Louise Chantepie

Loys Bodin

Julien Sarry

Florent Woloszyn

Julien Ruesche

Laurence Drouilhet

Stéphane Fabre
email: stephane.fabre@inra.fr

Presence of causative mutations affecting prolificacy in the Noire du Velay and Mouton Vendéen sheep breeds

Keywords: ovine, major gene, prolificacy, BMP15, B4GALNT2

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

In ovine breeds raised for meat purposes, numerical productivity represents an important technical and economic lever. The objective is to reach an optimum for the economic profitability of breeding. Improvement of this numerical productivity is achieved by increasing the number of lambs born per ewe at each lambing, i.e. the prolificacy, associated with the improvement of lamb viability as well as the maternal quality. This leads to increased post-natal survival and growth rate of the lambs. For decades, genetic selection efforts have been made particularly on improving prolificacy of sheep breeds. However, prolificacy is a weakly heritable polygenic trait (h 2 = 0.05 -0.2) (see the review by Bradford [START_REF] Bradford | Selection for litter size[END_REF]), allowing limited genetic gain.

Nevertheless in some breeds, a very large effect on ovulation rate (OR) and litter size (LS) due to single mutation in fecundity major genes (called Fec genes, reviewed in [START_REF] Fabre | Regulation of ovulation rate in mammals: contribution of sheep genetic models[END_REF]) has been demonstrated. The first evidence of the segregation of a prolificacy major gene was established in the early 1980's in Australian Booroola Merino. This was implicated by the observation of a large variability of LS and OR in this population and the presence of extremely prolific ewes in this low prolific breed [START_REF] Piper | Genetic segregation for fecundity in Booroola Merino sheep[END_REF][START_REF] Davis | Segregation of a major gene influencing fecundity in progeny of Booroola sheep[END_REF]. The causal mutation named FecB B was discovered 20 years later in the BMPR1B gene (Bone Morphogenetic Protein Receptor 1B) on the ovine chromosome 6 by several independent research groups (Wilson et al., 2001;[START_REF] Mulsant | Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes[END_REF][START_REF] Souza | The Booroola (FecB) phenotype is associated with a mutation in the bone morphogenetic receptor type 1 B (BMPR1B) gene[END_REF]. This mutation was thereafter introgressed in several ovine breeds around the world for research purposes or to improve their prolificacy although these latter programmes resulted in mixed outcomes (Walkden-Brown et al., 2009).

Up to now, many mutations were discovered worldwide in three other major genes namely BMP15 (known as FecX [START_REF] Galloway | Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner[END_REF]), GDF9 (known as FecG [START_REF] Hanrahan | Mutations in the genes for oocyte-derived growth factors GDF9 and BMP15 are associated with both increased ovulation rate and sterility in Cambridge and Belclare sheep (Ovis aries)[END_REF]) and B4GALNT2 (known as FecL [START_REF] Drouilhet | The Highly Prolific Phenotype of Lacaune Sheep Is Associated with an Ectopic Expression of the B4GALNT2 Gene within the Ovary[END_REF]). In France particularly, two genetic programmes were implemented to discover and to manage mutations with major effect in order to improve the prolificacy of commercial sheep populations [START_REF] Mulsant | Prolificacy genes in sheep: the French genetic programmes[END_REF][START_REF] Bodin | Etat des lieux et gestion pratique des gènes d'ovulation détectés dans les races ovines françaises[END_REF][START_REF] Martin | Effects of the FecL major gene in the Lacaune meat sheep population[END_REF] . The introgression of the Booroola FecB B mutation was started in Mérinos d'Arles in the 1980's. Experimental testing by the French agricultural institute INRA has estimated the effect of the mutated allele on prolificacy at one extra lamb per lambing [START_REF] Teyssier | Interet zootechnique du gene Booroola en race Mérinos d'Arles. In 4èmes Rencontres Recherches Ruminants[END_REF]. A controlled diffusion of genotyped animals in commercial flocks is now implemented in the Mérinos d'Arles population [START_REF] Teyssier | Biological and economic consequences of introgression of the FecB gene into the French Mérinos d'Arles sheep[END_REF]. In the Lacaune breed, two different mutations affecting prolificacy were discovered in the selection nucleus of the OVI-TEST cooperative, FecX L in the BMP15 gene on the chromosome X [START_REF] Bodin | A novel mutation in the bone morphogenetic protein 15 gene causing defective protein secretion is associated with both increased ovulation rate and sterility in Lacaune sheep[END_REF], and FecL L at the B4GALNT2 locus on the chromosome 11 [START_REF] Drouilhet | Fine mapping of the FecL locus influencing prolificacy in Lacaune sheep[END_REF][START_REF] Drouilhet | The Highly Prolific Phenotype of Lacaune Sheep Is Associated with an Ectopic Expression of the B4GALNT2 Gene within the Ovary[END_REF]. As soon as 2005, it was decided to eradicate the FecX L mutation inducing sterility at the homozygous state and to manage the FecL L mutation which increases LS by +0.5 lamb per lambing. The selection objective is to achieve 50% of heterozygous FecL L carrier ewes in the Lacaune OVI-TEST selection nucleus flocks [START_REF] Martin | Effects of the FecL major gene in the Lacaune meat sheep population[END_REF][START_REF] Raoul | Using a very low-density SNP panel for genomic selection in a breeding program for sheep[END_REF].

Beyond these genetic programmes, research of putative mutations affecting LS was undertaken in several French and foreign sheep populations, leading to the discovery of three original causal mutations affecting the BMP15 gene in the French Grivette, the Polish Olkuska and the Tunisian Barbarine breeds [START_REF] Demars | Genome-wide association studies identify two novel BMP15 mutations responsible for an atypical hyperprolificacy phenotype in sheep[END_REF][START_REF] Lassoued | FecXBar a Novel BMP15 mutation responsible for prolificacy and female sterility in Tunisian Barbarine Sheep[END_REF]. In contrast with the seven other known mutations in the BMP15 gene affecting prolificacy, the homozygous Grivette and Olkuska carrier ewes are not sterile but hyper-prolific [START_REF] Demars | Genome-wide association studies identify two novel BMP15 mutations responsible for an atypical hyperprolificacy phenotype in sheep[END_REF].

In the present paper, we present an analysis of LS data from 34 French and one Spanish meat sheep breeds highlighting the suspicion of a mutation in a major gene in two of them, the Noire du Velay and the Mouton Vendéen breeds. Through molecular genotyping we evidenced the segregation of mutations already known to control OR and LS in ovine breeds. Moreover, we give an early analysis of frequency and effects of these two mutations in commercial populations.

Material and methods

Data and statistical analysis

Relationship between mean and variance of LS. Data come from the OVALL French national database for meat sheep genetic evaluation and research managed by the Institut de l'Elevage (French Livestock Institute) and the Centre de Traitement de l'Information Génétique (Genetic Information Processing Center, Jouy-en-Josas, France) gathering about 12 million lambings from 1986 to 2016. We have extracted the lambing career of purebred females alive in 2005 from 34 different breeds, representing 2 353 324 natural LS obtained without hormonal synchronisation treatment of oestrus. Moreover, we have added LS data from the Spanish database for genetic evaluation of Rasa Aragonesa -UPRA-Grupo Pastores [START_REF] Fathallah | Relaciones entre los efectos del gen BMP15 y los efectos poligénicos sobre la prolificidad en la raza ovina Rasa Aragonesa[END_REF]. Basic statistical analysis (mean and variance of the observed LS) were used to characterize each breed as well as to select the animals entering in the genotyping programme.

Expected frequencies of LS and variance -expected career. A subsample of the 25 most numerically important French breeds gathering 88 428 ewes with at least 5 LS records each was considered to estimate the parameters of the LS distribution. As in Bodin and Elsen (Bodin et al., 1989), the second order regression coefficients of each LS frequency on the mean prolificacy of the breed were estimated on the subsample of all ewes with 5 records each, excluding the Noire du Velay and Mouton Vendéen breeds as well as those known to carry a major gene for OR. These coefficients (ai, b1i, b2i) permitted estimation of the expected frequencies of each LSi for a population of a given prolificacy (prol): LSi = ai + b1i prol + b2i prol² and consequently the expected variance which could be compared to the observed frequencies and variance. They were applied to a sample including 3 breeds without obvious major genes (Rava, Rouge de l'Ouest, Charollais), 2 breeds known to carry major genes (Lacaune and Grivette) and the two "suspected breeds" of the present study (Noire du Velay and Mouton Vendéen). According to the threshold model of LS [START_REF] Gianola | Assortative mating and the genetic correlation[END_REF], and using the expected frequencies of these 7 populations, it was also possible to simulate lifetime LS data of females with 5 records each. Thus, 200 000 careers were simulated with a repeatability on the underlying variate equal to 0.20 (i.e. ~0.15 on the observed scale). These simulations provided the expected percentage of animals with 5 lambings which exceed a given mean prolificacy (i.e. 3.0). As before, this gives the expected value in the absence of a major gene in the population was compared to the observed value.

Genetic parameter analyses. The test of deviation from the Hardy Weinberg equilibrium was performed using a Pearson chi square test, while the association between genotypes and prolificacy groups (defined bellow) was analysed using an exact Fisher test which can take into account the very small sample size in some categories of the contingency tables. Both tests were performed using specific functions of the R package (R Development Core Team, 2008).

The heritability [h 2 =

] and the repeatability [r= ] were calculated through the estimation of the additive genetic variance , the ( ) [START_REF] Gilmour | ASReml User Guide Release 4.1 Functional Specification[END_REF]. This model included the flock, the year, the season of lambing, the age and the genotype as fixed effects as well as random effects which were a permanent environmental effect, an animal effect whose terms were linked by a pedigree, and a residual effect. The estimation of the genotype effects were obtained from the same model by the predicted values of the genotype fixed effect.

2

Animals Initial sampling of Noire du Velay and Mouton Vendéen ewes based on extreme LS.

The first suspicions led to selecting very small samples of relevant animals which were genotyped for the 3 known mutations naturally segregating in the French populations (FecL L , FecX L in Lacaune and FecX GR in Grivette). The lists of extreme highly and lowly prolific animals regarding their natural mean LS (without hormonal treatments) over at least 3 lambings were first extracted from the OVALL national database to establish the prolificacy groups. Flocks with at least 5 extreme ewes still alive at that time were selected and blood samples were collected. For the Noire du Velay breed, the final list gathered 56 females in 8 different flocks, 35 high-prolific ewes (LS mean ≥ 2.0) and 21 low-prolific ewes used as control (LS mean ≤ 1.6). In the Mouton Vendéen breed, there were 114 samples from adult ewes with 87 high-prolific (LS mean ≥ 2.20) and 27 low-prolific (LS mean ≤ 1.20).

Samples for studies of the frequency and the effects of the encountered mutations. In order to avoid any bias due to selection, large cohorts of unselected animals were collected in both breeds. For the Noire du Velay breed, the estimation of allele frequencies was made on unselected adult ewes (n=2728) collected in 22 different well as the pedigree of the animals were extracted from the OVALL national database.

Genotypes were either unknown or determined by genotyping. The model included the flock (67 levels), the year of birth (17 levels), the age at lambing (10 levels), the season of lambing (3 levels) and the genotype (4 levels: ++, L+, LL or unknown) as fixed effects, and two random effects: a permanent environmental effect and the animal additive genetic effect.

In the Mouton Vendéen breed, blood sampling of the whole cohort of replacement ewe lambs (n=1200) belonging to 19 flocks of the selection nucleus was undertaken in 2016. A few months after sampling, these ewe lambs had their first lambing allowing estimation of the gene effect at this young age. A few adult sires were also genotyped (n=6) and the production of their daughters extracted from the national database. As for the Noire du Velay breed, the gene effect was estimated by a linear mixed model on the whole natural LS dataset of all ewes born after year 2000. These data (41269 records from 14550 females) as well as the pedigree of the animals were also extracted from the OVALL national database and the same model was applied. Levels for the fixed effects were 87 for the flock and 18 for the year of birth.

Blood sampling and KAPA-KASP genotyping. Blood samples (5 ml per animal) were collected from jugular vein by Venoject system containing EDTA in commercial flocks and directly stored at -20°C for further use. Genotyping was obtained by a first step of KAPA Blood PCR amplification of a specific fragment encompassing the mutation position (KAPA Biosystems). Primers used for PCR amplification were designed using Primer 3 software (

Results

Suspicion of mutation in major genes affecting prolificacy

As previously described, as long as there are less than 1% triplets in a sheep population, the distribution of LS approximately follows a binomial distribution for which the variance is directly linked to the mean (Bodin et al., 1989). For the breeds with a higher percent of triplets, the mean-variance relationship remains very strong. A plot of the relationship between mean and variance of LS following natural oestrus in 34

different French breeds and one Spanish breed is shown in Fig. 1. The breeds in which a mutation in a major gene is segregating were distinctly marked (Lacaune, Grivette and Rasa Aragonesa, black squares) and clearly stood out from the quadratic trendline (dashed line) which had a high r² (0.93). Excluding the three breeds with known mutations in major genes gave a quadratic trendline (plain line) with a higher r² (0.97).

Based on this second trendline, the figure 1 shows that the Noire du Velay and Mouton

Vendéen breeds (circles) also clearly deviated from their expected place, which could suggest the segregation of a mutation in a major gene in these two populations.

Following this first hint, we analysed the evolution of the mean prolificacy of the 34

French breeds between 1986 and 2016. In the figure 2, we have plotted the overall annual mean LS weighted by the number of individuals in each population. We observed a regular increase of the mean LS of these breeds during the last three decades corresponding to +0.70 lamb/100 ewes/year. In contrast to the increase observed for the Lacaune, Grivette and Noire du Velay breeds, the Mouton Vendéen breed showed a slight regular decrease of its mean LS. Remarkably, the Noire du Velay breed had a strong increase of the mean LS with +1.40 lambs/100 ewes/year, twice as fast as what was observed for the overall mean and even faster than the Lacaune and Grivette breeds (respectively +0.68 and +0.75 lambs/100 ewes/year), known to carry a mutation in a major gene increasing prolificacy. Even if we could suppose that a strong improvement of the environment had occurred for improving the prolificacy of the Noire du Velay breed during the last decades, we can also speculate on the segregation of mutation in major genes influencing this trait.

The ratio of the observed distribution of each LS class to its expectation (provided by regression coefficients estimated on a large dataset) is given by r in Table 2.

Estimations of LS frequencies were very close to the observed values for the Rava, the Rouge de l'Ouest and the Charollais breeds, as r ranged from 0.98 to 1.03. In contrast, r for the Noire du Velay and the Mouton Vendéen breeds ranged further apart from 1 (0.91 to 1.36). Similar results were obtained for the two breeds carrying a mutation in a major gene, Lacaune and Grivette (r ratio from 0.89 to 1.24).

Consequently, r ratios for LS variance were remarkably close to 1 for the non-carrier breeds in contrast to those of the Lacaune and Grivette as well as the Noire du Velay and Mouton Vendéen breeds (ranging from 1.08 to 1.32). Thus, the LS distributions observed in Noire du Velay and Mouton Vendéen breeds break the rules of homogenous populations and suggested for each breed a mixture of females with different prolificacy level.

The final hint was the excess of highly prolific animals in these breeds (Table 2). The observed number of females having a mean prolificacy higher than 3 on five records were generally low for all the main French breeds with mean LS under 2.0. Obviously, this parameter increased regularly when the mean prolificacy of the population increased, however it was higher for the breeds known to carry or suspected to carry a mutation in a major gene reaching 28‰ in Grivette, for example (Table 2).

Furthermore, r was close to 1 for the non-carrier breeds and higher for the other breeds. For the Noire du Velay breed, the number of females with a prolificacy higher than 3 was 16 times higher than expected for a comparable population without mutation in a major gene. Although this parameter was lower for the Mouton Vendéen breed, it was higher than for the non-carrier breeds.

Genotyping of known mutations affecting prolificacy in French ovine populations:

FecL L , FecX L and FecX Gr

Extremely low and high-prolific ewes from the Noire du Velay (n=56) and Mouton Vendéen (n=114) populations were genotyped for the 3 known mutations naturally segregating in the French populations i.e. FecL L , FecX L in Lacaune and FecX GR in Grivette (Table 3). The FecX L mutation was not found but the FecL L allele was found in the Noire du Velay high-prolific group and the FecX Gr allele was found in the highprolific group of Mouton Vendéen ewes (Table 3). All the low-prolific ewes from both breeds were wild-type at the genotyped loci. For both breeds, the exact Fisher test was highly significant (P < 0.001) showing a clear disequilibrium between prolificacy groups and genotypes of these mutations.

FecL L and FecX Gr genotype frequency and effect on prolificacy

Large cohorts of unselected animals were genotyped in order to accurately estimate the allele frequencies in the Noire du Velay and the Mouton Vendéen populations (Table 4). In Noire du Velay, the frequency of the L prolific allele at the FecL L locus was 0.11 with 20.1% heterozygous and 1.1% homozygous carriers. These frequencies are in Hardy Weinberg equilibrium (P = 0.36). Among the Mouton Vendéen replacement ewe lambs, the frequency of the Gr prolific allele at the FecX Gr locus was 0.05 and we observed 10.3% carrier ewes (+/Gr and Gr/Gr), only 3 animals being homozygous Gr/Gr. These frequencies were also in Hardy Weinberg equilibrium (P = 0.84).

The estimated genetic effects are presented in the Table 5. Genetic parameters were very similar in both breeds. Heritability (h 2 : 0.09) and repeatability (r; 0.10 to 0.14) were low and in full agreement with the classical values of these parameters for this species [START_REF] Janssens | Genetic parameters for litter size in sheep: natural versus hormone-induced oestrus[END_REF]. A single copy of the FecL L in Noire du Velay increased the mean prolificacy by 0.42 lamb per lambing (P < 0.001). The additional increase due to a second copy of the mutation in homozygous carriers was lower (0.13 ; P = 0.096). In the Mouton Vendéen breed, the effect of a single copy of the FecX Gr allele increased the prolificacy by 0.30 lamb per lambing (P < 0.001), while the effect of a second copy leading to a homozygous carrier, does not further increase the prolificacy (P = 0.485).

In both breeds, as expected, females of the unknown genotype group were slightly, although not significantly, more prolific than the corresponding females known as wildtype.

Discussion

Simple analyses of livestock industry data suggested the segregation of a mutation in a major gene affecting prolificacy of the Noire du Velay and Mouton Vendéen breeds.

Small samples of extremely high and low prolific ewes were then genotyped for known mutations and revealed the presence of causal mutations. Among the different strands of evidence, the deviation from the relationship between mean and variance of prolificacy for breeds with a mutation in a major gene comparing to other breeds was quite remarkable. Even if these parameters were calculated without any correction for variation factors, the very high number of observations for each breed, smoothed all the effects and led to a very strong relationship among non-carrying populations. Only LS after natural oestrus were extracted from the national database as using hormonal treatments to induce ovulations modifies the mean-variance relationship (Bodin et al., 1989). Moreover, the within breed variability of LS variance due to polygenic effects is generally very small (Bodin et al., 1989;[START_REF] Amer | Quantitative genetic selection for twinning rate in ewes[END_REF][START_REF] Cottle | Genetic selection for increased mean and reduced variance of twinning rate in Belclare ewes[END_REF] and could not affect the mean-variance relationship at a broad scale. Finally, the observed deviation was due to the mixture into the populations of two groups of animals widely differing in prolificacy as it had been already viewed in Lacaune [START_REF] Martin | Effects of the FecL major gene in the Lacaune meat sheep population[END_REF] and in Rasa Aragonesa breed [START_REF] Fathallah | Relaciones entre los efectos del gen BMP15 y los efectos poligénicos sobre la prolificidad en la raza ovina Rasa Aragonesa[END_REF].

The procedure used in the present study is relevant only for genes having large effect (about 0.5 standard deviation of the prolificacy mean). If the presence of known mutations was not found, the process would have been followed by a GWAS to compare allele frequencies between the few selected high (cases) and low (controls) prolific ewes. The selection of two small samples of very extreme animals and their analysis considering they are two states of a qualitative trait has been already successful and allowed the discovery of two new mutations for ovulation rate [START_REF] Demars | Genome-wide association studies identify two novel BMP15 mutations responsible for an atypical hyperprolificacy phenotype in sheep[END_REF]. However, as shown in the Table 3, some highly prolific Noire du Velay and Mouton Vendéen ewes were non-carriers of known prolific alleles. Their extreme prolificacy could be either explained by the polygenic determinism of this trait or by the segregation of another major mutation as already described in the Lacaune population carrying both FecX L and FecL L [START_REF] Bodin | A novel mutation in the bone morphogenetic protein 15 gene causing defective protein secretion is associated with both increased ovulation rate and sterility in Lacaune sheep[END_REF][START_REF] Drouilhet | Fine mapping of the FecL locus influencing prolificacy in Lacaune sheep[END_REF].

The frequency of carrier ewes is much higher in the Noire du Velay breed (~20%) than in the Mouton Vendéen breed (~10%) which can explain that the deviation from the mean-variance relationship is also higher in this breed. However, the Hardy Weinberg equilibrium is still very well conserved. This means that in both populations prolific allele frequencies are not strongly affected by selection, particularly in the Mouton Vendéen breed as shown by the mean LS evolution during the last three decades. In both breeds it seems that carrier ewes do not produce more replacement ewe lambs in spite of their higher prolificacy and that there is no preferential culling according to the genotype. In Lacaune, Hardy Weinberg equilibrium did not hold for a long time because between 1996 and 2010 the cooperative excluded animals that were too prolific and since 2011 the cooperative's aim has been to achieve 50% of L+ ewes [START_REF] Martin | Effects of the FecL major gene in the Lacaune meat sheep population[END_REF].

The effects of one copy of the FecL L mutation on LS is similar in the Noire du Velay (+0.42 lamb per lambing) and in the Lacaune breed (+0.47, [START_REF] Martin | Effects of the FecL major gene in the Lacaune meat sheep population[END_REF]), and is of the same order of magnitude as the effect of most of other known major genes for prolificacy [START_REF] Bodin | Etat des lieux et gestion pratique des gènes d'ovulation détectés dans les races ovines françaises[END_REF][START_REF] Jansson | Genes involved in ovulation rate and litter size in sheep[END_REF]. However, as it has been already noted, the effect of the FecL L mutation is much higher than the effect of the FecX Gr mutation observed in the Grivette population (+0.10 lamb per lambing, [START_REF] Demars | Genome-wide association studies identify two novel BMP15 mutations responsible for an atypical hyperprolificacy phenotype in sheep[END_REF]).

In Mouton Vendéen, the effect of one copy of the FecX Gr mutation (+0.30 ± 0.04 lamb per lambing) seems higher than the effect of the same mutation in the Grivette population, although in this latter population, the analysis of the allele effect was not conducted on a large sample. FecX Gr homozygous carrier ewes in the Grivette or the Mouton Vendéen population are as prolific, if not more, than the heterozygous ones (present work and [START_REF] Demars | Genome-wide association studies identify two novel BMP15 mutations responsible for an atypical hyperprolificacy phenotype in sheep[END_REF]), in contrast to most mutations of the BMP15 gene which induce sterility at the homozygous state [START_REF] Bodin | A novel mutation in the bone morphogenetic protein 15 gene causing defective protein secretion is associated with both increased ovulation rate and sterility in Lacaune sheep[END_REF][START_REF] Demars | Genome-wide association studies identify two novel BMP15 mutations responsible for an atypical hyperprolificacy phenotype in sheep[END_REF].

Conclusion

Based on an analysis of a very large LS dataset from 34 French meat sheep breeds and molecular genotyping, we have highlighted and evidenced the segregation of two mutations in the FecL and FecX major genes in the Noire du Velay and the Mouton Vendéen breeds. We have determined a fairly high frequency (0.05 to 0.11) and a rather strong effect (+0.3 to +0.4 lamb/lambing) of the FecL L and FecX Gr prolific alleles.

  genotyping, the allele frequencies were calculated on this sample. The gene effect was estimated by a linear mixed model on the whole natural LS dataset of all ewes born after year 2000. These data (111654 records from 26398 females) as

Figure captions Figure 1

 captions1 Figure captions

Figure 2

 2 Figure 2 Annual evolution of mean prolificacy in French sheep breeds

  Mut = Prolific mutation; LS = Litter size; Obs = observed distribution of the parameters (% or ‰); E = expected parameters estimated through the second order regression of the LS on the mean prolificacy; r = Obs / E

  Figure1

  This discovery should serve as a basis for implementing specific management programmes, including genotyping of reproducers, in relation with the Noire du Velay and Mouton Vendéen selection organizations in line with their breeding objective of prolificacy. Walkden-Brown SW, Wolfenden DH and Piper LR 2009. Use of the FecB (Booroola) gene in sheep-breeding programs. In Proceedings of Helen Newton Turner Memorial International Workshop Held Pune Maharashtra India 10-12 Novemb. 2008 (eds. S.W.

	Walkden-Brown, J.H.J. van der Werf, C. Nimbkar and V.S. Gupta), pp. 100-110.
	Australian Centre for International Agricultural Research.
	Wilson T, Wu XY, Juengel JL, Ross IK, Lumsden JM, Lord EA, Dodds KG, Walling GA,
	McEwan JC, O'Connell AR, McNatty KP and Montgomery GW 2001. Highly prolific
	Booroola sheep have a mutation in the intracellular kinase domain of bone
	morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and
	granulosa cells. Biology of Reproduction 64, 1225-1235.

Table 1

 1 List of PCR primers used in the study.

	Locus/	Primer sequence	Position 1	Application
	Chromosome	(variant allele underlined)	(start, bp)
	BMP15/	GGCACTTCATCATTGGACACT	50971433	KAPA PCR
	OARX	GGCAATCATACCCTCATACTCC	50970959	FecX Gr /FecX L
		TCTGATCCACCAGCTCACTG	50971066
		CATTGCTCCCCATCTCTATAC	50971170	KASP PCR FecX Gr
		CATTGCTCCCCATCTCTATAT	50971170
		GATGGGCCTGAAAGTAACCA	50971248
		ACCCGAGGACATACTCCCTTAC	50971137	KASP PCR FecX L
		ACCCGAGGACATACTCCCTTAT	50971137
	B4GALNT2/ OAR11	TGGTTCAAACTCCTACATGCAAGA TATGCATGGCATGTGATAGG	36938189 36938314	KAPA PCR FecL L
		TATGCATGGCATGTGATAGG	36938314
		GCAAGAAGCTGCGTGTGT	36938207	KASP PCR FecL L
		GCAAGAAGCTGCGTGTGA	36938207
	1 Start positions of primers (in base pair) are based on the OARv3.1 ovine genome assembly

Table 2

 2 Distributions of litter size in seven different breeds carrying -or not -mutation

	in major gene influencing prolificacy
								%LS=			‰♀with LS ≥ 3.0
				LS variance	1		2		3		over 5 lambings
	Breed	Mut	Mean LS	Obs	r	Obs	r	Obs	r	Obs	r	Obs	r
	Rava	no	1.50	0.31 1.01	52.9 1.00	44.4 1.00	2.6 0.98	0.0 E=0
	Rouge de l'Ouest	no	1.78	0.41 1.00	33.1 1.00	56.2 1.00	10.7 1.02	2.5 0.9
	Charollais	no	1.70	0.39 1.01	38.1 1.00	53.9 0.99	8.0 1.03	1.8 1.4
	Noire du Velay	?	1.62	0.43 1.23	46.7 1.07	46.1 0.91	7.7 1.36	5.7 16.2
	Mouton Vendéen	?	1.72	0.42 1.08	37.8 1.03	52.6 0.96	9.6 1.13	3.6 2.7
	Lacaune	yes	1.75	0.53 1.32	39.0 1.11	49.4 0.89	11.6 1.24	18.2 10.5
	Grivette	yes	1.92	0.56 1.24	28.7 1.12	54.3 0.92	17.8 1.09	28.3 3.07.

Table 3

 3 FecL L , FecX L and FecX Gr genotypes of few extreme ewes for each breed and prolificacy group

	Locus/ Genotype

Table 4

 4 FecL L and FecX Gr genotyping in the Noire du Velay and Mouton Vendéen populations

				Breed / Locus
		Noire du Velay / FecL (n=2728)	Mouton Vendéen / FecX (n=1200)
	Genotype	+/+	+/L	L/L	+/+	+/Gr	Gr/Gr
	Number	2151	548	29	1076	121	3
	Frequency ( % )	78.8	20.1	1.1	89.7	10.3
	SE 1 of frequency (%)	0.8	0.8	0.2	0.9	0.9
	Raw mean LS 2	1.58	2.03	2.18	1.55	2.01
	1 SE = Standard error
	2 LS = Litter size

Table 5

 5 Genetic parameters and allelic estimated values of litter size in Noire du Velay and Mouton Vendéen breeds Litter size; s²g = Additive genetic variance; s²p = Permanent environmental variance; s²e = Residual variance; h² = Heritability (standard errors are between brackets); r = Repeatability (standard errors are between brackets); zz = Unknown genotype; ++ = Homozygous wild type, M+: heterozygous, MM: homozygous mutated type. 1 M denotes the mutated prolific allele within each breed (FecL L or FecX Gr ) a,b Values within a row with different superscripts differ significantly at P<0.001.BY-NC-ND 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted July 11, 2018. ; https://doi.org/10.1101/367383 doi: bioRxiv preprint

				Genetic parameters			Allelic estimated values
	Breed	n LS	n ♀	s²g	s²p	s²e	h²	r	zz	+/+ +/M 1 M/M
	Noire du	111654 26398 0.036 0.017 0.334	0.09	0.14	1.60 a 1.57 a 1.99 b 2.12 b
	Velay						(0.005)	(0.003)
	Mouton	41269 14550 0.034 0.005 0.344	0.09	0.10	1.71 a 1.68 a 1.98 b 1.99 b
	Vendéen						(0.007)	(0.005)
	LS =

Acknowledgments

We thank Didier Cathalan from ROM Sélection managing the Noire du Velay population and Charline Rousseau from the Mouton Vendéen selection organization, for their precious help in the planning of blood sampling. We are grateful to the breeders who made their animals available for this study. LC was supported by a PhD grant co-funded by APIS-GENE through the Proligen project and the European Funds for Regional Development (FEDER) through the Interreg POCTEFA programme in the framework of the PIRINNOVI project (EFA103/15). Part of the Noire du Velay sampling was supported by the DEGERAM project co-funded by the FEDER Massif Central, the Régions: Aquitaine, Midi-Pyrénées, Limousin and Auvergne; and the French government.

Data repository resources

Declaration of interest

The authors declare that they have no competing interests

Ethics statement

The blood sampling procedure was approved (approval number 01171. Raw data from the OVALL database were managed by the Institut de l'Elevage (French Livestock Institute) and the Centre de Traitement de l'Information Génétique (Genetic Information Processing Center, Jouy-en-Josas, France). Derived data supporting the findings of this study are available from the corresponding author upon reasonable request.