Claude Universite

Bernard -Lyon

M Le Professeur

Frédéric Fleury

M Le Professeur Hamda

Ben Hadid

Didier Revel

Dominique Mme

Composantes Marchand

Sante

G Rode

D Bourgeois

Camille Rustenholz

Dominique Mouchiroud

Amin Madoui

Sandrine Hughes D'

Abdelaziz Heddi

Adil El Filali

Cristina Vieira

Richard Cordaux

Roman Hobza

Stefan Engelen

François, Grégoire, Raquel Eugénie

Rylan

Hélène, Louise Marie- Pauline Célia

Les Célinettes

Betty

Charles-Edouard Cécile

Coline, Diego, Émilie, Fabienne, Florine François D Claire

Oriane, Pauline, Solène, Sophie Nathalie Sophie

Les Musiciens Fantastiques De L'enm

Leslie En Tête Évidemment

Alice Alexis

Benoît

Cécile Blanche

Fred Thomas

Isa Mathias

Jérôme J'en Oublie

Keywords: population genetic approaches., Scientific computing system architecture, types of programming languages, data structures, algorithms and complexity, parallelism. Computing languages: Unix, Python, Perl, R, Scilab, LaTeX, HTML, CSS. Data analysis: Usage of a calculation cluster. Genome and transcriptome assembly. Mapping, genotyping angiosperms, sex determination, sex chromosomes, dioecy, long read sequencing, MinION, de novo genome assembly, Silene latifolia, Coccinia grandis angiospermes, déterminisme du sexe, chromosomes sexuels, dioécie, séquençage longs reads, MinION, assemblage de génome de novo, Silene latifolia, Coccinia grandis

Humaine

Ce manuscrit est le fruit de quatre ans de travail parfois acharné, parfois laborieux mais toujours passionnant, et il n'aurait pas vu le jour sans toutes les personnes qui m'ont aidée, encouragée et accompagnée. Merci à elles de m'avoir permis de ne pas suivre le conseil d'un vieil ami : "Fuyez pauvre fou !" 1 Je tiens à commencer par remercier Gabriel Marais, mon directeur de thèse, qui a su m'encourager et me guider dans mon travail avec beaucoup de bienveillance. Malgré les difficultés que j'ai pu rencontrer, je le remercie de m'avoir toujours soutenue et aidée.

Je voudrais ensuite remercier Alex Widmer et Pascal Touzet pour avoir accepté d'être rapporteurs de cette thèse. Merci pour l'ensemble de vos commentaires constructifs qui ont

COMPOSANTES ET DÉPARTEMENTS DE SCIENCES ET TECHNOLOGIE

Oral communications and Posters

Oral communication in international conferences with peer-review , la plupart des plantes à fleurs, les angiospermes * , sont hermaphrodites ; seulement 5% sont dioïques soit environ 15 000 espèces (Ming et al., 2011;Renner, 2014). Parmi ces dernières on retrouve des plantes cultivées telles que le palmier dattier Phoenix dactylifera, le kiwi Actinidia chinensis, le houblon Humulus lupulus ou encore les espèces du genre Pistacia. La dioécie peut être déterminée génétiquement, par exemple par la présence de chromosomes sexuels. Notre compréhension actuelle de l'apparition de la dioécie et de l'évolution des chromosomes sexuels * chez les plantes est lacunaire et ce que nous savons des chromosomes sexuels nous vient principalement de quelques espèces-modèles animales, chez lesquelles ces chromosomes sont en général vieux (∼250 Ma * chez les mammifères). Or les chromosomes sexuels des angiospermes ont souvent une origine plus récente que ceux des animaux (Muyle et al., 2017a), permettant une étude des premiers stades de l'évolution des chromosomes sexuels. Par ailleurs, de nombreux gènes du déterminisme du sexe restent encore à découvrir chez les plantes, seuls les gènes du déterminisme du sexe du kaki (Akagi et al., 2014), de l'asperge (Murase et al., 2017) et de la fraise [START_REF] Tennessen | Repeated translocation of a gene cassette drives sex chromosome turnover in strawberries[END_REF] étant connus à ce jour. Parmi les espèces de plantes dont les chromosomes sexuels ont été étudiés, Silene latifolia est l'une des plus connues (Bernasconi et al., 2009;Ming et al., 2011). S. latifolia est une caryophyllacée * dioïque avec des chromosomes sexuels XY hétéromorphes * , de taille et de contenus différents. Ces chromosomes représentent un sixième à un cinquième du génome de S. latifolia et sont en grande partie non recombinants * (voir encart 1, Bergero et al., 2013). Une autre espèce dioïque, Coccinia grandis, appartenant à la famille des cucurbitacées (qui comprend la pastèque, le melon, le concombre, la courgette. . . ), présente le système de chromosomes sexuels le plus hétéromorphe décrit jusqu'à présent, avec un chromosome Y quatre fois plus grand que le X. Les chromosomes sexuels de ces deux plantes sont d'origine relativement récente (3,5 Ma pour C. grandis et 5 à 10 Ma pour S. latifolia) et elles servent donc de modèle pour l'étude des premiers stades de l'évolution des chromosomes sexuels et notamment la mise en place de l'hétéromorphie des chromosomes sexuels. Les chromosomes sexuels diffèrent souvent de manière marquée des autosomes * tant pour ce qui est de leur structure que de leur évolution. Les chromosomes Y de C. grandis et S. latifolia sont particuliers en raison de leur grande taille, ce qui contraste avec les vieux chromosomes Y dégénérés et de petite taille observés chez les mammifères. La suppression de la recombinaison * sur le chromosome Y pourrait avoir conduit à l'accumulation de séquences répétées * et expliquerait ainsi leur grande taille. Cette étape serait la première du processus de dégénération qui mènerait in fine à un chromosome Y de petite taille comme chez les animaux.

• C.
La dioécie a vraisemblablement évolué plusieurs fois chez les angiospermes et il est possible que les gènes du déterminisme du sexe soient divers. Deux voies principales d'évolution vers la dioécie ont été proposées (voir figure 0.1). Les deux partent d'une espèce avec une fleur hermaphrodite, le régime de reproduction ancestral chez les angiospermes, et passent soit par un intermédiaire monoïque (espèce avec des fleurs unisexuées mâles et femelles sur le même individu) soit par un intermédiaire gynodioïque * (espèce avec des femelles et des individus ayant des fleurs hermaphrodites). Il est fort probable que les gènes du déterminisme du sexe soient différents pour la voie "monoïque" et la voie "gynodioïque".

Au cours de ma thèse, j'ai cherché à comparer le déterminisme du sexe dans deux systèmes de plantes représentant les deux voies mentionnées précédemment. Chez C. grandis, une cucurbitacée avec des chromosomes XY, l'évolution de la dioécie est passée par la monoécie * (Sousa et al., 2012).

Les gènes contrôlant la monoécie ont été identifiés chez le melon, dont C. grandis est un proche parent (Boualem et al., 2008;Boualem et al., 2015;Martin et al., 2009). La manipulation de ces gènes chez le melon et le concombre permet d'obtenir des femelles et des mâles, et il est possible que ces gènes correspondent aux gènes du déterminisme du sexe chez C. grandis. Chez S. latifolia, l'évolution de la dioécie s'est faite à partir de la gynodioécie (Desfeux et al., 1996;[START_REF] Marais | Multiple nuclear gene phylogenetic analysis of the evolution of dioecy and sex chromosomes in the genus Silene[END_REF]. Chez les silènes gynodioïques, les femelles résultent d'un facteur de stérilité mâle cytoplasmique * (CMS, Cytoplasmic Male Sterility) mitochondrial * dont l'action mâle stérilisante est contrecarrée (chez les individus hermaphrodites) par un gène restaurateur (Rf, Restorer of fertility) nucléaire * . Cela devrait contraindre fortement la nature des gènes qui sont recrutés pour participer à la détermination du sexe lors de la transition gynodioécie-dioécie (Fruchard et Marais, 2017). Mon hypothèse de travail est que les gènes recrutés pour le déterminisme du sexe sont différents chez C. grandis et S. latifolia, car liés à leur voie d'évolution vers la dioécie.

J'ai testé cette hypothèse de la façon suivante :

1) Le système C. grandis est un système d'étude récent dont les chromosomes sexuels sont à caractériser, ce que nous avons fait grâce à une approche déjà validée sur S. latifolia (Muyle et al., 2016), reposant sur la comparaison de données RNAseq * de plusieurs individus mâles et femelles apparentés. Nous avons ainsi identifié une liste de gènes liés au sexe, qui nous a permis de vérifier l'âge des chromosomes sexuels de C. grandis, d'étudier la dégénérescence du chromosome Y et d'établir l'existence d'une compensation de dosage * chez cette espèce. Nous avons ensuite testé si 2. RÉSULTATS xvii les orthologues * des gènes identifiés chez le melon étaient liés au sexe chez C. grandis en utilisant la liste des gènes liés au sexe fournie par notre travail de caractérisation du système.

2) Chez S. latifolia, trois grandes régions du déterminisme du sexe ont déjà été identifiées sur le chromosome Y [START_REF] Farbos | Sexual dimorphism in white campion: deletion on the Y chromosome results in a floral asexual phenotype[END_REF][START_REF] Lardon | Sexual Dimorphism in White Campion: Complex Control of Carpel Number Is Revealed by Y Chromosome Deletions[END_REF]. De nombreuses études portant sur les gènes liés au sexe ont été réalisées chez S. latifolia, principalement à partir de données d'expression * mais plus récemment avec l'assemblage partiel de son génome * , contenant environ 25% du chromosome Y. Nous avons utilisé une approche combinant des séquences de chromosomes Y isolés par cytométrie en flux * issues de différentes technologies de séquençage et des données RNAseq pour tenter d'assembler l'intégralité du chromosome Y de cette plante. L'objectif de ce travail était d'étudier les régions du déterminisme identifiées chez S. latifolia sur la carte du chromosome Y afin d'y trouver des gènes candidats du déterminisme du sexe dans cette espèce.

A. B.

Figure 0.2. Pipelines pour l'analyse des chromosomes sexuels de (A) C. grandis et (B) S. latifolia.

Résultats

2.1. Caractérisation des gènes liés au sexe de C. grandis : dégénérescence et compensation de dosage J'ai analysé un jeu de données RNAseq sur un croisement de C. grandis grâce à une méthode développée dans l'équipe, SEX-DETector (Muyle et al., 2016), voir figure 0.2. J'ai ainsi pu identifier 1 364 gènes liés au sexe chez cette espèce et utiliser cette liste pour caractériser les chromosomes sexuels de cette plante. La divergence de séquence entre les allèles * X et Y nous a permis d'estimer que l'âge de ces chromosomes était de 3,75 Ma, ce qui est conforme à l'âge prédit par les données phylogéniques * (Holstein and Renner, 2011;Sousa et al., 2012). Parmi les gènes situés sur les chromosomes sexuels, 168 sont X-hémizygotes * , ce qui signifie que la copie de ces gènes portée par le chromosome Y a été perdue ou n'est pas exprimée. La méthode utilisée ne permettant pas de tous les identifier, j'ai appliqué une correction et estimé que la perte totale de gènes sur le chromosome Y est de 40%. Cette perte est élevée si on considère que ce système de chromosomes sexuels est très jeune. J'ai observé une diminution de l'expression des gènes situés sur le chromosome Y. En outre, l'expression de 3 273 gènes est biaisée entre les mâles ou les femelles, et les chromosomes sexuels contiennent significativement plus de gènes biaisés que les autosomes.

Grâce au jeu de données dont je disposais chez C. grandis, j'ai pu étudier la présence et les modalités de la compensation de dosage, qui permet de restaurer une expression égale entre mâles et femelles, chez cette espèce. Je disposais d'une liste de gènes présents sur les chromosomes sexuels ainsi que de leurs niveaux d'expression, pour les copies X dans chaque sexe et les copies Y chez le mâles. J'ai pu observer que lorsque le Y dégénère et que son expression diminue, l'expression du X chez les mâles augmente, ce qui permet de maintenir des niveaux d'expression similaires entre les gènes XY des mâles et XX des femelles C. grandis. Cela confirme qu'il existe une compensation de dosage chez cette espèce. Les gènes X-hémizygotes étaient séparables en deux groupes dont l'un présentait une expression égale entre mâles et femelles, malgré l'absence d'expression des copies présentes sur le chromosome Y chez les mâles. Cela suggère que certains de ces gènes sont sensibles au dosage alors que d'autres ne le sont pas. Conformément à cela, nous avons réalisé une analyse de la fonction de ces gènes. Les gènes X-hémizygotes qui subissent une compensation étaient significativement enrichis en gènes impliqués dans des complexes protéiques. Leur compensation s'esplique donc, car ces gènes requièrent un dosage bien réglé entre les différents composants pour permettre leur fonctionnement optimal.

C. grandis est un nouveau système d'étude avec beaucoup d'atouts : cette espèce est proche du melon dont le génome a été séquencé, son petit génome est en cours de séquençage, ses chromosomes X et Y sont jeunes mais déjà très hétéromorphes, et des gènes candidats ont été proposés pour le déterminisme du sexe. En effet, trois gènes ont été identifiés chez le melon (Boualem et al., 2008;Boualem et al., 2015;Martin et al., 2009) : CmWIP1 dont l'expression inhibe le développement des carpelles * entraînant alors le développement d'une fleur mâle ; CmACS7 qui peut inhiber le développement des étamines * ; et CmASC11 qui inhibe l'expression de CmWIP1 au niveau des ramifications de la plante et y entraîne le développement de fleurs femelles. En combinant différents allèles pour ces trois gènes, il est possible de créer artificiellement des espèces dioïques et de proposer un modèle d'évolution du sexe chez les plantes, qui va des espèces hermaphrodites aux dioïques en passant par le stade intermédiaire des espèces monoïques (Boualem et al., 2015). Nous sommes actuellement en train d'identifier des gènes candidats dans le cadre de cette hypothèse, en particulier pour tester si le gène WIP1 est présent sur les chromosomes sexuels de C. grandis.

Assemblage du chromosome Y de S. latifolia

Plusieurs approches pour le séquençage des chromosomes sexuels et de leurs gènes ont été développées, l'une d'entre elles se basant sur l'acquisition de données de troisième génération permettant de résoudre les problèmes d'assemblage liés aux nombreuses répétitions présentes sur le chromosome Y (voir encart 2). En utilisant de la cytométrie en flux, nous avons obtenu de l'ADN de chromosomes Y de S. latifolia. J'ai eu recours à une méthode hybride, combinant des reads * courts de seconde génération (Illumina) à du séquençage de troisième génération (Oxford Nanopore et PacBio). J'ai mis en place un pipeline pour l'analyse de ces données, décrit dans la figure 0.2.

Mes travaux m'ont permis de déterminer les points clés d'un assemblage hybride réussi, le premier étant la qualité des séquences utilisées. En effet, il est déterminant de ne garder que les données nettoyées pour que le pipeline aboutisse à un assemblage complet et suffisamment continu pour permettre des analyses biologiques. J'ai réduit mon jeu de données Illumina en enlevant successivement les séquences de mauvaise qualité et les doublons de PCR * . J'ai conservé 94,44% de ces séquences nettoyées en effectuant un enrichissement en séquences Y selon une méthode appliquée pour le séquençage du chromosome Y du gorille [START_REF] Tomaszkiewicz | A time-and cost-effective strategy to sequence mammalian Y chromosomes: An application to the de novo assembly of gorilla Y[END_REF], montrant que la contamination lors de l'étape de cytométrie en flux est faible. Pour les données issues de méthodes dites de troisième génération, qui produisent des séquences longues (>1kb * ) à très longues (>50kb), la correction à l'aide des séquences courtes Illumina est une étape cruciale, en raison de leur taux d'erreur élevé (10 à 15%). Au cours du processus d'assemblage, il est important de détecter et de retirer du jeu de données les contaminants bactériens, qui constituaient environ 10% de nos séquences PacBio. Enfin, les étapes de scaffolding * , qui permettent de concaténer des fragments courts à l'aide des séquences longues, et de gap-filling * , qui remplissent les bases inconnues générées lors de cette concaténation, permettent de tirer profit de la taille des séquences de troisième génération. Notre assemblage final, obtenu par nos collaborateurs de l'Université de Poitiers, Mohammed-Amine Chebbi et Richard Cordaux, et annoté par Adil El Filali, a une taille de 563 Mb * pour un N50 * de 6 114 bases, et contient 16 219 gènes annotés de novo dont 6 770 gènes codant pour des protéines.

Mes premiers résultats d'assemblage ont également été utilisés pour une étude sur la compensation de dosage chez S. latifolia (Muyle et al., 2017b), réalisée principalement par Aline Muyle et l'équipe d'Alex Widmer à Zürich. Les résultats de cette étude montrent que la compensation de dosage chez S. latifolia est due à une augmentation de l'expression du chromosome X maternel chez les mâles et les femelles, ce qui permet de restaurer l'expression ancestrale chez les mâles pour les gènes ayant une copie Y dégénérée.

Conclusion

Apports généraux de mes travaux

Au cours de ma thèse, j'ai produit la première liste de gènes liés au sexe chez C. grandis, et j'ai utilisé cette liste pour évaluer la dégénérescence du chromosome Y de cette plante. J'ai ainsi pu montrer la présence de compensation de dosage chez cette espèce, la deuxième plante chez laquelle ce phénomène est décrit après S. latifolia. Le séquençage MinION, en cours de développement pendant ma thèse, s'annonce prometteur pour l'analyse de parties du génome jusqu'à présent difficiles à séquencer et assembler, telles que les chromosomes sexuels. Ma thèse a permis le développement d'une nouvelle méthode de séquençage au sein de mon laboratoire d'accueil. J'ai ainsi pu produire un jeu de données et réaliser un assemblage du chromosome Y de S. latifolia.

Déterminisme du sexe chez les plantes : où en est-on ?

Les questions de l'identité des gènes du déterminisme du sexe et de l'évolution des chromosomes sexuels sont étroitement liées. Les chromosomes sexuels portent des régions spécifiques à chaque sexe qui ne sont que peu explorées chez les plantes. Les seules espèces chez lesquelles le chromosome Y est séquencé sont la papaye, Carica papaya, et S. latifolia en partie, mais les gènes du déterminisme n'ont pas été identifiés chez ces espèces.

La caractérisation du contenu des chromosomes sexuels et des gènes différentiellement exprimés en fonction du sexe permettent une meilleure compréhension du déterminisme du sexe chez les plantes. Cependant, des approches gènes-candidats permettent également de trouver les gènes du déterminisme. Chez S. latifolia par exemple, les régions du déterminisme pourront être explorées grâce à une carte physique et des collections de mutants (Bergero et al., 2008;Kazama et al., 2016). Au cours de ces dernières années, de nouveaux gènes du déterminisme ont été identifiés chez les plantes (Akagi et al., 2014;Murase et al., 2017;[START_REF] Tennessen | Repeated translocation of a gene cassette drives sex chromosome turnover in strawberries[END_REF] et révèlent une diversité des systèmes qui appelle à davantage de découvertes. Grâce aux progrès des nouvelles technologies de séquençages, de nouveaux modèles vont pouvoir être explorés et nous mener à une meilleure compréhension de l'évolution du déterminisme du sexe chez les plantes. Le chromosome Y non recombinant dégénère à cause d'effets collectivement connus sous le nom d'effets de Hill-Robertson (Bachtrog, 2013;Charlesworth and Charlesworth, 2000) : les gènes sont transmis en blocs à la progéniture et la sélection agit globalement sur les mutations. Par exemple, lorsqu'une mutation neutre ou légèrement avantageuse est génétiquement liée à une mutation fortement délétère, la sélection supprimera les deux (modèle «ruby-in-the-waste» présenté dans Bergero et al., 2013), réduisant la capacité du chromosome Y à s'adapter. Le chromosome X recombine chez les femelles et est donc protégé contre la dégénérescence. Les différentes étapes de la suppression de recombinaison au cours du temps conduisent à la formation de strates évolutives le long du chromosome Y. Les processus dégénératifs conduisent à une perte de gènes sur le chromosome Y. La sélection est moins efficace sur le chromosome Y pour les mutations légèrement délétères dans les régions non recombinantes, ce qui est favorable à l'accumulation d'éléments répétitifs pouvant mener à une augmentation de la taille du Y comme on l'observe chez certaines plantes (Bachtrog, 2008;Hughes and Rozen, 2012). Au final, cette théorie prédit que les chromosomes Y devraient perdre des gènes et accumuler des éléments transposables (TE).

Encart 2. Assemblage de novo.

La réalisation d'un assemblage est comparable à la reconstitution d'un livre dont on aurait découpé plusieurs copies de manières différentes. L'ensemble des pièces résultant de ces découpes doit être rassemblé en une seule version du livre. Cette tâche est compliquée par le fait que le livre original peut contenir plusieurs paragraphes répétés à l'identique ou qui se ressemblent beaucoup, et que certains des lambeaux peuvent avoir été modifiés pendant le découpage et la lecture, et comportent alors des fautes de frappe. La complexité de l'assemblage dépend de plusieurs facteurs, dont les principaux sont : le nombre de fragments et leurs longueurs, ainsi que la taille totale de l'assemblage : plus le livre est gros, plus il est difficile de le reconstituer.

Le séquençage de novo consiste à obtenir la séquence d'un organisme sans utiliser de référence. Il s'agit donc de l'assemblage de données de séquences d'un génome inconnu. Les outils bioinformatiques actuellement disponibles pour l'assemblage de novo utilisent le chevauchement des séquences pour la construction d'un nombre limité de contigs de taille la plus large possible. Ce processus est facilité par la production d'un mélange de reads courts et de reads longs. En effet, les fragments courts comme les longs présentent des défauts. Des fragments plus nombreux et plus longs permettent une meilleure identification des chevauchements de séquences, mais comportent souvent plus d'erreurs, ce qui augmente la complexité de l'assemblage. Des fragments, ou séquences plus courtes sont certes plus rapides à aligner, mais elles compliquent la procédure d'assemblage car elles ne permettent pas de distinguer les motifs répétés les uns des autres. L'idéal serait évidemment d'utiliser des reads longs et fiables, mais tant que les technologies de séquençage de troisième génération, qui produisent ces reads longs, auront des taux d'erreur élevés, l'utilisation de reads courts ou de grandes quantités de reads longs permettant leur autocorrection restera nécessaire. L'assemblage hybride permet de tirer profit des deux types de séquences.

List of Figures

2.S3

Mapping statistics of all the samples used for de novo transcriptome assembly.

2.S4 Number of Differentially Expressed Genes (DEG) per SEX-DETector categories.

3.1 Summary of all the raw and processed S. latifolia Y chromosome sequencing datasets produced and used for assembling.

3.2 Assembly statistics of the final assembly.

3.3 Assembly quality assessment with a reference transcriptome, sex-linked genes and Y-linked BAC sequences.

3.4 Results of EuGene gene finder applied to the final assembly.

3.S1 Long read sequencing runs specifications.

A The evolution of sex determination in plants 3

The following manuscript has been published as a book chapter in "Evolutionary Developmental Biology -A Reference Guide" edited by Dr. Laura Nuño de la Rosa (UPV/EHU University of the Basque Country, Spain) and Prof. Gerd B. Müller (University of Vienna, Austria) in the section "Plant evo-devo" with section editor Dr. Charles P. Scutt (ENS de Lyon, France), published under the imprint Springer, for which Gabriel Marais and myself were invited to write a chapter on the evolution of sex determination in plants.

Introduction

Albeit rare, dioecious plant species, that is, species with male and female individuals (as typically found in most animal species) exist in flowering plants and represent 15,600 species (i.e., 5-6% of all species, Renner, 2014). Sexual systems are very diverse in plants, but the most common sexual system is hermaphroditism with bisexual flowers, which suggests that this is the ancestral sexual system [START_REF] Barrett | The evolution of plant sexual diversity[END_REF].

It is thus assumed that dioecy has evolved from hermaphroditism and it has done so repeatedly, as dioecy is widespread in plants and found in 43% of all plant families (Renner, 2014). A total of 871 to 5,000 independent origins of dioecy in plants have been estimated (Renner, 2014). In most cases, these events are very recent and sometimes how dioecy has evolved can be tracked. Why dioecy has remained at a low frequency in angiosperms, whereas its frequency is much higher in animals (> 95%, see Table 1.1) and in other land plant lineages such as gymnosperms (36%), mosses (50%), and liverworts (75%) is not yet clear (Käfer et al., 2017).

In animals, a range of sex determination mechanisms have been described (Bachtrog et al., 2014): genetic (sex chromosomes, polygenic systems, single gene) or environmental (e.g., temperature-dependent), and some information on the sex determination mechanism is known for >90% of animal species (Table 1.1).

In plants, we know much less on sex determination. Sex chromosomes have been reported in ∼40 species out of the 871 to 5,000 independent dioecious systems (Ming et al., 2011;Muyle et al., 2017a;Renner, 2014). Dozens of master sex-determining genes such as SRY, the maledetermining gene in mammals, have been identified in animals (Bachtrog et al., 2014), and the gene network for sex determination is well characterized in several groups such as vertebrates (Matson and Zarkower, 2012). By contrast in plants only two master sex-determining genes have been recently identified (Akagi et al., 2014;Murase et al., 2017) 1 Genetic or environmental, presence of sex chromosomes. From Bachtrog et al. (2014), [START_REF] Barrett | The evolution of plant sexual diversity[END_REF], Ming et al. (2011), Muyle et al. (2017a), Renner (2014), and[START_REF] Weeks | The role of androdioecy and gynodioecy in mediating evolutionary transitions between dioecy and hermaphroditism in the animalia[END_REF].

This current lack of knowledge strongly limits our understanding of dioecy both at functional and evolutionary levels, which we will see is based on a few well-studied species and some elegant theories, but with little empirical evidence to support them. This problem has major implications for the understanding of dioecy in crops, as ∼ 20% of all crop species are dioecious, or derive from a dioecious progenitor. Examples of dioecious crops are kiwi, asparagus, hop, cannabis, date-palm, and persimmons, while crops such as papaya, grapevine, and strawberries are dioecious-derived. In species where only one sex (female in general) has an agricultural utility, the lack of genetic markers for sexing to select out the useless sex can generate huge costs, especially in trees where sexual maturity (and the opportunity of sexing individuals by looking at flowers) is reached only after several years. Moreover, our lack of knowledge on sex-determining genes prevents us from controlling the sexual system of crops.

Here we will present the data and theories that are currently available for dioecious plants. The first part of our chapter concerns theoretical aspects of dioecy and sex determination in plants, which have been developed despite the paucity of concrete data in this field of research. The second part concerns the data that have been accumulating at a slow pace for a long time. However, this situation is changing and we will discuss recent important empirical findings in plant sex determination.

Dimorphic individuals

Monomorphic individuals (two or more distinct forms) ( The main evolutionary routes to dioecy. Two main routes from hermaphroditism (the assumed ancestral sexual system in angiosperms) to dioecy have been proposed: the gynodioecy-dioecy route (A) and the monoecydioecy route (B). It has been recently highlighted that dioecy may frequently revert to hermaphroditism (C, D), hence its rareness (Käfer et al., 2017). In the gynodioecy-dioecy route, inconstant males (males producing few viable seeds) may help reversions to hermaphroditism as drawn in C [START_REF] Ehlers | Inconstant males' and the maintenance of labile sex expression in subdioecious plants[END_REF]. In the monoecy-dioecy route, there may be cycles between monoecy and dioecy (D, E). Some work suggests that in this case, androdioecy (males and monoecious individuals) and gynodioecy (females and monoecious individuals) may also evolve and may represent a situation of the re-evolution of dioecy after it has reverted to monoecy (e.g., [START_REF] Pannell | Regional variation in sex ratios and sex allocation in androdioecious Mercurialis annua[END_REF]. See text for details

Theories on the Evolution of Dioecy

The Routes to Dioecy

By both looking at the sexual system of close relatives of dioecious species and reasoning on how dioecy could evolve using population genetics, it was suggested several decades ago that dioecy has probably not evolved directly from hermaphroditism, but indirectly along various routes via different intermediates with distinct sexual systems (all sexual systems found in angiosperms and their frequencies are shown in Table 1.2). Two main routes have been proposed [START_REF] Barrett | The evolution of plant sexual diversity[END_REF], see Figure 1.1 A and B): the first of these involves a monoecious intermediate (monoecy = presence of separate male and female flowers on the same individual in a population, [START_REF] Renner | Dioecy and its correlates in the flowering plants[END_REF], while the second involves a gynodioecious intermediate (gynodioecy = presence in a population of individuals with bisexual flowers and others with female flowers, [START_REF] Charlesworth | A Model for the Evolution of Dioecy and Gynodioecy[END_REF]. The gynodioecy-dioecy pathway has been well studied theoretically [START_REF] Charlesworth | A Model for the Evolution of Dioecy and Gynodioecy[END_REF]Charlesworth, 1999), and there is some phylogenetic evidence suggesting that many dioecious species may have evolved through a gynodioecious intermediate [START_REF] Dufay | An angiosperm-wide analysis of the gynodioecy-dioecy pathway[END_REF]. This pathway starts with a hermaphroditic population in which a male-sterility mutation producing females appears (Fig. 1.1 A). Those females will reallocate all their reproductive energy to producing seeds and may produce more seeds than hermaphrodites. If the hermaphroditic population suffers from inbreeding depression, due for example to some self-fertilization, the females may not only produce more seeds but also seeds of better quality through obligate outcrossing. At some point, a female-sterility mutation producing males might appear. Those males will reallocate all their reproductive energy to producing pollen and will outcompete the hermaphrodites in fertilizing the females. The population has become dioecious.

A strong co-occurrence of monoecy and dioecy in the angiosperms phylogeny suggests that the monoecy-dioecy pathway is widespread among angiosperms, perhaps more widespread than the gynodioecy-dioecy pathway (Renner, 2014;[START_REF] Renner | Dioecy and its correlates in the flowering plants[END_REF]. However, much less has been done on modeling the transition from hermaphroditism to monoecy and then to dioecy, and in particular no population genetics model is available. In this pathway, the hermaphroditic population first evolves to a monoecious population in which both unisexual flower types can be found on the same individual (Fig. 1.1 B). Then, it is assumed that disruptive selection (in which extreme values for a trait are favored over intermediate values) on the quantitative genetic variation in floral sex ratios within a monoecious population will gradually increase sex specialization, culminating in female and male individuals [START_REF] Charnov | The theory of sex allocation[END_REF].

The androdioecy-dioecy pathway (androdioecy = presence in a population of individuals with bisexual flowers and others with male flowers) in which males would have evolved first in a hermaphroditic population is considered very unlikely. First, contrary to monoecy and gynodioecy, androdioecy is extremely rare in angiosperms (Table 1.2). Second, in a hermaphroditic population where the individuals can self-fertilize, this pathway would require the production of a huge amount of pollen for the males to spread. The spread of females is much easier. A single example of androdioecy that may have evolved from hermaphroditism is found in Phillyrea angustifolia [START_REF] Saumitou-Laprade | A Self-Incompatibility System Explains High Male Frequencies in an Androdioecious Plant[END_REF], though for most androdioecious species, the scenario is believed to be different [START_REF] Pannell | Regional variation in sex ratios and sex allocation in androdioecious Mercurialis annua[END_REF].

In Mercurialis annua, one of the best-studied androdioecious systems, it seems that androdioecy arose during cycles of evolution between dioecy and monoecy, and is an intermediate in populations evolving back to dioecy (Fig. 1.1 D, E).

Reversions from dioecy, as shown in Figure 1.1 C and D, are probably much more frequent than we used to think and these could explain why dioecy is so rare in angiosperms (Käfer et al., 2017). Dioecy would be very easy to evolve (hence the numerous independent evolution of dioecy in angiosperms) but also easy to lose, in particular when population density drops and mates are difficult to find (Käfer et al., 2017).

The Genetics of the Transition to Dioecy

Only the genetics of the gynodioecy-dioecy pathway have been modeled. In the most popular model, both male-sterility and femalesterility mutations are nuclear [START_REF] Charlesworth | A Model for the Evolution of Dioecy and Gynodioecy[END_REF]. If the male-fertility and female-fertility loci where these mutations appear are on the same chromosome, then selection will suppress recombination between these loci, which will create proto-sex chromosomes (Fig. 1.2 A). A recessive male-sterility mutation and a dominant female-sterility mutation would typically create an XY system [START_REF] Charlesworth | A Model for the Evolution of Dioecy and Gynodioecy[END_REF], and once such a system is established, the sex chromosomes may then differentiate over time (Muyle et al., 2017a).

Another possibility for the generation of sex chromosomes is Cytoplasmic Male Sterility (= CMS ), which is well known in plants. Females carry a male-sterility mutation in the mitochondrial genome, the CMS mutation, and hermaphrodites either have a male-fertile cytotype or carry a nuclear restorer-of-fertility (= Rf ) gene that counteracts the CMS factor. It has been shown through modeling and computer simulations that the evolution of dioecy is less restrictive in nucleo-cytoplasmic than in nuclear gynodioecy [START_REF] Maurice | Evolution of dioecy: can nuclear-cytoplasmic interactions select for maleness?[END_REF][START_REF] Schultz | Nucleo-Cytoplasmic male sterility and alternative routes to dioecy[END_REF]. CMS -Rf systems are engaged in evolutionary arms races. CMS -Rf systems arise from conflicts over what is the optimal reproductive system for mitochondrial genes versus nuclear genes. Mitochondrial genes are transmitted only through females, and a CMS The dominant model involves two nuclear mutations, which if on the same autosome will evolve into sex chromosomes (a). Mf male fertility gene (dominant), Ff female fertility gene (recessive), Msm male sterility mutation (recessive), Fsm female sterility mutation (dominant). Proto-X and proto-Y = nascent sex chromosomes. (b) An alternative model involves nucleo-cytoplasmic mutations, with gynodioecy being determined by a cytoplasmic-male-sterility factor (CMS) and a nuclear restorer of male fertility gene (Rf ) system, in which case dioecy is determined by cryptic CMS, fixed in the population. If the Rf gene and a female fertility gene are on the same autosome, a proto-Y chromosome comprising the Rf gene (dominant) and a female sterility mutation (Fsm, dominant) may evolve mutation will clearly increase their transmission; females will spread in the population. Nuclear genes are transmitted through both males and females, and once CMS has evolved, there will be a strong selective pressure for Rf genes to evolve, increasing the frequency of the hermaphrodites in the population. Gynodioecy will be maintained through episodic invasions of a new CMS mutation followed by evolution of a new Rf gene and so on or through balanced polymorphism due to Rf genes having some costs that prevent their fixation [START_REF] Delph | Merging theory and mechanism in studies of gynodioecy[END_REF]. These dynamics can occasionally generate peaks of female frequency in the population, an ideal situation for males to spread, and simulations have shown that the evolution of dioecy from nucleocytoplasmic gynodioecy is, in fact, easier than from purely nuclear gynodioecy [START_REF] Maurice | Evolution of dioecy: can nuclear-cytoplasmic interactions select for maleness?[END_REF][START_REF] Schultz | Nucleo-Cytoplasmic male sterility and alternative routes to dioecy[END_REF]. This scenario too could create sex chromosomes (Fig. 1.2 B). The genetics of the dioecy is expected to be different in the fully nuclear model (Fig. 1.2 A) and in the nucleocytoplasmic model (Fig. 1.2 B). If a dioecious species with an XY system evolved from a nucleo-cytoplasmic gynodioecious precursor, a CMS cytotype should be present in the dioecious species, in both sexes. CMS would generate the females; the males would be determined by a Y chromosome including a Rf gene (counteracting CMS ) and a female-sterility gene (Fig. 1.2 B). The CMS would be cryptic as the sex ratio would be balanced (not femalebiased as in gynodioecy). However, some simulations suggest that dioecy might be unstable when arising from a nucleo-cytoplasmic context; males may disappear when a new unrestored CMS cytotype appears, if some inconstant males/hermaphrodites (still producing seeds) are present when this happens [START_REF] Schultz | Nucleo-Cytoplasmic male sterility and alternative routes to dioecy[END_REF]. This is why the evolution of dioecy through nucleo-cytoplasmic gynodioecy is usually considered less likely on theoretical ground (Charlesworth, 1999), although empirical tests of these models are needed to tell how restricted/widespread they are.

Empirical Data on Dioecious Plants

Evolution of Dioecy Through Gynodioecy

Available data suggest that gynodioecy and dioecy are found associated in the same genus more than expected by chance in angiosperms [START_REF] Dufay | An angiosperm-wide analysis of the gynodioecy-dioecy pathway[END_REF]. Some case-studies have provided clear phylogenetic evidence that dioecy can evolve through gynodioecy, as in the Silene genus, for example (see below). The expected reallocation from female to male functions in hermaphrodites co-occurring with females has been documented in several subdioecious (anatomically cosexual, but functionally male or female) and gynodioecious species and suggests an ongoing transition to dioecy [START_REF] Spigler | Gynodioecy to dioecy: are we there yet?[END_REF].

There are two types of gynodioecy: nuclear and nucleo-cytoplasmic, on which the current models are based (see previous section and Figure 1.2 and Table 1.3). Despite the fully nuclear model being the dominant model in the literature on dioecy, this type of gynodioecy is rare; nucleo-cytoplasmic gynodioecy is much more common [START_REF] Delph | Merging theory and mechanism in studies of gynodioecy[END_REF]. Species showing nucleo-cytoplasmic gynodioecy typically have a CMS -Rf system [START_REF] Touzet | Cytoplasmic male sterility and mitochondrial metabolism in plants[END_REF].

Silene latifolia, a dioecious plant, is often presented as the typical example of the evolution of dioecy through gynodioecy (Fig. 1.3). It has indeed several gynodioecious relatives and dioecy has probably followed the gynodioecy pathway in the Silene genus (Desfeux et al., 1996). Silene latifolia has a X/Y chromosome pair, which is probably ∼ 5 million years old (Rautenberg et al., 2010). These sex chromosomes are very large (X: 400 Mb, Y: 550 Mb) and still not fully sequenced (Papadopulos et al., 2015). Although sex-determining genes are still unknown in S. latifolia, three sex-determining regions of a few Mb have been identified on the Y chromosome using a mutant collection (Fig. 1.3 C, Zluvova et al., 2007), among which are a female-sterility region and a male-fertility region, as would be expected from theory (Fig. 1.2). The gynodioecious Silene species are of the nucleo-cytoplasmic type (e.g., Silene vulgaris, Silene nutans, Silene acaulis), but their CMS and Rf genes have not yet been identified (Bernasconi et al., 2009). There are several indirect lines of evidence indicating cryptic CMS in S. latifolia, including biased sex ratios in crosses with close dioecious relatives [START_REF] Taylor | The genetic basis of sex ratio in Silene alba (= S. latifolia)[END_REF]). One of the sex-determining regions (MFF, see Figure 1.3 C), when deleted, gives a phenotype that closely resembles that of females in gynodioecious close relatives; they undergo anomalous anther development, with problems of cell proliferation in the tapetum (a cell layer feeding the developing pollen grains), which results in pollen-less anthers [START_REF] Zluvova | Early events in the evolution of the Silene latifolia Y chromosome: Male specialization and recombination arrest[END_REF]. Also, a cross of a S. latifolia female and a Silene viscosa hermaphrodite resulted in a 100% female F1 [START_REF] Zluvova | The inter-specific hybrid Silene latifolia x S. viscosa reveals early events of sex chromosome evolution[END_REF], which can be easily explained by CMS transmitted by the S. latifolia mother for which the S. viscosa father did not have the corresponding Rf.

Sexual systems Sex determination Genes

Gynodioecy

Fragaria (strawberry) is another genus in which dioecy probably evolved through the gynodioecy-dioecy pathway. The gynodioecious Fragaria vesca subsp. bracteata has a CMS -Rf system involving several Rf and Rf inhibiting loci (Ashman et al., 2015). Interestingly, one of the chromosomes harboring these loci has evolved into ZW chromosomes in the dioecious F. virginiana and F. chiloensis, which raises the possibility that these Rf loci are involved in sex determination in those dioecious relatives. et al., 2007). (C) The S. latifolia Y chromosome and the three sex-determining regions identified using mutants. These regions comprise two male-fertility regions: MFF male fertility factor, SPF stamen promoting factor, and one female-sterility region: GSF gynoecium sterility factor. Well-known Y-linked genes are indicated. PAR pseudoautosomal region (X-Y recombining region)

Evolution of Dioecy Through Monoecy

Phylogenetic evidence that monoecy and dioecy are associated is very strong as shown by angiosperm-wide, plant-family-focused and case studies [START_REF] Barrett | The evolution of plant sexual diversity[END_REF]Käfer et al., 2017;Renner, 2014;[START_REF] Renner | Dioecy and its correlates in the flowering plants[END_REF]. It is quite clear that the model developed for the gynodioecy-dioecy pathway does not apply to the monoecy-dioecy pathway (Golenberg and West, 2013). In monoecious plants, male and female flowers do not develop randomly on the plant, instead the sex of the flowers depends on their location. The sexual identity of flowers appears to result from gene networks that are responsive to information on the position of the flower in the plant, which are mediated by hormones. This is a starting point for evolving master sex-determining genes that is very different from that in the gynodioecy-dioecy pathway (Golenberg and West, 2013). The genetics of monoecy is starting to be deciphered, in cucurbits and in grasses, and both groups also include dioecious species.

In melon (Cucumis melo) and cucumber (Cucumis sativus), three key genes, the (andromonoecious (M), androecious (A) and gynoecious (G) genes, interact to determine the sexual identity of a given flower with respect to its position on the plant. Combinations of mutations of these genes produce female individuals, male individuals, and hermaphroditic individuals with bisexual flowers, and it was possible to create an artificially dioecious population in melon using these mutations (Boualem et al., 2015). The M gene inhibits the development of stamens (male organs), which results in a female flower. The M gene is inhibited by the G gene, which also suppresses the development of carpel (female organs). When expressed, the G gene turns a flower into a male one. The A gene inhibits the G gene and thus produces female flowers. Its expression is probably under the influence of the information on the position of the flower in the plant. If the M gene is nonfunctional (and A and G are functional), hermaphroditic flowers are produced. Both A and M genes encode for enzymes (ACS-11 and ACS-7, respectively) which are involved in ethylene biosynthesis, an important hormone that regulates many plant processes. The G gene encodes a C2H2 zinc finger transcription factor (WIP1 ). These three genes thus work coordinately to control the sexual phenotype of flowers in monoecious Cucumis species (Boualem et al., 2015).

Maize (Zea mays) is monoecious with two types of unisexual inflorescences located, respectively, at the plant apex (tassels = male inflorescences) and leaf axils (ears = female inflorescences). Many mutants affecting the sexual phenotype of flowers have been identified, contributing to the choice of this species for studies of the molecular mechanisms of flower development in grasses (Li et al., 2017a). The Silkless gene sk1 was identified as the master gene for pistil identity. This gene interacts with the Tasselseed family genes TS1 and TS2, which are responsible for the arrest of pistil development. On the other hand, male flower development is controlled by the tasselseed4 microRNA, a member of the miR172 family of microRNAs (miRNAs), which regulates the APETALA2 family of transcription factors and inhibits pistil development. A genetic hierarchy is beginning to emerge from the analysis of single and double mutants of these genes.

The Master Sex-Determining Genes in Dioecious Plants

The only sex determination mechanism known thus far in flowering plants is based on sex chromosomes, which have been reported in a minority of dioecious plants (< 1%, Tables 1.1 and 1.3). For the vast majority of dioecious plants, the basis of sex determination, either genetic or environmental, is unknown. Both XY systems, in which males are heterogametic (have different sex chromosomes), and ZW systems, in which females are heterogametic, have been found, although the latter seem to be rarer than the former (Ming et al. 2011). Of the ∼40 sex chromosome systems currently described, ∼20 are heteromorphic (i.e., sex chromosomes are clearly distinguishable using cytogenetics). Examples of heterogametic systems are S. latifolia and Coccinia grandis, a dioecious cucurbit with an XY system which happens to have the most heteromorphic sex chromosomes known in plants (Sousa et al., 2012). In these two species, the Y is larger than the X, a situation that has not been encountered in any animal species thus far. The remaining ∼20 known plant sex chromosome systems are homomorphic (i.e., the sex chromosomes are indistinguishable using cytogenetics). One example is Carica papaya (papaya), the only plant species for which the sex chromosomes (XY as it happens) are fully sequenced and assembled (Wang et al., 2012). Interestingly, in this species a modified Yh determines XYh hermaphrodites in addition to XY males and XX females, and this Yh was probably selected during the domestication process of papaya (Vanburen et al., 2015). It is possible that many dioecious plants have yet unidentified homomorphic sex chromosomes, which are more difficult to detect, as in many cases dioecy has evolved recently and young sex chromosomes tend to be homomorphic (Ming et al., 2011;Muyle et al., 2017a). Importantly, among the ∼40 plant species with known sex chromosomes, in almost all cases the master sex determining genes have not yet been identified.

The only dioecious plant species for which master sex-determining genes have been identified are persimmons (Akagi et al., 2014) and possibly asparagus (Murase et al., 2017) (Table 1.3). In persimmons (Diospyros lotus), sex determination is driven by a Y-specific sex determinant, OGI (Japanese for "male tree"), which encodes a small RNA, and its target, the autosomal MeGI gene (Japanese for "female tree"), a homeodomain transcription factor that regulates anther fertility. Transformed tobacco and Arabidopsis thaliana plants clearly suggest that MeGI is a feminizing gene and OGI acts by suppressing MeGI expression. In XY individuals, OGI is present and inhibits the expression of MeGI, which results in anther development and the arrest of carpel development, and the individuals are consequently male. In XX individuals, MeGI is expressed, carpel development proceeds, and anther development is repressed, resulting in female individuals. The OGi-MeGI system seems to be conserved in several Diospyros species and could be >50 million years old.

In asparagus (Asparagus officinalis), a plant with X and Y chromosomes, sex determination is controlled by a single locus, the Mating (M) locus. Transcriptome and RT-PCR analysis showed that a myeloblastosis-like (MYB) gene, Male Specific Expression 1 (MSE1), is specifically expressed in males during early anther development and exhibits tight linkage with the Y chromosome, as well as loss-of-function on the X chromosome (Murase et al., 2017). Knockout of MSE1 orthologue in A. thaliana produces female plants, as expected for a maledetermining gene. MSE1 has a male-specific expression pattern in several dioecious asparagus species, which suggests it plays a master male-determining role in these species too. It is also present in hermaphroditic species, which suggests that another master sex-determining gene, suppressing the female development program, is yet to be discovered in asparagus.

In asparagus, dioecy may have evolved through gynodioecy [START_REF] Dufay | An angiosperm-wide analysis of the gynodioecy-dioecy pathway[END_REF] and current data are consistent with more than one master sex determining gene, which is expected from models for the genetics of the gynodioecydioecy pathway. In persimmons, in which dioecy may have evolved through the monoecydioecy pathway (Renner, 2014), a Y-autosomal gene duet seems to be enough to determine male and female, and there seems to be a single master sex determining gene on the Y, as is the case for mammals, in which Sry is the master control gene. These considerations suggest that the genetics of the monoecy-dioecy and gynodioecy-dioecy pathways may be different [START_REF] Renner | Pathways for making unisexual flowers and unisexual plants: Moving beyond the Two mutations linked on one chromosome model[END_REF].

Conclusions and Perspectives

At this stage, it is of course difficult to draw general conclusions about sex determination in plants as genetic, molecular, and developmental data are too scarce. This scarcity is partly explained by the difficulty in studying sex chromosomes in plants. First, it is likely that many dioecious plants have young and homomorphic sex chromosomes, which cannot be identified by simply looking at male and female karyotypes and requires more sophisticated methods. Second, sequencing sex chromosomes is notoriously difficult, even in animals (Muyle et al., 2017a). However, new approaches to sequence sex chromosomes have been developed recently, and this could boost the field of plant sex determination (Muyle et al., 2017a).

To understand better the gynodioecy-dioecy pathway, more master sex-determining genes of dioecious species that evolved from this pathway should be identified, in particular to understand whether fully nuclear or nucleocytoplasmic mutations have been more common. The monoecy -dioecy route is well established, although monoecy could also evolve from dioecy. More theoretical work is needed to understand the evolutionary forces underlying the monoecy-dioecy pathway. The genetics of this pathway is not clear either, but recent findings on genes controlling monoecy will surely give a boost to this field of research. Also, some dioecious plants might have evolved via minor pathways, i.e., androdioecy or heterodistyly. Study of these cases will certainly be needed to form a global picture of the evolution of dioecy and sex determination in plants.

Dioecy is rare in angiosperms, but this is not the case in other land plant lineages such as gymnosperms, mosses and liverworts. In haplodiploid plants, males and females can be haploid and carry a third type of sex chromosomes called UV (Muyle et al., 2017a). In order to gain an even wider picture of sex determination in plants, these groups must also be studied.

Thesis objectives and outline

Der Anfang ist immer dunkel.

Mondenkind 1

1 Die unendliche Geschichte, Michael Ende 17

The aim of my PhD was to study sex determination, and in particular the sex-determining genes in plant systems, in order to understand the genetic mechanisms involved in the evolution of dioecy.

As discussed in Chapter 1, dioecy likely evolved several times independently in angiosperms, suggesting that sex-determining genes might be of diverse origins. Hermaphroditism is the predicted ancestral state of the angiosperm flower. Two main pathways have been identified that explain the evolution of hermaphroditism towards dioecy: either through a gynodioecious state (with females and individuals having hermaphroditic flowers) or a monoecious state (with both unisexual male and female flowers on the same individual). I focused my work on two plant systems representing each one of these two pathways: in Coccinia grandis, a Cucurbitaceae with an XY chromosome system, dioecy evolved through monoecy; and in Silene latifolia, a well-studied dioecious plant with XY sex chromosomes, dioecy evolved through gynodioecy.

Testing sex-determining candidate genes in C. grandis, an emerging model for the study of plant sex chromosomes

C. grandis, commonly known as ivy gourd, grows in the wild in many tropical and subtropical regions in Asia [START_REF] Shaina | Morphological variation and evolutionary significance of Coccinia grandis (L.) Voigt: An under-exploited cucurbitaceous vegetable crop[END_REF]) and all over India. It is a dioecious member within Cucurbitaceae, and has a genetic system of sex determination with male heterogamety and heteromorphic sex chromosomes, as shown in Figure 1.4. C. grandis displays the largest difference in size between sex chromosomes currently known in plants, with a Y chromosome that is four times larger than the X (Sousa et al., 2012). X-Y divergence is ∼3My old (Sousa et al., 2012) and the Y chromosome was shown to play a decisive role in determining maleness [START_REF] Roy | Mechanism of sex determination in Coccinia indica[END_REF].

C. grandis belongs to a small genus (25 species) that is phylogenetically close to Cucumis, the genus containing cucumber and melon [START_REF] Schaefer | Phylogenetic relationships in the order Cucurbitales and a new classification of the gourd family (Cucurbitaceae)[END_REF]. Its relatively small genome size, around 1 Gb, large Y chromosome and phylogenetic proximity to the fully sequenced Cucumis sativus and Cucumis melo make C. grandis an interesting model to study sex chromosome evolution.

Studies by Boualem et al. (2008) and Boualem et al. (2015) and Martin et al. (2009) discovered three important genes for flower sex determination in monoecious melon, Cucumis melo: the 'A' androecious gene encoding of a ACS-11 protein; the 'M' andromonoecious gene encoding for a ACS-7 protein; and the 'G' gynoecious gene encoding for a WIP1 protein (see Chapter 1 for details). Figure 1.5 shows how different mutations in CmACS-11 sequence, combined with CmACS-7 and CmWIP1 expression, control the sex of flowers in C. melo. Interestingly, Boualem et al. (2015) were able to create artificial dioecious melon plants with a sex ratio 1:1 from a monoecious parental by generating plant populations with different combinations of acs11 and wip1 alleles (Figure 1.5). The authors propose that these three genes are the sex-determining genes in cucurbits. C. grandis, a dioecious cucurbit, appears as the perfect model for testing this hypothesis, as it is closely related to melon and has heteromorphic sex chromosomes. According to the model showed in Figure 1.5, we predict that WIP1 is located on the sex chromosomes of C. grandis, because the model requires a different allele of WIP1 being present in males compared to females. The acs11 recessive allele needs to be fixed in the whole population, so it can be located on any chromosome. We therefore chose a candidate-gene approach in this species.

Very few genetic and genomic data are available in C. grandis, and we chose to use SEX-DETector, a probabilistic method that uses RNA-seq data to genotype parents and their offspring. This method infers sex-linked genes and does not require a reference genome (Muyle et al., 2016). The idea here was to identify a set of genes that are present on the X and Y chromosomes in C. grandis, which could then be used for the description of this system (see Chapter 2). We used this dataset to test for the presence of WIP on the sex chromosomes of C. grandis, which should be located on the Y according to our prediction.

A. B.

C. 2. Sequencing the Y chromosome of S. latifolia, a well studied XY plant system

The genus Silene includes about 700 species, most of which are hermaphroditic or gynodioecious. However there are two groups of dioecious species that have proved valuable for the study of sex-determining mechanisms. These dioecious species belong to two different Silene groups: section Melandrium (e.g. S. latifolia, S. dioica, S. diclinis) and section Otites (e.g. S. otites, S. colpophylla), out of which 20 are dioecious and 5 have known sex chromosomes [START_REF] Slancarova | Evolution of sex determination systems with heterogametic males and females in silene[END_REF]. S. latifolia is a flower from the Caryophyllaceae family, which has been studied for having heteromorphic sex chromosomes (Bernasconi et al., 2009). Both X and Y are the largest chromosomes in the karyotype, and the heteromorphic sex chromosomes are easily recognized. S. latifolia has therefore emerged as the model plant sex chromosome system for early cytogenetic research in the 1920s and later in the 1990s. S. latifolia's whole haploid genome is around 3 Gb, the Y chromosome being 550 Mb (see Figure 1.6). The repeated content is very high on both sex chromosomes (TEs, NUMTs, NUPTs and other repeats). The Y chromosome has expanded to nearly 1.5 times the length of the X and is full of satellite DNA [START_REF] Kubat | Microsatellite accumulation on the Y chromosome in Silene latifolia[END_REF], nuclearintegrated organellar DNA [START_REF] Kejnovsky | Accumulation of chloroplast DNA sequences on the Y chromosome of Silene latifolia[END_REF], and active transposons [START_REF] Pritham | An Ac-like transposable element family with transcriptionally active Y-linked copies in the white campion, Silene latifolia[END_REF].

We identified candidate genes for sex-determination in species of the gynodioecious pathway, as presented in Fruchard and Marais (2017), see Chapter 1. Three regions involved in sex determination were identified on a genetic map of the Y chromosome of S. latifolia (Figure 1.7, Bergero et al., 2008). Many sex-linked gene lists, of hundreds to thousands of genes, have already been made available in this plant (Bergero and Charlesworth, 2011;Chibalina and Filatov, 2011;Muyle et al., 2012;Muyle et al., 2016), but their positions on the sex chromosomes remain unknown. Papadopulos et al. (2015) sequenced a male and a female genome of S. latifolia and produced an assembly with a total length of 665 Mb, or 25% of the genome comprising 71% of the 248 ultraconserved core eukaryotic genes, which suggests that they mainly assembled gene-rich regions. But this assembly lacked long Y chromosome scaffolds. Despite these efforts, the sex-determining genes have not been found yet in S. latifolia. There is need for assembled genomic data to fill the gaps between the markers from the genetic map and to locate the genes.

During my PhD, I sequenced and assembled the Y chromosome of S. latifolia, aiming at identifying a list of candidate genes for sex determination. In the course of this work, I produced a first assembly with Illumina-only next-generation sequencing (NGS), using both DNA and RNA sequencing to retrieve a list of Y-linked genes that were used for a paper presented in Appendix B.

To overcome the difficulty of assembling reads with many repeats, I used a combination of MinION and PacBio third-generation sequencing. I produced a dataset using the Oxford Nanopore Min-ION sequencer with Y DNA. Chapter 3 describes our assembling process using the three datasets (Illumina, MinION and PacBio), as well as the assessment and annotation of this assembly.

A. B.

C.

Glossary

• Dioecy: in plants, when the male and female reproductive organs are on separate plants of the same species. Males and females are therefore separated individuals. • Dosage compensation: mechanism which allows to equalize expression of sex chromosomes relatively to the ancestral state, and results in male and female equivalent expression.

• Evolutionary strata: regions with distinct X-Y divergence resulting from the stepwise recombination suppressions, at different time points, between both sex chromosomes. • Gynodioecious: having bisexual flowers on some plants and only female flowers on other plants of the same species. • Heterogametic sex: refers to the sex of a species in which the sex chromosomes are not the same. For example in humans: males, with an X and a Y sex chromosome are the heterogametic sex, and females having two X sex chromosomes are the homogametic sex.

• Heteromorphic sex chromosomes: sex chromosomes that can be distinguished in cytogenetical analysis (different size and gene content). • Sex chromosomes: specialized chromosomes that determine the sex of an individual; e.g., XY is male and XX is female. • X-hemizygous gene: X-linked gene with a lost Y copy, therefore present in a single copy in males.

Part 2

Results

CHAPTER 2

An approach to identify sex-determining genes in the dioecious Coccinia grandis

Curiosity is not a sin, but we should exercise it with caution.

Albus Dumbledore 1

1 Harry Potter and the Goblet of Fire, J. K. Rowling.

29

In this chapter, I present the characterization of the sex chromosomes of Coccinia grandis. I describe the sequencing project (RNA-seq) of samples provided by Anjan Banerjee (Pune, India), for which I performed the read quality check and cleaning, reference transcriptome assembly, mapping, SEX-DETector analysis, functional annotation, expression analysis and estimated gene loss as well as the age of the sex chromosome system in this plant.

This part of my PhD project took me more time than anticipated to obtain the data. We first planned to produce a cross of C. grandis in Munich (Germany) in collaboration with Susanne Renner, but after waiting for 2 years, we realized that the plants did not flower well enough in Munich for sampling to be made there. C. grandis being a tropical plant, we had better chances of producing seedlings and see them flower rapidly by working with collaborators in subtropical regions. Thanks to Abdelhafid Bendhamane from IPS2 (Paris), we were able to contact the team of Anjan Banerjee in Pune (India) in Fall 2015. The F1 seedlings were planted in Fall 2015 and the male flowers started flowering in May 2016. The female flowers took a little longer, and all individuals had started flowering when the rainy season was at its fullest, in August 2016, so that their sex could be identified. The RNA samples were sent to IPS2 (Paris) for sequencing during Fall 2016, and the sequences were sent to us in July 2017.

Since then, I have assembled the transcriptome of C. grandis with the help of Aline Muyle. Aline performed the cleaning of the assembly with PRINSEQ and riboPicker as well as the final assembly steps with Trinity TransDecoder and Cap3. I then mapped the reads on the assembly, genotyped the individuals and made the segregation inference with SEX-DETector. Hélène Badouin annotated the transcriptome, and Bénédicte Rhoné provided her scripts for the differential expression analysis. I performed all downstream analyses presented in this chapter, with the help and guidance of Hélène Badouin, who also helped me writing this chapter.

Characterization of the Highly Heteromorphic Sex Chromosome System of the Dioecious Cucurbit Coccinia grandis

Authors: Cécile Fruchard 1 , Hélène Badouin 1 , Aline Muyle 1 , Bénédicte Rhoné 1 , Anjan K Banerjee 2 , Abdelhafid Bendahmane 3 , Gabriel AB Marais 1

Affiliations:

1 Université de Lyon, F-69000, Lyon ; Université Lyon 1 ; CNRS, UMR5558, Laboratoire de Biométrie et Biologie Evolutive, F-69622, Villeurbanne, France.

2 Biology Division, Indian Institute of Science Education and Research (IISER), Pune, Pune, Maharashtra, India. Abstract. Sex chromosomes have evolved several times independently in animals and plants. Plant systems are however poorly described compared to animal ones. While transitions towards separate sexes (= dioecy) occurred hundreds possibly thousands of times in plants, sex chromosomes have been studied and identified in only a handful of species and only one Y chromosome, the one of papaya, has been fully sequenced. More model systems are needed to have a global picture of the evolution of sex chromosomes in plants. Here we aimed at describing a new plant sex chromosome system: Coccinia grandis, a close dioecious relative of monoecious melon and cucumber with the most heteromorphic sex chromosomes known in plants so far. Using a high-throughput segregation analysis, we identified 1,364 sex-linked and 2,437 autosomal genes. Using X-Y divergence, we found that recombination arrest between the X and Y chromosomes happened recently in this plant, ∼3.75 My ago, as previous work based on cucurbit phylogenies suggested. We observed that the genes on the Y are genetically degraded, with in particular the Y allele being less expressed than the X in males. We found that X expression in males increased with decreasing Y expression, which resulted in similar expression levels of sex-linked genes in both sexes, providing evidence for dosage compensation in C. grandis, the second plant system with heteromorphic sex chromosomes in which dosage compensation has been observed. We found 3,273 sex-biased genes and showed that sex-biased genes are more common amongst sex-linked than autosomal genes. Our results support a young but already degenerated sex chromosome system, in which dosage compensation has evolved.

Introduction

Separate sexes (dioecy) occur less frequently in plants than in animals: about 6% of angiosperms are dioecious, amounting to 15,600 species from 987 genera included in 175 families (Renner, 2014). Transitions towards dioecy occurred between 871 and 5,000 times independently in angiosperms (Renner, 2014), but very few models have been studied yet. Sex chromosomes have been identified using cytogenetic and molecular evidence in only 39 plant species from 15 families (Ming et al., 2011). Study of sex chromosomes in plants showed similarities with animal models. There is evidence that plant sex chromosomes derive from a pair of autosomes as animal ones do [START_REF] Bergero | The evolution of restricted recombination in sex chromosomes[END_REF]Filatov, 2005;[START_REF] Nicolas | A gradual process of recombination restriction in the evolutionary history of the sex chromosomes in dioecious plants[END_REF]. But contrary to animals, plant sex chromosomes appear to have evolved multiple times independently and are usually younger than the animal ones [START_REF] Charlesworth | Plant contributions to our understanding of sex chromosome evolution[END_REF]Muyle et al., 2017a). For this reason, plant sex chromosomes are particularly informative about the early stages of sex chromosome evolution. In heteromorphic sex chromosome systems, the sex-specific Y chromosome shows non-recombining regions that tend to expand over time [START_REF] Bergero | The evolution of restricted recombination in sex chromosomes[END_REF] resulting in the formation of evolutionary strata (i.e., genomic regions that show discrete divergence times, as a result of stepwise recombination suppression). For example, in Silene latifolia there have been at least two recombination suppression events (Bergero et al., 2008;[START_REF] Nicolas | A gradual process of recombination restriction in the evolutionary history of the sex chromosomes in dioecious plants[END_REF] as genes fall into at least two categories of X/Y divergence. In plants, recombination suppression seems to be associated with chromosomal rearrangements, in particular with inversions, as in animals. For example, in Carica papaya, two large inversion events appear to define the two strata present in this species (Wang et al., 2012). Genetic degeneration occurs in plant sex chromosomes in the same way as in animal systems: accumulation of deleterious mutations and gradual loss of functional genes from the Y, due to reduced efficacy of selection [START_REF] Bachtrog | Reduced adaptation of a non-recombining neo-Y chromosome[END_REF]Bachtrog et al., 2014;Bergero et al., 2015), but plant sex chromosomes appear to be younger and less degenerate than animal sex chromosomes [START_REF] Charlesworth | The status of supergenes in the 21st century: Recombination suppression in Batesian mimicry and sex chromosomes and other complex adaptations[END_REF].

Weak degeneration might be due in some cases to the small size of the non-recombining regions, as degeneration will be slower when a non-recombining region includes few genes under selection (Bachtrog, 2008;Charlesworth and Charlesworth, 2000). In young plant sex chromosomes, genetic degeneration has been studied in a few systems including white campion, S. latifolia (Bergero and Charlesworth, 2011;Chibalina and Filatov, 2011); C. papaya (Wang et al., 2012) and Rumex species [START_REF] Beaudry | Genomic Loss and Silencing on the Y Chromosomes of Rumex[END_REF][START_REF] Crowson | Purifying and Positive Selection Influence Patterns of Gene Loss and Gene Expression in the Evolution of a Plant Sex Chromosome System[END_REF]Hough et al., 2014). In papaya, the Y-linked region is approximately 8 Mb, but only approximately 1.6 Mb is nonrepetitive, and gene density is very low ( Vanburen et al., 2015). Higher accumulation of non-synonymous substitutions compared to Xlinked alleles is detectable in S. latifolia (Bergero and Charlesworth, 2011;Chibalina and Filatov, 2011;Muyle et al., 2012) and R. hastatulus (Hough et al., 2014), consistent with degeneration; the Y-linked sequences also have lower expression than their X-linked alleles (Bergero and Charlesworth, 2011;Chibalina and Filatov, 2011;Muyle et al., 2012).

Genetic degeneration on the Y results in an imbalance of X genes: one copy is present in males when there are two in females, which can be deleterious, in particular for dosagesensitive genes. This can lead to the evolution of dosage compensation, a mechanism through which equal expression is restored between males and females. Dosage compensation has been well characterized in animals, where it ranges from complete chromosome compensation to active compensation of only a minority of dosage-sensitive genes (Ercan, 2015;Mank, 2013;Pessia et al., 2014;Veitia et al., 2015), but is far less known in plants (Muyle et al., 2017a). Dosage compensation has been shown in plants for the first time in S. latifolia (Muyle et al., 2012;Papadopulos et al., 2015) where it is achieved by a genomic imprinting mechanism in which the maternal X chromosome is upregulated in both males and females (Muyle et al., 2017a). In S. latifolia, studies focusing on X-hemizygous genes, i.e. genes with a lost Y copy due to the degeneration of the Y chromosome, showed a significant difference between male and female expression, i.e. no dosage compensation (Chibalina and Filatov, 2011;Kazama et al., 2012). However, another study that focused on sex-linked genes with a preserved Y copy observed that males and females maintained similar expression levels even when Y expression was reduced due to degeneration (Muyle et al., 2012). Bergero et al. (2015) found that male expression was globally halved compared with females for X-hemizygous genes, as expected under no dosage compensation, but observed that for X/Y genes with a degenerated Y copy, the male over female expression level ratio was globally higher than the value expected without dosage compensation. Another analysis of sex-linked genes in S. latifolia compared male and female expression levels for the X chromosome (Papadopulos et al., 2015). Due to limitations of the RNA-seq approach in inferring X-hemizygous genes, results on their dosage compensation were contradictory, as explained in the Supplementary materials of Muyle et al. (2017b), see Appendix C. Using a genomic approach, Papadopulos et al. (2015) found a much higher and reliable fraction of Xhemizygous genes than previous studies. Two clusters of X-hemizygous genes were identified: one cluster had a ratio of male X expression over female XX expression close to 1, suggesting dosage compensation, whereas another cluster of X-hemizygous genes had a ratio of around 0.5, suggesting no dosage compensation. The study thus confirmed complete compensation for some genes in S. latifolia, including X-hemizygous genes. In R. hastatulus, genes with a preserved but degenerated Y copy maintain similar male and female expression levels, suggesting dosage compensation, as shown in Hough et al. (2014), Figure 3, although the claim that dosage compensation exists in R.hastatulus has never been made. Overall, these results support the existence of dosage compensation in some plants, and that de novo evolution of dosage compensation is possible in quite recent systems such as the 5-10 My old S. latifolia. But only two heteromorphic systems have been studied yet, and new models would help clarifying the picture.

Dioecy facilitates the evolution of sexual dimorphism, i.e., the expression of different male versus female phenotypes [START_REF] Barrett | Sexual dimorphism in flowering plants[END_REF]Charlesworth, 1999;[START_REF] Moore | Sexual selection in plants[END_REF]. Sex-biased selection occurs when females and males have different optima for the same trait. Sexual dimorphism can evolve through sex-limited or sex-biased expression of genes that are present in both sexes (Ellegren and Parsch, 2007;[START_REF] Parsch | The evolutionary causes and consequences of sexbiased gene expression[END_REF], but can also be facilitated by the evolution of sex-specific non-recombining regions on sex chromosomes, when conflicts between male and female reproductive functions exist [START_REF] Charlesworth | Evolution of recombination rates between sex chromosomes[END_REF]. Male-biased genes are expected to accumulate on the Y chromosome, or on the X when they bear a recessive mutation, and female-biased dominant mutations are expected to accumulate on the X chromosome [START_REF] Parsch | The evolutionary causes and consequences of sexbiased gene expression[END_REF]. Plant sex chromosomes can therefore be involved in the resolution of sexual conflicts, but there are too few plant systems where sex-linkage of sexbiased genes was investigated for this observation to be conclusive: in Populus gunnanensis and Populus tremula, sex-biased genes are not enriched in the sex-specific non-recombining region [START_REF] Jiang | Transcriptional profiling analysis in Populus yunnanensis provides insights into molecular mechanisms of sexual differences in salinity tolerance[END_REF][START_REF] Robinson | Populus tremula (European aspen) shows no evidence of sexual dimorphism[END_REF], whereas in S. latifolia, sex-biased genes are significantly enriched among sex-linked genes compared with autosomal genes (Zemp et al., 2016). Therefore, we need more plant systems in which the frequency of sex-biased genes on sex chromosomes is analyzed.

Here, we aimed at describing a new system from Cucurbitaceae, a plant family with ∼800 species, out of which 460 are monoecious and 340 are dioecious. But the characteristics of sex chromosomes and their degeneration are poorly known and described in cucurbits. Only two cucurbit sex chromosome systems have been characterized cytologically and for their repetitive content: Bryonia dioica [START_REF] Oyama | A specific insertion of a solo-LTR characterizes the Y-chromosome of Bryonia dioica (Cucurbitaceae)[END_REF] and Coccinia grandis (Sousa et al., 2012;[START_REF] Sousa | Analysis of transposable elements and organellar DNA in male and female genomes of a species with a huge Y chromosome reveals distinct Y centromeres[END_REF][START_REF] Sousa | Cytogenetic comparison of heteromorphic and homomorphic sex chromosomes in Coccinia (Cucurbitaceae) points to sex chromosome turnover[END_REF]. C. grandis, a dioecious Cucurbitaceae commonly known as ivy gourd, is a perennial, tropical plant with a relatively short time before maturity (1 year from seed to seed). The Coccinia genus is entirely dioecious (Holstein and Renner, 2011), but C. grandis males and females show no morphological differences except in their flowers, so sexual dimorphism is limited. The process of stamen initiation occurs in wild type females even in the absence of a Y chromosome but the further development of stamen is stopped before their full maturity, so sex-biased gene expression is expected to occur mainly in flower buds at an advanced developmental stage.

C. grandis displays the largest difference in size (i.e. heteromorphy) between sex chromosomes currently known in plants, the 200 Mb chromosome Y being four times bigger than the X chromosome (Sousa et al., 2012). The large size of the C. grandis Y chromosome is mainly due to the accumulation of transposable elements (TEs) and satellite repeats [START_REF] Sousa | Analysis of transposable elements and organellar DNA in male and female genomes of a species with a huge Y chromosome reveals distinct Y centromeres[END_REF] and results in a 10% difference in the size of male and female genomes (Sousa et al., 2012). Plastid and mitochondrial-like sequences also accumulated on the C. grandis Y chromosome. The repetitive fraction of both male and female genomes of C. grandis is mainly composed of Ty1/copia and Ty3/gypsy LTR elements. Of these elements, 5 are found in much greater abundance on the Y than on other chromosomes [START_REF] Sousa | Analysis of transposable elements and organellar DNA in male and female genomes of a species with a huge Y chromosome reveals distinct Y centromeres[END_REF]. Other elements, satellites and rDNA are much less Y-specific than in R. acetosa and S. latifolia, where many Y-specific tandem repeats have been identified, as reviewed in [START_REF] Hobza | Impact of repetitive DNA on sex chromosome evolution in plants[END_REF]. Paradoxically, despite the important heteromorphy between both C. grandis sex chromosomes, X/Y divergence is estimated to be only 3.5 My old based on phylogenetic data (Holstein and Renner, 2011;Sousa et al., 2012). Differential expression of genes in male and female flower buds has been already studied in C. grandis. Between 2 to 8% of genes were differentially expressed between males and females (Devani et al., 2017;Mohanty et al., 2017). But no identification of sex-linked genes has been done so far and no genomic data is available. C. grandis is an interesting model to study sex chromosome evolution due to 1) the relatively small genome size of this species for a plant genome (around 1Gb) 2) its large heteromorphic Y chromosome and 3) its phylogenetic proximity to the fully sequenced Cucumis sativus and Cucumis melo. This system raises the question of how quickly heteromorphic sex chromosome evolve in plants, since recombination arrest is predicted to have occurred only recently (3.5 My ago), and whether the high heteromorphy is only due to repetitive sequences or if the gene contents also diverged. It is also not known whether sex-biased genes are more numerous on the sex chromosomes in C. grandis.

We aimed at (1) identifying sex-linked genes in Coccinia grandis, (2) verifying the predicted young age for this sex chromosome system that is paradoxically already highly heteromorphic (3) characterizing genetic degeneration of the Y chromosome (4) investigating the presence of dosage compensation in this possibly young sex chromosome system and (5) shedding new light onto male and female expression differences and determining the abundance of sexbiased genes on the sex chromosomes.

Gathering sequencing data is necessary to address these questions, but except for Carica papaya (Wang et al., 2012) and a partial sequencing of S. latifolia (Papadopulos et al., 2015), no complete sequence of plant sex chromosomes is available. The amount of repetitive DNA present in non-recombining Y chromosomes makes the standard WGS approach not feasible (Muyle et al., 2017a) and producing high-quality assemblies using BACs is very expensive. Several large-scale analyses have focused on segregation of SNPs identified from RNA-seq data to identify new sex-linked genes. Initially, this was done using empirical approaches but more recently, a more sensitive model-based approach, SEX-DETector, has been developed (Muyle et al., 2016). We used RNA sequencing of floral buds of a cross to identify sex-linked transcripts with SEX-DETector. Once sex-linked genes were identified, we performed a molecular clock analysis of X/Y sequences to investigate the age of recombination arrest. We estimated gene loss on the Y and compared male and female expression. We investigated the presence of dosage compensation, and searched for enriched functions that could show a biased retention of Y copies during the degeneration process. The RNA-seq data were also used to analyze expression differences between males and females to allow for the identification of sex-biased genes. We analyzed sex-biased expression in regards to the sex-linkage of the genes.

Material and Methods

Plant material

RNA sequencing data was obtained from a cross between a distantly related wild type male and female of the dioecious plant C. grandis, both grown in the experimental fields of IISER Pune, India. Seeds from the cross were collected as soon as the fruits matured. 24 seedlings were raised from these seeds and took around 4 to 7 months to enter flowering stage, which allowed for sexing. Flower buds at early developmental stages 3-4 (defined in [START_REF] Ghadge | Flower development, pollen fertility and sex expression analyses of three sexual phenotypes of Coccinia grandis[END_REF] were sampled from plants being grown in a temperature-controlled greenhouse at IISER Pune. RNAs were isolated from 5 males (sons) and 5 females (daughters) from the F1 generation as well as from their parents.

RNA sequencing

The samples were sent to IPS2 Paris, France using RNA later ICE kits by Thermo Fisher. Total RNA was extracted from 12 flower bud samples using Agilent's spin column purification method, mRNA were isolated with Oligo-dT Beads from NEB and RNAseq libraries constructed with the Directional Kit from NEB. Sequencing was performed at IPS2 Paris, France with Illumina NextSeq500 following a paired-end protocol of library preparation (fragment lengths 100-150 bp, 75 bp sequenced from each end). RNA samples were checked for quality, individually tagged and sequenced (see Supplementary Table 2.S1 for library sizes).

De novo transcriptome assembly

A reference transcriptome was built for C. grandis using Trinity [START_REF] Haas | De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis[END_REF] on the combined libraries of the 12 individuals described previously. For each individual, 100% identical reads, assumed to be PCR duplicates, were filtered out using the ConDeTri v2.3 trimming software [START_REF] Smeds | ConDeTri-a content dependent read trimmer for Illumina data[END_REF]. Reads were then filtered out for sequencing adapters and low quality using ea-utils FASTQ processing utilities v1.04.636 (Aronesty, 2011). Cleaned reads from all male and female individuals were combined and assembled using Trinity version 2.4.0 with default settings [START_REF] Haas | De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis[END_REF]. 276,225 transcripts were obtained. Poly-A tails were removed from transcripts using PRINSEQ v0.20.4 (Schmieder and Edwards, 2011) with parameters -trim\_tail\_left 5 -trim\_tail\_right 5. rRNA-like sequences were removed using ri-boPicker version 0.4.3 [START_REF] Schmieder | Identification and removal of ribosomal RNA sequences from metatranscriptomes[END_REF] with parameters -i 90 -c 50 -l 50 and the following databases: SILVA Large subunit reference database, SILVA Small subunit reference database, the GreenGenes database and the Rfam database. In order to ensure that X and Y gametologs are assembled in consensus transcripts, Trinity components were merged using Cap3 [START_REF] Huang | CAP3: A DNA sequence assembly program[END_REF], with parameter -p 90 and custom perl scripts. Coding sequences were predicted using Trinity TransDecoder version 3.0.1 [START_REF] Haas | De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis[END_REF] and including Pfam domain searches as ORF retention criteria. This assembly included 128,904 ORFs. In order to correctly predict X/Y SNPs, we needed to reduce the redundancy of Trinity predictions, to avoid mapping X and Y reads on separate contigs. We chose to work on the longest ORF predicted per Tritinty isoform, which resulted in a final assembly of 82,699 isoforms (see Table 2.1). BUSCO v3.0.2 (Benchmarking Universal Single-Copy Orthologs) was used to assess the completeness of our transcriptome according to conserved gene content from the Plant Dataset [START_REF] Simao | BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs[END_REF]. Results are shown for full assembly with all ORFs and for the longest ORF per Trinity isoform, now referred to as our reference transcriptome (128,904 and 82,699 contigs respectively) in Supplementary Table 2.S2.

Functional annotation and Gene Ontology enrichment analysis

De novo annotation of our transcriptome was performed using Trinnotate v3.1.0 [START_REF] Haas | De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis[END_REF] and resulted in 59,319 annotations for 82,699 transcripts (71.73%). Gene Ontology (GO) was assessed using GOSeq [START_REF] Young | Gene ontology analysis for RNA-seq: accounting for selection bias[END_REF] version 1.30.0 on R version 3. 4.3 (2017-11-30) to identify over or underrepresented GO terms (p-value cutoff 0.05).

Inference of sex-linked contigs

The raw Illumina reads were mapped on the reference transcriptome using BWA mapping [START_REF] Li | Fast and accurate short read alignment with Burrows-Wheeler transform[END_REF] (Gayral et al., 2013;Tsagkogeorga et al., 2012), option -aeb that allows alleles to have different expression levels, and -par 0 to avoid removal of paralogous positions by the paraclean program that tends to filter out X/Y SNPs. SEX-DETector (Muyle et al., 2016) version 1.0 (03rd September 2017) was used to infer contig segregation types using a stochastic expectation maximization (SEM) algorithm. The detected SNPs were filtered using Perl scripts to retrieve the autosomal or sex-linked SNPs, when their posterior probability to be respectively autosomal or sex-linked was higher than 0.8. A contig was then inferred as sex-linked if its global probability of being sex-linked was higher than the probability of it being autosomal, and the contig had at least one sex-linked SNP without genotyping error. Amongst sexlinked genes, X-linked genes without a detectable homologous Y-linked copy are called X-hemizygous. Sex-linked contigs with no Y expression were considered as X-hemizygous, the rest as X/Y.

SEX

Correction of reference mapping bias

In order to avoid biases towards the reference allele in expression level estimates, a second mapping was done with GSNAP (Wu et al. 2010), a SNP-tolerant mapping software (see Supplementary Table 2.S3). A SNP file of X/Y SNPs identified in the first run of SEX-DETector was produced with home-made perl scripts as described in Muyle et al. (2017b). Raw Illumina reads were mapped with GN-SAP version 2017-11-15 and parameters -m 10 and -N 1. Only uniquely mapped and concordant paired reads were kept for expression analysis. SEX-DETector was run a second time on this new mapping and the new inferences were used afterwards for all analyses. No significant difference in the number of sex-linked inferences was observed: 1,196 X/Y and 168 X-hemizygous contigs were found as shown in Table 2.2.

Estimation of the age of recombination suppression

The X and Y ORF sequences were produced by SEX-DETector using only X/Y segregating SNPs, and pairwise dS was estimated by the codeml program implemented in the PAML suite [START_REF] Yang | PAML 4: phylogenetic analysis by maximum likelihood[END_REF] version 4.8 (see Supplementary Figure 2.S5). The age of recombination arrest was estimated from the divergence times of the contig pairs, which were determined using the maximum synonymous substitution rate of several molecular clocks: 1.4e-8 to 2.2e-8 substitution/synonymous site/year for chalcone synthase (Chs) and alcohol dehydrogenase (Adh) in Arabidopsis and Arabis taxa [START_REF] Koch | Comparative evolutionary analysis of chalcone synthase and alcohol dehydrogenase loci in Arabidopsis, Arabis, and related genera (Brassicaceae)[END_REF], 7.1e-9 ± 0.7e-9 substitutions/site/generation determined on 30 generations of Arabidopsis thaliana [START_REF] Ossowski | The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana[END_REF]) and 4e-9 substitutions/synonymous site/year based on a phylogeny of Brassicaceae (Beilstein et al., 2010).

Estimation of gene loss

X-hemizygous contigs are inferred by SEX-DETector from X polymorphism, as explained in Muyle et al. (2017b), whereas X/Y contigs rely on fixed mutations. X-hemizygous contigs are therefore harder to detect because they can only be detected in contigs with X polymorphism, and their number is thus underestimated. Corrected gene loss was estimated using the number of X-hemizygous contigs (168) relative to X/Y contigs with X polymorphism (424) that were listed in the output of SEX-DETector. Prematures stop codons were detected using a custom script on X and Y alleles.

Analysis of expression level difference between X and Y alleles

2.9.1. Measure of allelic expression Normalized allelic expression of sex-linked contigs was computed as in Muyle et al. (2017b) from SEX-DETector outputs. Expression of X and Y alleles was computed at the level of diagnostic X/Y SNPs, and were normalized using the library size and the number of studied SNPs in the contig.

Normalized expression levels appeared to be lower in males compared to females in autosomal contigs, as seen in Supplementary Figure 2.S2. This may be due to the fact that a small subset of genes are very highly expressed in developing male organs, as observed in other plants [START_REF] Badouin | The sunflower genome provides insights into oil metabolism, flowering and Asterid evolution[END_REF], resulting in apparent lower expression of housekeeping genes after normalization for total library size. We applied a correction coefficient to male expression using the ratio of median male autosomal expression over median female autosomal expression, in order to have a comparable median expression in males and females for autosomal contigs. The same ratio was then applied to sex-linked contigs. The results of this correction are shown in Supplementary Figures 2.S2, 2.S3 and 2.S4.

Analysis of dosage compensation in X-hemizygous contigs

To test for dosage compensation, we filtered genes based on their log 2 male-to-female expression ratio, as done in [START_REF] Mullon | Evolution of dosage compensation under sexual selection differs between X and Z chromosomes[END_REF]. Contigs with Xmale/2Xfemale ratios above 8 or under 0.125 were excluded like in Muyle et al. (2012) because we do not expect dosage compensation to occur in these sex-biased genes.

X-hemizygous contigs which showed the same expression level in males and in females, i.e. with of log 2 (male/female) of 0 ± 0.2 were considered as compensated and X-hemizygous contigs with twice as much expression in females compared to males, i.e.

with of log 2 (male/female) of -1 ± 0.2 were considered as non-compensated (see Figure 2.2). A wider range of log 2 (male/female) of ± 0.5, as used in [START_REF] Zimmer | Compensation of Dosage-Sensitive Genes on the Chicken Z Chromosome[END_REF] for dosage-compensated contigs, had the same GO enrichment categories. A smaller range of ± 0.1 had not enough contigs per category to allow for a significant GO enrichment analysis.

Identification of contigs with sex-biased expression

We used R packages DESeq2 [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]) version 1.18.1, edgeR (McCarthy et al., 2012;Robinson et al., 2010) version 3.20.8 and Limma-Voom [START_REF] Law | voom: Precision weights unlock linear model analysis tools for RNAseq read counts[END_REF](Law et al., ) version 3.34.8 on R version 3.4.3 (2017-11-30) -11-30) to perform biased gene expression analysis between males and females. DGE analysis was performed on the raw read counts (untransformed, not normalized for sequencing depth). Contigs with a CPM (Count Per million) lower than 0.5 (corresponding to a count of 10) were filtered out. Regularized log-transformation of the DESeq2 package was used to reduce variance of low read counts. Normalization with edgeR was made with a tagwise dispersion and GLM normalization method (calcNormFactors, estimateTagwiseDisp and glmLRT functions). With LimmaVoom, counts were fitted to a linear model and differential expression was computed by empirical Bayes (lmFit and eBayes functions).

Contigs identified in at least two methods with an FDR cutoff of 0.0001 were retained as differentially expressed (see Supplementary Figure 2.S1 and Supplementary Table 2.S4). This union was kept because the 3 methods do not have the same constraints, and intersections are more robust. DESeq2 and edgeR are based on the same method, but edgeR has less false positives. LimmaVoom is very specific (see Supplementary Figure 2.S1), and has therefore very few false negatives. The union of intersections limits false negatives but retains false positives that LimmaVoom discards.

Statistics and graph section

Unless stated otherwise in the relevant sections, all statistical analyses and graphs were done with R version 3.4.3 (R Development Core Team, 2015). Fisher's exact tests were two-tailed and p-values were adjusted with the FDR method (Bonferroni and Hochberg). Exact adjusted p-values are provided for each test.

Results

We assembled a de novo transcriptome for C. grandis with male and female reads (82,699 contigs, see Table 2.1). We used RNA sequencing data from a cross of C. grandis mapped to our reference transcriptome to identify genes located on the sex chromosomes (Supplementary Table 2.S1). The raw reads were mapped on open reading frames (ORF). A total of 45.76% reads were mapped with standard mapping, and 49.24% with SNP-tolerant mapping (see Supplementary Table 2.S3). The male offspring libraries had higher PCR duplication rates than the female libraries (35.52-0.49% and 18.62-25.83% of raw reads respectively, see Supplementary Table 2.S1) resulting in lower mapping rates (36.49 compared to 49.42% with SNP-tolerant mapping, see Supplementary Table 2.S3). We divided the contigs expressed in buds into autosomal, sex-linked X/Y (defined as contigs having both X-and Y-linked alleles) and X-hemizygous contigs (sex-linked, but with no Y-copy expression). These categories were inferred from single nucleotide polymorphisms (SNPs) segregating in a family, using a probabilistic model (Muyle et al., 2016). Out of the 82,699 transcripts, 3,801 had enough informative SNPs to be assigned to a segregation type, 2,437 were inferred as autosomal (64.11% of transcripts with enough informative SNPs), 1,196 as X/Y (31.47%) and 168 as Xhemizygous (4.42%) (see Table 2.2).

Age estimates are based on the divergence between X and Y copies and various molecular clocks to date the system. We measured a maximum dS of 0.17 in all contigs, and of 0.13 in contigs longer than 1kb. With the molecular clock of Beilstein et al. (2010), we estimated that the first recombination arrest event happened 3.75 My ago. Total Y and X read numbers were summed at sex-linked SNP locations for each contig and normalized for each male separately (see Methods section 2.9), then averaged across males to obtain the Y/X ratio. The median is shown in red.

We looked for patterns of degeneration in our dataset. Males showed lower gene expression than females for most of the genes, because a small subset of genes are very highly expressed in developing male flower buds resulting in apparent lower mean expression after normalization for total library size (Supplementary Figure 2.S2). After correcting for this expression bias between males and females (see Methods section 2.6), we found that sex-linked genes are less expressed in males than in females (see Supplementary Figures 2.S2 and 2.S4, Wilcoxon ranked test p-value = 2.57e-8). In order to refine our analysis of Y chromosome degeneration in C. grandis, we analysed the allelic expression of genes inferred as sexlinked, and found that Y-linked alleles were significantly less expressed than X-linked alleles in males (see Figure 2.1, Wilcoxon ranked test p-value = 3.06e-13). Lost Y genes can be detected by SEX-DETector when the Y copy is absent or unexpressed and are assigned as Xhemizygous. But given that X-hemizygous contigs can be inferred from segregation patterns only if there is polymorphism on the X chromosome (Bergero and Charlesworth, 2011;Muyle et al., 2017b), they can be less frequently detected than X/Y contigs that rely on fixed differences between the X and Y that are more numerous. X-hemizygous gene numbers are thus underestimated when segregation patterns are analyzed. To correct for this bias, we counted the number of X/Y contigs with X polymorphism (424) and compared the number of Xhemizygous genes (168) to all sex-linked genes with X polymorphism, which gives a corrected rate of gene loss of 39.62%. Another marker of degeneration is the presence of premature stop codons. We detected 17 X alleles (1.4%) and 56 Y alleles (4.7%) with a premature stop, which are significantly more abundant on Y alleles (Fisher's exact test p-value = 6.90e-6). These observations suggest there is degeneration on the Y chromosome as observed in several other plant systems. In order to determine whether some genes are dosage-compensated, we first studied the log 2 fold change between male and female expressions. In the absence of dosage compensation, the Xmale/2Xfemale expression ratio is expected to be 0.5, so the log 2 of the ratio is expected to be -1, because males (XY) have one X-linked copy and females (XX) have two. This is what we observed for contigs that do not show reduced expression of the Y-linked allele relative to the X-linked allele, i.e., that have a Y/X expression ratio close to 1 (median of log 2 Xmale/2Xfemale ratio is -1.29 for contigs with Y/X>1; see Figure 2.2). For contigs with reduced Y expression (low Y/X ratios), we observed a higher Xmale/2Xfemale expression ratio, which suggests that dosage compensation occurs for some genes (median of contigs with Y/X<0.5 is -0.85; see Figure 2.2). Finally, in X-hemizygous contigs, the distribution of Xmale/2Xfemale expression ratio was bimodal, with a set of 31 contigs centered on a log 2 Xmale/2Xfemale ratio of -1 (no compensation), and 23 contigs centered on 0 (total compensation). This suggests that these two sets of genes exhibited respectively no compensation and total compensation resulting in equal expression in males and females. This trend is also present in a less visible pattern for X/Y contigs with low Y expression (see Figure 2.2). To investigate dosage compensation further, we compared expression of X-linked and Y-linked alleles in males and females for different Y/X expression ratio categories (Figure 2.3), using female expression as a reference. We excluded 1% of the sex-linked contigs that showed either an elevated Y expression (high Y/X ratios) or sex-biased X expression (very high or very low Xmale/2Xfemale ratios). The Y/X ratio was computed in C. grandis males and averaged between individuals, and used as a proxy for Y degeneration. In the absence of dosage compensation Xmale/XXfemale expression ratio is expected to be 0.5. On the contrary, we found that X expression in males increases with decreasing Y expression, which results in similar expression levels of sex-linked contigs in both sexes and provides further evidence for dosage compensation in C. grandis. X/Y genes appeared to be depleted in hormonerelated functions such as response to ethylene and negative regulation of ethylene biosynthetic process (GO:0009723 and GO:0010366). In contrast, X-hemizygous genes were depleted in dosage-sensitive functions such as macromolecular complex, intracellular ribonucleoprotein complex, ribonucleoprotein complex, transferase complex, membrane protein complex (GO:0032991, GO:0030529, GO:1990904, GO:1990234 and GO:0098796). These functions were enriched in X-hemizygous genes that show dosage compensation when compared to all sex-linked genes, which suggests favored compensation for dosage-sensitive genes.

We detected 3,453 sex-biased genes with edgeR, 4,881 with DESeq and 538 with LimmaVoom (Supplementary Figure 2.S1). To establish a robust set of sex-biased genes we retained genes that were identified as sex-biased in at least two methods (3,273 genes). Among these, 2,682 (81.94%) were male-biased and 591 (18.06%) female-biased. Genes with sex-biased expression were significantly over-represented among sex-linked genes (see Supplementary Table 2.S4, Fisher's exact test p-value < 2.2e-16), with 241 out of 1,364 sex-linked genes being sex-biased (17.67% of sex-linked genes, with respectively 13.42% and 4.25% having male and female-biased expression), and 206 out of 3,706 autosomal genes being sex-biased (5.56% of them, 4.45% with male and 1.11% with femalebiased expression). Out of the sex-biased genes that were localized on the sex chromosomes, 228 (94.61%) had a X/Y segregation inference (181 male-biased and 47 female-biased), and only 11 male-biased genes and 2 female-biased were X-hemizygous. X-hemizygous contigs were not enriched for differentially expressed genes when compared to autosomal contigs (see Supplementary Table 2.S4, Fisher's exact test p-value = 0.2302), which might be due to the small sample of X-hemizygous genes in our study. Gene ontology analysis revealed several biological processes that are significantly overrepresented among female-biased genes and under-represented among male-biased genes, or vice versa. GO categories related to pollen production were enriched in male-biased genes, such as pectin catabolic process, pollen wall assembly, sporopollenin biosynthetic process, pollen exine formation, pectin metabolic process, pollination, anther wall tapetum development, pollen sperm cell differentiation, anther development, rejection of self pollen and regulation of pollen tube growth (GO:0045490, GO:0010208, GO:0080110, GO:0010584, GO:0045488, GO:0009856, GO:0048658, GO:0048235, GO:0048653, GO:0060320 and GO:0080092). Functions related to hormone signaling were enriched in female-biased genes, such as response to auxin, auxin-activated signaling pathway, regulation of ethyleneactivated signaling pathway, brassinosteroid mediated signaling pathway, auxin polar transport, auxin mediated signaling pathway involved in phyllotactic patterning, ethylene receptor activity, ethylene binding, auxin transport, jasmonic acid and ethylene-dependent systemic resistance, ethylene mediated signaling pathway (GO:0009733, GO:0009734, GO:0010104, GO:0009742, GO:0009926, GO:0060774, GO:0038199, GO:0051740, GO:0038199, GO:0051740, GO:0060918 and GO:0009871). These functional enrichments suggest that sex-biased expression may have evolved to support contrasting biological functions in C. grandis females and males.

Discussion

Our segregation analysis provides the first set of sex-linked genes identified in C. grandis with a total of 1,364 transcripts including 1,196 X/Y and 168 X-hemizygous transcripts. 2,437 transcripts were inferred as autosomal, allowing for a wide-scale comparison between sex-linked and autosomal genes. Many contigs remained uncategorized when using SEX-DETector (78,898), which might be due to a lack of SNPs, the father and mother being related, which reduces overall polymorphism. This reduced polymorphism results in a lack of power to detect autosomal and X-hemizygous genes, the latter relying on the presence of polymorphism on the X chromosome. As we detected only a subset of X-hemizygous, we corrected for their under-representation when calculating gene loss (see Methods section 2.8). The identification of X-hemizygous genes relies on transcripts missing from the Y chromosome, that either correspond to genes that are deleted or not expressed in our sample. With the use of RNA sequencing, lowly expressed Y genes can be erroneously inferred as X-hemizygous. Low polymorphism between the parents of the cross does not reduce the power to detect X/Y genes, because their detection is based on fixed mutations between the X and Y gametologs. Sensitivity for X/Y genes can be reduced for genes from very recent evolutionary strata, which might be lacking fixed sexlinked SNPs and cannot therefore be inferred correctly by SEX-DETEctor. The main limitation for the detection of X/Y genes is that the segregation analysis cannot detect highly divergent X/Y gametologs, which are assembled separately in our de novo transcriptome assembly. To solve these issues and avoid false negatives in the X/Y dataset, one can either compare X-hemizygous genes with the rest of the transcriptome, as was done in Bergero et al. (2015), or with male-specific transcripts as in Blavet et al. (2015). SEX-DETector lacks the possibility to detect Y-specific genes and such genes were not identified in this study. The estimated divergence time between the X and Y chromosomes in C. grandis being small (3.5 My), we do not expect to have many such false negatives if this estimate is correct. This results in a robust dataset of X/Y genes in this study, despite the eventual lacking of Yspecific genes.

The divergence between X and Y copies allowed us to estimate an age of recombination arrest. With a molecular clock also used in other plant systems (see Table 2.3), we found that the divergence between the X and Y chromosomes started around 3.75 My ago. We used the maximal divergence for this estimation, but the maximum dS can be underestimated in regions where the Y copy diverges too much from the X copy. The consensus assembly then contains two separate X and Y alleles, or even worse only the X allele because Y reads are under-represented (1/4 of all male and female X and Y reads come from Y chromosomes). This can lead us to underestimate the age of the sex chromosomes. On the other hand, molecular clocks are based on autosomal divergence, and sex chromosomes might evolve much faster, especially the Y chromosome, so using these clocks might result in an overestimated divergence time. Despite these limitations, our dating confirms the recent origin of C. grandis sex chromosomes, which was already suggested by phylogenetic studies (Holstein and Renner, 2011;Sousa et al., 2012). Since C. grandis displays the largest heteromorphy currently known in plants, this raises the question of why some plant sex chromosomes quickly evolve heteromorphy when others stay homomorphic much longer. In addition to the changes in TE content between the young X and Y chromosomes of C. grandis that have been previously described, I provided new information regarding the changes in gene contents and expression, as well as for the rapid evolution of dosage compensation in this system. C. grandis has recent XY chromosomes that share characteristics with much older systems such as mammalian sex chromosomes, and shows that plant sex chromosomes can evolve very quickly.

Our results suggest that the Y chromosome of the diploid C. grandis is undergoing genetic degeneration and not only repeat accumulation. Out of the 1,364 sex-linked genes, 168 did not have a Y-linked homolog, and we estimated a total gene loss of about 40%. This rate is similar to rates observed in Carica papaya (Wang et al., 2012), the old XY system of R. hastatulus (Hough et al., 2014), S. latifolia (Papadopulos et al., 2015) and M. annua [START_REF] Ridout | Hallmarks of early sexchromosome evolution in the dioecious plant[END_REF], for which the arrest of recombination between sex chromosomes was evaluated to have happened respectively 7 My, 9-16 My, 5-10 My and 1.5 My ago, as detailed in Table 2.3. This suggests an important gene loss from the C. grandis Y chromosome despite the young age of the system, only 3.75 My old. Moreover, C. grandis Y alleles had significantly more premature stop codons than X alleles. Expression of sex-linked genes was significantly lower in males than in female. In males, Y alleles were significantly less expressed than X alleles, which could be due to DNA methylation. One explanation could be than C. grandis sex chromosomes are young but highly silenced. Methylation has been described as a silencing defense, in particular against TE invasion which are abundant on the Y chromosome of C. grandis [START_REF] Sousa | Analysis of transposable elements and organellar DNA in male and female genomes of a species with a huge Y chromosome reveals distinct Y centromeres[END_REF]. Methylation spreading over the Y chromosome would explain a low Y/X expression ratio. High heteromorphy can cause methylation to spread over sex chromosomes [START_REF] Root | Evolution of dioecy and sex chromosomes via methylation driving Muller's ratchet[END_REF], and silenced Y copies can lead to an overestimation of gene loss through RNA-based methods. In C. grandis, Y-specific heterochromatic patterns have been detected (Sousa et al., 2012).

We observed an increase in the expression of the X allele in males for genes that had a reduced Y expression (Figure 2.3). This confirms that a subset of sex-linked genes undergo dosage compensation, as seen in S. latifolia (Bergero et al., 2015;Muyle et al., 2012;Papadopulos et al., 2015). Similarly to what was seen in S. latifolia (Papadopulos et al., 2015), we observed a subset of X-hemizygous genes in C. grandis that displayed equal expression in males and females due to an increase in the expression of the X allele in males compared to 2X in females. We investigated a biased retention or compensation of genes on the Y chromosome, and observed that compensated X-hemizygous genes are significantly enriched in functions concerning molecular complexes. As these functions are known to be dosage sensitive (Pessia et al., 2014;Veitia et al., 2015), this suggests that dosage compensation for some X-hemizygous genes in C. grandis is driven by dosage constraints. We were limited by the absence of outgroup in our studies, which did not allow us to orient changes and analyze more than sex equality. With an outgroup, it is possible to investigate for the restoring of ancestral expression levels, which is an important marker of dosage compensation and was studied in S. latifolia (Muyle et al., 2017b;Zemp et al., 2016), where it was shown that dosage compensation was achieved by an increase in the expression of the maternal X in both males and females. Overall, we observed very similar dosage compensation patterns for X/Y genes as in S. latifolia, the other heteromorphic system were dosage compensation is found in plants, but found differences in terms of dosage compensation of X hemizygous genes, which suggests that balance constraints drive dosage compensation in a subset of C. grandis genes. Our results show that dosage compensation can evolve very quickly in young heteromorphic plant sex chromosome systems. We found that 4% of the genes expressed in C. grandis floral buds are sex-biased (Supplementary Table 2.S4), with a total of 3,273 sexbiased genes identified (2,682 male-biased and 591 female-biased), similarly to what was previously found in Devani et al. (2017) and Mohanty et al. (2017), but also in other plants like S. latifolia (Zemp et al., 2016) and M. annua [START_REF] Ridout | Hallmarks of early sexchromosome evolution in the dioecious plant[END_REF]. Like Mohanty et al. (2017) and Devani et al. (2017), our results support enrichment for pollen-production related functions in male-biased genes as found in C. grandis and other plant systems. Femalebiased genes, on the other hand, were significantly enriched for hormone signaling functions. X/Y genes appeared to be depleted in hormone-related functions that were enriched in female-biased genes. We found a significant enrichment for sex-biased genes among sexlinked genes in C. grandis compared to autosomal genes (Fisher's exact test p-value < 2.2e-16), similarly to what has been described in S. latifolia (Zemp et al., 2016). This enrichment could be due to sex chromosome degeneration, but most sex-biased genes are male-biased in our C. grandis dataset, whereas degeneration mechanisms generally involve a decreased expression on the Y and in males. The most probable hypothesis is that antagonistic selection favors sex-linkage of sex-biased genes involved in sexual dimorphism, and further work on the transcriptome of C. grandis should involve its testing.

Species

Conclusion and Perspectives

In this study, we obtained the first set of sexlinked genes in the most heteromorphic plant sex chromosome system characterized to date. We confirmed the young age of the sex chromosomes in C. grandis, but showed that gene loss is already equivalent to that of other older systems. We showed that Y degeneration occurs in C. grandis and that dosage compensation has already evolved in this young system. As this species is closely related to monoecious cultivated species, cucumber and melon, this will help deciphering the mechanism of sex chromosome evolution in the Cucurbitaceae family. More detailed orientation of expressional changes requires an outgroup, and we could use closely related cucurbits such as melon and cucumber which both have sequenced and annotated genomes, to better decipher the mechanisms of dosage compensation in C. grandis. Supplementary Figure 2.S3. Autosomal gene expression is similar for males and females after correction. Distribution of normalized expression difference between males and females in autosomal contigs. Total read numbers were summed at autosomal SNP locations for each contig and normalized for each individual separately; then averaged across sexes to obtain the male/female ratio. Expression was corrected using the autosomal median male/female ratio. Distribution is shown in log 2 scale with its density curve. The median is shown in red. Grégoire Siekaniec, whom I supervised during his Master's internship, worked with me on decontaminating the datasets and testing assembly pipelines. Mohammed-Amine Chebbi from the University of Poitiers made the read processing, read correction, assembling and post-processing for the final assembly presented in this chapter. The transcriptomic dataset used to assess the assemblies was provided by Aline Muyle. Adil El-Filali annotated the final assembly.

Introduction

Separate sexes are more common in animals than plants, but up to 6% of all angiosperm species and 20% of all crops are dioecious.

The genetic basis of separate sexes has been investigated abundantly in animals, and remains vastly unknown in plants. Three master sex-determining genes have been identified in plants so far, in persimmons (Akagi et al., 2014), asparagus (Murase et al., 2017) and strawberry [START_REF] Tennessen | Repeated translocation of a gene cassette drives sex chromosome turnover in strawberries[END_REF], but in the vast majority of dioecious species, those genes are unknown. Only one angiosperm Y chromosome has been fully sequenced yet, the one of Carica papaya (Wang et al., 2012). Reasons for this lack of Y sequences are: (1) a poor X-Y coverage compared to the autosomes in diploid species, making it difficult to assemble correctly the heteromorphic sex, so that most sequencing projects are carried on XX females; and (2) non-recombining Y displays very repeated contents, containing many transposable elements (TEs) and amplicons, which makes them difficult to assemble (Hughes and Rozen, 2012;Muyle et al., 2017a).

Novel sequencing technologies have opened opportunities to make Y chromosome sequencing faster and more affordable. One such approach sequences both male and female genomes and uses a differential analysis to identify Y-linked contigs [START_REF] Carvalho | Efficient identification of Y chromosome sequences in the human and Drosophila genomes[END_REF][START_REF] Vicoso | Reversal of an ancient sex chromosome to an autosome in Drosophila[END_REF]. However, this approach still requires substantial amounts of wholegenome sequencing and the building of a reference genome. For systems where no reference is available, bacterial artificial chromosome (BAC) sequencing has been used to provide sex chromosome sequences. Single-haplotype iterative mapping and sequencing (SHIMS), relies on BAC sequencing and was used to sequence the human, chimpanzee, macaque, and mouse Y chromosomes [START_REF] Hughes | Chimpanzee and human Y chromosomes are remarkably divergent in structure and gene content[END_REF](Hughes et al., , 2012;;[START_REF] Skaletsky | The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes[END_REF][START_REF] Soh | Sequencing the mouse y chromosome reveals convergent gene acquisition and amplification on both sex chromosomes[END_REF]. The only plant Y chromosome sequenced until now, the one of C. papaya (Wang et al., 2012) Oxford Nanopore's main problem is handling homopolymers: as soon as there are more than 5 repeated bases, base-calling with 5-mers (or 6 in case of 6-mer calling) will not be able to take them into account because the ion current does not change. Base-calling will therefore not detect any base added to the DNA after 5 repeated bases [START_REF] Lu | Oxford Nanopore MinION Sequencing and Genome Assembly[END_REF]. PacBio has the same limitations as Illumina regarding GC-rich regions, which are more difficult to handle for the polymerase [START_REF] Chen | Effects of GC bias in next-generation-sequencing data on de novo genome assembly[END_REF]. Long reads were nevertheless used to assemble Gorilla gorilla, Drosophila melanogaster and Anopheles gambiae sex chromosomes [START_REF] Hall | Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes[END_REF][START_REF] Krsticevic | Long-Read Single Molecule Sequencing to Resolve Tandem Gene Copies: The Mst77Y Region on the Drosophila melanogaster Y Chromosome[END_REF][START_REF] Tomaszkiewicz | A time-and cost-effective strategy to sequence mammalian Y chromosomes: An application to the de novo assembly of gorilla Y[END_REF], as they stretch over repetitions and solve miss-assembled duplicated genes.

S. latifolia is one of the most studied dioecious plants, in particular for its sex chromosomes (Bernasconi et al., 2009). The identification of sex-determining genes in S. latifolia is particularly difficult given that the non-recombining region is very large, likely comprising many more genes than in papaya. Lists of sex-linked genes have been produced in S. latifolia (Bergero and Charlesworth, 2011;Chibalina and Filatov, 2011;Muyle et al., 2012). A recently developed probabilistic approach, SEX-DETector (Muyle et al., 2016), produced a dataset of 1,025 sex-linked genes using allele transmission patterns from RNAseq SNPs in a cross to infer sex-linkage. Y chromosome specific BAC sequences have been obtained by Blavet et al. (2015). Moreover, three candidate regions for sex determination have already been identified and located on a physical map of the Y chromosome of S. latifolia (Bergero et al., 2008): gynoecium suppressing factor (GSF ), stamen promoting factor (SPF ), and male fertility factor (MFF ) (see Chapter 1 for details). These three regions can help shortlist the candidate sex-determining genes. Until now, the approaches that provided sex-linked gene lists did not give the genomic position of the sequenced genes. Knowing precisely these genes and their location on the Y chromosome will be necessary to find the sex-determining genes.

For these reasons, we need the DNA sequences of the Y chromosome of S. latifolia. Full sex chromosome sequences are also needed if we want to be able to study Y gene loss and degeneration, X-Y divergence, evolutionary stratas, sex chromosome differentiation, and Y-specific genes.

A recent study combined high-throughput genome and transcriptome sequencing to assemble the S. latifolia genome (Papadopulos et al., 2015). To identify sex-linked genes, regardless of their expression, the authors sequenced male and female genomes of S. latifolia and produced an assembly with a total length of 665 Mb, 25% of the S. latifolia genome, comprising 71% of the 248 ultra-conserved core eukaryotic genes, which suggests that they mainly assembled gene-rich regions. Computational "subtraction" of the female genome from the sequences of the male allowed to reconstruct male-specific, putatively Y-linked contigs on which 931 genes were annotated, including 201 genes with known positions in the X chromosome genetic map. These sequences represent ∼25% of the total expected size of the Y chromosome, but given that the Y is highly fragmented, some of the genes were probably not sequenced, and the scaffolds are not large enough to cover the markers that would allow linking genes to the three known sex-determining regions.

In this paper, we present our strategy to sequence and assemble the Y chromosome of S. latifolia. We aimed at identifying the genetic contents of this particuliar chromosome. We combined chromosome sorting using flow cytometry with whole genome amplification (WGA) to procure sufficient material for sequencing. We used Illumina next-generation sequencing, as well as MinION and PacBio third-generation sequencing (TGS), and performed read cleaning, correction and decontamination. Assembling, scaffolding and gap-filling were finelly tuned to obtain an assembly of the Y chromosome of S. latifolia. We mapped Y chromosome specific BAC sequences from Blavet et al. (2015) against the assembly to evaluate its completeness. We also used a list of sex-linked X/Y genes, X hemizygous genes, i.e. genes with a lost Y copy, and autosomal genes inferred by Muyle et al. (2016) to test whether we found them on our Y chromosome assembly. Finally, we annotated our final assembly de novo.

Material and Methods

Sorting of Y chromosomes using flow cytometry

Y chromosome DNA was isolated for sequencing using flow cytometry. We isolated metaphasic chromosomes from S. latifolia root-tip meristems after inducing artificial cell synchrony using DNA synthesis inhibitor hydroxyurea. Labeling of Y-specific sequences decreased contamination rate under 5% [START_REF] Vrana | Flow Analysis and Sorting of Plant Chromosomes[END_REF]. Flow-cytometry Y chromosome isolation was performed after the protocol described in [START_REF] Vrana | Flow Analysis and Sorting of Plant Chromosomes[END_REF]. DNA from flow sorted chromosomes is a suitable template for whole-genome amplification (WGA) using Phi29 high-fidelity polymerase. With this method, we produced microgram quantities of DNA with a low amplification bias. The product we obtained ranged from 5 to 20 kb in size, which was suitable for most nextgeneration sequencing (NGS) technologies. We performed amplification using a GenomiPhi V2 kit (GE Healthcare), which relies on multiple displacement amplification (MDA) through Degenerate-oligonucleotide-primedpolymerase-chain-reaction (DOP-PCR) of linear amplification. This method was tested in wheat [START_REF] Capal | Multiple displacement amplification of the DNA from single flow-sorted plant chromosome[END_REF] and about 99% of genes were found in the amplified sequences. All flow cytometric experiments were performed on FACSAria II SORP flow cytometer (BD Biosciences, San José, Calif., USA).

Illumina sequencing and read processing

Sequencing was performed with 2x100bp pairend (PE) Illumina Hiseq 2000, using fragment lengths of 150 to 250 bp, resulting in a dataset of 436,648,776 PE reads of 104 bp (80X coverage). We checked the raw reads quality using FastQC (version 0.11.5, [START_REF] Andrews | FastQC: a quality control tool for high throughput sequence data[END_REF] and discarded adapters and low quality bases with Trimmomatic (version 0.33, [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] with parameters Illuminaclip:adapters.fasta:2:30:10 SlindingWindow:4:10 Leading:20 Trailing:20 and MinLen:100. A total of 73,972,600 reads (16.94%) were removed from the dataset. We then removed 233,801,574 (64.47% of reads remaining after quality check) PCR duplicates with PRINSEQ-lite (version 0.20.3, Schmieder and Edwards, 2011). We used KmerGenie (Chikhi and Medvedev, 2014) to estimate the best k-mer length (15 bp) and removed further contamination with RecoverY [START_REF] Rangavittal | RecoverY: k-mer-based read classification for Ychromosome-specific sequencing and assembly[END_REF], resulting in a final Illumina dataset of 121,713,370 uncontaminated Y reads (94.44% of cleaned reads).

Library preparation for MinION (Oxford Nanopore) long-read sequencing

We analyzed the fragmentation of our S. latifolia Y chromosome DNA with an Agilent High Sensitivity DNA assay. The highest molarity (939.9 pmol/l) was obtained at 2,418 bp and highest concentration (2,816.66 pg/μl) at 5,712 bp. All bead purification steps were done at a 0.8 bead to DNA ratio to favor longer fragments in our remaining purified DNA, and eluted in nuclease free water. The output material of WGA typically tends to be more complex than standard double strand DNA and shows branching, which makes it impracticable for long-read sequencing. We therefore had to remove branches and repair the DNA to obtain double stranded DNA suitable for sequencing. When debranching was performed (see Supplementary Table 3.S1), sorted Y chromosome DNA was incubated with Phi29 DNA Polymerase (250 units, 10,000 U/mL, Biolabs) supplemented with 200 μg/mL with BSA and 200 μM dNTPs and 1 X reaction buffer for 2 hours at 30 • C followed by 3 minutes at 65 • C. We then added S1 nuclease (SIGMA-ALDRICH) for digestion and incubated at 37 • C for 30 minutes. Subsequently, the debranched DNA was repaired with NEBNext FFPE DNA Repair Kit (New England Biolabs) followed by a clean up with AMPure XP Beads. We then performed NEBNext UltraII End Repair/dA-Tailing treatment (New England Biolabs) following manufacturer instructions and subjected the sample to AMPure XP bead clean up again. The end repaired and adenylated DNA was then prepared following Oxford Nanopore protocols (see Supplementary Table 3.S1): Adapter Mix ligation with NEB Blunt/TA Master Mix (New England Biolabs), Low-input Hairpin Tether (LIT) ligation and purification with MyOne C1 Streptavidin beads. We kept the final sequencing library on ice until sequencing.

The MinION sequencer reads the nucleotide sequence of individual DNA strands as they are driven through biological nanopores with an applied electric field. Sequencing starts from the 5' end of the leader adapter. The motor protein unwinds the dsDNA allowing single-stranded DNA to pass through the pore. A hairpin adapter links both template and complement strands, allowing them to be sequenced through the same pore. The 1D base calls are inferred separately for the template and complement signals, and the 2D reads are build from a consensus signal from a template and a complement strand of dsDNA going through the same pore. The MinION MkIB is the most recent version of the MinION, and was released in May 2016. It follows the MkI that was released in May 2015. MinION MkIB is based on its predecessor but uses different flow cell chemistries, the main difference being the speed of sequencing. The electromagnetic shield used in R9 flow cells provides a significant boost in accuracy. Raw 1D reads from the MinION sequencer have a reported error rate of 25-35%, while higher quality 2D reads have an error rate of 12-20% [START_REF] Ashton | MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island[END_REF][START_REF] Ip | MinION Analysis and Reference Consortium: Phase 1 data release and analysis[END_REF][START_REF] Laver | Assessing the performance of the Oxford Nanopore Technologies MinION[END_REF], which is why we chose not to work with MkI 1D reads. Our low Q-score were probably due to the basecaller not being trained for plant DNA [START_REF] Ip | MinION Analysis and Reference Consortium: Phase 1 data release and analysis[END_REF][START_REF] Loman | A complete bacterial genome assembled de novo using only nanopore sequencing data[END_REF]. R9 flowcell upgrade showed an important improvement in the quality of 1D reads, their error rates dropping to 10% [START_REF] Lu | Oxford Nanopore MinION Sequencing and Genome Assembly[END_REF], which is why only 1D reads were sequenced with MkIB.

Library preparation for PacBio sequencing and read processing

We performed Phi29 debranching on our sample before sending them to the Quebec Sequencing Genomes platform where the sequencing was done. We prepared the library as following: a starting 4 μg DNA were debranched with Phi29 and digested with S1 nuclease, then repaired with FFPE DNA Repair Kit (New England Biolabs) and purified with AMPure beads at 1.8 beads to DNA ratio, to retain as much DNA as possible. After purification, we resuspended the remaining 2 μg of DNA in Elution Buffer NE and sent it to the sequencing platform. PacBio P6-C4 chemistry was used with collection protocol MagBead OneCellPerWell v1, DNA Template Prep Kit 3.0 and DNA/Polymerase Binding Kit P6 v2. The 10 SMRT (Single Molecule Real-Time) cells outputted a total of 16 Gb of reads (see Table 3.1) that were called with BasecallerV1.

PacBio SMRT (Single-Molecule Real Time) sequencing operates within a silicon chip (a SMRTcell) which contains a large number of microscopic holes, the zero-mode waveguides (ZMW). Within a ZMW, the target DNA is circularized in a molecule called a SMRTbell, consisting of the double-stranded DNA insert and single stranded hairpin loops. Each polymerase read is partitioned to form one or more subreads, which contain sequence from a single pass of a polymerase on a single strand of an insert within a SMRTbell template. The subreads taken from a single ZMW are used to reconstruct circular consensus (CCS) reads. CCS reads require at least two full-pass subreads from the insert. Base error rates for PacBio reads are slightly better than for Oxford Nanopore's first chemistries, reportedly in the range of 10-15% [START_REF] Rhoads | PacBio Sequencing and Its Applications[END_REF].

Long-read error correction

2D MkI and 1D MkIB MinION reads were filtered based on quality and trimmed for adapters using NanoFilt (parameters q 9 and readtype 1D, version 1.5.2, De Coster et al., 2018) and Porechop (version 0.2.3, Wick, 2017). We corrected these MinION processed reads and the PacBio subreads that were not included in the circular consensus sequences (CCS), hereafter called "no CCS-like", as shown in Figure 3.1. We used LoRDEC (Long-Read DBG Error Correction, version 0.4), developed by [START_REF] Salmela | LoRDEC: Accurate and efficient long read error correction[END_REF], a hybrid error correction method that builds a succinct de Bruijn graph (DBG) using Illumina short reads, and seeks a corrective sequence for each erroneous region in the long reads by choosing paths in the graph. We tested k values from 13 to 21 using steps of 2 and obtained our best result with a k-mer size of 17. We used corrected but untrimmed read for further steps. Our correction rate was of 88.28% for MinION reads and 81.78% for PacBio subreads.

Assembly

We performed a first assembly with the hybrid assembler MasurCA (version 3.2.2, [START_REF] Zimin | The MaSuRCA genome assembler[END_REF] using Illumina PE short reads and PacBio high quality CCS long reads. The generated unitigs were then reassembled using a long read dataset of CCS over 500 pb, corrected PacBio "no-CCS-like" subreads over 1kb and MinION reads over 1kb (see Figure 3.1) with a second hybrid assembler, DBG2OLC [START_REF] Ye | DBG2OLC: Efficient Assembly of Large Genomes Using Long Erroneous Reads of the Third Generation Sequencing Technologies[END_REF] with following parameters: k 17 AdaptiveTh 0.001 KmerCovTh 2 MinOverlap 10 RemoveChimera 0.

Scaffolding and post-processing

To help further assemble the genes present and expressed on the Y chromosome, we used the S. latifolia transcriptome from Muyle et al. (2016). The scaffolding was performed with L_RNA_scaffolder (Xue et al., 2013) which uses BLAT (version 36x2, Kent, 2002) to map the RNAseq data on the genomic assembly, and then reconstructs exon liaisons from RNAseq reads mapping on several genomic contigs. The long read dataset (CCS over 500 pb, corrected PacBio subreads over 1kb and MinION reads over 1kb, see Figure 3.1) was then used for gap filling with PBJelly 2 [START_REF] English | Mind the Gap: Upgrading Genomes with Pacific Biosciences RS Long-Read Sequencing Technology[END_REF]. We used BlobTools (version 0.9.17, [START_REF] Kumar | Blobology: exploring raw genome data for contaminants, symbionts and parasites using taxon-annotated GC-coverage plots[END_REF] to determine possible contaminants based on GC per cent, Illumina read coverage and taxonomic information from BLAST (version 2.2.30+, [START_REF] Camacho | BLAST+: architecture and applications[END_REF] and Diamond (version 0.7.12.61, [START_REF] Buchfink | Fast and sensitive protein alignment using DIAMOND[END_REF]. Ten assemblies (in green) are nested within each other, from which five with decontaminated reads (light green). The best assembly is framed in red. Illumina, MinION, and PacBio data are highlighted in different shades of blue. For each assembly, the total length, the number of contigs/scaffolds and the N50 are shown. The N50 is a weighted median statistic such that 50% of the entire assembly is covered by contigs equal to or larger than this value.

Our long reads dataset was then remapped on the whole assembly and we used BlobTools results to remove contaminant reads. Up to 6% of the PacBio subreads and 23% of the CCS, as well as 0.07% of the MinION reads were discarded due to contamination. All assembly steps starting with DGB2OLC were then run with the decontaminated long read dataset, as shown in Figure 3.1.

Assessment

To assess the completeness of our final assembly, we mapped Y-specific BAC clone sequences from Blavet et al. (2015) with Gmap (version 2017-11-15, Wu andWatanabe, 2005, default parameters). We also mapped the reference transcriptome from Muyle et al. (2016) with Gmap and used a list of sex-linked and autosomal genes inferred by SEX-DETector from Muyle et al. (2016), to test whether they were present in the final assembly.

Annotation

We used EuGene (version 4.2 with egnep-1.4, [START_REF] Sallet | EuGene-PP: a next-generation automated annotation pipeline for prokaryotic genomes[END_REF] to predict coding sequences in the final assembly. EuGene uses a graph based model to predict genes, covering Exon, Intron, UTR and UTR introns. The resulting mRNAs were blasted against non-redudant protein database (evalue cutoff 10e-5) and converted into GO terms using blast2GO (version 5, Conesa et al., 2005).

Results

We sorted Y chromosomes from S. latifolia using flow cytometry. We combined three sequencing technologies (Illumina, PacBio and MinION) along with several data processing approaches and assembly tools (see Figure 3.1). We obtained datasets with different characteristics, in particular regarding read length, as all the next-generation Illumina reads are 104 bp long whereas the third generation sequencing (TGS) reads range from 14 to 18,347 bp for circular consensus sequences (CCS, see Methods section 2.4 for details), 50 to 46,748 bp for subreads and 5 to 216,612 bp for MinION reads (see Table 3.1 and Figure 3.2). We chose to work only with MinION reads and PacBio subreads longer than 1kb. We trimmed and removed adapters, duplicated and low quality sequences from the Illumina PE dataset, reducing the number of reads by ∼70%. As a result, we obtained 60 million pairs of reads for the Illumina dataset. Processing both short and long reads is a major step of our pipeline, even when it removes a significant part of the dataset, as hybrid assembling does not require quantity as much as quality.

Looking at the GC content of all the sequencing technologies we used, it appears that when PacBio and Illumina produce reads of approximately the same GC per cent (see Table 3.1), MinION reads have a higher GC. This can be due both to PacBio and Illumina enzymebased methods having trouble handling GCrich regions, and MinION making deletions in homopolymers such as A-stretches. Overall, MkIB reads (second version of the MinION sequencer) have a closer GC to Illumina reads, which suggests that the improvement in the Oxford Nanopore technology enabled a better sequencing. MkIB reads are of a more even distribution than MkI ones and are globally longer than the PacBio reads, as shown in Figure 3.2. MinION sequencing is clearly progressing and future outputs hold great promises, in regard of yield and read quality (Supplementary Table 3.S1).

Another very important prerequisite for hybrid assembly is the error-correction of long reads. MinION and PacBio "no CCS-like" subreads longer than 1 kb were error corrected with LoRDEC [START_REF] Salmela | LoRDEC: Accurate and efficient long read error correction[END_REF] using the processed Illumina dataset as a reference for correction, resulting in 2,464,154 error-corrected long reads (8,773). Our correction rate was of 88.28% for MinION reads and 81.78% for PacBio subreads. Until we met such high levels of correction, all further assembly steps were failing. Many critical steps were required to obtain a high quality dataset that can be used for assembly. We needed to remove a lot of data before being able to assemble, our philosophy shifting from "the more the better" towards "the cleaner the better" as recommended in recent reviews [START_REF] Li | Genome Sequencing and Assembly by Long Reads in Plants[END_REF][START_REF] Sohn | The present and future of de novo whole-genome assembly[END_REF].

Care also needs to be brought to the contents of our sequencing datasets, in particular regarding possible contaminants. RecoverY [START_REF] Rangavittal | RecoverY: k-mer-based read classification for Ychromosome-specific sequencing and assembly[END_REF], a method to remove contaminants from flow sorted data, showed that our Illumina dataset contained ∼95% pure S. latifolia Y chromosomes (see Methods section 2.2). For our long reads, we performed decontamination with BlobTools [START_REF] Kumar | Blobology: exploring raw genome data for contaminants, symbionts and parasites using taxon-annotated GC-coverage plots[END_REF] after a first round of assembly, to determine possible contaminants based on GC per cent, Illumina read coverage and taxonomic information. Up to 6% of the PacBio subreads and 23% of the CCS, as well as 0.07% of the MinION reads were discarded due to contamination. The main contaminant in the PacBio dataset came from the bacteria Elizabethkingia anophelis, and its absence in our other datasets suggested specific contamination during the PacBio library preparation. Differences between sequencing technologies may thus be due to external contaminations. We therefore need to be careful when processing sequencing data, testing for possible contaminants and properly cleaning the datasets before assembling.

Our best assembly had a total length of 563 Mb with a scaffold N50 of 6,114 bp. Assuming that the size of the 3.1 for the workflow and Methods section 2.6 and 2.7 for details) was obtained after processing and decontaminating the reads, assembling with MasurCA and DBG2OLC, scaffolding with L_RNA_Scaffolder, filling the gaps with PBJelly and decontaminating with Blobtools. Only scaffolds longer than 500 bp were retained). Statistics: total number scaffolds, total size of the dataset, range of read lengths (minimum and maximum), GC content, number and percentage of reads longer than 1kb and 10kb, mean read length and N50 (weighted median statistic such that 50% of the entire assembly is covered by read equal to or larger than this value) and percentage of unknown nucleotides after scaffolding (Ns).

display its full gene content. The final assembly was generated using both short reads (Illumina data) and long reads (PacBio and MinION data), applying a succession of assembly and scaffolding tools that led to assembly improvement at each step (Figure 3.1). Specifically, we evaluated the performance of these approaches and tools in terms of total length assembled, N50 (Figure 3.1 and Table 3.2), and number of genes recovered (Table 3.3). To perform our assembly, we first used the MasurCA genome assembler [START_REF] Zimin | The MaSuRCA genome assembler[END_REF], a hybrid assembler that relies on both de Bruijn graph and Overlap-Layout-Consensus (OLC) approaches.

The PE reads and the high quality PacBio CCS were assembled into unitigs, contigs that are fully consistent with the data and in particular with overlaps. This helped reduce the complexity in our short read and high-quality CCS dataset, so that a first consensus could be reached. We then reassembled the unitigs obtained with MasurCA with the error-corrected long reads and CCS longer than 500bp with DBG2OLC [START_REF] Ye | DBG2OLC: Efficient Assembly of Large Genomes Using Long Erroneous Reads of the Third Generation Sequencing Technologies[END_REF]. This second assembler's main advantage is that it reduces computing time and complexity when dealing with error-prone reads. DBG2OLC provided the main improvement in our pipeline, with a major N50 increase (see Figure 3.1) which validated our long read approach.

We then moved to scaffolding and gap-filling, which are necessary post-assembly cleaning and polishing steps: (1) RNAseq data was used to join, order and orient the genomic fragments. We ran L_RNA_Scaffolder (Xue et al., 2013), which used transcriptomic data to improve the DBG2OLC assembly by merging scaffolds using exons and (2) we applied PBJelly [START_REF] English | Mind the Gap: Upgrading Genomes with Pacific Biosciences RS Long-Read Sequencing Technology[END_REF] to align the corrected long reads to fill the gaps in the assembly. The combination of L_RNA_Scaffolder and PBJelly produced an improvement in scaffold N50 from 6,089 to 6,204 bp (Figure 3.1), and decontamination with Blobtools lead to the final N50 of 6,114 bp. We named the resulting assembly "final assembly" (Figure 3.1 and Table 3.2 for description). Long-reads appear to be very useful for highly repeated contents, but they require thorough processing and polishing. 2016), sex-linked genes are divided into X/Y genes and X-hemizygous genes (with no Y copy). Y-linked BAC sequences from Blavet et al. (2015). Mapping rates were obtained with Gmap (Wu and Watanabe, 2005), see Methods section 2.8).

BAC sequences

Usually the completeness of a de novo genome assembly is assessed in terms of gene contents using comparison tools such as BUSCO (Benchmarking Universal Single-Copy Orthologs ). The BUSCO dataset contains universal genes that are expected to be found in any complete genome assembly. But here, given that we worked on a single chromosome and not on the entire genome, we did not expect to find all the core eukaryotic genes (CEGs) that BUSCO or CEGMA (Core Eukaryotic Genes Mapping Approach) approaches look for. We therefore needed to use a set of genes that we expected to find on the Y chromosome of S. latifolia, to test the quality of our assembly. We mapped Y chromosome specific BAC sequences from Blavet et al. (2015) against the assembly. Out of the 85 Y-specific BAC sequences, 73 (85.88%) mapped onto our assembly. Each mapping BAC had hits with 1 to 94 different scaffolds from our assembly (median number of hits: 4, see Table 3.3). This shows that our assembly is fairly complete with only 12 missing BAC sequences, but that it remains fragmented, given the high number of scaffolds each BAC mapped onto.

We used a list of genes from Muyle et al. (2016) to test for their presence on our Y chromosome assembly. Table 3.3 shows the mapping rates for sex-linked X/Y genes, X hemizygous genes, i.e. genes that show no expression on the Y, and autosomal genes, and unassigned genes (due to a lack of informative SNPs). From the sex-linked genes inferred from SNP segregation in Muyle et al. (2016), we found 810 out of 1,048 X/Y genes (77.29%), which suggests that some genes are still missing from our dataset. We also detected matches with X-hemizygous genes: 185 out of 337 mapped (54.90%), which would suggest that these genes are silenced but not lost from the Y chromosome. Surprisingly, 2,705 out of 14,082 genes (19.21%) that were inferred as autosomal from transcriptomic data were found in our assembly. From the full transcriptome, 9,249 genes (19.55%) mapped on our final assembly (Table 3.3).

To explore this further, we performed de novo annotation of our assembly with EuGene, and predicted a total of 16,219 genes on the Y chromosome of S. latifolia (see Table 3.4), amongst which 6,770 protein coding genes and 9,449 non-coding genes. But these predicted genes seem to be fragmented, as 42% of the coding ones did not show any introns (Table 3.4) and they had a mean of 1.89 exons per proteincoding gene. GO term analysis showed the presence of several interesting functions such as plant hormone signaling especially auxin (GO:0009734), pollinic functions (GO:0009856) and embryonic development (GO:0009790), as shown in Supplementary Figure 3.1. Overall, we predicted a high number of genes on the Y chromosome of S. latifolia. Statistics of the de novo annotations performed with EuGene-PP on the S. latifolia Y chromosome, characterizing protein-coding genes, non-coding genes and intergenic regions.

Discussion

Our strategy for sequencing and assembling the Y chromosome of S. latifolia shows that plant sex chromosome sequencing can be achieved even for large and repetitive genomes. We used flow sorting based on chromosome size as well as pre-and post-processing steps to enrich for Y chromosome DNA. We determined the necessary conditions for a successful assembly: sufficient read processing and decontamination, long error-prone read correction with at least 10-15X long reads remaining after cleaning, and subsequent polishing steps. We verified that flow sorting of S. latifolia Y chromosomes only resulted in a 5% contamination rate in our Illumina reads, after removing low quality reads and PCR duplicates. PacBio contamination was mainly due to E. anophelis and specific to this sequencing. The strategy presented here is more affordable than a strategy based on sequencing of both male and female genomes for the same species [START_REF] Carvalho | Efficient identification of Y chromosome sequences in the human and Drosophila genomes[END_REF][START_REF] Vicoso | Reversal of an ancient sex chromosome to an autosome in Drosophila[END_REF]. Our strategy remains less accurate and produces a more fragmented assembly than SHIMS, but is cheaper by a factor of 10 to 100. By combining short-and long-read technologies, we are able to obtain high coverage while also increasing assembly contiguity, compared with the use of short reads only. Our study provides a workflow for future sequencing projects of sex chromosomes. We show that ∼10-15X sequencing depth of processed MinION and/or PacBio already provides a substantial improvement of Illumina assemblies for Y chromosomal data.

To achieve the highest N50, the most recent MinION and/or PacBio chemistries should be used, and fewer long reads are preferred to many shorter ones. Improvement in long read data analysis tools, which are undergoing active development, could make both MinION and PacBio-only assemblies an attractive option in the future.

To evaluate this assembly, we used BAC sequences from Blavet et al. (2015), and found that 85.88% of them were mapped on our assembly, showing that amplification and sequencing biases did not cause too much missing data. Each BAC sequences mapped on a median of 4 chromosomic scaffolds, showing that our assembly is still fragmented. With a hybrid sequencing approach of flow sorted chromosomes, we were thus able to produce a full, although fragmented assembly of the S. latifolia Y chromosome. We then analyzed Y chromosome content and gene loss using transcriptomic data from Muyle et al. (2016) to test if predicted sex-linked genes were present in our data, and found 77.29% of X/Y genes from this paper (Table 3.3). We detected matches for 54.80% of X-hemizygous genes, which would suggest that these genes are silenced but not lost from the Y chromosome. We also found 19.21% of autosomal genes mapping on our assembly of the Y chromosome. These genes could be part of the pseudo autosomal region (PAR) that is present on both X and Y chromosomes and still recombines. Previous studies in S. latifolia showed that the PAR amounts to one third of the X and Y chromosomes, which make up to one seventh of the genome. It is therefore not too surprising to find autosomal genes, but we would expect around 5% of the autosomal genes to be in the PAR, and not around 20%. This raises the question of the specificity of our mapping, which needs to be tuned so that we can identify the genes from the PAR properly. It will be very interesting to study those genes in the light of sex chromosome evolution, as they give insights in the shared content between both sex chromosomes [START_REF] Otto | About PAR: the distinct evolutionary dynamics of the pseudoautosomal region[END_REF].

If we assume that the S. latifolia genome contains as many genes as the A. thaliana genome, that is 28,000 genes, and given that the X chromosome represents one seventh of the S. latifolia genome, we can expect around 4,000 genes on the Y before recombination arrest (Bergero and Charlesworth, 2011). This is consistent with the first genome assembly of S. latifolia in Papadopulos et al. (2015) who assembled 25% of the X chromosome and identified around 1,000 X-linked genes, but less genes and genes with bigger introns on the Y. However, a total of 9,249 genes from the transcriptome of Muyle et al. (2016) mapped on our final assembly, which is a surprisingly high number when compared to these predictions, but might be due to this transcriptome being fragmented or redundant. To explore this further, we performed de novo annotation of our assembly with Eu-Gene [START_REF] Sallet | EuGene-PP: a next-generation automated annotation pipeline for prokaryotic genomes[END_REF], and predicted a total of 16,219 genes on the Y chromosome of S. latifolia, amongst which 6,770 protein-coding genes and 9,449 non-coding genes, confirming our transcriptomic mapping results. But about half of these coding genes only had one exon, when around 5 exons are expected for most plant coding genes [START_REF] Deutsch | Intron-exon structures of eukaryotic model organisms[END_REF], which suggests that de novo gene prediction might have been disturbed by the fragmented state of our final assembly. The 6,770 proteincoding genes that we found on the Y chromosome of S. latifolia exceed expectations made by comparison with A. thaliana and by the full genome sequencing of Papadopulos et al. (2015). This assembly constitutes an interesting starting point of further studies of the genetic contents of the Y chromosome of S. latifolia.

Conclusion and Perspectives

We achieved the first complete assembly of the Y chromosome of S. latifolia. Our assembly had a total length of 563 Mb with a scaffold N50 of 6,114 bp. But this first draft remains fragmented and needs to be scaffolder further, to better characterize this chromosome. First, we can still improve scaffolding, by using different independent available data sources such as the Y-linked BACs we used to assess our assembly (Blavet et al., 2015) and a physical map of the Y chromosome with a few known markers (Kazama et al., 2016). Annotation can be improved with the help of the reference transcriptome from Muyle et al. (2016), in which a number of Y-linked genes had already been identified. This will help us evaluate our annotation, which we haven't done here.

Secondly, biological analyses can help us explore our assembly. Markers from the physical map of the Y chromosome can help us get a list of candidate genes for the three sexdetermining regions that were identified on the Y chromosome of S. latifolia. X-hemizygous genes can be confirmed from our data since we will be able to tell apart silenced and deleted genes. We will be able to assess the presence on the Y of male specific genes from differential expression studies performed in S. latifolia (Zemp et al., 2016). Finally, we can study the abundance of repeats for which the nature have been identified, but quantities are yet to assessed [START_REF] Macas | Next generation sequencing-based analysis of repetitive DNA in the model dioceous plant silene latifolia[END_REF]. Knowing the location of genes on the Y will allow us to study the PAR and the non-recombining Y regions. Their precise size and localization will give us major inputs to study recombination arrests and degeneration processes of the sexchromosomes in S. latifolia. Given that the physical map contains about 100 markers and our assembly 100,000 scaffolds, it is very unlikely that we succeed in ordering our scaffolds, which will require more sequencing.

Lastly, new data can be acquired to refine the assembly. This can be done using recent technologies such as Hi-C, optical mapping, and long-read sequencing with MinION and PacBio with high coverage (100X). WGA, unavoidable when working with limited material, might introduce artificial junctions of DNA fragments [START_REF] Lasken | Mechanism of chimera formation during the Multiple Displacement Amplification reaction[END_REF]. Although it has been demonstrated that such artifacts are rare [START_REF] Tomaszkiewicz | A time-and cost-effective strategy to sequence mammalian Y chromosomes: An application to the de novo assembly of gorilla Y[END_REF], the WGA step should be omitted if the material is more abundant. A new library of very long fragment sequencing is going to be produced, with the help of full nucleus sorting. Using MinION sequencing on these ultra-long DNA fragments should give us a very interesting genomic library for S. latifolia, from which the Y sequences can be extracted with what has already been sequenced, and further help assembly and scaffolding processes. We also aim at sequencing the X chromosome of S. latifolia to perform a comparative analysis.

B.

A. 103,163,644 148,354,895 205,770,135 418,196,448 55,795,720 36,593, 212,175,794 186,743,213 469,433,508 423,486,789 273,143,246 282,971

Run

General Conclusion and Perspectives

Sex chromosomes and sex determination in Silene latifolia and Coccinia grandis

Describing the haystack on our way to the needle

Reminder of the objectives

The starting point of this thesis was to study sex determination in plants, under the hypothesis that the pathways towards dioecy influence the genes that are recruited for sex determination (see Chapter 1 for details). Two main pathways have been identified for the evolution from hermaphroditism towards dioecy, which is why we focused on Silene latifolia to represent the gynodioecious pathway, and Coccinia grandis for the monoecious pathway.

During my PhD, I have worked with sequence data from both plant systems in order to describe the contents of their sex chromosomes and find the "needle in the haystack", i.e. their master sex-determining genes. Reviews by Muyle et al. (2017a) and [START_REF] Tomaszkiewicz | Y and W Chromosome Assemblies: Approaches and Discoveries[END_REF] describe the difficulties of assembling Y chromosomes, in particular in plants. Due to these difficulties, we lack insights into the diversity of plant sex chromosomes, particularly into the mechanisms of their evolution. In this thesis, I focused on two approaches: (1) describing the genetic contents of the sex chromosomes of C. grandis, for which no sex-linked genes were known before my work, and

(2) producing a genomic assembly of the Y chromosome of S. latifolia, in which sex-linked genes have been intensively studied and segregation inference methods based on RNAseq data have been applied, as is presented in a paper that I co-authored (Appendix B). But their positions on the X and Y chromosomes remain unknown, except for some genes that have been positioned on a genetic map of the X (Papadopulos et al., 2015).

Novel outcomes from my results and perspectives

2.1. Adding one more system for the comparative analysis of sex chromosomes in plants In Chapter 2, I described my findings in C. grandis, a Cucurbitaceae with the most heteromorphic sex chromosomes known so far in plants. Our segregation analysis provided the first set of sex-linked genes identified in C. grandis with a total of 1,364 transcripts. We also found 3,273 sex-biased genes, and showed that sex-biased expression is commoner among the sex-linked contigs than in autosomal contigs. The divergence between X and Y copies allowed us to estimate an age of recombination arrest of 3.75 My. We observed Y degeneration patterns and estimated a total gene loss on the Y chromosome of about 40%. Y alleles have significantly more premature stop codons than X alleles. Expression of sex-linked genes was significantly lower in males than in female. In males, Y alleles were significantly less expressed than X alleles. Thus, the Y chromosome of C. grandis bears the hallmarks of ongoing degeneration.

As seen in Chapter 2, we observed that dosage compensation occurs in C. grandis, making it the second plant system where this phenomenon has been described. Indeed, when Y expression decreased, expression of the X increased in males resulting in equal expression between sexes. C. grandis and S. latifolia show that both high heteromorphy and dosage compensation can arise very quickly, as they are observed in these young sex chromosome systems. We also identified 23 91 X-hemizygous contigs (with no Y expression) for which expression is expected to be unbalanced between males and females. But these genes displayed a compensation for the absence of Y expression, showing an increased X expression in males. Non-compensated X-hemizygous genes in C. grandis and in S. latifolia are depleted in protein complex functions, which are known to be dosage sensitive since the stoichiometry between their components have to be finely tuned. The next step in studying dosage compensation, which has been applied to S. latifolia in Muyle et al. (2017b, see Appendix B), is to compare expression changes to a close outgroup, which can be done in C. grandis with the fully annotated melon genome. This approach gave insight into the mechanisms of dosage compensation in S. latifolia (see Appendix B).

My study of C. grandis is a remainder of how easy it is to conduct a SEX-DETector analysis in a new system. Such tool, and other similar tools, will certainly help performing the large scale comparative analysis that we currently lack and help gaining a global picture on the evolution of plant sex chromosomes.

Towards testing sex-determining gene candidates in C. grandis

According to the model of artificial dioecy in melon proposed by Boualem et al. (2015), we predict that WIP1 is located on the sex chromosomes of C. grandis (see Chapter 1 and Introduction). I thus checked whether WIP1 was among the sex-linked genes. CmWIP1 sequences from GenBank accession numbers GQ870274.1 and GQ870275.1 published by Martin et al. (2009) were used. I aligned these sequences on the transcriptome of C. grandis with Gmap (Wu and Watanabe, 2005). A total of 16 contigs mapped with the candidate gene, out of which only two were classified as sex-linked by SEX-DETector, the rest being autosomal or unclassified (Muyle et al., 2016, see Chapter 2). These two sex-linked genes mapped on the putative transposase gene that is associated with WIP1. Interestingly, one of unclassified contigs had 7 X-Y SNPs with genotyping errors, and mapped specifically on WIP1.

Due to the errors preventing SEX-DETector from inferring a segregation type correctly, it was unclear if this contig was indeed sex-linked (see Table A.1). These errors could be due either to mapping problems, or to unexpressed alleles in the father at the time of sampling. To address this issue, we reconstructed separate male and female transcriptomes from public datasets (Devani et al., 2017;Mohanty et al., 2017) and identified the X and Y alleles, shown in Figure A.1. This analysis is ongoing and the identified SNPs and contigs will be further tested by our collaborators Anjan Kumar Banerjee (IISER, Pune, India) and Abdelhafid Bendahmane (IPS2, Orsay, France).

The WIP1 allele that we found on the Y chromosome needs to be functionally tested, to assess its role in the mechanisms of sex determination in C. grandis. If WIP1 is not confirmed as a master sex-determining gene, our dataset will allow us to look for other possible candidates. For example, we could use our differential expression analysis to shortlist male-specifically expressed genes that could be sex-determining genes, and to cross those genes with genomic data of Y chromosomes, which will be provided by our collaborator Susanne Renner (Ludwig Maximilian University, Munich, Germany) as done in Akagi et al. (2014). 2.3. Long-read sequencing and assembly of the Y chromosome in S. latifolia My work helped produce an assembly of the Y chromosome in S. latifolia, which had a total length of 563 Mb with a scaffold N50 of 6,114 bp. During my PhD, I prepared samples and performed MinION sequencing, a third-generation sequencing platform relying on physical sequencing of single molecules. MinION sequencing is still undergoing major developments and we took part in the testing of this novel sequencer. The use of third generation long reads clearly made the difference in obtaining the first complete assembly of the Y chromosome of S. latifolia. I assessed this assembly using previous BAC sequencing data (Blavet et al., 2015) and other independent datasets (see Chapter 3). Annotation is ongoing in our team, performed by Adil El Filali.

Despite our assembly being fragmented, we can nevertheless use it to perform some analyses. In addition to identifying sex-determining genes (see following section), we can use our dataset to confirm the presence of X-hemizygous genes on the Y, to tell apart silenced and deleted genes. We can also identify scaffolds of the PAR, by looking at scaffolds containing only genes with an autosomal segregation type. Finally, the repetitive contents in terms of quantities and distribution over the Y chromosome can be assessed.

To get a higher quality assembly, several possibilities exist (see Chapter 3 for details). This will allow us to identify the sex-determining genes if we have failed to find them with the first draft, and to further explore the sex chromosomes of S. latifolia, defining the evolutionary strata, the pseudo-autosomal region (PAR) and for example by identifying inversions or other chromosomal rearrangements between the X and Y chromosomes.

Looking for sex-determinating genes in S. latifolia

We identified candidate genes for sex determination in species of the gynodioecious pathway, as presented in Fruchard and Marais (2017). In particular, we expect a restorer-of-fertility to be on the Y chromosome (see Chapter 1 for details). A majority of Restorers of fertility (Rf ) genes have been shown to encode pentatricopeptide-repeat proteins (PPR). PPRs are a large protein family in plants that are targeted to mitochondria or chloroplasts and influences the expression of their genome. PPR-Rfs act by specifically suppressing the expression of sterility-causing mitochondrial transcripts [START_REF] Dahan | The Rf and Rf-like PPR in higher plants, a fast-evolving subclass of PPR genes[END_REF], for example in strawberries (Ashman et al., 2015) and rice [START_REF] Huang | Pentatricopeptide-repeat family protein RF6 functions with hexokinase 6 to rescue rice cytoplasmic male sterility[END_REF]. We intend to use protein family annotations to test for the presence of PPR-Rf genes in our Y chromosome assembly. Pfam annotations have been produced with hmmscan (hmmer version 3.1b2, [START_REF] Eddy | Profile hidden Markov models[END_REF] by Adil El Filali and analysis is ongoing.

As mentioned in Chapter 1 and in the Introduction, three sex-determining regions have been located onto a physical map of the Y chromosome of S. latifolia. Radim Cegan and Roman Hobza, our collaborators in Czech Republic, are currently checking whether they can find the markers of the physical map on the Y scaffolds that we assembled. We are particularly interested in the scaffolds that include markers close to the three sex-determining regions. The genes that will be found in those scaffolds are potential candidate master sex-determining genes in S. latifolia. These candidates can be compared with our list of PPR-Rfs genes to narrow down a list of genes for functional validation. If we fail to validate our candidates, it will be possible to study Y-specific genes and their role in male-determination using the Y assembly and RNAseq data.

Conclusion

In this thesis, I described our search for sex-determining genes in plants and provided new insights into two very interesting systems of young plant sex chromosomes. The resources that I have produced are useful to identify the sex-determining genes in those systems, which was my primary goal, although more work is clearly needed. They are also useful for other purposes and for addressing other interesting questions about sex chromosome evolution in plants, as I have tried to show for C. grandis.

Part 4 Appendix

In this chapter, I present the paper "Genomic imprinting mediates dosage compensation in a young plant XY system" Muyle et al. (deposited on BioRxiv1 , submitted to PCI Evol Biol and to Nature Plants.

I worked with Clothilde Deschamps (Engineer for the Bioinformatics Pole at LBBE) on cleaning the data and obtaining a first assembly using SOAPdenovo2, as well as the first steps of scaffolding this assembly. I finished the scaffolding on my own and provided Aline Muyle with a dataset of Y chromosome scaffolds that were used for this paper.

One Sentence Summary: In the plant Silene latifolia, reduced expression from the Y chromosome is compensated by increased expression from the maternal X in both sexes.

Sex chromosomes have repeatedly evolved from a pair of autosomes. Consequently, X and Y chromosomes initially have similar gene content, but ongoing Y degeneration leads to reduced Y gene expression and eventual Y gene loss (Bachtrog, 2013). The resulting imbalance in gene expression between Y genes and the rest of the genome is expected to reduce male fitness, especially when protein networks have components from both autosomes and sex chromosomes. A diverse set of dosage compensating mechanisms that alleviates these negative effects has been described in animals [START_REF] Charlesworth | The evolution of chromosomal sex determination and dosage compensation[END_REF][START_REF] Gu | Evolution of Sex Chromosome Dosage Compensation in Animals: A Beautiful Theory, Undermined by Facts and Bedeviled by Details[END_REF]Mank, 2013). However, the early steps in the evolution of dosage compensation remain unknown and dosage compensation is poorly understood in plants (Muyle et al., 2017a). Here we show a novel dosage compensation mechanism in the evolutionarily young XY sex determination system of the plant Silene latifolia. Genomic imprinting results in higher expression from the maternal X chromosome in both males and females. This compensates for reduced Y expression in males but results in X overexpression in females and may be detrimental. It could represent a transient early stage in the evolution of dosage compensation. Our finding has striking resemblance to the first stage proposed by Ohno for the evolution of X inactivation in mammals.

In Drosophila, the X chromosome is upregulated specifically in males, resulting in complete dosage compensation through both ancestral expression recovery in males and equal expression between the sexes (hereafter sex equality) [START_REF] Kuroda | Dosage Compensation in Drosophila-a Model for the Coordinate Regulation of Transcription[END_REF]. In Caenorhabditis elegans, both X chromosomes are downregulated in XX hermaphrodites resulting in sex equality, but only a few genes have their X expression doubled for ancestral expression recovery [START_REF] Albritton | Sex-biased gene expression and evolution of the x chromosome in nematodes[END_REF]. In placental mammals, including humans, one X chromosome is randomly inactivated in XX females, resulting in sex equality but without recovering the ancestral expression of sex chromosomes, except for a few dosage-sensitive genes whose X expression was doubled in both sexes [START_REF] Disteche | Dosage compensation of the sex chromosomes[END_REF]Ercan, 2015;[START_REF] Julien | Mechanisms and evolutionary patterns of mammalian and avian dosage compensation[END_REF]Pessia et al., 2012Pessia et al., , 2014)). In the marsupials, the paternal X chromosome is consistently inactivated in XX females [START_REF] Whitworth | The X factor: X chromosome dosage compensation in the evolutionarily divergent monotremes and marsupials[END_REF]. Differential expression that depends on the parent of origin is known as genomic imprinting [START_REF] Patten | The evolution of genomic imprinting: theories, predictions and empirical tests[END_REF], and this mechanism also operates in the mouse placenta [START_REF] Wagschal | Genomic imprinting in the placenta[END_REF].

Despite the plethora of studies on gene expression on sex chromosomes, it is not yet clear if genomic imprinting is commonly involved in the early steps of dosage compensation evolution. In a seminal work, Ohno hypothesized a two-step process for the evolution of dosage compensation [START_REF] Ohno | Sex Chromosomes and Sex-Linked Genes[END_REF]. In the first step, expression from the X is doubled, thereby mediating the recovery of ancestral expression in XY males. Second, the resulting overexpression in XX females selects for X inactivation. This scenario is consistent with the fact that sexual selection is often stronger on males than on females. Under this scenario, selection on XY males to upregulate their single X chromosome should be stronger than selection on females, leading to overexpression in females until a second correcting mechanism evolves (Mank, 2013). However, in order to understand these early steps of dosage compensation evolution, species with young sex chromosomes must be studied.

The plant Silene latifolia is an ideal model to study early steps of sex chromosome evolution thanks to its pair of X/Y chromosomes that evolved ∼4 Mya (Rautenberg et al., 2010). Dosage compensation is poorly understood in plants (Muyle et al., 2017a). Thus far only sex equality has been studied. Equal expression levels were observed for males and females for some genes despite Y expression degeneration (Bergero and Charlesworth, 2011;Bergero et al., 2015;Chibalina and Filatov, 2011;Kazama et al., 2012;Muyle et al., 2012;Papadopulos et al., 2015). However, the mechanisms through which sex equality is achieved -and whether ancestral expression is recovered in S. latifolia males -remain unknown. To address these questions, we have developed an approach relying on (i) the use of an outgroup without sex chromosomes as an ancestral autosomal reference (Muyle et al., 2017a) in order to determine whether X chromosome expression increased or decreased in S. latifolia, (ii) the application of methods to study allele-specific expression while correcting for reference mapping bias (Muyle et al., 2017a), and (iii) a statistical framework to quantify dosage compensation (Muyle et al., 2017a). Because only ∼25% of the large and highly repetitive S. latifolia genome has been assembled so far (Papadopulos et al., 2015), we used an RNA-seq approach based on the sequencing of a cross (parents and a few offspring of each sex), to infer sex-linked contigs (i.e. contigs located on the non-recombining region of the sex chromosome pair) (Muyle et al., 2016). X/Y contigs show both X and Y expression, while X-hemizygous contigs are X-linked contigs without Y allele expression. We made inferences separately for three tissues: flower buds, seedlings and leaves (Supplementary Table B.S2). Results are consistent across tissues and flower buds and leaves are shown in Supplementary Materials. In seedlings, ∼1100 sex-linked contigs were inferred. Among these, 15% of contigs with significant expression differences between males and females were removed for further analyses (Supplementary Table B.S2 and Materials and Methods). These are likely involved in sex-specific functions and are not expected to be dosage compensated (Ellegren and Parsch, 2007). This was done as a usual procedure for studying dosage compensation, however the resulting trends and significance levels are not affected. About half of the non sex-biased sex-linked contigs could be validated by independent data using three sources: literature, a genetic map and sequence data from Y flow-sorted chromosomes (see Supplementary Table B.S2 and Materials and Methods). X-hemizygous contigs are more difficult to identify than X/Y contigs using an RNA-seq approach (see Supplementary Text 1). This explains conflicting earlier results on dosage compensation in S. latifolia (Muyle et al., 2017a). A study using genomic data (i.e. not affected by the aforementioned ascertainment bias) found sex-equality in approximately half of the studied X-hemizygous genes (Papadopulos et al., 2015). In our set of X-hemizygous contigs, no evidence for dosage compensation was found (Supplementary Text 1 ), in agreement with previous work relying on an RNA-seq approach (Bergero et al., 2015;Chibalina and Filatov, 2011). This could be due to an over-representation of dosage insensitive genes in our set of X-hemizygous contigs (Supplementary Text 1). If Δ is lower, higher or equal to zero, then expression in S. latifolia is respectively lower, higher or equal to the outgroup (See Materials and Methods for details). For all contig categories, Δ was compared to zero using a Wilcoxon test. The median Δ, confidence intervals and p-values adjusted for multiple testing using a Benjamini and Hochberg correction are shown (***: p-value < 0.001; **: p-value < 0.01, *: p-value < 0.05). Allelic expression at SNP positions was averaged for each contig separately and the Y/X ratio was used as a proxy for Y degeneration to group contigs. Contigs with sex-biased expression were removed, as well as contigs with Y/X expression ratios above 1.5. Sample sizes for the different contig categories are: autosomal: 200; 1-1.5:148; 0.75-1:139; 0.5-0.75:160; 0.25-0.5:114; 0-0.25:79 (we randomly selected 200 autosomal contigs to ensure similar statistical power among gene categories).

We estimated paternal and maternal allele expression levels in males and females for sexlinked and autosomal contigs in S. latifolia after correcting for reference mapping bias (Materials and Methods). We then compared these allelic expression levels to one or two closely related outgroups without sex chromosomes in order to polarise expression changes in S. latifolia. For autosomal contigs, expression levels did not differ between S. latifolia and the outgroups (Figure B.1). This is due to the close relatedness of the outgroups (∼5My, Supplementary Figure B.S1), and validates their use as a reference for ancestral expression levels. We used the ratio of Y over X expression levels in S. latifolia males as a proxy for Y degeneration and then grouped contigs on this basis. As expression of the Y allele decreased (paternal allele in blue in S2-B.S7) establishes a role for genomic imprinting in dosage compensation. In order to statistically test this inference at the SNP level, we used a linear regression model with mixed effects (Materials and Methods). Outgroup species were used as a reference and expression levels in S. latifolia were then analyzed while accounting for the variability due to contigs and individuals. The joint effect of the parental origin and the degeneration level was estimated, which allowed computing expression differences between maternal and paternal alleles in females for different Y/X degeneration categories (Figure B.2). Maternal and paternal alleles of autosomal SNPs were similarly expressed in females, indicating a global absence of genomic imprinting for these SNPs. However, for X/Y SNPs, the difference between the maternal and paternal X in females increased with Y degeneration. These results were confirmed in two other tissues and when analyzing independently validated contigs only (although statistical power is sometimes lacking due to the limited number of validated contigs, Supplementary Figures B.S8-B.S13).

Previous studies that showed sex equality in S. latifolia could have been explained by simple buffering mechanisms, where one copy of a gene is expressed at a higher level when haploid than when diploid, due to higher availability of the cell machinery or adjustments in gene expression networks [START_REF] Malone | Mediation of Drosophila autosomal dosage effects and compensation by network interactions[END_REF]Papadopulos et al., 2015;Veitia et al., 2015). However, the upregulation of the X chromosome we reveal here in S. latifolia males cannot be explained by buffering mechanisms alone, as the maternal X in females would otherwise not be upregulated. Instead, our findings indicate that a specific dosage compensation mechanism relying on genomic imprinting has evolved in S. latifolia. This apparent convergent evolution with marsupials is mediated by different mechanisms (in marsupials the paternal X is inactivated [START_REF] Whitworth | The X factor: X chromosome dosage compensation in the evolutionarily divergent monotremes and marsupials[END_REF], while in S. latifolia the maternal X is upregulated). An exciting challenge ahead will be to understand how upregulation of the maternal X is achieved in S. latifolia males and females at the molecular level. Chromosome staining suggests that DNA methylation is involved. Indeed, one arm of one of the two X chromosomes in females was shypomethylated, as well as the same arm of the single X in males (Siroky et al., 1998) (Figure B.3 and Supplementary Figure B.S14). Based on our results, we hypothesize that the hypomethylated X chromosome corresponds to the maternal, upregulated X. Unfortunately, parental origin of the X chromosomes was not established in this study (Siroky et al., 1998). It would be of interest in the future to study DNA methylation patterns in S. latifolia paternal and maternal X chromosomes, along with the homologous pair of autosomes in a closely related species without sex chromosomes. The methylation pattern observed by chromosome staining suggests that dosage compensation in S. latifolia could be a chromosome arm-wide phenomenon. To test this hypothesis with expression data, positions of genes along the X chromosome remain to be elucidated.

Our study is the first to establish female upregulation of the X chromosome compared to autosomes, as predicted by Ohno. An earlier report in Tribolium castaneum was later shown to be due to biases from inclusion of gonads in whole body extracts [START_REF] Gu | Evolution of Sex Chromosome Dosage Compensation in Animals: A Beautiful Theory, Undermined by Facts and Bedeviled by Details[END_REF]. X overexpression in females may be deleterious. Its occurence suggests that reduced expression of sex-linked genes in males is more deleterious than overexpression in females. This potentially suboptimal situation may be transitory and a consequence of the young age of S. latifolia sex chromosomes. Sex equality may evolve at a later stage, following the evolutionary path trajectory originally proposed by Ohno for placental mammals [START_REF] Ohno | Sex Chromosomes and Sex-Linked Genes[END_REF].

Online Methods

Sequence data and inference of sexlinkage. RNA-seq data was generated in S. latifolia for a cross (parents and progeny) for three tissues (seedlings, leaves and flower buds) and analysed using the SEX-DETector pipeline (Muyle et al., 2016). RNA-seq data was also generated for two outgroup species (S. viscosa and S. vulgaris). Reference mapping bias was corrected using the program GSNAP (Wu and Nacu, 2010). Inferences of sex-linked contigs were validated using three sources of information (literature, a genetic map and flow-sorted Y chromosome sequences). See Supplementary Text 2 for details.

Allelic expression levels.

Contigwise autosomal, X, Y, X+X and X+Y normalised allelic expression levels were computed by summing read numbers for each X-linked or Y-linked alleles for filtered SNPs of the contigs (Supplementary Text 2) for each individual separately and then normalised using the library size and the number of studied sex-linked SNPs in the contig:

(1) E = r n × l With E = normalised expression level for a given individual, r = sum of total read counts, n = number of studied SNPs, l = library size of the individual (number of mapped reads). Allelic expression levels were then averaged among individuals for each contig. In order to make S. latifolia expression levels comparable to S. viscosa and S. vulgaris, S. vicosa and S. vulgaris expression levels were estimated using only the filtered SNP positions used in S. latifolia. Normalised expression levels computed as explained in equation ( 1) in the two outgroups were then averaged together for leaves and flower buds as expression levels are highly correlated (R 2 0.7 and 0.5 for flower buds and leaves respectively and p-value < 2 • 10 -6 in both cases). Averaging expression levels between the two outgroups allows to get closer to the ancestral autosomal expression level.

Sex-biased expression. Sex-biased contigs were inferred as in Zemp et al. (2016). See Supplementary Text 2 for more detail.

Expression divergence between S. latifolia and the two outgroups at the contig level. The normalised difference in allelic expression between S. latifolia and the two outgroups (hereafter Δ) was computed in order to study how sex chromosome expression levels evolved in S. latifolia compared to autosomal expression levels in the two outroups: Δ is equal to zero if S. latifolia and the outgroups have equal expression levels, Δ is positive if S. latifolia has higher expression levels compared to the outgroups and Δ is negative otherwise:

(2) Δ = (S. latifolia exp. lvl -outgroup exp. lvl) (outgroup exp. lvl)

Sex-linked contigs were grouped by categories of degeneration level using the average Y over X expression ratio in males. 200 autosomal contigs were randomly selected in order to have similar statistical power among gene categories. Δ values for each allele (maternal and paternal in males and females) and each gene category were compared to zero using a Wilcoxon test. P-values were corrected for multiple testing using a Benjamini and Hochberg correction. The seedlings was done using Y specific markers [START_REF] Hobza | Efficient molecular sexing in dioecious Silene latifolia and S. dioica and paternity analysis in F(1) hybrids[END_REF]) that were amplified with the direct PCAR KAPA3G Plant PCR Kit (however male number 3 was later shown to be a female). High quality RNA (RIN > 8.5) was extracted using the total RNA mini kit from Geneaid. Twelve RNA-seq libraries were produced using the Truseq kit v2 from Illimina. Libraries were tagged individually and sequenced in two Illumina Hiseq 2000 channels at the D-BSSE (ETH Zürich, Switzerland) using 100 bp paired-end read protocol.

S. viscosa seeds were received from botanical gardens or collected in the wild by Bohuslav Janousek and grown under controlled conditions in a greenhouse in Eschikon (Switzerland) and Lyon (France). Similarly to (Zemp et al., 2016), flower buds after removing the calyx and leaves were collected. Total RNA were extracted through the Spectrum Plant Total RNA kit (Sigma, Inc., USA) following the manufacturer's protocol and treated with a DNAse. Libraries were prepared with the TruSeq RNA sample Preparation v2 kit (Illumina Inc., USA). Each 2 nM cDNA library was sequenced using a paired-end protocol on a HiSeq2000 sequencer. Demultiplexing was performed using CASAVA 1.8.1 (Illumina) to produce paired sequence files containing reads for each sample in Illumina FASTQ format. RNA extraction and sequencing were done by the sequencing platform in the AGAP laboratory, Montpellier, France (http://umr-agap.cirad.fr/).

A female individual from an interspecific S. latifolia cross (C1_37) was back crossed with a male from an 11 generation inbred line (U10_49). The offspring (hereafter called BC1 individuals) were grown under controlled conditions in a greenhouse in Eschikon (Switzerland). High quality RNA from flower buds as described in [START_REF] Zemp | Identification of internal reference genes for gene expression normalization between the two sexes in dioecious white Campion[END_REF] was extracted from 48 BC1 individuals (35 females and 13 males). 48 RNA-seq libraries were produced using the Truseq kit v2 from Illimina with a median insert size of about 200 bp. Individuals were tagged separately and sequenced in four Illumna Hiseq 2000 channels at the D-BSSE (ETH Zürich, Switzerland) using 100bp paired-end read protocol. The parents used for this back cross had previously been sequenced in a similar way (Muyle et al., 2012;Zemp et al., 2016).

2.1.2. DNA-seq data from filtered Y chromosome Y chromosome DNA was isolated using flow cytometry. The samples for flow cytometric experiments were prepared from root tips according to [START_REF] Vrana | Flow cytometric chromosome sorting in plants: the next generation[END_REF] with modifications. Seeds of S. latifolia were germinated in a petri dish immersed in water at 25 • C for 2 days until optimal length of roots was achieved (1 cm). The root cells were synchronized by treatment with 2mM hydroxyurea at 25 • C for 18h. Accumulation of metaphases was achieved using 2.5μM oryzalin. Approximately 200 root tips were necessary to prepare 1ml of sample. The chromosomes were released from the root tips by mechanical homogenization using a Polytron PT1200 homogenizer (Kinematica AG, Littau, Switzerland) at 18,000rpm for 13 s. The crude suspension was filtered and stained with DAPI (2μg/ml). All flow cytometric experiments were performed on FAC-SAriaII SORP flow cytometer (BD Biosciences, San José, Calif., USA). Isolated Y chromosomes were sequenced with 2×100bp PE Illumina HiSeq.

RNA-seq PacBio data

Plants from an 11 generation inbred line were grown under controlled conditions in a greenhouse in Eschikon (Switzerland). One male (U11_02) was randomly selected. High quality RNA (RIN > 7.5) were extracted using the total RNA mini kit of Geneaid from very small flower buds, small and large flower buds, flowers before anthesis without calyces, rosette leaves, seedlings (4 leaves stage) and pollen. RNA of the different tissues was equally pooled and cDNA was produced using the Clontech SMARTer Kit. The cDNA pool was then normalized using a duplex specific endonuclease of the Evrogen TRIM-MER kit. Two ranges were selected (1-1.3 kb and 1.2 -2 kb) using the Pippin Prep (Sage Science). Two SMRTbell libraries were prepared using the C2 Pacific Biosciences (PacBio) chemistry and sequenced with two SMRT Cells runs on a PacBio RS II at the Functional Genomic Center Zurich (FGCZ).

RNA-seq 454 data

Previously generated 454 data was used (Muyle et al., 2016;[START_REF] Zemp | Identification of internal reference genes for gene expression normalization between the two sexes in dioecious white Campion[END_REF].

Reference trancriptome assembly

The same reference transcriptome as in Muyle et al. (2012) and Zemp et al. (2016) was used.

Inference of sex-linked contigs

Autosomal and sex-linked contigs were inferred as in Muyle et al. (2012) and Zemp et al. (2016). Illumina reads from the individuals of the cross were mapped onto the assembly using BWA (Li and Durbin, 2009a) version 0.6.2 with the following parameters: bwa aln -n 5 and bwa sampe. The libraries were then merged using SAMTOOLS version 0.1.18 [START_REF] Li | The Sequence Alignment/Map format and SAMtools[END_REF]. The obtained alignments were locallyrealigned using GATK IndelRealigner (DePristo et al., 2011) and were analysed using reads2snps (Tsagkogeorga et al., 2012) version 3.0 with the following parameters:

-fis 0 -model M2 -output$\_$genotype best -multi$\_$alleles acc-min$\_$coverage 3 -par false. This allowed to genotype individuals at each loci while allowing for biases in allele expression, and without cleaning for paralogous SNPs. Indeed, X/Y SNPs tend to be filtered out by paraclean, a program which removes paralogous positions (Gayral et al., 2013). A second run of genotyping was done with paraclean in order to later remove paralogous SNPs from autosomal contigs only. SEX-DETector (Muyle et al., 2016) was then used to infer contig segregation types after estimation of parameters using an SEM algorithm. Contig posterior segregation type probabilities were filtered to be higher than 0.8. Because the parents were not sequenced for the leaf and seedling datasets, SEX-DETector was run using the flower bud data for the parents.

Reference mapping bias correction

In order to avoid biases towards the reference allele in expression level estimates, a second mapping was done using the program GSNAP (Wu and Nacu, 2010) with SNP tolerant mapping option. A GSNAP SNP file was generated by home-made perl scripts using the SEX-DETector SNP detail output file. Shortly, for each polymorphic position of all contigs, the most probable posterior SNP type was used to extract the possible alleles and write them to the GSNAP SNP file. This way, reference mapping bias was corrected for both sex-linked and autosomal contigs. Only uniquely mapped and concordant paired reads were kept after this. See Supplementary Table B.S1 for percentage of mapped reads. SEX-DETector was run a second time on this new mapping and the new inferences were used afterwards for all analyses (see Supplementary Table B.S2 for inference results).

Validation of sex-linked contigs 2.5.1. Detection of false X-hemizygous contigs

Erroneous inference of X-hemizygous contig can be due to a true X/Y gene which X and Y copies were assembled into different contigs. In order to detect such cases, X-hemizygous contigs were blasted [START_REF] Altschul | Basic local alignment search tool[END_REF] with parameter -e 1E-5 against RNA-seq contigs that have male-limited expression (see Section 2.7 below for how male-limited contigs were inferred). These cases were removed from the analyses presented here.

2.5.2. Validation using data from literature A few sex-linked and autosomal genes in S. latifolia have already been described in the literature (see Supplementary Table B.S3).

Validation using a genetic map

A genetic map was built and contigs from the X linkage group were used to validate SEX-DETector inferences. RNA-seq reads from the flower bud S. latifolia full-sib cross (hereafter CP) and backcross (hereafter BC1) were mapped against the reference transcriptome using BWA (Li and Durbin, 2009a) with a maximum number of mismatch equal to 5. Libraries were merged and realigned using GATK [START_REF] Depristo | A framework for variation discovery and genotyping using nextgeneration DNA sequencing data[END_REF] and SNPs were analysed using reads2snps (Tsagkogeorga et al., 2012). Using a customized perl script, SNP genotypes from the parents and the offspring as well as the associated posterior probabilities were extracted from the reads2snps output file. Only SNPs with a reads2snps posterior genotyping probability higher than 0.8 were kept for further analyses. Then, only informative SNPs were kept: both parents had to be homozygous and different between father LEUK144-3 and mother U10_37 in a first generation backcross population design (BC1) and at least one allele had to be different between mother C1_37 and father U10_49 in the cross-pollinator (CP). Filtered SNPs were then converted into a JoinMap format using a customized R script. If more than one informative SNP per contig was present, the SNP was used with less segregation distortion and less missing values. This led to 8,023 BC1 and 16,243 CP markers.Loci with more than 10 % missing values were excluded, resulting in 7,951 BC1 and 15,118 CP markers. Linkage groups were identified using the default setting of JoinMap 4.1 [START_REF] Van Ooijen | Multipoint maximum likelihood mapping in a full-sib family of an outbreeding species[END_REF]. Robustness of the assignment of the linkage groups was tested using LepMap [START_REF] Rastas | Lep-MAP: fast and accurate linkage map construction for large SNP datasets[END_REF]. Blasting the contigs against known sex-linked genes allowed the identification of the X chromosome linkage group. Contigs could not be ordered along the linkage groups due to the too limited number of individuals that prevented the convergence of contig order. However, contigs were reliably attributed to linkage groups.

Validation using isolated Y chromosome DNA-seq data

Filtered Y chromosome DNA-seq reads were filtered for quality and Illumina adapters were removed using the ea-utils FASTQ processing utilities (Aronesty, 2011). The optimal kmer value for assembly was searched using KmerGenie (Chikhi and Medvedev, 2014). Filtered reads were assembled using soapden-ovo2 [START_REF] Luo | SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler[END_REF] with kmer=49, as suggested by KmerGenie. The obtained assembly was highly fragmented, therefore RNA-seq data was used to join, order and orient the genomic fragments with L_RNA_scaffolder (Xue et al., 2013). The following RNA-seq reads were used (see section 2.1): one sample of male flower buds sequenced by 454, 6 samples of male flower buds sequenced by Illumina paired-end, 4 samples of male leaves sequenced by Illumina paired-end and one sample of male pooled tissues sequenced by PacBio. The genomic assembly was successively scaffolded with L_RNA_scaffolder using RNA-seq samples one after the other, first 454 samples then Illumina and finally PacBio. The obtained contigs were filtered to be longer than 200pb.

Set of validated sex-linked and autosomal contigs

The three sources of data (litterature, genetic map and filtered Y sequence data) were compared to SEX-DETector inferred sex-linked RNA-seq contigs using BLAST [START_REF] Altschul | Basic local alignment search tool[END_REF] with parameter -e 1E-5. Blasts were filtered for having a percentage of identity over 90%, an alignment lengthover 100bp and were manually checked. If a sex-linked RNA-seq contig blasted against a sequence from one of the three data sources (literature, X genetic map or filtered Y DNA-seq) it was then considered as validated. See Supplementary Table B.S2 for numbers of validated sex-linked contigs.

Expression level estimates

2.6.1. whole contig expression levels Whole contig mean expression levels were obtained for each individual using GATK DepthOfCoverage [START_REF] Depristo | A framework for variation discovery and genotyping using nextgeneration DNA sequencing data[END_REF] as the sum of every position coverage, divided by the length of the contig. Normalised expression levels, in RPKM [START_REF] Oshlack | From RNA-seq reads to differential expression results[END_REF], were then computed for each individual by dividing by the value by the library size of the individual (total number of mapped reads), accounting for different depths of coverage among individuals. Whole contig mean male and female expression levels were then computed by averaging male and female individuals for each contig.

Allelic expression levels filtering

In order to study separately X and Y allele expression levels in males and females, expression levels were studied at the SNP level. In S. latifolia, for each sex-linked contig expression levels were estimated using read counts from both X/Y and X-hemizygous informative SNPs. SNPs were attributed to an X/Y or X hemizygous segregation type if the according posterior probability was higher than 0.5. SNPs are considered informative if the father is heterozygous and has a genotype that is different from the mother (otherwise it is not possible to tell apart the X from the Y allele and therefore it is not possible to compute X and Y expression separately). X/Y SNPs for which at least one female had over two percent of herreads belonging to the Y allele were removed as unlikely to be true X/Y SNPs. Informative autosomal SNPs from autosomal contigs were used in a similar way.

For contigs that only have X/X SNPs (SNPs for which the father's X is different to both Xs from the mother), Y expression level is only computed from the father as all males are homozygous in the progeny. Such contigs were therefore removed when having under 3 X/X SNPs to avoid approximations on the contig mean Y/X expression level (39 contigs removed in the flower buds dataset, 44 in the leaves dataset and 40 in the seedlings dataset).

In order to make S. latifolia expression levels comparable to S. viscosa and S. vulgaris for sex-linked contigs, S. vicosa and S. vulgaris expression levels were estimated using only the positions used in S. latifolia (informative X/Y or X-hemizygous SNPs). The read count of every position in every contig and for every S. viscosa and S. vulgaris individual was given by GATK DepthOfCoverage [START_REF] Depristo | A framework for variation discovery and genotyping using nextgeneration DNA sequencing data[END_REF]. Only positions corresponding to informative autosomal, X/Y or X-hemizygous SNPs in S. latifolia were used to compute the expression level for each contig and each individual as explained in equation (1).

Contigwise S. latifolia autosomal, X, Y, X+X, X+Y allelic expression levels were then averaged among individuals. Autosomal normalised expression levels in the two outgroups (S. vulgaris and S. viscosa) were averaged together.

Identification of contigs with sexbiased expression

The analysis was done separately for the three tissues (flower buds, seedling and rosette leaves) as in Zemp et al. (2016) using the R package edgeR (Robinson et al., 2010). See Supplementary Table B.S2 for number of sex-biased contigs removed in order to study dosage compensation. Male-limited expressed contigs were identified by calculating the mean expression values (FPKM) in both sexes and selecting those which were exclusively expressed in males.

•

  Fruchard, N. Burlet, R. Cegan, M.A. Chebbi, R. Cordaux, R. Hobza, G. Marais, "Assembling a large plant Y chromosome using long read sequencing", DynaGev (Dynamics of plant genome), June 2017, Montpellier • C. Fruchard, "Assembling a large plant Y chromosome", JOBIM (Open Days in Biology, Computer Science and Mathematics), June 2016, Lyon • C. Fruchard et N. Burlet, "Sequencing a large plant chromosome using the MinION", London Calling: Oxford Nanopore conference, May 2015, London Posters • C. Fruchard, N. Burlet, R. Cegan, M.A. Chebbi, R. Cordaux, R. Hobza, G. Marais, "Assembling a large plant Y chromosome using long read sequencing", Plant Genome Evolution, October 2017, Barcelona • C. Fruchard, "Sequencing a large plant Y chromosome using the MinION", EMBO Practical course, May 2016, Heraklion (Crete) Oral communication in internal seminars • NGS Group, LBBE, October 2016, Lyon • Internal seminar, ISEM (Montpellier Institute of Evolutionary Sciences), November 2015, Montpellier Attended Conferences • EvoLyon, November 2015, Lyon • SMBE (Society for Molecular Biology and Evolution), July 2015, Vienna • Plant Genomics, May 2015, London Teaching 2017-2018 Teaching assistant (ATER) at the University Lyon 1 and ENS Lyon with a contract of 96 hours per year 2014-2017 Teaching assistant (PhD) at the University Lyon 1 and ENS Lyon with a contract of 64 hours per year Bachelor Biostatistics and Bioinformatics, tutorial classes • Computer Sciences applied to Biology, tutorial classes, ENS Lyon • Mathematics applied to Biology, tutorial classes • Population Genetics, practical and tutorial classes Master • Adaptation, article analysis, ENS Lyon • Methods for the Assembly of Genomic Data, practical classes • Evolutionary Genetics and Genomics, PAML practical classes • Genomics and molecular genetics, NGS and Galaxy tutorial classes, ENS Lyon • Molecular Evolution, analysis of a transcriptomics dataset with Galaxy, ENS Lyon • Phylogenetics, lecture, ENS Lyon des gymnospermes * sont monoïques * (espèces avec des fleurs unisexuées mâles et femelles sur le même individu) ou dioïques * (espèces avec des individus mâles et des individus femelles)

Figure 0 . 1 .

 01 Figure 0.1. Voies d'évolution vers la dioécie chez les angiospermes.

-

  Scaffolding : concaténation de contigs en laissant des trous correspondant à des bases inconnues.-Gap-filling : remplissage des bases inconnues dans les scaffolds.-kb, Mb : unités de mesure en génétique. Nombres de paires de bases d'un segment génomique : kilobases (milliers de bases), megabases (millions de bases). -N50 : longueur de segment telle que 50% des bases de l'assemblage sont comprises dans des segments de longueur supérieure à cette valeur (N50 = 50% du nombre de bases). -Ma : million d'années. • Structures cellulaires -Cytoplasme : ensemble des éléments qui se trouvent à l'intérieur de la cellule, à l'exclusion du noyau. -Mitochondrie (adj. mitochondrial) : élément cellulaire responsable des processus énergétiques de la respiration. Elles sont transmises de la mère à ses descendants. -Noyau (adj. nucléaire) : structure cellulaire contenant le génome nucléaire, constitué d'ADN. Encart 1. Évolution des chromosomes sexuels. Chez les mammifères, l'apparition d'une région déterminante mâle (sex-determining region Y, SRY ) a probablement initié l'évolution des chromosomes sexuels à partir d'une paire d'autosomes. Il peut se produire une accumulation d'allèles favorables à un sexe et désavantageux pour l'autre autour de cette région. Comme le Y n'est présent que chez les mâles, il est avantageux qu'il concentre les gènes bénéfiques aux mâles et délétères aux femelles. La sélection agit alors en faveur d'un arrêt de la recombinaison. Cela permet de conserver un lien génétique entre le gène déterminant le sexe et les mutations avantageuses chez les mâles (Charlesworth, 2013; Muyle et al., 2017a). De cette manière, les gènes mâles-bénéfiques sont liés aux gènes spécifiant la masculinité. Le Y contient alors à la fois les gènes permettant de devenir un mâle et les gènes mâle-bénéfiques. Le X et le Y cessent de recombiner au niveau d'une région incluant le gène SRY et probablement d'autres gènes, formant ainsi des régions spécifiques du X et du Y. Ces régions s'agrandissent au cours de l'évolution suite à des événements de suppression de recombinaison supplémentaires et divergent progressivement. Les régions pseudo-autosomiques (PARs) continuent de recombiner, mais dans sa région non-recombinante spécifique, le chromosome Y des mammifères a perdu la plupart de ses gènes.

  .1 SNPs for transcript TRINITY_DN20534_c4_g2_i1 B.S1 Library sizes (number of reads) of each individual and mapping statistics. xxxii B.S2 Number of contigs after SEX-DETector inferences, removal of sex-bias and selection of validated contigs in the three tissues. xxxiii B.S3 List of known sex-linked genes in S. latifolia and associated literature references. xxxv

Figure

  Figure1.1. The main evolutionary routes to dioecy. Two main routes from hermaphroditism (the assumed ancestral sexual system in angiosperms) to dioecy have been proposed: the gynodioecy-dioecy route (A) and the monoecydioecy route (B). It has been recently highlighted that dioecy may frequently revert to hermaphroditism (C, D), hence its rareness(Käfer et al., 2017). In the gynodioecy-dioecy route, inconstant males (males producing few viable seeds) may help reversions to hermaphroditism as drawn in C[START_REF] Ehlers | Inconstant males' and the maintenance of labile sex expression in subdioecious plants[END_REF]. In the monoecy-dioecy route, there may be cycles between monoecy and dioecy (D, E). Some work suggests that in this case, androdioecy (males and monoecious individuals) and gynodioecy (females and monoecious individuals) may also evolve and may represent a situation of the re-evolution of dioecy after it has reverted to monoecy (e.g.,[START_REF] Pannell | Regional variation in sex ratios and sex allocation in androdioecious Mercurialis annua[END_REF]. See text for details

Figure 1 . 2 .

 12 Figure 1.2. The genetics of the evolution to dioecy from gynodioecy. The dominant model involves two nuclear mutations, which if on the same autosome will evolve into sex chromosomes (a). Mf male fertility gene (dominant), Ff female fertility gene (recessive), Msm male sterility mutation (recessive), Fsm female sterility mutation (dominant). Proto-X and proto-Y = nascent sex chromosomes. (b) An alternative model involves nucleo-cytoplasmic mutations, with gynodioecy being determined by a cytoplasmic-male-sterility factor (CMS) and a nuclear restorer of male fertility gene (Rf ) system, in which case dioecy is determined by cryptic CMS, fixed in the population. If the Rf gene and a female fertility gene are on the same autosome, a proto-Y chromosome comprising the Rf gene (dominant) and a female sterility mutation (Fsm, dominant) may evolve

Figure 1 . 3 .

 13 Figure 1.3. The dioecious plant Silene latifolia. (A) Flowers from male and female individuals. (B) A karyotype of a S. latifolia male individual (from Hobzaet al., 2007). (C) The S. latifolia Y chromosome and the three sex-determining regions identified using mutants. These regions comprise two male-fertility regions: MFF male fertility factor, SPF stamen promoting factor, and one female-sterility region: GSF gynoecium sterility factor. Well-known Y-linked genes are indicated. PAR pseudoautosomal region (X-Y recombining region)

Figure 1

 1 Figure 1.4. C. grandis female (A) and male (B) flowers and idiogram of chromosomes (C). Autosome pairs are ordered according to their relative length. Female karyotype is (22 + XX) and male karyotype (22 + XY). Adapted from Ghadge et al. (2014), image source: commons.wikimedia.org

Figure 1 . 5 .

 15 Figure 1.5. Sex-determining genes and artificial dioecy in melon. (A) Model of the sex determination pathway in melon integrating CmACS-11, CmWIP1 and CmACS-7. Male flowers result from nonexpression of CmACS-11, which permits CmWIP1 expression. Female flowers develop on the branches because of the expression of CmACS-11 that represses the expression of CmWIP1. Consequently, the non-expression of CmWIP1 releases the expression of CmACS-7 that inhibits the stamen development. If nonfunctional CmACS-7 is expressed, hermaphroditic, instead of female, flowers develop. (B) Engineering dioecy from monoecious melon controlling acs11 and wip1 genes segregation. Source: Boualem et al. (2015)

Figure 1

 1 Figure 1.6. S. latifolia female (A) and male (B) flowers and idiogram of chromosomes (C). Autosome pairs are ordered according to their relative length. Female karyotype is (22 + XX) and male karyotype (22 + XY). Adapted from Lengerova et al. (2004), image source: http://www.plantsystematics.org

  3 IPS2, INRA, CNRS, University Paris Sud, University of Evry, University Paris Diderot, University of Paris Saclay, Batiment 630, 91405 Orsay, France.

Figure 2

 2 Figure 2.1. Y alleles are less expressed than X alleles in the flower buds of C. grandis males.Distribution of normalized expression ratio between X and Y alleles. Total Y and X read numbers were summed at sex-linked SNP locations for each contig and normalized for each male separately (see Methods section 2.9), then averaged across males to obtain the Y/X ratio. The median is shown in red.

Figure 2 . 2 .

 22 Figure2.2. With Y degeneration, X allele expression increases in males compared to females. Distribution of the ratio between the expression of the single X in males and the two X copies in females (log 2 Xmale/2Xfemale) for all sex-linked contigs. Distributions are shown for Y/X expression ratio categories in males: (A) Y/X>1; (B) X/Y in ]0.5-1]; (C) X/Y in ]0-0.5]; (D) X-hemizygous genes (no Y copy expression, see Methods section 4). Total X read numbers were summed at sex-linked SNP locations in each contig and normalized for each individual separately, then averaged among males and females to get the Xmale/2Xfemale ratio (see Methods section 2.9). Distribution is shown in log 2 scale with its density curve. Contigs with Xmale/2Xfemale ratios above 8 or under 0.125 were excluded, which reduced the dataset to 1,351 sex-linked contigs. Sample sizes are: 0, 168; 0-0.5, 460 (8 outliers); 0.5-1, 389 (2 outliers); >1, 334 (3 outliers). Medians are indicated for each Y/X ratio category.

Figure 2 . 3 .

 23 Figure 2.3. When the Y allele expression decreases, expression of the X increases in males to compensate. Expression levels of sex-linked contigs in both sexes for different Y/X expression ratio categories. Total read numbers were summed at sex-linked SNP locations in each contig and normalized for each individual separately; medians for all contigs and individuals of the same sex were then obtained. XX females, median expression level of both X-linked alleles in females; X males, median expression level of the single X-linked allele in males; Y males, median expression level of the Y-linked allele in males; XY males, median expression level of the X-linked plus Y-linked alleles in males. To compare different Y/X expression ratio categories, medians were normalized using the XX expression levels in females. Sample sizes are: 0, 168; 0-0.25, 207; 0.25-0.5, 261; 0.5-0.75, 243; 0.75-1, 148; 1-1.25, 108. Error bars indicate 95% confidence intervals of the median.

Figure 3 . 1 .

 31 Figure 3.1. Workflow applied for the Y chromosome assembly. Ten assemblies (in green) are nested within each other, from which five with decontaminated reads (light green). The best assembly is framed in red. Illumina, MinION, and PacBio data are highlighted in different shades of blue. For each assembly, the total length, the number of contigs/scaffolds and the N50 are shown.The N50 is a weighted median statistic such that 50% of the entire assembly is covered by contigs equal to or larger than this value.

Figure 3 . 2 .

 32 Figure 3.2. Read length distributions for the S. latifolia Y chromosome long read sequencing datasets produced and used for assembling. MinION MkI 2D and MkIB 1D, and PacBio reads production is described in the Methods section 2.3 and 2.4. MkI and MkIB are two versions of the MinION, the main difference being the sequencing speed (see Methods section 2.3).

Figure A. 1 .

 1 Figure A.1. Alignment of WIP1 in C. melo and C. grandis. The unclassified transcript, TRINITY_DN20534_c4_g2_i1, was produced with both male and female reads (see Chapter 3). Positions 801 to 900 are shown. Male and female contigs were identified using separate male and female transcriptomes reconstructed from RNAseq data from public datasets (Devani et al., 2017; Mohanty et al., 2017). X/Y SNPs are highlighted in red, and correspond to the positions identified by SEX-DETector (Muyle et al., 2016) as shown in Table A.1, confirming the sex-linkage of this transcript in C. grandis. WIP1 reference allele from C. melo was published by Martin et al. (2009).

Figure B. 1 .

 1 Figure B.1. Normalised difference (hereafter Δ) in allelic expression levels between S. latifolia and the outgroup without sex chromosomes, S. vulgaris, in autosomal and sex-linked contigs for the seedling tissue.If Δ is lower, higher or equal to zero, then expression in S. latifolia is respectively lower, higher or equal to the outgroup (See Materials and Methods for details). For all contig categories, Δ was compared to zero using a Wilcoxon test. The median Δ, confidence intervals and p-values adjusted for multiple testing using a Benjamini and Hochberg correction are shown (***: p-value < 0.001; **: p-value < 0.01, *: p-value < 0.05). Allelic expression at SNP positions was averaged for each contig separately and the Y/X ratio was used as a proxy for Y degeneration to group contigs. Contigs with sex-biased expression were removed, as well as contigs with Y/X expression ratios above 1.5. Sample sizes for the different contig categories are: autosomal: 200; 1-1.5:148; 0.75-1:139; 0.5-0.75:160; 0.25-0.5:114; 0-0.25:79 (we randomly selected 200 autosomal contigs to ensure similar statistical power among gene categories).

  Figure B.1), expression of the corresponding X allele in males increased (maternal allele in red in Figure B.1). This is the first direct evidence for ancestral expression recovery in S. latifolia, i.e. ancestral expression levels are reestablished in males despite Y expression degeneration. In females, expression of the maternal X allele also increased with Y degeneration (gray bars in Figure B.1), similarly to the maternal X allele in males. The paternal X alleles in females, however, maintained ancestral expression levels, regardless of Y degeneration (black bars in Figure B.1). Consequently, sex equality is not achieved in S. latifolia due to upregulation of sex-linked genes in females compared to ancestral expression levels. These results were confirmed in two other tissues and when analysing independently validated contigs only (although statistical power is sometimes lacking due to the limited number of validated contigs, Supplementary Figures B.S2-B.S7). Upregulation of the maternal X allele both in males and females of S. latifolia (Figure B.1 and Supplementary Figures B.

Figure B. 2 .

 2 Figure B.2. Normalised expression difference between maternal and paternal alleles in S. latifolia females in autosomal and sex-linked SNPs in the seedling tissue. The Y axis unit is the normalised allelic read count difference in log scale. A linear regression model with mixed effects was used to estimate the normalised difference between the effect of paternal and maternal origin of alleles in interaction with the contig status (autosomal or sex-linked with various levels of Y degeneration), while accounting for inter-contig and inter-individual variability (see Materials and Methods for details). The analysis is SNP-wise and reveals consistent patterns across SNPs. See Fig. B.1 legend for sample sizes for the different contig categories and statistical significance symbols

Figure B. 3 .

 3 Figure B.3. Illustration of DNA methylation staining results in S. latifolia from Siroky et al. (1998) (Siroky et al., 1998) See Supplementary Figure B.S14 for the original Figure. One arm of one of the two X chromosomes in females was hypomethylated, as well as the same arm of the single X in males.

données d'expression

  : espèce chez laquelle les organes reproductifs mâles et femelles sont sur des plantes séparées. Les mâles et les femelles sont ainsi des individus disctincts. -Monoïque (n. monoécie) : espèce chez laquelle les organes reproductifs mâles et femelles sont dans des fleurs séparées de la même plante. Les individus ont ainsi des fleurs mâles et des fleurs femelles. : séquençage de l'acide ribonucléique (ARN). L'ARN est en général synthétisé à partir d'une matrice d'ADN dont il est une copie. Les cellules utilisent en particulier l'ARN comme un support intermédiaire des gènes pour synthétiser les protéines dont elles ont besoin. -Read : une séquence d'un fragment d'ADN ou d'ARN. -Contig : séquence continue générée par l'alignement de fragments (reads) qui se chevauchent.

	Lexique
	• Chromosomes
	-Chromosomes sexuels : chromosomes spécialisés qui déterminent le sexe d'un
	individu, par exemple XX pour les femelles et XY pour les mâles.
	-Chromosomes hétéromorphes : chromosomes sexuels qui peuvent être distin-
	gués lors d'analyses cytogénétiques (chromosomes X et Y de tailles et contenus
	génétiques différents). -Gynodioïque (n. gynodioécie) : Espèce avec des femelles (individus ayant -Autosomes : chromosomes non sexuels. uniquement des fleurs femelles) et des hermaphrodites (individus ayant des fleurs • Classification bisexuées). -Angiosperme : plante à fleurs, à ovules enclos et à graines enfermées dans des • Séquençage : détermination de l'ordre linéaire des composants d'une macromolécule, fruits. par exemple l'ordre d'enchaînement des nucléotides d'un fragment d'ADN donné. -Gymnosperme : plante à l'ovule nu, porté par une feuille fertile. -DNAseq : séquençage de l'acide désoxyribonucléique (ADN), consistant à déter--Caryophyllacée : famille de plantes à fleurs, comprenant des arbustes et des miner l'ordre d'enchaînement des nucléotides pour un fragment d'ADN donné. plantes herbacées annuelles ou vivaces majoritairement de l'hémisphère nord. Leur fleur est composée de cinq pétales. -RNAseq,
	-PCR (Polymerase Chain Reaction) : réaction en chaîne par polymérase,
	méthode de biologie moléculaire d'amplification génique qui permet de dupliquer
	en grand nombre (avec un facteur de multiplication de l'ordre du milliard) une
	séquence d'ADN ou d'ARN, à partir d'une faible quantité.
	• Pièces florales

• Génétique -Génome : l'ensemble du matériel génétique d'une espèce. -Allèle : version variable d'un même gène qui peut être distinguée par des variations de sa séquence. -Orthologues (n. ou adj.) : gènes issus d'un ancêtre commun, suite à une spéciation. -Recombinaison (adj. recombinant) : mécanisme d'échange entre molécules d'ADN qui permet la réorganisation du génome. Ce phénomène conduit à l'apparition, dans une cellule ou dans un individu, d'une association des gènes différente de celle observée chez les cellules ou individus parentaux. -Gène X-hémizygote : gène lié au chromosome X mais dont la copie Y a été perdue, qui n'est donc présent qu'en un seul exemplaire chez les mâles. -Compensation de dosage : mécanisme qui permet d'égaliser l'expression des gènes situés sur les chromosomes sexuels entre mâles et femelles, ou de restaurer un état d'expression ancestral malgré la dégénerescence du chromosome Y. -Éléments répétés : séquences qu'on retrouve en de multiples copies dans le génome. Parmi ces éléments plusieurs familles existent, dont les éléments transposables. • Méthodes -Phylogénie (adj. phylogénique) : étude des liens existant entre espèces apparentées. Elle permet de retracer les principales étapes de l'évolution des organismes depuis un ancêtre commun. -Cytométrie en flux : technique permettant de mesurer les caractéristiques individuelles de particules (cellules, bactéries, parasites, billes..) telles que leur taille, leur forme ou la présence d'un composé fluorescent, et de les séparer en fonction d'un critère prédéfini. -Carpelle : enveloppe protectrice enfermant les ovules chez les angiospermes. Le carpelle se transforme en fruit après la fécondation. -Étamine : partie de l'appareil reproducteur mâle chez les plantes à fleurs. • Régimes de reproduction -Dioïque (n. dioécie) -Scaffold : ensemble de contigs orientés et ordonnés. Les trous sont de longueur connue, et correspondent à des séquences qui ne chevauchent pas avec d'autres et ne peuvent donc pas entrer dans un contig.

  Results of the SEX-DETector pipeline on the C. grandis dataset.

	0.1 Voies d'évolution vers la dioécie chez les angiospermes. 1.1 Frequency of species with separate male and female individuals and the current state of	xv
	0.2 Pipelines pour l'analyse des chromosomes sexuels de C. grandis et S. latifolia. knowledge on sex determination in animals and flowering plants.	xvii
	1.2 Frequency of sexual systems in angiosperm species.
	1.1 The main evolutionary routes to dioecy. 1.3 Sex determination mechanisms in dioecy and in gynodioecious and monoecious	7
	1.2 The genetics of the evolution to dioecy from gynodioecy. intermediates.	9
	1.3 The dioecious plant Silene latifolia. 2.1 Transcriptome assembly statistics of C. grandis flower buds.	1 1
	1.4 C. grandis female and male flowers and idiogram of chromosomes. 2.2	19
	1.5 Sex-determining genes and artificial dioecy in melon.	20
	1.6 S. latifolia female and male flowers and idiogram of chromosomes.	22
	1.7 Deletion map of the constructed Y chromosome of S. latifolia.	2 3
	/Y
	contigs.	55
	3.1 Workflow applied for the Y chromosome assembly.	67
	3.2 Read length distributions for the S. latifolia Y chromosome long read sequencing datasets
	produced and used for assembling.	68
	3.S1 Summary of the gene ontology (GO) annotations as defined by blast2GO.	78
	A.1 Alignment of WIP1 in C. melo and C. grandis.	9 4
	B.1 Normalised difference in allelic expression levels between S. latifolia and the outgroup
	without sex chromosomes, S. vulgaris, in autosomal and sex-linked contigs for the seedling
	tissue.	ix

2.1 Y alleles are less expressed than X alleles in the flower buds of C. grandis males. 39 2.2 With Y degeneration, X allele expression increases in males compared to females in C. grandis. 40 2.3 When the Y allele expression decreases, expression of the X increases in males to compensate. 42 2.S1 Distinct methods for the detection of differentially expressed genes are conservative. 51 2.S2 Sex-linked genes are under-expressed in males compared to females, even after correcting for a similar expression in males and females for autosomal genes. 52 2.S3 Autosomal gene expression is similar for males and females after correction. 53 2.S4 Sex-linked genes are under-expressed in males compared to females even after correction. 54 2.S5 Distribution of pairwise synonymous divergence (dS) between X and Y alleles in XB.2 Normalised expression difference between maternal and paternal alleles in S. latifolia females in autosomal and sex-linked SNPs in the seedling tissue. xi B.3 Illustration of DNA methylation staining results in S. latifolia. xii B.S1 Relatedness among the three studied species. xxiii xxix List of Tables 2.3 Comparison of X/Y divergence, gene loss and estimated age of sex chromosomes in plant systems. 2.S1 Library sizes and statistics for RNA sequencing from C. grandis male and female flower buds. 2.S2 BUSCO results for C. grandis flower bud transcriptomes.

Frequency of species with separate male and female indi- viduals and the current state of knowledge on sex determination in animals and flowering plants.

  Table 1.1.

		Animals Flowering plants
	% of species with seperate sexes	95%	5-6%
	% of species with rough information > 90%	< 1%
	on sex determination mechanisms 1

. We do not yet know how widespread sex chromosomes are in plants, or whether other genetic mechanisms and environmental sex determination have evolved in this taxon, and we have not yet identified the vast majority of the molecular players involved in plant sex determination.

Table 1 .2. Frequency of sexual systems in angiosperm species. Adapted from

 1

	only one phenotype)

Käfer et al. (2017)

.

Sex determination mechanisms in dioecy and in gynodi- oecious and monoecious intermediates. 1

		Mostly nucleo-cytoplasmic	CMS : Chimeric mitochondrial genes
			Rf : Pentatricopeptide-repeat (PPR) family
			proteins
		Phytohormonal
		Cucurbits: Ethylene pathway	Cucurbits: Genes of the ethylene pathway
	Monoecy		(2 enzymes, 1 transcription factor)
		Maize: Gibberellic acid pathway Maize: Silkless gene sk1, Tasselseed family
			genes TS1 and TS2, tasselseed4 microRNA
	Dioecy	Sex chromosomes (XY, ZW) 1	Persimmon: OGI small RNA/MeGI Asparagus: MYB transcription factor
	Table 1.3. In XY systems, the males are
	heterogametic (have a pair of nonidentical sex chromosomes), while in ZW sys-
	tems, it the females that are heterogametic.

  Golenberg, E. M. and N. W. West. "Hormonal interactions and gene regulation can link monoecy and environmental plasticity to the evolution of dioecy in plants". In: American Journal of Botany 100.6 (2013), pp. 1022-1037. Hobza, R., E. Kejnovsky, B. Vyskot, and A. Widmer. "The role of chromosomal rearrangements in the evolution of Silene latifolia sex chromosomes". In: Molecular Genetics and Genomics 278.6 (2007), pp. 633-638. Käfer, J., G. A. Marais, and J. R. Pannell. "On the rarity of dioecy in flowering plants". In: Mol. Ecol. 26.5 (2017), pp. 1225-1241. Li, R., J. Li, G. Yin, J. Yang, W. Zou, and

L. Bai. "A male-specific SCAR DNA marker and sex ratio of seedlings in salak (Salacca zalacca var. zalacca)". In: Journal of Forestry Research 28.1 (2017a), pp. 47-50. Matson, C. K. and D. Zarkower. "Sex and the singular DM domain: insights into sexual regulation, evolution and plasticity". In: Nat. Rev. Genet. 13.3 (2012), pp. 163-174.

Transcriptome assembly statistics of C. grandis flower buds.

  Statistics in the final Trinity transcriptome and the working transcriptome containing the longest ORF predicted per Tritinty isoform.

		Full Transcriptome Longest ORF per isoform
	Total transcripts	128,904	82,699
	Total assembled bases (bp)	103,275,123	27,290,670
	Median contig length	552	836
	Average contig length	801.18	836.83
	Maximum contig length	16,296	16,296
	Minimum contig length	297	297
	N50	1,029	1,086
	Total contigs longer than 1kb	30,795	21,587
	GC content (%)	42.96	42.96
	Table 2.1.

  version 0.7.15 with following commands: bwa aln -n 5 and bwa sampe). Mapping statistics are shown in Supplementary Table 2.S3. Mapped reads were kept with Samtools version 1.3.1 and individual genotypes were predicted with reads2snps version 2.0.64 with paralog detection

Results of the SEX-DETector pipeline on the C. grandis dataset

					-DETector	with	SEX-DETector	with
					BWA mapping		GSNAP SNP-tolerant
							mapping
	Transcripts in final assembly		82,699		82,699
	Transcripts with enough coverage to	82,689		70,298
	be studied
	Transcripts with enough informa-	4,320		3,801
	tive SNPs to compute a segregation
	probability
	Transcripts assigned to an autosomal	2,889		2,437
	segregation type
	Transcripts assigned to a X-Y	1,239		1,196
	segregation type
	Transcripts	assigned	to	a	192		168
	X-hemizygous segregation type
	Table 2.2.

. Number of contigs assigned by SEX-DETector to an autosomal, X/Y or X-hemizygous segregation type (see Methods section 2.5) before and after SNP-tolerant mapping (see Methods section 2.6).

Table 2 .3. Comparison of X/Y divergence, gene loss and estimated age of sex chromosomes in plant systems. Charac

 2 teristics of plant sex-chromosome in which molecular clocks were used to give an estimate of the time since recombination arrest between X and Y gene copies. Used molecular clocks are all based on Brassicaceae (see Methods section 2.7)

Table 3 .1. Summary of all the raw and processed S. latifolia Y chromo- some sequencing datasets produced and used for assembling.

 3

	Statistics:

  S. latifolia Y chromosome is approximately 550 Mb, this assembly should

	Number of Scaffolds	100,050
	Total bases (bp)	563,320,934
	Sequence length (bp)	502 -47,836
	%GC	34
	Number of sequences >1kb	99,833
	% sequences >1kb	99.8%
	Number of sequences >10kb	8,463
	% sequences >10kb	8.5%
	Mean length (bp)	5,630
	N50 (bp)	6,114
	% N	0.01%
	Table 3.2. Assembly statistics of the final assembly. The final assembly
	(see Figure

  Percentage of transcripts or BAC sequences mapping on the final assembly (see Figure3.1). Full transcriptome and inferences fromMuyle et al. (

				Transcriptome
			Full	Sex-linked	Autosomal
				X/Y X-hemizygous
	Total	85	47,301	1,048	337	14,082
	Mapped	73	9,249	810	185	2,705
	% Mapped	85.88%	19.55% 77.29%	54.90%	19.21%
	Mean number of hits	11.14	5.51	7.18	3.51	3.89
	Median number of hits	4	2	3	2	2
	Table 3.3. Assembly quality assessment with a reference transcrip-
	tome, sex-linked genes and Y-linked BAC sequences.

Table 3

 3

	Total number of genes	16,219
	Protein coding genes
	Number of protein coding genes	6,770
	Mean gene length (bp)	595.37
	Mean CDS length (bp)	354.29
	Mean number of exons per gene	1.89
	Mean exon length (bp)	200.9
	Mean number of introns per gene	0.89
	Mean intron length (bp)	206.98
	Per cent genes with introns	58
	Per cent genes with 5' UTR	8
	Per cent genes with 3' UTR	7
	Non protein coding genes
	Number of non protein coding genes	9,449
	Mean ncRNA gene length (bp)	1,198.78
	Per cent ncRNA genes with introns	0
	Mean exon number per ncRNA gene	1
	Intergenic (inter protein-coding genes)
	Mean length	33,785.16

.4. Results of EuGene gene finder applied to the final assembly.

Gandalf, La Communauté de l'Anneau, J.R.R. Tolkien.

. THE EVOLUTION OF SEX DETERMINATION IN PLANTS

THE EVOLUTION OF SEX DETERMINATION IN PLANTS

SEQUENCING THE Y CHROMOSOME OF S. LATIFOLIA

SEX-LINKED GENES IN COCCINIA GRANDIS

ASSEMBLING THE Y CHROMOSOME OF SILENE LATIFOLIA USING LONG READ SEQUENCES

https://www.biorxiv.org/content/biorxiv/early/2017/09/20/179044.full.pdf

Remerciements

Acknowledgments

The authors thank Jos Käfer and Editor Charlie Scutt for comments and suggestions for improving this manuscript. We apologize to all colleagues whose work could not be cited due to number of references restrictions.

Acknowledgments

This project was supported through ANR grant NGSex to GABM and AB.

Acknowledgements

This project was supported through ANR grant NGSex to GABM. We thank Corinne Cruaud and Stefan Engelen from the Genoscope (Evry, France) for their help with Min-ION sequencing, as well as Grégoire Siekaniec for his work on decontaminating the datasets and testing assembly pipelines.

Acknowledgments

This project was supported through an ANR grant ANR-14-CE19-0021-01 to GABM, SNSF grant 160123 to AW and Czech Science Agency grant number 16-08698S to RH. We thank the EMBL Genomics Core Facility (EMBL, Heidelberg) for Y chromosome sequencing. We thank Brandon Gaut, Tatiana Giraud and Judith Mank for comments on the manuscript.

Author contributions

The study was conceived and coordinated by GABM and AB. The cross was done by AKB. Samples were collected by AKB. Sequencing was done by AB. CF and AM built the reference transcriptome. HB performed the GO annotation. BR provided scripts and advice for interpreting for the differential expression analysis. CF made all subsequent analysis and prepared the figures. Data analysis and interpretation was done by CF, HB and GABM. CF wrote the manuscript with input from HB and GABM. Supplementary Figure 2.S1. Distinct methods for the detection of differentially expressed genes are conservative. Venn diagram of Differentially Expressed Genes identified by DESeq2, edgeR and LimmaVoom. Number of genes with FDR < 0.0001 for each method. 3,273 differentially expressed genes were identified by at least 2 methods. Supplementary Figure 2.S2. Sex-linked genes are under-expressed in males compared to females, even after correcting for a similar expression in males and females for autosomal genes. Expression levels of autosomal and sex-linked contigs in both sexes. Total read numbers were summed at SNP locations in each autosomal (A and C) and sex-linked (B and D) contig and normalized for each individual separately; medians for all individuals of the same sex were then obtained. Expression was corrected (C and D) using the autosomal median male/female ratio (see Methods section 2.9.1). Supplementary Figure 2.S4. Sex-linked genes are under-expressed in males compared to females even after correction. Distribution of normalized expression difference between males and females in sex-linked contigs. Total read numbers were summed at sex-linked SNP locations for each contig and normalized for each individual separately; then averaged across sexes to obtain the male/female ratio. Expression was corrected using the autosomal median male/female ratio. Distribution is shown in log 2 scale with its density curve. The median is shown in red.

Supplementary Figures and Tables

Supplementary Figure 2.S5. Distribution of pairwise synonymous divergence (dS) between X and Y alleles in X/Y contigs. The vertical line indicates contigs longer than 1kb.

Supplementary Table 2.S1. Library sizes and statistics for RNA sequencing from C. grandis male and female flower buds. Dataset characteristics before and after filtering out for sequencing adapters and low quality.

CHAPTER 3

Assembling the Y chromosome of Silene latifolia using long read sequences

Probleme lassen sich lösen, wenn man ein paar Ideen hat, das ist ja klar. 1 1 Die Freiheit der Krokodile, Friedrich von Borries.

59

Author contributions

The study was conceived and coordinated by GABM. Plant material collection and flow cytometry were done by RC and RH. CF and NB performed MinION sequencing, and CF prepared the library for PacBio sequencing which was done at the Quebec Sequencing Genomes platform. MAC and RC developed the pipeline for the assembly, CF evaluated the final assembly and AEF did the annotation. CF made the figures and wrote the manuscript with input from GABM.

Supplementary Figures and Tables

A.

Supplementary Figure 3.S1. Summary of the gene ontology (GO) annotations as defined by blast2GO. The S. latifolia Y chromosome genes are categorized according to biological processes (A) and molecular function (B). 3.S1. Long read sequencing runs specifications. (A) MinION MkI and MkIB run outputs. MkI and MkIB are two versions of the MinION sequencer. A basecalled read is classified as a "failed" read if base-calling failed, or the read had a mean base quality score 9. Statistics: total number of reads and total number of bases sequenced during the run, mean and maximum read size for the run, N50 (weighted median statistic such that 50% of the entire dataset is contained in reads equal to or larger than this value), average quality (Q-score; developed by Oxford Nanopore). (B) MinION MkI and MkIB run dates, library preparation kits and software versions used. (C) PacBio run outputs. Two runs were perfomed on two separate lanes with respectively 3 and 7 wells. The subreads are used to reconstruct CCS (Circular Consensus Sequence) reads. Statistics: total number of reads and total number of bases sequenced during the run, mean read size for the run.

Supplementary Table

APPENDIX A

List of Abbreviations

Expression differences between maternal and paternal alleles at the SNP level.

Maternal and paternal alleles expression were compared in S. latifolia for autosomal and sexlinked SNPs. In order to deal with the difference in numbers of autosomal versus sex-linked contigs (Supplementary Table B.S2), 200 autosomal contigs were randomly selected in order to keep comparable powers of detection. Allelic expression levels in S. latifolia for each individual at every SNP position were analysed using a linear regression model with mixed effects with the R package lme4. We assumed a normal distribution of the read count data after log transformation. In order to account for inter-individual and inter-contig variability, a random "individual" and a random "contig" effect were included in the model. The aim of this modeling framework was to estimate the joint effect of the chromosomal origin of alleles (paternal or maternal in males or females) and the status of the gene (autosomal or sex-linked with various levels of Y degeneration defined by the average Y over X expression ratio in males). Two fixed effects with interaction were therefore considered in the model, see equation (3).

In order to estimate the changes in sex-linked gene expression levels since the evolution of sex chromosomes, we used the average of the two outgroup expression levels as a reference (offset) for every SNP position, divided by two in order to be comparable to S. latifolia allelic expression levels.

(3)

All effects of the model (fixed or random) were proved highly significant (p-values < 2.2•10 -16 ) using comparison of the fit of model (3) to simpler nested models (removing one effect at a time in model ( 3)). In order to statistically test whether there was a difference between the effects of paternal and maternal alleles in females in different degeneration categories we used the contrasts provided by the lmerTest package in R. This strategy provided estimates, confidence intervals and p-values of the difference between the two effects of paternal and maternal origin in females in interaction with degeneration levels, while normalizing by the expression of the two outgroups. Moreover, the presence of random effects allows to account for inter-individual and inter-contig variability.

Author information

The authors declare no competing interests.

Correspondence and requests for materials should be addressed to Aline Muyle (email: muyle.aline@gmail.fr).

Supplementary materials for Appendix B

1. Supplementary Text S1: Dosage compensation in X-hemizygous genes

The first papers on dosage compensation in S. latifolia were contradictory because they focused on different gene sets. Muyle et al. (2012) focused on X/Y gene pairs while other papers focused on Xhemizygous genes (Bergero et al., 2015;Chibalina and Filatov, 2011). However, the X-hemizygous gene sets returned by the RNA-seq approach used in those papers is less reliable than the X/Y gene sets (Blavet et al., 2015). A gene might be inferred as Xhemizygous simply because the -still functional -Y copy is not expressed in the tissue sampled for RNAseq. In S. latifolia, X-hemizygous genes tend to be less expressed than X/Y genes and are less likely to be detected by segregation analysis as efficient SNP calling requires a certain read depth, see (Blavet et al., 2015). Moreover, X-hemizygous genes are inferred from X polymorphisms while X/Y genes can be detected both with X and X/Y polymorphisms, which are more numerous. Another inherent bias to X-hemizygous contig inference comes from the assembly step. If the X and the Y copy are too divergent to be assembled together, the X contig will be wrongly inferred as X-hemizygous because Y alleles will be absent from the contig (this bias was at least partly corrected in the analyses presented here, see Material and Method section 2.5.1). The inferences of X-hemizygous genes using the RNA-seq approach (including SEX-DETector) imply a higher rate of both false positives and false negatives than those for X/Y gene pairs. In Papadopulos et al. (2015), 25% of the X/Y chromosomes were sequenced using a genomic approach. A much higher fraction of X-hemizygous genes was found than in previous RNA-seq papers (Bergero et al., 2015;Chibalina and Filatov, 2011). Papadopulos et al. (2015) did find evidence for dosage compensation in approximately halfof X-hemizygous genes (see their figure 3D). Due to limitations of the RNA-seq approach in inferring X-hemizygous genes, results on X-hemizygous contigs are analysed separately here.

Poor dosage compensation of X-hemizygous contigs compared to X/Y contigs with high Y degeneration was observed across all tissues (Supplementary Figures B.S2 to B.S7). Also, the parental origin of the X chromosome has limited to no effect on female X expression levels for X-hemizygous contigs, unlike X/Y contigs (Supplementary Figures B.S8 to B.S13). A reason that could explain such a different pattern for X-hemizygous genes compared to X/Y genes is the possible dosage insensitivity of X-hemizygous genes. X-hemizygous genes could have lost their Y copy because dosage was not important for them and selection neither slowed down the loss of the Y copy nor selected for dosage compensation when degeneration inevitably occurred (Muyle et al., 2017a). A well described characteristic of dosage sensitive genes is that they tend to code proteins involved in large complexes (Pessia et al., 2012). Gene Ontology was studied using the Blast2GO PRO version 2.7.2 (Conesa et al., 2005) as in Zemp et al. (2016). Using the GOterm analysis, our set of X-hemizygous contigs were found to be significantly depleted in ribosomal protein coding genes compared to autosomal genes (pvalue 1.3 • 10 -4 ), which is consistent with the global dosage insensitivity of X-hemizygous genes in S. latifolia. This depletion in large protein complexes was not found when comparing X/Y genes to autosomal genes.

Supplementary Text S2:

2.1. Plant material and sequencing 2.1.1. RNA-seq Illumina data RNA-seq data from previous studies were used (the GEO database GEO Series GSE35563, European Nucleotide Archive PRJEB14171), it included flower buds and leaf tissues from individuals of a cross in S. latifolia as well as individuals in S. vulgaris. In addition to these preexisting data, RNA-seq reads were generated in a comparable way for seedlings of a controlled cross using the same parents in S. latifolia, four males and four females were sampled. Seedlings were also sequenced for S. vulgaris. Seedlings were grown in a temperature controlled climate chamber in Eschikon (Switzerland) using the same conditions as in Zemp et al. (2016). The S. latifolia and S. vulgaris seedlings were collected without roots at the four-leaf stage. The sexing of the S. latifolia Δ was computed as follows: Δ =(allelic expression in S. latifoliaallelic expression in the outgroup) / allelic expression in the outgroup). If Δ is lower, higher or equal to zero, then expression in S. latifolia is respectively lower, higher or equal to the outgroup. For all contig categories, Δ was compared to zero using a Wilcoxon test. The median Δ, confidence intervals and p-values adjusted for multiple testing using a Benjamini and Hochberg correction are shown (***: p-value < 0.001; **: p-value < 0.01, *: p-value < 0.05). The Y/X ratio was computed in S. latifolia males and averaged among individuals to use as a proxy for Y degeneration. X-hemizygous contigs have a Y/X ratio equal to zero. Contigs with sex-biased expression were removed, as well as contigs with Y/X expression ratios above 1.5. Sample sizes for the different contig categories are: autosomal:200; 1-1.5:148; 0.75-1:139; 0.5-0.75:160; 0.25-0.5:114; 0-0.25:79; 0:205 (note that 200 autosomal contigs were randomly selected in order to have similar statistical power among gene categories). In the absence of dosage compensation, the single X in males should be expressed at levels similar to the outgroup that does not have sex chromosomes, in other words, without dosage compensation Δ should be close to zero for the maternal allele in males (red bars). Results show that the maternal allele is hyper-expressed in S. latifolia when the Y chromosome is degenerated, both in males and females. :200;0:275 Supplementary Table B

Pipin

End? No, the journey doesn't end here.

Gandalf 1

1 The Lord of the Rings, J.R.R. Tolkien