Morgane Robles
email: morgane.robles@gmail.com

Pauline M Peugnet

Sarah A Valentino

C Dubois

Michèle Dahirel

Marie-Christine Aubrière

Fabrice Reigner

D Serteyn

L Wimel

Anne Couturier-Tarrade

M.-C Aubrière

A Tarrade

P Chavatte-Palmer

Placental alterations in structure and function in intra-uterine growth-retarded horses

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Introduction

Epidemiological studies in humans [START_REF] Hanson | Early developmental conditioning of later health and disease: physiology or pathophysiology?[END_REF] and experiments in non-equine animals [START_REF] Mcmullen | Animal models for the study of the developmental origins of health and disease[END_REF][START_REF] Chavatte-Palmer | Breeding animals for quality products: not only genetics[END_REF][START_REF] Wu | Intrauterine growth retardation: Implications for the animal sciences[END_REF][START_REF] Chavatte-Palmer | Diet before and during pregnancy and offspring health: The importance of animal models and what can be learned from them[END_REF] have shown that preconceptional and gestational environmental conditions induce physiological adaptations in the offspring that persist postnatally and increase the risks of developing non-communicable diseases at adulthood. A U-shaped curve of risk has been observed, with both individuals born small and large being affected [START_REF] Stettler | Infant weight gain and childhood overweight status in a multicenter, cohort study[END_REF][START_REF] Gluckman | Maternal constraint of fetal growth and its consequences[END_REF]. These Developmental Origins of Health and Disease (DOHaD) also apply to the horse [START_REF] Fowden | Nutritional Programming of Intrauterine Development: A Concept Applicable to the Horse?[END_REF][START_REF] Peugnet | Management of the pregnant mare and long-term consequences on the offspring[END_REF][START_REF] Coverdale | Nutritional programming and the Version postprint Comment citer ce document[END_REF][START_REF] Satterfiled | Nutrition, epigenetic mechanisms and human disease[END_REF].

The placenta plays a pivotal role in transmitting stimuli from the maternal environment to the fetus [START_REF] Fowden | The placenta and intrauterine programming[END_REF][START_REF] Belkacemi | Maternal Undernutrition Influences Placental Fetal Development[END_REF][START_REF] Reynolds | Role of the pre-and post-natal environment in developmental programming of health and productivity[END_REF][START_REF] Barker | Placental programming of chronic diseases, cancer and lifespan: A review[END_REF]. Placental function varies according to placental size, morphology, blood flow, nutrient transporter abundance and intrinsic nutrient consumption and production [START_REF] Fowden | The placenta and intrauterine programming[END_REF][START_REF] Belkacemi | Maternal Undernutrition Influences Placental Fetal Development[END_REF][START_REF] Jansson | Role of the placenta in fetal programming: underlying mechanisms and potential interventional approaches[END_REF]. In equids, placentation is non-invasive, diffuse and epitheliochorial.

Microcotyledons with intense branching maximise the contact surface between endometrium and trophoblast for haemotrophic exchanges. Histotrophic exchanges take place in-between microcotyledonary units, where pseudo-stratified trophoblastic cells are specifically adapted to absorb the exocrine material from the uterine glands [START_REF] Steven | Anatomy of the placental barrier in the mare[END_REF][START_REF] Macdonald | Scanning electron microscopy of the microcotyledonary placenta of the horse (Equus caballus) in the latter half of gestation[END_REF][START_REF] Allen | Fetomaternal interactions and influences during equine pregnancy[END_REF].

An estimated 21,359 equine embryos were transferred in the world in 2015 [START_REF] Perry | Statistics of embryo collection and transfer in domestic farm animals[END_REF]. Recipient mares are mostly chosen for economical or practical (character, availability) reasons and are often of a different size and breed than the embryo. Manipulation of fetal growth through embryo transfers between ponies and Thoroughbreds showed that metabolic and stress responses are affected in growth-enhanced and growth-restricted newborn foals [START_REF] Forhead | Postnatal insulin secretion and sensitivity after manipulation of fetal growth by embryo transfer in the horse[END_REF][START_REF] Giussani | Postnatal cardiovascular function after manipulation of fetal growth by embryo transfer in the horse[END_REF], with effects on body size persisting up to three years of age [START_REF] Allen | Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero[END_REF][START_REF] Allen | The influence of maternal size on pre-and postnatal growth in the horse: III postnatal growth[END_REF]. Placental structure was pointed out as the primary control mechanism for the observed alterations in foal growth and

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metabolism [START_REF] Allen | Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero[END_REF] but functional mechanisms associated with these structural changes were not investigated.

The current study follows up from previous work where pony or Saddlebred embryos were transferred into draught mares and Saddlebred embryos transferred into pony mares [START_REF] Peugnet | Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses[END_REF]. Postnatally, enhanced growth pony foals remained taller than pony controls until 18 months of age whereas restricted Saddlebred foals partially caught up to Saddlebred controls [START_REF] Peugnet | Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses[END_REF][START_REF] Peugnet | Longitudinal Study of Growth and Osteoarticular Status in Foals Born to Between-Breed Embryo Transfers[END_REF]. Early insulin-resistance and subsequent lower basal glycaemia were observed in enlarged ponies compared to pony controls. In contrast, restricted Saddlebred foals initially developed increased insulin sensitivity that persisted until 7 months. They had increased basal glucose concentration compared to Saddlebred controls until 18 months of age [START_REF] Peugnet | Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses[END_REF].

Here, we evaluated placental adaptations (placental biometry, histological structure, expression of genes involved in growth, vascularisation and nutrient transfer processes) to possibly relate them to the post-natal development of foals.

Materials and methods

The study was conducted over 2 successive breeding seasons (2011 and 2012 foalings) (Table 1). Welsh Pony mares (P, n = 27) were housed and P embryos (n = 61) were produced in experimental farm 1 (47°32'36.12''N, 0°44'38.96''E, 120 m). Anglo-Arabian Saddlebred (S, n = 28) and draught Breton (D, n = 22) mares were housed and S embryos (n = 48) were produced in the experimental farm 2 (45°34'55.17''N, 1°43'16.29''E, 442 m). Age, parity and size of the mares are shown in Table 1. The experimental protocol is summarised in Figure 1 as described previously [START_REF] Peugnet | Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses[END_REF].

Saddlebred embryos were produced by two different stallions depending on the breeding season but both stallions produced experimental and control foals. Briefly, embryos were recovered 7 days after ovulation, washed in Emcare Holding ® solution (ICPbio), transported in Equitainer ®1 to the other experimental farm (3-4 hours) and transferred immediately into synchronised recipients 5 to 7 days after ovulation. Pregnancy was diagnosed 7 days after ET by ultrasonography.

Management of mares and placental sampling

From ovulation, mares were kept in pasture with free access to mineral salts. From the 5 th gestational month, they were housed in boxes and fed a diet based on straw and hay with concentrates in farm 1 and homemade pellets and hay in farm 2, with free access to mineral salts (Supplementary Item1).

At delivery, all placentas (allantochorion) were sampled near the umbilical cord: 1 cm² fragments were fixed in 4% formaldehyde for histological analysis and 3 mm² fragments were snap-frozen in liquid nitrogen for molecular analysis. The foal and the placenta associated to amnion and cord (total fetal membranes -TFM) were weighed the next morning. The gross

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placental surface area was measured by applying a clear Plexiglas sheet marked with 10 cm

x 10 cm squares on placentas laid in the "F" configuration, revealing both uterine horns and the uterine body (Fig 2) [START_REF] Allen | Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero[END_REF]. Photographs were taken and the placental surface subsequently measured using the ImageJ ® software 2 . What corresponded to 10 cm in the picture was set as a pixel scale. The placental surface was subsequently measured as the area calculated by drawing the contour of the placenta, multiplied by 2.

Histological and stereological analyses

Placental samples were embedded in paraffin, cut in 7 µm sections, stained with haematoxylin/eosin and scanned using NanoZoomer Digital Pathology ®3 . Stereology is a quantitative method that enables the calculation of three dimensional structural quantities (number, length, area and volume) from two-dimensional sectional images. Relative surface (Sv, μ -1 ) and relative volume (Vv, %) of the different components of the placentas were measured using the One Stop Stereology software (MercatorPro ®4 [START_REF] Reed | One-stop stereology: the estimation of 3D parameters using isotropic rulers[END_REF]).

As presented in Figure 3, the components measured were:

-For the microcotyledonary part: Both the haemotrophic and histotrophic trophoblasts labelled as "trophoblast", the microcotyledonary vessels and the whole microcotyledons (by adding the measures of the trophoblasts and the microcotyledonary vessels)

-For the allantoic part: the allantoic tissue vessels and the allantoic connective tissue.

Gene expression analysis

Total RNA from placental samples was isolated as previously described [START_REF] Chomzynski | Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction[END_REF] and purified using the RNeasy ® Mini kit 5 according to the manufacturer's instructions. Reverse transcription and real time quantitative PCR were performed as previously described [START_REF] Tarrade | Sexual dimorphism of the feto-placental phenotype in response to a high fat and control maternal diets in a rabbit model[END_REF]. Vascularisation Vascular endothelial growth factor A (VEGFA), that promotes angiogenesis and vasculogenesis, vascular endothelial growth factor receptor 1 (FLT1) and vascular endothelial growth factor receptor 2 (KDR), receptors of VEGFA that regulate negatively and positively its action, respectively, and endothelial NOS (eNOS), an enzyme that catalyses the production of nitric oxide gaseous vasodilator.

Data were analysed using QbasePLUS ®6 . Calibrated normalised relative quantities (CNRQ) were calculated as previously described [START_REF] Tarrade | Sexual dimorphism of the feto-placental phenotype in response to a high fat and control maternal diets in a rabbit model[END_REF]. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Ribosomal Protein L32 (RPL32) and Secretory Carrier Membrane Protein 3 (SCAMP3) [START_REF] Brosnahan | IL-22 Is Expressed by the Invasive Trophoblast of the Equine (Equus caballus) Chorionic Girdle[END_REF], as selected by geNorm, were used as reference genes.

Comment

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Data analysis

Results are expressed as medians with interquartile range and presented as boxplots (minimum to maximum). Statistical analyses were performed using R software 7 (version 3.3.0) using linear models to examine associations between breed (P-P vs. S-S);

Results

Median, interquartile range and p-value of statistically significant co-factors are presented in Supplementary Item 2.

Pregnancies (Table 1)

Altogether, 21 P-P and 28 S-S controls and 6 P-D, 6 S-P and 8 S-D experimental foals were delivered spontaneously during spring and summer (6 April -13 August) with >75% in May/June, all within the normal gestational age range.

Pregnancy length was not statistically different between P-D and P-P nor between S-D and S-S pregnancies. In contrast, pregnancy was 13 days longer in S-P vs. S-S (p<0.001) and 16 days longer in S-P vs. S-D (p<0.01).

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Foals (Figure 4A)

All foals were healthy at birth, but for 3 of 6 S-P foals that needed assistance to stand and suckle immediately after birth and were bottle-fed with horse colostrum and ewe milk until first suckling. These foals presented signs of dysmaturity such as silky hair coat and joint and tendon laxity [START_REF] Rossdale | A clinician's view of prematurity and dysmaturity in thoroughbred foals[END_REF].

P-D foals were heavier than P-P foals (+57.3%, p<0.0001). S-P and S-D foals were respectively lighter (-31.2%, p<0.0001) and heavier (+8.9%, p<0.001) than S-S foals. S-P foals were lighter by 36.8% than S-D foals (p<0.0001).

Placental and fetal membranes biometry (Figures 4B, 4C and 4D)

TFM were heavier (p<0.0001) and the placenta larger (p<0.0001) in P-D compared to P-P. The foal/TFM weight ratio tended to be smaller in P-D compared to P-P groups (p = 0.07). TFM weight and placental surface were respectively reduced (p<0.001, p<0.0001, respectively) and increased (p<0.01, p = 0.02, respectively) in S-P and S-D compared to S-S controls. Both TFM weight and placental surface area were reduced in S-P compared to S-D (p<0.0001, p<0.0001). Nevertheless, the foal to TFM weight ratio was not different between S-P, S-S and S-D.

Placental morphometry (Supplementary Item 3 and Fig 5)

Only the parameters that differed within a breed are presented in Figure 5. There were no gross or histologic differences observed between controls and experimental groups on histological sections.

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Placental gene expression (Table 3 and Figure 6)

Only the parameters that differed within a breed are presented in Figure 6.

Growth and development:

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Discussion

Embryo transfer into a breed of a larger/smaller size induced fetal enhanced growth or growth restriction, respectively, as observed by others [START_REF] Allen | Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero[END_REF][START_REF] Tischner | Embryo recovery from Polish pony mares and preliminary observations on foal size after transfer of embryos to large mares[END_REF][START_REF] Tischner | The development of Polish ponies born after embryo transfer to large recipients[END_REF][START_REF] Walton | The maternal effects on growth and conformation in Shire horse-Shetland pony crosses[END_REF]. Enhanced growth was associated with increased placental size and weight in ponies and Saddlebreds, whereas growth restriction was associated with a reduction in both parameters and 3 cases of dysmaturity. In terms of placental features, enhanced growth was associated with a decrease in the surface density of trophoblast in ponies and of allantoic vessels in Saddlebred placentas, both with little gene expression modifications. In contrast, growth restriction was associated with an increase in trophoblast surface density and volume fraction together with a strong reduction in the expression of genes involved in growth and development and nutrient transfer in Saddlebred placentas.

Intrauterine enhanced growth did not affect gestational length in pony and Saddlebred foals. Enhanced growth was associated with heavier TFM and a larger placenta in both groups. These results are consistent with previous studies using between-breed embryo transfer and crossbreeding in horses, pigs and cows [START_REF] Allen | Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero[END_REF][START_REF] Tischner | The development of Polish ponies born after embryo transfer to large recipients[END_REF][START_REF] Walton | The maternal effects on growth and conformation in Shire horse-Shetland pony crosses[END_REF][START_REF] Ashworth | Embryo survival and conceptus growth after reciprocal embryo transfer between Chinese Meishan and Version postprint Comment citer ce document[END_REF][START_REF] Ferrell | Maternal and fetal influences on uterine and conceptus development in the cow.2. Blood flow and nutrient flux[END_REF][START_REF] Biensen | The impacts of uterine environment and fetal genotype on conceptus size and placental vascularity during late gestation in pigs[END_REF]. Placental efficiency (as estimated by the foal/TFM weight ratio) tended to be decreased in P-D foals maybe because placental growth was driven by uterine space. As the power of the statistical test was only 36%, these differences may have become significant if there had been more animals in the P-D group. Placental efficiency was not modified in S-D foals, probably because of the relatively smaller difference between S and D size.

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Pregnancy was longer in restricted S-P, as if time was unsuccessfully given to the foal to try and grow to its genetic potential. In humans, intra uterine growth retardation (IUGR) is defined as a birth weight below the tenth percentile for gestational age [START_REF]The World Health Report 1995: bridging the gaps[END_REF]. Here, the weight of S-P foals was 7.20 kg below the 10th percentile of S-S foals' birthweight and 8.54 kg below the 10th percentile of birth weight of 115 S-S foals from the same breed born between 2011 and 2015 in the same farm. Restricted S-P foals can thus be clinically considered as IUGR, which could explain the observed dysmaturity (in addition to clinical signs of dysmaturity at birth, foals presented an immature pancreatic function at 3 days of age) [START_REF] Chomzynski | Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction[END_REF][START_REF] Brosnahan | IL-22 Is Expressed by the Invasive Trophoblast of the Equine (Equus caballus) Chorionic Girdle[END_REF][START_REF] Rossdale | A clinician's view of prematurity and dysmaturity in thoroughbred foals[END_REF]. Indeed, their pancreatic function was immature when evaluated post-natally [START_REF] Peugnet | Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses[END_REF]. In Allen et al. experiments, IUGR foals also had symptoms of dysmaturity [START_REF] Allen | Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero[END_REF].

IUGR was associated with lighter TFM and a smaller placenta, consistent with other work in horses and pigs [START_REF] Allen | Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero[END_REF][START_REF] Ashworth | Embryo survival and conceptus growth after reciprocal embryo transfer between Chinese Meishan and Version postprint Comment citer ce document[END_REF]. The lack of difference in placental efficiency (as estimated by the foal/TFM ratio, as used in other studies in horses and pigs [START_REF] Allen | Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero[END_REF][START_REF] Biensen | The impacts of uterine environment and fetal genotype on conceptus size and placental vascularity during late gestation in pigs[END_REF][START_REF] Veronesi | A comparative stereological study of the term placenta in the donkey, pony and Thoroughbred[END_REF]) between restricted foals and controls indicates that the placenta of Saddlebred foals was unable to overcome maternal uterine size constraints by structural or functional adaptations, which in turn prevented a possible dystocia.

In contrast to what was observed by others [START_REF] Allen | Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero[END_REF], trophoblast and microcotyledon surface density were reduced with fetal enhanced growth and increased in cases of IUGR.

The reduced density in cases of enhanced growth may reflect placental adaptations to limit fetal growth beyond a certain threshold. This could explain why P-D foals tended to have a lower placental efficiency compared to P-P foals. Moreover, the work of Constancia et al. in mice elegantly showed that the placenta will respond to fetal demand and adjust nutrient transfer [START_REF] Constância | Adaptation of nutrient supply to fetal demand in the mouse involves interaction between the Igf2 gene and placental transporter systems[END_REF]. In the current situation, however, the demand from the pony foetuses may not

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collected placental samples [START_REF] Allen | Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero[END_REF]. Here, placental structure was only analysed in one area, near the umbilical cord, to improve standardisation (in all the pregnancies, umbilical cord was attached at the base of the pregnant horn). This may be a major limitation of the current work as in humans, microcotyledonary development and gene expression vary between placental areas [START_REF] Bacon | Regional Anatomy of the Term Human Placenta[END_REF][START_REF] Boyd | Quantitative Structural Differences Within the Normal Term Human Placenta: A Pilot Study[END_REF][START_REF] Schuhmann | Regional Ultrastructural Differences in Placental Villi in Cotyledons of a Mature Human Placenta[END_REF][START_REF] Wyatt | The correlation between sampling site and gene expression in the term human placenta[END_REF], but so far this variability has not been explored in the horse. The difference in gene expression between placentas from enhanced growth and control foetuses can be considered as not biologically relevant as only one gene, SLC38A1, was decreased in P-D vs. P-P placentas but with a fold-change of x1.04. Nevertheless, term placentas only reflect the terminal result of the pregnancy and one cannot preclude that adaptations did not take place earlier in pregnancy as shown in response to adverse nutritional conditions in mice [START_REF] Sferruzzi-Perri | An obesogenic diet during mouse pregnancy modifies maternal nutrient partitioning and the fetal growth trajectory[END_REF]. Alternatively, foetal growth may have mainly been driven by the mechanical increase in placental size due to the increased uterine capacity.

In restricted S-P pregnancies, the expression of genes involved in growth and development (H19, IGF2, IGF1R and IGF2R) and nutrient transfer (SLC2A1, SLC38A2 and LPL) was reduced. These results are well associated with the lowest TFM weight and the growth restriction of the foals at birth. H19 and IGF2 are major regulators of placental and fetal growth, especially through IGF1R and IGF2R [START_REF] Fowden | Programming placental nutrient transport capacity[END_REF][START_REF] Gicquel | Hormonal Regulation of Fetal Growth[END_REF]. A decrease in their expression might then be linked with the IUGR in the S-P group. Moreover, the fact that H19, IGF2 and its receptor IGF2R are imprinted genes may have increased their susceptibility to be affected because of their monoallelic expression. As intrauterine growth of foals requires a high amount of glucose [START_REF] Fowden | Ontogenic and nutritionally induced changes in fetal metabolism in the horse[END_REF][START_REF] Fowden | Comparative aspects of fetal carbohydrate metabolism[END_REF] and as GLUT1 and GLUT3 are glucose transporters in the horse placenta [START_REF] Wooding | Separate Sites and Mechanisms for Placental Transport of Calcium, Iron and Glucose in the Equine Placenta[END_REF], a decrease in SLC2A1 placental expression might also be linked with a lower

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amount of glucose transferred to the foal for growth. These results might also be linked with the delayed pancreatic maturation observed in foals from S-P group, 3 days after birth [START_REF] Peugnet | Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses[END_REF].

Moreover, IGF2 is known to regulate the nutrient availability and to control the expression of glucose and amino acids transporters [START_REF] Fowden | Programming placental nutrient transport capacity[END_REF]. Thus, as observed in other species, the decrease in the expression of IGF2 is linked with the decrease of the expression of SLC2A1 and SLC38A1 [START_REF] Fowden | Programming placental nutrient transport capacity[END_REF]. Moreover, these results are in agreement with data in other species where IUGR has been associated with similar changes in humans, sheep and rodents [START_REF] Avagliano | Placental amino acids transport in intrauterine growth restriction[END_REF][START_REF] Fowden | The insulin-like growth factors and feto-placental growth[END_REF].

One main limitation of this work is that control embryos were produced using artificial insemination. Nevertheless, in horses, data collected from foals and placentas born after ET or in vivo produced embryos or artificial insemination did not demonstrate any difference in placental nor foal size and weight at birth [START_REF] Lanci | Preliminary descriptive study of equine placenta generated after transfer of in vivo and in vitro produced embryos[END_REF].

The other limitation is that ponies were not housed in the same farm as Saddlebred and draught mares. Nevertheless, they were all fed concentrates and hay in late gestation and their body weight and body condition remained constant throughout gestation [START_REF] Peugnet | Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses[END_REF]. Data from our own laboratory as well as others indicate that only severe restriction in maternal nutrition affect foal birthweight and placental size [START_REF] Peugnet | Effects of moderate amounts of barley in late pregnancy on growth, glucose metabolism and osteoarticular status of pre-weaning horses[END_REF][START_REF] Wilsher | Effects of a Streptococcus equi infection--mediated nutritional insult during mid-gestation in primiparous Thoroughbred fillies. Part 1: placental and fetal development[END_REF][START_REF] Thorson | Effect of selenium supplementation and plane of nutrition on mares and their foals: Foaling data[END_REF][START_REF] Robles | Effects of maternal nutrition in latepregnancy on placental structure and function and the neonatal foal's response to glucose[END_REF][START_REF] Banach | Effects of inadequate energy during gestation and lactation on the estrous cycle and conception rates of mares and on their foal weights[END_REF]. Thus, it may be considered that the effects of the different location may be negligible compared to effects due to differences in maternal and fetal size and breed.

Finally, age and parity are known to affect fetal and placental development with smaller foals and placentas being born to young primiparous mares [START_REF] Wilsher | The effects of maternal age and parity on placental and fetal development in the mare[END_REF]60]. Consequently, parity was always added to the statistical model. Results indicate that even though draught mares were significantly younger than Saddlebred and pony mares and that parity was higher in Saddlebred vs. pony and draught mares, foals born to draught mares were heavier with heavier placentas compared to the other groups. Moreover, when foal weights were corrected for parity (+0.7 kg per parity unit [60]), differences in foal birth weight remained statistically significant.

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Conclusion

Using ET between breeds of different size and weight, it was demonstrated that foal birthweight was directly related to placental weight and gross placental surface. Placental adaptations in restricted pregnancies were not sufficient to overcome the restriction imposed by the reduced maternal size, which also protected the mare and foal from severe risks of dystocia. Conversely, the reduced microcotyledonary exchange surface area in enhanced growth foetuses may also have prevented excess foetal oversize and dystocia.

Authors' declaration of interests

No competing interests have been declared.

Ethical animal research

Animal studies were approved by the local animal care and use committee and received ethical approval from the local ethics committee (approval No5-2013-5).

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Candidate gene Forward and reverse primers Accession number

Growth and development This article is protected by copyright. All rights reserved.

H19 F 5'-GGACCCCAAGAACCCTCAAG-3' NR_027326 R 5'-GGGACTTGAAGAAGTCCGGG-3' IGF2 F 5'-TTTCTTGGCTTTTGCCTCGT-3' NM_001114539.1 R 5'-CCTGCTGAAGTAAAAGCCGC-3' IGF1R F 5'-CGAGAAGACCACCATCAACAAC-3' XM_001489765.2 R 5'-TGGCAGCACTCGTTGTTCTC-3' IGF2R F 5'-GTCGGCTTGCCAGATGAGAT-3' XM_001491469.2 R 5'-TACTGATGGAGACGGCCTCA-3' TGFβ1 F 5'-TTGATGTCACCGGAGTCGTG-3' NM_001081849.1 R 5'-CCACGCGGAGTGTGTTATCT-3' EGFR F 5'-GTCTGGAAGTTTGCGGATGC-3' XM_001497730.2 R 5'-CTTGGGCCCATTTCTTGCAC-3' Nutrient transfer SLC2A1 F 5'-TGTGCTCATGACCATCGCC-3' NM_001163971.1 R 5'-AAGCCAAAGATGGCCACGAT-3' SLC38A1 F 5'-AAATGAACTACCCTCCGCCA -3' XM_001489402. R 5' ACACAGAGGGAGAATTATGCCAA-3' SLC38A2 F 5'-ACAGCTCGAACAGCGACTTCA-3' NM_001081849.1 R 5'-TTCTTCCCCAAATTCGATTCA-3' CD36 F 5'-GGTCTACGCCGTGTTTGGAG-3' XM_005609038.1 R 5'-CCGTGCAGAAGCAGTGGTTA-3' LPL F 5'-AGTTGGGTGCCAAAACTTGTG-3' XM_001489577.2 R 5'-GCTTGGTGTACCCCGCAGAC-3' Vascularization VEGFα F 5'-TACCTCCACCATGCCAAGTG -3' NM_001081821.1 R 5'-GTCTCGATTGGACGGCAGTA -3' Flt1 F 5'-AGTGTGAGCGGCTCCCTTATG-3' XM_003363176.1 R 5'-ATGCCAAATGCAGATGCTTG-3' KDR F 5'-CAGTGGGCTGATGACCAAGA-3' XM_001916946.2

  Comment citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses. Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761

  citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses. Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761

  maternal environment (P-P vs. P-D and S-P vs. S-S vs. S-D), age, dam parity and foal sex and feto-placental biometry parameters (foals' birthweight, TFM weight, placental surface area, foal birthweight/placental surface area ratio), volume fraction and surface density of all the components of the placenta (trophoblast, microcotyledonary vessels, microcotyledons, allantoic tissue vessels, allantoic connective tissue) and the placental expression of genes involved in growth and development (IGF2, IGF1R, IGF2R, TGFβ1, H19), nutrient transfer (SLC2A1, SLC38A1, SLC38A2, LPL, CD36) and vascularisation (VEGFA, FLT1, KDR, eNOS). The effect of the breeding season could not be studied because all S-D foals were born during breeding season 1 (2011), and only one P-D foal was born during breeding season 2 (2012). Effects were considered significant when p≤0.05.

  Comment citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses. Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761

  Comment citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses. Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761

P

  -D versus P-P controls: The relative trophoblastic surface density was significantly decreased in P-D vs. controls (p = 0.03) and the microcotyledonary surface density tended to be reduced in P-D (p = 0.056).S-P versus S-S controls:The relative surface density and volume fraction of microcotyledonary vessels were significantly increased in S-P vs. controls (p = 0.01 and p = 0.03, respectively). The microcotyledonary surface density tended to be increased in S-P compared to controls (p = 0.058).S-D versus S-S controls:The relative surface density of the allantoic vessels was reduced in S-D compared to S-S placentas (p = 0.05). The relative surface density of microcotyledonary vessels and trophoblast were reduced in S-D compared to S-P (p<0.01, p = 0.02, respectively), resulting in reduction of the microcotyledonary surface density in S-D versus S-P (p = 0.01).

  The expression of H19, IGF2, IGF1R and IGF2R was decreased in S-P vs. S-S placentas (fold-change = 0.71, p = 0.03; fold-change = 0.53, p<0.01; fold-change = 0.67, p<0.01; fold-change = 0.62, p = 0.05, respectively). In S-P placentas, the expression of H19 and IGF2 was reduced compared to S-D placentas (foldchange = 0.56, p = 0.02 and fold-change = 0.63, p = 0.01, respectively). Nutrient transfer: The expression of SCL38A1 was increased in P-D vs. P-P (foldchange = 1.04, p = 0.02). The expression of SLC2A1, SLC38A2 and LPL was decreased in S-P vs. S-S (fold-change = 0.71, p = 0.01; fold-change = 0.58, 0.03; fold-change = 0.71, p = 0.02, respectively). Finally, CD36 gene expression was not different between groups. Comment citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses. Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761

Vascularisation:

  KDR gene expression was increased in S-D compared to S-S placentas (fold-change = 1.34, p = 0.04). The expression of FLT1 and KDR was reduced in S-P vs. S-D (fold-change = 0.76, p = 0.03; fold-change = 0.70, p = 0.05, respectively).

  Comment citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses. Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761

  have been sufficient to increase placental capacity. Conversely, the increased exchange surface in restricted Saddlebred foetuses may have improved nutrient supply to support fetal development. In their embryo transfer experiments, Allen et al. observed increased surface density in case of enhanced growth and reduced density in case of IUGR, using 10 randomly Comment citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses. Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761

  The sampling in only one part of the placenta may thus not represent the overall placental structure and function. Moreover, both trophoblasts (haemotrophic and histotrophic trophoblasts) have been taken into account for the calculation of the relative surface and volume of the microcotyledons, unlike in the work of Allen et al. This difference of measure might also explain the difference of results between both studies.

  Comment citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses. Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761

  Comment citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses. Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761

Fig 3 :

 3 Fig 3: Structure of the horse placenta at term. AC: Allantoic connective tissue, AV: Allantoic

Fig 4 :

 4 Fig 4: Foal birth weight (A), total fetal membranes (TFM) weight (B), placental surface area

Fig 5 :

 5 Fig 5: Surface density and volume fraction of the different components of the placenta. P-P

(n = 28 )

 28 : Saddlebred-in-Saddlebred; S-D (n = 8): Saddlebred-in-Draught. Different letters indicate p-value ≤0.05.

Fig 6 :

 6 Fig 6: Calibrated normalised relative quantity (arbitrary unit) for differentially expressed

b

  Comment citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses. Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761 ccepted ArticleThis article is protected by copyright. All rights reserved.

R 5 ' 3

 53 -TCCACCGAAGATTCCATGCC-3' eNOS F 5'-TTCGGGAGAGTGAGCTGGTA-3' XM_001504650.Version postprint Comment citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses. Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761 ccepted ArticleThis article is protected by copyright. All rights reserved.

3 '

 3 Comment citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses. Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761 ccepted Article

Table 1 :

 1 Number of pregnancies with maternal age and withers height (median [quartile 1-

	quartile 3], parity and gestational length within the six groups for each experimental year
	(2011 and 2012).
	Different letters inside each sub-table indicate p-value≤0.05.

Table 2 :

 2 Gene-specific primers and accession number or reference.

Table 3 :

 3 Gene expression within the 6 groups of placentas. A. Calibrated normalised relative Comment citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses.

	quantity (CNRQ) for differentially expressed genes. Data are presented as median [quartile 1
	-quartile 3].

Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761 ccepted Article This article is protected by copyright. All rights reserved.

Table 1 . Number of pregnancies with maternal age and withers height (median [quartile 1-quartile 3], parity and gestational length within the six groups for each experimental year (2011 and 2012).

 1 Different letters inside each sub-table indicate p-value<0.05.

	Mares and recipient mares

b S-P 8.6 [4.6-9.3]

Table 2 . Gene-specific primers and accession number or reference.

 2

Table 3 .

 3 Gene expression within the 6 groups of placentas. A. Calibrated normalized relative quantity (CNRQ) for differentially expressed genes. Data are presented as median [quartile 1quartile 3].

	CNRQ

This article is protected by copyright. All rights reserved.

Acknowledgments

The authors are grateful to Joseph Bellonie, Patrice Dupuis, Philippe Barrière, Thierry Gascogne, Thierry Blard, Yvan Gaudé and François Stieau for care and management of the mares and foals.

Source of funding

This work was funded through a grant from the Institut Français du Cheval et de l'Equitation (IFCE) under the grant name "FOETALIM" and through funding from INRA Dept of Physiology and Breeding Systems. Version postprint Comment citer ce document : Robles, M. (Auteur de correspondance), Peugnet, P. M., Valentino, S. A., Dubois, C., Dahirel, M., Aubrière, M.-C., Reigner, F., Serteyn, D., Wimel, L., Tarrade, A., Chavatte Palmer, P. (2018). Placental alterations in structure and function in intra-uterine growth-retarded horses. Equine Veterinary Journal, 50 (3), 405-414 . , DOI : 10.1111/evj.12761

Accepted Article

This article is protected by copyright. All rights reserved. http://www.ncbi.nlm.nih.gov/pubmed/12875326. 60. Elliott, C., Morton, J. and Chopin, J. (2009) Factors affecting foal birth weight in Thoroughbred horses. Theriogenology 71, 683-689.

Accepted Article

This article is protected by copyright. All rights reserved.

Accepted Article

This article is protected by copyright. All rights reserved.

Supplementary information

Supplementary Item 1: Nutritional values of pregnant mares' diets.

Supplementary Item 2: Co-factors associated with foal birthweight and placental biometry, morphometry and gene expression.

Supplementary Item 3: Histological sections of term allantochorion.

Figure and table legends


IRHS Pauline Lasserre-Zuber

Valérie

René Stievenard

Arnaud Lemarquand

Valérie Caffier
email: valerie.caffier@inra.fr

Pyramiding Quantitative Resistance with a Major Resistance Gene in Apple: from Ephemeral to Enduring Effectiveness in Controlling Apple Scab

Keywords: apple, Malus x domestica, scab, Venturia inaequalis, durability, sustainability, Quantitative Trait Locus, major resistance gene, pyramiding

Genetic resistance is a useful strategy to control plant disease, but its effectiveness may be reduced over time due to the emergence of pathogens able to circumvent the defences of the plant.

However, the pyramiding of different resistance factors in the same plant can improve the effectiveness and durability of the resistance. To investigate the potential for this approach in apple to control scab disease we surveyed scab incidence in two experimental orchards located more than 300 km apart planted with apple genotypes carrying quantitative resistance and major gene resistance alone or in combination. Our results showed that the effectiveness of pyramiding in controlling scab was dependent on the site and could not be completely explained by the effectiveness level of the resistances alone.

Comment citer ce document : Lasserre-Zuber, P., Caffier, V., Stievenard, S., Lemarquand, A., Le Cam, B., Durel, C. E. (2018). Pyramiding quantitative resistance with a major resistance gene in apple: from ephemeral to enduring effectiveness in controlling scab. Plant Disease, 102 (11), 2220-2223. , DOI : 10.1094/PDIS-11-17-1759-RE

Valérie Caffier 3 Plant Disease

The control of plant diseases by chemical products presents risks to human health and the ecological environment. Furthermore, these treatments can be ineffective if they are not used at the appropriate time or if the pathogens adapt and become resistant. Plant genetic resistance constitutes an environmentally friendly way to control disease and to reduce chemical inputs. Plant resistance can be described by two traits: (1) its phenotype with either a complete or partial level of resistance, and (2) its mode of heredity with either a continuous or a bimodal distribution between resistant and susceptible phenotypes in a progeny [START_REF] Niks | Quantitative resistance to biotrophic filamentous plant pathogens: concepts, misconceptions, and mechanisms[END_REF]. Plant resistance is generally classified in two extreme categories: qualitative resistance conditioned by a single gene (i.e. major gene) and quantitative resistance conditioned by multiple genes of partial effect (i.e. Quantitative Trait Locus = QTL), with possibilities of intermediate types of resistances between these two categories [START_REF] Poland | Shades of gray: the world of quantitative disease resistance[END_REF]Stuthman et al. 2007). Genetically homogeneous crops with qualitative resistance induce strong directional selection on pathogen populations when they are deployed at a large scale and can result in resistance breakdown (Bus et al. 2011;[START_REF] Peressotti | Breakdown of resistance to grapevine downy mildew upon limited deployment of a resistant variety[END_REF][START_REF] Rouxel | A 10-year survey of populations of Leptosphaeria maculans in France indicates a rapid a daptation towards the Rlm1 resistance gene of oilseed rape[END_REF]. Quantitative resistance has been frequently considered to be more durable than qualitative resistance [START_REF] Lindhout | The perspectives of polygenic resistance in breeding for durable disease resistance[END_REF][START_REF] Parlevliet | Durability of resistance against fungal, bacterial and viral pathogens; present situation[END_REF]Stuthman et al. 2007). However, it was postulated that quantitative resistance might also exert selection on pathogen populations [START_REF] Gandon | Evolution of parasite virulence against qualitative or quantitative host resistance[END_REF][START_REF] Parlevliet | The integrated concept of disease resistance: a new view including horizontal and vertical resistance in plants[END_REF]. Indeed, it was shown experimentally in several pathosystems that quantitative resistance can also be eroded, for instance for Mycosphaerella graminicola on wheat [START_REF] Cowger | Aggressiveness of Mycosphaerella graminicola isolates from susceptible and partially resistant wheat cultivars[END_REF], lettuce mosaic virus on lettuce [START_REF] Pink | Novel pathotypes of lettuce mosaic virus -breakdown of a durable resistance?[END_REF], Phytophthora infestans on potato [START_REF] Andrivon | Adaptation of Phytophthora infestans to partial resistance in potato: Evidence from French and Moroccan populations[END_REF][START_REF] Montarry | Adaptation to the most abundant host genotype in an agricultural plant-pathogen system -potato late blight[END_REF], Plasmopara viticola on grape [START_REF] Delmas | Adaptation of a plant pathogen to partial host resistance: selection for greater aggressiveness in grapevine downy mildew[END_REF][START_REF] Delmotte | Rapid and multiregional adaptation to host partial resistance in a plant pathogenic oomycete: evidence from European populations of Plasmopara viticola, the causal agent of grapevine downy mildew[END_REF] and Venturia inaequalis on apple [START_REF] Caffier | Erosion of quantitative host resistance in the apple -Venturia inaequalis pathosystem[END_REF][START_REF] Caffier | Slow erosion of a quantitative apple resistance to Venturia inaequalis based on an isolate-specific Quantitative Trait Locus[END_REF]). As a consequence, both types of resistance may be eroded or overcome more or less rapidly according to the evolutionary potential of the pathogen, the host characteristics and the agronomic practices. It is therefore necessary to develop strategies of resistance gene management that can improve the durability of resistance [START_REF] Fabre | Durable strategies to deploy plant resistance in agricultural landscapes[END_REF][START_REF] Mundt | Durable resistance: a key to sustainable management of pathogens and pests[END_REF][START_REF] Sapoukhina | Spatial deployment of gene-for-gene resistance governs evolution and spread of pathogen populations[END_REF][START_REF] Sapoukhina | Quantitative plant resistance in cultivar mixtures: wheat yellow rust as a modeling case study[END_REF].

Pyramiding genes of resistance in one cultivar is one of these strategies and is now feasible by marker-assisted breeding [START_REF] Pilet-Nayel | Quantitative resistance to plant pathogens in pyramiding strategies for durable crop protection[END_REF][START_REF] Pink | Strategies using genes for non-durable disease resistance[END_REF]. Several studies have demonstrated that combining resistance factors increases resistance effectiveness, either by cumulating major genes [START_REF] Hittalmani | Fine mapping and DNA markerassisted pyramiding of the three major genes for blast resistance in rice[END_REF]Tan et al. 2010;[START_REF] Koide | Development of pyramided lines with two resistance genes, Pish and Pib, for blast disease (Magnaporthe oryzae B. Couch) in rice (Oryza sativa L.)[END_REF], QTL [START_REF] Castro | Pyramiding quantitative trait locus (QTL) alleles determining resistance to barley stripe rust: Effects on resistance at the seedling stage[END_REF][START_REF] Hamon | A complex genetic network involving a broad-7 spectrum locus and strain-specific loci controls resistance to different pathotypes of Aphanomyces 8 euteiches in Medicago truncatula[END_REF][START_REF] Laloi | Efficiency of pyramiding of three quantitative resistance loci to apple scab[END_REF][START_REF] Richardson | Pyramiding and dissecting disease resistance QTL to barley stripe rust[END_REF] or major genes and QTL [START_REF] Eibach | The use of molecular markers for pyramiding resistance genes in grapevine breeding[END_REF]Tabien et al. 2002). It has been experimentally observed that combinations of major genes and QTL can increase both resistance effectiveness and durability, for instance for Leptosphaeria maculans on rapeseed [START_REF] Plant Disease Delourme | Quantitative resistance affects the speed of frequency increase but not the diversity of the virulence alleles overcoming a major resistance gene to Leptosphaeria maculans in oilseed rape[END_REF], Microcyclus ulei on rubber [START_REF] Le Guen | A rubber tree's durable resistance to Microcyclus ulei is conferred by a qualitative gene and a major quantitative resistance factor[END_REF], Globodera pallida on potato [START_REF] Fournet | Selection of nematodes by resistant plants has implications for local adaptation and cross-virulence[END_REF], Meloidogyne species on pepper [START_REF] Barbary | The plant genetic background affects the efficiency of the pepper major nematode resistance genes Me1 and Me3[END_REF] [START_REF] Palloix | Durability of plant major resistance genes to pathogens depends on the genetic background, experimental evidence and consequences for breeding strategies[END_REF][START_REF] Disease Quenouille | Quantitative trait loci from the host genetic background modulate the durability of a resistance gene: a rational basis for sustainable resistance breeding in plants[END_REF]. Indeed, major genes are totally effective to control the avirulent fraction of the pathogen population, whereas QTL reduce the pathogen population size, which limits the selection for virulent isolates and maintains a partial effectiveness of the resistance towards the virulent isolates [START_REF] Plant Disease Delourme | Quantitative resistance affects the speed of frequency increase but not the diversity of the virulence alleles overcoming a major resistance gene to Leptosphaeria maculans in oilseed rape[END_REF]. Theoretical studies indicate that durability of cultivars pyramiding resistances will depend on the way that the resistance genes are employed [START_REF] Bourget | Timing of pathogen adaptation to a multicomponent treatment[END_REF]. If each resistance gene is used separately, concomitantly or before the use of cultivars pyramiding resistances, then the pathogen will be able to overcome the resistance by incrementally stacking virulences [START_REF] Ayliffe | Durable resistance to wheat stem rust needed[END_REF][START_REF] Pink | Strategies using genes for non-durable disease resistance[END_REF]Stuthman 2002) either by independent successive gain of virulence in the same strain or by sexual recombination between mono-virulent strains. It should be noted that the durability of resistance pyramiding may depend on the genetic structure of the pathogen populations and ability of pathogen populations carrying single mutations to recombine, but this point has not yet been explored experimentally.

Here we investigated the effectiveness and durability of combining one major gene with two QTL in the apple scab pathosystem. Apple scab, caused by the fungus Venturia inaequalis, is one of the most important diseases in commercial orchards in all temperate countries, because it strongly reduces fruit quality which is vital for fresh market sales. Disease control today generally depends on the use of fungicides, spraying up to 20 treatments per year [START_REF] Sauphanor | Analyse comparative de différents systèmes en arboriculture fruitière[END_REF]). The perennial trait of apple necessitates genotypes that can maintain resistance over many years. Major resistance genes have been identified in wild or domesticated apple species (Bus et al. 2011;[START_REF] Gessler | Venturia inaequalis resistance in apple[END_REF][START_REF] Soufflet-Freslon | Inheritance studies of apple scab resistance and identification of Rvi14, a new major gene that acts together with other broadspectrum QTL[END_REF]. The Rvi6 (= Vf) major resistance gene has been widely used in apple breeding programs during the last 50-60 years [START_REF] Laurens | Review of the current apple breeding programmes in the world: objectives for scion cultivar improvement[END_REF], but this monogenic resistance has been overcome by the pathogen (Bus et al. 2011;[START_REF] Leroy | When virulence 9 originates from nonagricultural hosts: evolutionary and epidemiological consequences of introgressions following secondary contacts in Venturia inaequalis[END_REF]. Many QTL, including specific and broad-spectrum QTL, have also been identified [START_REF] Calenge | Quantitative trait loci (QTL) analysis reveals both broad-spectrum and isolate-specific QTL for scab resistance in an apple progeny challenged with eight isolates of Venturia inaequalis[END_REF][START_REF] Durel | Genetic dissection of partial resistance to race 6 of Venturia inaequalis in apple[END_REF]Lê Van et al. 2013 ;Liebhard et al. 2003a;[START_REF] Soufflet-Freslon | Inheritance studies of apple scab resistance and identification of Rvi14, a new major gene that acts together with other broadspectrum QTL[END_REF]. The combination of two broad-spectrum resistance QTL, F11 and F17, has already been shown to be eroded in experimental trials [START_REF] Caffier | Erosion of quantitative host resistance in the apple -Venturia inaequalis pathosystem[END_REF]). Thus, our aim was to evaluate the utility of pyramiding Rvi6 with QTL F11 and F17, in two unsprayed orchards where resistance effectiveness of these QTL and this major gene has already been challenged.

Materials and Methods

Plant material. A cross between cv. 'Prima' and cv. 'Fiesta' was carried out by the former CPRO-DLO, which is now part of Wageningen-UR Plant Breeding, The Netherlands [START_REF] Maliepaard | Aligning male and female linkage maps of apple (Malus pumila Mill.) using multi-allelic markers[END_REF]. These progeny (referred to as the "J progeny") segregate for two major genes, Rvi1 (= Vg) and Rvi6 (= Vf), and two broad-spectrum QTL, since cv. 'Prima' carries Rvi1 and Rvi6 [START_REF] Durel | Localisation of a major gene for apple scab resistance on the European genetic map of the Prima x Fiesta cross[END_REF][START_REF] Maliepaard | Aligning male and female linkage maps of apple (Malus pumila Mill.) using multi-allelic markers[END_REF]) and cv. 'Fiesta' carries two QTL on linkage groups 11 and 17 (referred to as F11 and F17 in the present study) [START_REF] Durel | An overview of the position and robustness of scab resistance QTLs and major genes by aligning genetic maps of five apple progenies[END_REF][START_REF] Durel | Genetic dissection of partial resistance to race 6 of Venturia inaequalis in apple[END_REF]Liebhard et al. 2003b). Using available molecular markers for the initial and curated marker positions published in [START_REF] Maliepaard | Aligning male and female linkage maps of apple (Malus pumila Mill.) using multi-allelic markers[END_REF] and Schouten et al. ( 2012) respectively, we selected 22 apple genotypes that carried either no favourable allele for QTL or for Rvi6 (Class-0), two favorable alleles for both QTL F11 and F17 only (Class-QTL), favourable allele for Rvi6 only (Class-R), and favorable alleles for F11, F17 and Rvi6 (Class-RQTL), from the 161 available progenies. As Rvi1 is largely overcome in V. inaequalis populations, we considered that this major gene had no effect, and that the classes with Rvi1 and Rvi6 represented the effect of Rvi6. The number of genotypes per combination varied from five to seven (Table 1).

Orchard network. During winter 2003During winter -2004, each genotype was planted in two experimental orchards located more than 300 km apart, representing two different environments and climates (Angers in north-western France, and Villeneuve d'Ascq in the north of France, later referred to as "Villeneuve"). In each orchard, three replicates per genotype were planted in a randomised threeblock design after grafting onto the apple rootstock, 'Pajam 2'. The distance between the trees on a row was 1.5 m in Villeneuve and 1 m in Angers; the distance between the rows was 4 m. Pesticides were applied when necessary, but no fungicide treatment was applied against scab.

Disease assessment. Scab incidence was scored on each tree in June after the period of primary infection, in 2006-2008 at both sites. In Villeneuve, data from 2007 could not be interpreted because of a very low level of disease on the susceptible trees. In Angers, where the orchard was maintained after the end of the project, an additional assessment was carried out in 2012. On each tree, disease incidence was scored using an ordinal scale adapted from [START_REF] Lateur | Screening fruit tree genetic resources in Belgium for disease resistance and other desirable characters[END_REF].

This scale consists of nine levels, described as intervals around the following values: 0%, 1%, 3%, 12.5%, 25%, 37.5%, 50%, 75% and 90% of scabbed leaves.

Statistical analysis.

All statistical analyses were performed on the variable "disease incidence". Within each site and each year, the four classes of genotypes were compared for their disease incidence using a linear mixed effect model LME [START_REF] Pinheiro | Mixed-effects models in S and S-PLUS[END_REF]. The 'genotype' was treated as a random factor nested in the class of genotypes, and 'block' as a fixed factor. The model was fitted by maximizing the log-likelihood and the best model was selected using the Bayesian Information Criterion after including each factor ('class of genotypes' and 'block') through an ascendant selection [START_REF] Pinheiro | Mixed-effects models in S and S-PLUS[END_REF]. The data were transformed into arcsine square root to normalise and homogenise the residuals. The resistance of Class-R, Class-QTL or Class-RQTL was considered as effective if "disease incidence" was significantly different from "disease incidence" of Class-0. All statistical analyses were performed with R software, version 3.1.2 (R Core Team 2016), 1 using the 'nlme' package [START_REF] Pinheiro | Nlme: linear and nonlinear mixed effects models[END_REF]. 2 3

Results

4

In both sites, the level of disease on the control (Class-0) was high with fluctuations from year 5 to year ranging from 51% to 83% scabbed leaves. In Angers, the resistance based on Rvi6 (Class-R) 6 was completely effective in 2006 with no scabbed leaves (fig. 1a). This resistance was still effective in 7

2007 but a higher level of disease was observed (22% scabbed leaves). This resistance became 8 ineffective by 2008 (67% scabbed leaves). The QTL combination F11F17 (Class-QTL) significantly 9 reduced the disease each year compared to Class-0, even if the level of control decreased slowly over time (not shown in the present study, see [START_REF] Caffier | Erosion of quantitative host resistance in the apple -Venturia inaequalis pathosystem[END_REF]. In 2006, the pyramiding of Rvi6 and F11F17 (Class-RQTL) was completely effective with no scabbed leaves, due to the effectiveness of Rvi6. In 2007, this pyramiding was significantly more effective (6% scabbed leaves) than the resistance factors Rvi6 (Class-R with 22% scabbed leaves) or F11F17 (Class-QTL with 55% scabbed leaves) alone. In 2008 and 2012, the resistance of Class-RQTL was still effective with 20% and 64% scabbed leaves respectively, compared to 75 and 83% for Class-0. However, the added value of Rvi6 was lost from 2008, while the effect of F11F17 remained with no significant difference between Class-RQTL and Class-QTL in 2008 and in 2012. By contrast, in Villeneuve, both the major resistance Rvi6 (Class-R) and the QTL combination F11F77 (Class-QTL) were ineffective from the beginning of the experiment with more than 40% scabbed leaves. However, the apple genotypes carrying the pyramiding of Rvi6 and F11F17 (Class-RQTL) were significantly less diseased than Class-0 with less than 20% scabbed leaves for the two observed years 2006 and 2008 (fig. 1b).

Discussion

We evaluated the effectiveness of pyramiding quantitative resistance with a major resistance gene in two contrasting situations: one site (Angers) where the resistance factors alone were progressively overcome during the experiment trial and one site (Villeneuve) where the resistance factors alone were already overcome when the experiment started [START_REF] Caffier | Erosion of quantitative host resistance in the apple -Venturia inaequalis pathosystem[END_REF]. Our results showed the effectiveness of pyramiding resistances for controlling scab in both sites, but to a different extent depending on the site. In Angers, the added value of pyramiding quantitative resistance with a major gene to control scab was ephemeral and disappeared when the level of disease control based on resistance factors alone decreased, whereas in Villeneuve, the pyramiding (Class-RQTL) remained effective both in 2006 and 2008, even if the resistance factors alone where not effective compared to Class-0. These results agree with other studies suggesting pyramiding QTL with major resistance 1 genes improves effectiveness and increases durability of resistance [START_REF] Plant Disease Delourme | Quantitative resistance affects the speed of frequency increase but not the diversity of the virulence alleles overcoming a major resistance gene to Leptosphaeria maculans in oilseed rape[END_REF]Le Guen et 2 al. 2011;[START_REF] Disease Quenouille | Quantitative trait loci from the host genetic background modulate the durability of a resistance gene: a rational basis for sustainable resistance breeding in plants[END_REF]. The effectiveness of pyramiding may be due to the low probability 3 of a pathogen strain to accumulate independent mutations necessary to overcome all resistance 4 genes in the pyramid [START_REF] Mundt | Durable resistance: a key to sustainable management of pathogens and pests[END_REF], the reduction in the pathogen size population or the 5 diversification in the selection pressures exerted on the pathogen population (Pilet-Nayel et al. 6 2017). It is surprising however that the added value of pyramiding was maintained in Villeneuve 7 whereas it was lost in Angers. Indeed, at the Villeneuve site, the effectiveness of pyramiding the Rvi6 8 gene with QTL was observed although apple genotypes with single non-effective resistance factors 9 (Class-R or Class-QTL) were planted in the same orchard, which putatively facilitates stacking pathogenicity loci by mating. In such a situation, we could have expected disease control associated with pyramiding to decrease or be completely lost over time [START_REF] Ayliffe | Durable resistance to wheat stem rust needed[END_REF][START_REF] Pink | Strategies using genes for non-durable disease resistance[END_REF]Stuthman 2002), which was not observed. We assume that the effectiveness of resistance observed at this site was probably due to a lack of recombination between pathogen populations growing on class-R and populations growing on class-QTL. Indeed, a strong genetic differentiation was observed in the northwest of France between populations virulent towards Rvi6 and populations avirulent towards Rvi6 [START_REF] Gladieux | 2 Emergence of novel fungal pathogens by ecological speciation: importance of the reduced viability of 3 immigrants[END_REF]. At the Angers site, the improved resistance associated with pyramiding instead of single resistance factors was also observed in 2007 at the beginning of Rvi6 breakdown, but was lost from 2008, with the level of disease control associated with pyramiding QTL and the major gene (Class-RQTL) becoming similar to the level of disease control observed with the QTL (Class-QTL) alone. At this site, [START_REF] Leroy | When virulence 9 originates from nonagricultural hosts: evolutionary and epidemiological consequences of introgressions following secondary contacts in Venturia inaequalis[END_REF] showed a low genetic differentiation between populations virulent towards Rvi6 and populations avirulent towards Rvi6 due to recombination between the two populations. Such a recombination may have resulted in the development of strains that were well adapted to all resistance factors incorporated in the pyramiding. Our results suggest that the effectiveness of pyramiding resistance genes not only depends on the way the resistance genes are used [START_REF] Ayliffe | Durable resistance to wheat stem rust needed[END_REF][START_REF] Bourget | Timing of pathogen adaptation to a multicomponent treatment[END_REF][START_REF] Pink | Strategies using genes for non-durable disease resistance[END_REF]Stuthman 2002) but also on the capacity of pathogen populations carrying virulence and aggressiveness traits to recombine. We assume that the more gene flow that occurs between populations able to overcome different resistances the more quickly the resistance of the pyramiding will be eroded.

Even in case of ephemeral effectiveness, these results are promising for the development of environmentally friendly strategies to control apple scab and to reduce chemical treatments. Indeed, in a previous study, [START_REF] Didelot | Sustainable management of scab control through the integration of apple resistant cultivars in a low-fungicide input system[END_REF] showed that the breakdown of the major resistance Rvi6 by virulent isolates could be delayed by the removal of leaf litter in autumn associated with spraying of fungicides in case of moderate or high risks of scab infection. We can expect that the association of all these control methods, i.e. pyramiding of quantitative resistance with major resistance genes,

Figure 1 .9

 1 Figure Legend

  and Potato

	virus Y on pepper
	Valérie Caffier	4
	Plant Disease	Comment citer ce document :
	Lasserre-Zuber, P., Caffier, V., Stievenard, S., Lemarquand, A., Le Cam, B., Durel, C. E.
	(2018). Pyramiding quantitative resistance with a major resistance gene in apple: from ephemeral to
	enduring effectiveness in controlling scab. Plant Disease, 102 (11), 2220-2223. , DOI :
		10.1094/PDIS-11-17-1759-RE

  Comment citer ce document : Lasserre-Zuber, P., Caffier, V., Stievenard, S., Lemarquand, A., Le Cam, B.,Durel, C. E. (2018). Pyramiding quantitative resistance with a major resistance gene in apple: from ephemeral to enduring effectiveness in controlling scab. Plant Disease, 102 (11), 2220-2223. , DOI : 10.1094/PDIS-11-17-1759-RE and chemical treatment applied only in case of great risk of scab infection, will provide a sustainable management of disease in a low-fungicide input system, which is crucial for a perennial plant.Lê Van, A., Caffier V., Lasserre-Zuber P., Chauveau A., Brunel D., Le Cam B.,Durel C., 2013. 1 Differential selection pressures exerted by host resistance quantitative trait loci on a pathogen 2 population: a case study in an apple x Venturia inaequalis pathosystem. New 4 Lemaire, C., De Gracia M.,Leroy T., Michalecka M., Lindhard-Pedersen H., Guerin F., Gladieux P., Le Stuthman, D.D., 2002. Contribution of durable disease resistance to sustainable agriculture.Stuthman, D.D., Leonard K.J., Miller-Garvin J., 2007. Breeding crops for durable resistance to disease.

	Euphytica 124:253-258.
	Adv. Agron. 95:319-367.
	Valérie Caffier Valérie Caffier Valérie Caffier	8 12 15
	Plant Disease Plant Disease Plant Disease

sanitation Comment citer ce document : Lasserre-Zuber, P., Caffier, V., Stievenard, S., Lemarquand, A., Le Cam, B., Durel, C. E. (2018). Pyramiding quantitative resistance with a major resistance gene in apple: from ephemeral to enduring effectiveness in controlling scab. Plant Disease, 102 (11), 2220-2223. , DOI : 10.1094/PDIS-11-17-1759-RE Comment citer ce document : Lasserre-Zuber, P., Caffier, V., Stievenard, S., Lemarquand, A., Le Cam, B., Durel, C. E. (2018). Pyramiding quantitative resistance with a major resistance gene in apple: from ephemeral to enduring effectiveness in controlling scab. Plant Disease, 102 (11), 2220-2223. , DOI : 10.1094/PDIS-11-17-1759-RE

Plant Disease "First Look" paper • http://dx.doi.org/10.1094/PDIS-11-17-1759-RE • posted 04/25/2018 This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.

Acknowledgements

We thank Wageningen-UR Plant Breeding for the availability of the apple progeny of cv. 'Prima' x cv.

'Fiesta' and especially Eric van de Weg for the sharing/use of molecular marker data for this family.

We thank Eric van de Weg and Jason Shiller for helpful comments on the manuscript. We thank Mathieu Lascostes from CRRG for his contribution to the planting of the experimental orchard at Villeneuve d'Ascq and the phenotyping. We thank Philippe Billy, Christophe Brouard, Serge Brunet, Christophe Vilfroy from INRA Experimental Unit UEH for participating to the phenotyping and taking care of the experimental orchard located in Angers. We thank Abou Kouassi and Amidou N'Diaye for their contribution in the phenotyping during their post-doc stay at INRA-Angers. We also thank Fabienne Mathis and Caroline Denancé from IRHS for complementary genotyping. This work was funded by the CTPS programme "Durabilité des résistances variétales à des bioagresseurs 2006-2008" and the ANR grant, "ANR-05-PADD-05, CEDRE".

Version preprint

Comment citer ce document : Lasserre-Zuber, P., Caffier, V., Stievenard, S., Lemarquand

Resistance factors

Class-0

Class-R

a Absence of the resistance factor b Presence of the resistance factor c Apple progenies of the cross between cv. 'Prima' and cv. 'Fiesta'