Carole Moreno

Design and Characterization of a 52K SNP Chip for Goats

The success of Genome Wide Association Studies in the discovery of sequence variation linked to complex traits in humans has increased interest in high throughput SNP genotyping assays in livestock species. Primary goals are QTL detection and genomic selection. The purpose here was design of a 50–60,000 SNP chip for goats. The success of a moderate density SNP assay depends on reliable bioinformatic SNP detection procedures, the technological success rate of the SNP design, even spacing of SNPs on the genome and selection of Minor Allele Frequencies (MAF) suitable to use in diverse breeds. Through the federation of three SNP discovery projects consolidated as the International Goat Genome Consortium, we have identified approximately twelve million high quality SNP variants in the goat genome stored in a database together with their biological and technical characteristics. These SNPs were identified within and between six breeds (meat, milk and mixed): Alpine, Boer, Creole, Katjang, Saanen and Savanna, comprising a total of 97 animals. Whole genome and Reduced Representation Library sequences were aligned on >10 kb scaffolds of the de novo goat genome assembly. The 60,000 selected SNPs, evenly spaced on the goat genome, were submitted for oligo manufacturing (Illumina, Inc) and published in dbSNP along with flanking sequences and map position on goat assemblies (i.e. scaffolds and pseudo-chromosomes), sheep genome V2 and cattle UMD3.1 assembly. Ten breeds were then used to validate the SNP content and 52,295 loci could be successfully genotyped and used to generate a final cluster file. The combined strategy of using mainly whole genome Next Generation Sequencing and mapping on a contig genome assembly, complemented with Illumina design tools proved to be efficient in producing this GoatSNP50 chip. Advances in use of molecular markers are expected to accelerate goat genomic studies in coming years.

Selection Signatures in Worldwide Sheep Populations

The diversity of populations in domestic species offers great opportunities to study genome response to selection. The recently published Sheep HapMap dataset is a great example of characterization of the world wide genetic diversity in sheep. In this study, we re-analyzed the Sheep HapMap dataset to identify selection signatures in worldwide sheep populations. Compared to previous analyses, we made use of statistical methods that (i) take account of the hierarchical structure of sheep populations, (ii) make use of linkage disequilibrium information and (iii) focus specifically on either recent or older selection signatures. We show that this allows pinpointing several new selection signatures in the sheep genome and distinguishing those related to modern breeding objectives and to earlier post-domestication constraints. The newly identified regions, together with the ones previously identified, reveal the extensive genome response to selection on morphology, color and adaptation to new environments.

A genome scan for QTL affecting resistance to Haemonchus contortus in sheep.

Gastrointestinal nematodes are one of the main health issues in sheep breeding. To identify loci affecting the resistance to Haemonchus contortus, a genome scan was carried out using 1,275 Romane × Martinik Black Belly backcross lambs. The entire population was challenged with Haemonchus contortus in 2 consecutive experimental infections, and fecal egg counts (FEC) and packed cell volumes were measured. A subgroup of 332 lambs with extreme FEC was necropsied to determine the total worm burden, length of female worms, sex ratio in the worm population, abomasal pH, and serum and mucosal G immunoglobulins (IgG) responses. Pepsinogen concentration was measured in another subset of 229 lambs. For QTL detection, 160 microsatellite markers were used as well as the Illumina OvineSNP50 BeadChip that provided 42,469 SNP markers after quality control. Linkage, association, and joint linkage and association analyses were performed with the QTLMAP software. Linkage disequilibrium (LD) was estimated within each pure breed, and association analyses were carried out either considering or not the breed origin of the haplotypes. Four QTL regions on sheep chromosomes (OAR)5, 12, 13, and 21 were identified as key players among many other QTL with small to moderate effects. A QTL on OAR21 affecting pepsinogen concentration exactly matched the pepsinogen (PGA5) locus. A 10-Mbp region affecting FEC after the 1st and 2nd infections was found on OAR12. The SNP markers outperformed microsatellites in the linkage analysis. Taking advantage of the LD helped to refine the locations of the QTL mapped on OAR5 and 13.

Which PrP haplotypes in a French sheep population are the most susceptible to atypical scrapie

SummaryA French sheep case control study has been organised to estimate the effects of the PrP haplotypes on resistance to atypical scrapie. The ALHQ and AFRQ haplotypes are significantly more susceptible than the others.

Assessment of genetic variation explained by markers for wool traits in sheep via a segment mapping approach.

A first step towards the genetic dissection of quantitative traits is to perform a genome scan with DNA markers evenly spaced throughout the genome. However, and despite recent advancements in typing technology, a dense genome scan remains a lengthy and very expensive task. An alternative approach is to perform a preliminary typing with a reduced number of markers, not necessarily covering all chromosomes, in order to assess the fraction of total genetic variation explained by typed markers and to identify the most relevant chromosomes to pursue genotyping. The segment mapping strategy (Pe ́rez-Enciso and Varona 2000) is particularly well suited to this end, as it allows us to analyze simultaneously the whole genome. In short, the method consists of partitioning the genome into a series of segments, delimiting specific genome regions that we are interested in analyzing. The method allows us to model the segment as a fixed effect, as in crosses between inbred lines, as a random effect, e.g., in purebred analysis, or as a mixed model, which is useful in analyzing crosses between divergent breeds. The fraction of total genetic variance explained by each segment is subsequently estimated. In this approach, the most relevant question is the fraction of genetic variance explained by the whole segment; we are not interested so much in how many quantitative trait loci (QTLs) there are or in their exact position. We illustrate the proposed strategy in a halfsib design experiment in sheep for QTL detection in wool traits (Allain et al. 1998). The population analyzed came from the synthetic breed INRA401. This breed is a composite Romanov (prolific breed) and Berrichon du Cher (meat breed). It was created in France in 1980 (Ricordeau et al. 1992). The main selection objective is reproductive performance, and there is no selection on wool characteristics. The experimental resource population for the QTL study comprised 30 rams and 690 ewes, which produced 1109 lambs (694 males and 415 females). The number of offspring per sire ranged from 31 to 45. The number of ewes mated to a given ram also ranged from 31 to 45. Most of the ewes were mated to a single ram, but 78 of them were mated to two different rams. Thus, all individuals in the pedigree were indirectly related. Mid-side wool samples were taken from the offspring lambs at a fixed weight, 32 and 38 kg for females and males respectively (about 3 months of age). Staple length (SL) was measured. Then, after a washing treatment, wool samples were analyzed in a textile laboratory (ITF, France) by Optical Fiber Diameter Analysis for determining the mean (MFD) and the coefficient of variation (CVFD) of the fiber diameter. Fiber diameter was assessed according to the IWTO-4795 standard method. In total, 4000 fiber snippets per individual were measured. All sires, dams, and offspring were typed for the 40 microsatellites listed in Fig. 1, distributed among 20 out of the 26 ovine autosomes. The total length covered by all markers was about 600 cM, plus eight chromosomes where only one marker was available. The total length of the sheep genome is about 2500 cM. DNA was extracted from the blood, and PCR-amplified microsatellites were analyzed on a capillary electrophoresis equipment with fluorescent detection. Genetic distances between microsatellites within the same linkage group were estimated with the CRIMAP software. The linear model used to analyze the data was:

Identification of New Quantitative Trait Loci (Other Than the PRNP Gene) Modulating the Scrapie Incubation Period in Sheep

Although susceptibility to scrapie is largely controlled by the PRNP gene, we have searched for additional genomic regions that affect scrapie incubation time in sheep, using two half-sib families with a susceptible PRNP genotype and naturally infected by scrapie. Quantitative trait loci were detected on OAR6 and OAR18.

Genetic parameters for resistance to the Salmonella abortusovis vaccinal strain Rv6 in sheep

An experimental population (1216 lambs from 30 sires) of the Inra401 sheep was created in an Inra flock to allow QTL detection for susceptibility to Salmonella infection, wool and carcass traits. The Inra401 is a sheep composite line developed from two breeds: Berrichon du Cher and Romanov. At 113 days of age on average, the lambs were inoculated intravenously with 108Salmonella abortusovis Rv6 (vaccinal strain). They were slaughtered 10 days after the inoculation. Several traits were measured at inoculation and/or slaughtering to estimate the genetic resistance of the lambs to Salmonella infection: specific IgM and IgG1 antibody titres, body weight loss, spleen and pre-scapular node weights and counts of viable Salmonella persisting in these organs. This paper presents a quantitative analysis of the genetic variability of the traits related to salmonellosis susceptibility. The heritabilities of the traits varied between 0.10 and 0.64 (significantly different from zero). Thus, in sheep as well as in other species, the determinism of resistance to Salmonella infection is under genetic control. Moreover, the correlations between the traits are in agreement with the known immune mechanisms. The genetic variability observed should help QTL detection.

A study on associations between PrP genotypes and meat traits in French sheep breeds

In this study the potential association of PrP genotypes with meat traits has been investigated. The data included young rams from individual testing stations of three breeds: Ile de France, Prealpes du Sud and Blanc du Massif Central. These breeds were chosen due to their large number of available animals (with performance records and PrP genotypes) and their differential ARR haplotype frequency. Two analyses differing in the PrP genotype classes considered were carried out. Firstly, animals were categorized into three classes: ARR homozygous, ARR heterozygous, and animals without the ARR haplotype. The data for this analysis included 725, 534 and 832 animals for Ile de France, Prealpes du Sud and Blanc du Massif Central breeds, respectively. Secondly, as the two predominant haplotypes in these breeds are ARR and AR- (and AR- includes ARQ and ARH haplotypes), the effect of substituting 1 or 2 ARR haplotypes for AR- haplotypes was studied. These comparisons involved three genotype classes: ARR homozygous, ARR/AR-, and AR-homozygous. The data for this analysis included 532, 509 and 620 animals of Ile de France, Prealpes du Sud and Blanc du Massif Central breeds, respectively. Meat traits were analysed using an animal model (where the PrP genotype was included as a fixed effect) and they included growth rate, ultrasonic fat depth, and ultrasonic muscle depth. The results of this study indicate no evidence of association between PrP genotypes and the meat traits studied in these sheep breeds.

A joint analysis to identify loci underlying variation in nematode resistance in three European sheep populations.

Gastrointestinal nematode infections are one of the main health/economic issues in sheep industries, worldwide. Indicator traits for resistance such as faecal egg count (FEC) are commonly used in genomic studies; however, published results are inconsistent among breeds. Meta (or joint)-analysis is a tool for aggregating information from multiple independent studies. The aim of this study was to identify loci underlying variation in FEC, as an indicator of nematode resistance, in a joint analysis using data from three populations (Scottish Blackface, Sarda × Lacaune and Martinik Black-Belly × Romane), genotyped with the ovine 50k SNP chip. The trait analysed was the average animal effect for Strongyles and Nematodirus FEC data. Analyses were performed with regional heritability mapping (RHM), fitting polygenic effects with either the whole genomic relationship matrix or matrices excluding the chromosome being interrogated. Across-population genomic covariances were set to zero. After quality control, 4123 animals and 38 991 SNPs were available for the analysis. RHM identified genome-wide significant regions on OAR4, 12, 14, 19 and 20, with the latter being the most significant. The OAR20 region is close to the major histocompatibility complex, which has often been proposed as a functional candidate for nematode resistance. This region was significant only in the Sarda × Lacaune population. Several other regions, on OAR1, 3, 4, 5, 7, 12, 19, 20 and 24, were significant at the suggestive level.

Quantitative trait loci linked to PRNP gene controlling health and production traits in INRA 401 sheep

In this study, the potential association of PrP genotypes with health and productive traits was investigated. Data were recorded on animals of the INRA 401 breed from the Bourges-La Sapinière INRA experimental farm. The population consisted of 30 rams and 852 ewes, which produced 1310 lambs. The animals were categorized into three PrP genotype classes: ARR homozygous, ARR heterozygous, and animals without any ARR allele. Two analyses differing in the approach considered were carried out. Firstly, the potential association of the PrP genotype with disease (Salmonella resistance) and production (wool and carcass) traits was studied. The data used included 1042, 1043 and 1013 genotyped animals for the Salmonella resistance, wool and carcass traits, respectively. The different traits were analyzed using an animal model, where the PrP genotype effect was included as a fixed effect. Association analyses do not indicate any evidence of an effect of PrP genotypes on traits studied in this breed. Secondly, a quantitative trait loci (QTL) detection approach using the PRNP gene as a marker was applied on ovine chromosome 13. Interval mapping was used. Evidence for one QTL affecting mean fiber diameter was found at 25 cM from the PRNP gene. However, a linkage between PRNP and this QTL does not imply unfavorable linkage disequilibrium for PRNP selection purposes.