprotein network (Figure 32 c). They might be co-regulators of glabrous stems in roses with other important genes (as AFO, YAB2, YAB5, KNAT1, TCP4, NTT, ROXY1 homologues), indicating that a loss of function of these genes may cause a defect in cell fate, and thereby, may cause prickle initiation. As previously, RcMMD1 can be a negative regulator of this gene network. We proposed that RcMMD1 is expressed at the early stage of prickle initiation and may control (by transcriptional repression) the inhibitory network. In the glabrous stems, where RcMMD1 is not expressed, and the repressive network is active and no prickle can be initiated.

In the OW population, we found that prickle density is controlled by QTLs on LG3, 4 and 1 (Chapter 3, Figure 25). As for trichomes in Arabidopsis, the density of prickles can be explained by a complex regulation with negative feedback loops and communication between neighboring cells. In Arabidopsis, trichome formation is promoted by a trimeric activator complex (MBW, see Figure 5 in Chapter 1). This complex is activated by R3MYB proteins such as TRY, which can move to neighboring cells and repress the formation of trichomes [START_REF] Hülskamp | Plant trichomes: a model for cell differentiation[END_REF]. try mutants present a higher density of trichomes (nested trichome, [START_REF] Hülskamp | Genetic dissection of trichome cell development in Arabidopsis[END_REF]). A similar process can be suggested for prickles in rose. The negative regulators present below the QTL3 and 4 can be part of this negative regulatory network. A precise spatial and temporal expression analysis of these genes and their interaction is necessary to test this hypothesis.

Combination of the different alleles at these different loci may contribute to the diversity of prickle density.

Prickles in Rosa and Rubus: comparison of the different studies

Prickles have been little studied in roses and Rubus. However, some publications so exist. We compared our results with the results of these publications (Figure 36).

Prickle morphology

Prickle types

In a previous study on Rubus and roses [START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF], glandular trichomes (GTs), non-glandular and glandular prickles (NGPs and GPs) were described as the different developmental stages for the same type of prickle. Their conclusions were based on the observations of late developmental stages on the stem.

In the rose cultivar 'Radtko', NGPs and GPs were present at the same time and at the same location of the developed stem. NGPs were proposed for the later stage of GPs (or GTs) with the fall of the glandular head from the stalk. Then, following growth, the GPs (or GTs) are converted into NGPs. However, no morphological evidence was provided for the early stage since prickle initiation begins at the shoot tip. In our study, we carried out a comprehensive morphological study. We found that GPs had a glandular head or several glands positioned along the prickle. These glands are present throughout their lifetime. In contrast, NGPs did not present any glandular structure. NGPs and GPs have their own developmental process (Chapter 2, Figure 13 and 14), and clearly represent different types of prickles but not different stages of the same prickle. Thus, we classified them into two categories: NGPs and GPs. We also found several types of prickles in wild roses that were classified into the sub-categories of NGPs and GPs, depending on if the prickles are branched or unbranched and if their surface is naked or covered with hairs (hairy).

Prickle origin

In the previous studies, prickles were described as epidermal appendages that evolved from epidermis and modified glandular trichomes in raspberry and rose [START_REF] Kellogg | Morphological studies of developing rubus prickles suggest that they are modified glandular trichomes[END_REF], in blackberry [START_REF] Coyner | Thornlessness in blackberries[END_REF] and in grapes (Ma et al., 2016b). However, this result was not supported by any anatomical evidence.

In our study, we found that NGPs evolve from multiple cells of the ground meristem under the protoderm. For GPs, multiple cells of the ground meristem first give rise to a bump, and then the protoderm (or epidermis)

of the bump differentiates into gland cells (Chapter 2, Figure 14m-r).

QTL analysis

We compared our results with previous genetic studies by associating genetic markers and the reference genome sequences. In the tetraploid K5 population, three QTLs on LG3, 4 and 6 were detected (Bourke et al., 2018a). Two SNP peak markers, K7826_576 and K5629_995 were reported. The first one is located within our Chr3 cQTL interval region (36,517,224 -46,440,369 bp), and the second is very close to our Chr4

Old questions and new insight: prickles and trichomes

Homology

Several homologies and resemblances exist between prickles and trichomes. First, concerning their morphology, both trichomes and prickles are non-glandular or glandular, can be branched or unbranched, and neither have any vascular bundle. Second, considering their function and their co-evolution with herbivores, prickles and trichomes may have a certain homology. Non-glandular trichomes (NGTs) are normally thought to have evolved primarily as physiological barriers against extreme environmental conditions and herbivores (insects) [START_REF] Kariyat | Plant spines deter herbivory by restricting caterpillar movement[END_REF]. The earliest evidence of glandular trichomes (GTs) comes from the fossils of the late carboniferous (Stephanian stage, 290 Mya) where they are modified from NGTs in the process of their co-evolution with insects [START_REF] Krings | Trichomes of the seed fern blanzyopteris praedentata: implications for plant-insect interactions in the late carboniferous[END_REF][START_REF] Lange | The evolution of plant secretory structures and emergence of terpenoid chemical diversity[END_REF]. Similarly, prickles are assumed to be an adaptation of rose against herbivores (insects and mammals) [START_REF] Chaloner | The rise of the first land plants[END_REF][START_REF] Kariyat | Plant spines deter herbivory by restricting caterpillar movement[END_REF]. A question therefore arises: are prickles or certain features of prickles inherited from trichomes? Combining morphological, ultrastructural, chemical and molecular evidence could help to answer this question. For example, from the morphology, NGPs and GPs are more closely related to NGTs and GTs, respectively.

Distinction

Trichomes, glandular or non-glandular, are epidermal appendages that originate from one or more protoderm (or epidermis) cell(s) only [START_REF] Esau | Plant anatomy[END_REF][START_REF] Werker | Trichome diversity and development[END_REF]. Non-glandular prickles originate from the tissue under the protoderm, that we refer to as the ground meristem here (Figure 14). Glandular prickles also originate from the ground meristem, but their glands evolve from the protoderm of the stalk (or epidermis)

during prickle development (Chapter 2). Usually, cells on the different positions perceive different signals, respond through intracellular signaling pathways and eventually adopt a specific cell fate, followed by the production of different organs or tissues. Thus, for trichomes and prickles, the tissue they evolve from is different (epidermis vs. sub-epidermis). Different gene networks may control prickle and trichome initiation and development. This hypothesis is supported by the results we obtained in the candidate genes (Chapter 3) and transcriptomic (Chapter 4) approaches. Since we proposed that the NGP may be more homologous to NGT than to GT, we identified in rose the homologues of genes involved in NGT initiation and development in Arabidopsis. In Chapter 3, we annotated 15 rose TFs that, based on similarity, can be involved in trichome development in rose: RcGL1, RcMYB82, RcMYB61,RcCPC,RcTRY,RcGL3,RcTT8,RcMYC1,RcTTG1,RcTTG2,RcZFP5,RcGIS3,RcGIS2,RcZFP1 and RcGL2 (Supplementary Table A.3).

Among them, several were located in the confidence interval of the detected QTLs: RcMYB61 and RcTTG2

for the QTL on LG3; RcGIS3 for the QTL on LG4; and RcCPC, RcTRY and RcMYC1 for the QTL on LG1.

RcZFP5 (Chr04: 57,125,905 bp) is out of the QTL interval on LG4 in the OW progeny, but close to the LOD peak marker K5629_995 of a prickle QTL in the K5 population (Chr04: 57,791,999 bp) (Bourke et al., 2018a). These genes were considered as good candidates for the detected QTLs, but we cannot rule out the fact that these co-localizations may be just a coincidence. This coincidence is magnified,

Appendices

A Supplementary tables and figures associated with Chapter 3

The number of NGPs on 4 internodes [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF]. e Genome location The gene Location on the Rosa chinensis Genome v1.0 (Hibrand-Saint [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF]. Green The rose gene ID were named in Rosa chinensis 'Old Blush' homozygous Genome v2.0 [START_REF] Raymond | The Rosa genome provides new insights into the domestication of modern roses[END_REF]. A

Supplementary Table

Raw reads (Trimmomatic)

Mapping

(STAR)

BAM(alignment file)

. (h) Amidase signature family proteins. The 'Old Blush' candidate genes are in bold-red and their duplications are in bold-black. The A. thaliana homologs are in blue, the protein name corresponds to the TAIR database (https://www.arabidopsis.org/). For rose, 'RCxxxxxxxxx' protein corresponds to the reference genome of the haploid of 'Old blush' [START_REF] Oyant | A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits[END_REF], 'RchiOBHm_Chrxxxxxxxxx' protein to the haploid genome of 'Old blush' [START_REF] Raymond | The Rosa genome provides new insights into the domestication of modern roses[END_REF]. The other rose genomes: R. multiflora [START_REF] Nakamura | Genome structure of Rosa multiflora, a wild ancestor of cultivated roses[END_REF]), R. xanthina, R. rugosa, R. persica (Hibrand-Saint Oyant et al., 2018). The other Rosaceae species protein obtained from R. occidentalis genome [START_REF] Vanburen | A near complete, chromosome-scale assembly of the black raspberry (Rubus Occidentalis) genome[END_REF], M. domestica Borkh genome [START_REF] Daccord | High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development[END_REF], P. communis genome [START_REF] Linsmith | Pseudo-chromosome-length genome assembly of a double haploid "bartlett" pear (Pyrus communis l.)[END_REF], P. armeniaca genome [START_REF] Jiang | The apricot (Prunus armeniaca l.) genome elucidates Rosaceae evolution and beta-carotenoid synthesis[END_REF], P. avium 'Tieton' [START_REF] Wang | A de novo assembly of the sweet cherry (Prunus avium cv. tieton) genome using linked-read sequencing technology[END_REF], P. persica genome [START_REF] Verde | The peach v2.0 release: High-resolution linkage mapping and deep resequencing improve chromosome-scale assembly and contiguity[END_REF]. The read (or fragment) was assigned to a gene feature in the annotation file. b Unassigned_NoFeature The fragment mapped to a region that is not annotated in the annotation file. c Unassigned_Ambiguity The fragment might originate from gene A or gene B, and it is not clear which gene it originated from.

bai IGV samtools

RSeQC

Bibiography