Mapping the four‐horned locus and testing the polled locus in three Chinese sheep breeds

Four‐horned sheep are an ideal animal model for illuminating the genetic basis of horn development. The objective of this study was to locate the genetic region responsible for the four‐horned phenotype and to verify a previously reported polled locus in three Chinese breeds. A genome‐wide association study (GWAS) was performed using 34 two‐horned and 32 four‐horned sheep from three Chinese indigenous breeds: Altay, Mongolian and Sishui Fur sheep. The top two significant single nucleotide polymorphisms (SNPs) associated with the four‐horned phenotype were both located in a region spanning positions 132.6 to 132.7 Mb on sheep chromosome 2. Similar locations for the four‐horned trait were previously identified in Jacob, Navajo‐Churro, Damara and Sishui Fur sheep, suggesting a common genetic component underlying the four‐horned phenotype. The two identified SNPs were both downstream of the metaxin 2 (MTX2) gene and the HOXD gene cluster. For the top SNP—OAR2:g.132619300G>A—the strong associations of the AA and AG genotypes with the four‐horned phenotype and the GG genotype with the two‐horned phenotype indicated the dominant inheritance of the four‐horned trait. No significant SNPs for the polled phenotype were identified in the GWAS analysis, and a PCR analysis for the detection of the 1.8‐kb insertion associated with polled sheep in other breeds failed to verify the association with polledness in the three Chinese breeds. This study supports the hypothesis that two different loci are responsible for horn existence and number. This study contributes to the understanding of the molecular regulation of horn development and enriches the knowledge of qualitative traits in domestic animals.     

Polyceraty (multi‐horns) in Damara sheep maps to ovine chromosome 2

Polyceraty (presence of multiple horns) is rare in modern day ungulates. Although not found in wild sheep, polyceraty does occur in a small number of domestic sheep breeds covering a wide geographical region. Damara are fat‐tailed hair sheep, from the south‐western region of Africa, which display polyceraty, with horn number ranging from zero to four. We conducted a genome‐wide association study for horn number with 43 Damara genotyped with 606 006 SNP markers. The analysis revealed a region with multiple significant SNPs on ovine chromosome 2, in a location different from the mutation for polled in sheep on chromosome 10. The causal mutation for polyceraty was not identified; however, the region associated with polyceraty spans nine HOXD genes, which are critical in embryonic development of appendages. Mutations in HOXD genes are implicated in polydactly phenotypes in mice and humans. There was no evidence for epistatic interactions contributing to polyceraty. This is the first report on the genetic mechanisms underlying polyceraty in the under‐studied Damara.     

SNP-based association mapping of the polled gene in divergent cattle breeds

Naturally, hornless cattle are called polled. Although the POLL locus could be assigned to a c. 1.36‐Mb interval in the centromeric region of BTA1, the underlying genetic basis for the polled trait is still unknown. Here, an association mapping design was set up to refine the candidate region of the polled trait for subsequent high‐throughput sequencing. The case group comprised 101 homozygous polled animals from nine divergent cattle breeds, the majority represented by Galloway, Angus, Fleckvieh and Holstein Friesian. Additionally, this group included some polled individuals of Blonde d’Aquitaine, Charolais, Hereford, Jersey and Limousin breeds. The control group comprised horned Belgian Blue, Fleckvieh, Holstein Friesian and Illyrian Bu?a cattle. A genome‐wide scan using 49 163 SNPs was performed, which revealed one shared homozygous haplotype block consisting of nine neighbouring SNPs in all polled animals. This segment defines a 381‐kb interval on BTA1 that we consider to be the most likely location of the POLL mutation. Our results further demonstrate that the polled‐associated haplotype is also frequent in horned animals included in this study, and thus the haplotype as such cannot be used for population‐wide genetic testing. The actual trait‐associated haplotype may be revealed by using higher‐density SNP arrays. For the final identification of the causal mutation, we suggest high‐throughput sequencing of the entire candidate region, because the identification of functional candidate genes is difficult owing to the lack of a comparable model.     

Polledness in Argentinean Creole cattle,five centuries surviving

Polledness has been shown to have autosomal Mendelian inheritance, with the polled locus being dominant to the horned locus. This trait was mapped to the BTA1 centromeric end in several breeds. One of the distinctive attributes of Creole cattle, such as the Argentinean Creole, is the presence of long, lyre‐shaped horns. However, polled native animals were reported before the introduction of modern selected European breeds. Here, we studied the origin of the polled mutation, either independent or introgressed, in a Creole line from the Creole cattle founder group at the IIACS‐INTA Leales Experimental Station (northwest Argentina). The study sample (65 animals: 26 horned and 39 polled) was genotyped using high‐density SNP microarrays and three previously reported genetic markers (P_202ID, P_80kbID and P_G). A genome‐wide association study, selection signatures, linkage disequilibrium analysis and copy number variations were used to detect the responsible region and the segregating haplotypes/alleles. The interval mapped in the Leales herd (1.23–2.13 Mb) overlapped with the region previously reported in several European cattle breeds, suggesting that the same locus could be segregating in this population. The previously reported variants P_F and P_G were not detected, thus dismissing the Holstein‐Friesian and Nellore origins of the polled phenotype in this native breed. Conversely, the presence of the Celtic variant P_C suggests an almost complete co‐segregation. The cluster analysis rejected the hypothesis of recent introgression, which is compatible with the historical record of polled Creole cattle in northwest Argentina.     

Runs of homozygosity and signatures of selection: a comparison among eight local Swiss sheep breeds

A dataset consisting of 787 animals with high‐density SNP chip genotypes (346 774 SNPs) and 939 animals with medium‐density SNP chip genotypes (33 828 SNPs) from eight indigenous Swiss sheep breeds was analyzed to characterize population structure, quantify genomic inbreeding based on runs of homozygosity and identify selection signatures. In concordance with the recent known history of these breeds, the highest genetic diversity was observed in Engadine Red sheep and the lowest in Valais Blacknose sheep. Correlation between F_PED and F_ROH was around 0.50 and thereby lower than that found in similar studies in cattle. Mean F_ROH estimates from medium‐density data and HD data were highly correlated (0.95). Signatures of selection and candidate gene analysis revealed that the most prominent signatures of selection were found in the proximity of genes associated with body size (NCAPG, LCORL, LAP3, SPP1, PLAG1, ALOX12, TP53), litter size (SPP1), milk production (ABCG2, SPP1), coat color (KIT, ASIP, TBX3) and horn status (RXFP2). For the Valais Blacknose sheep, the private signatures in proximity of genes/QTL influencing body size, coat color and fatty acid composition were confirmed based on runs of homozygosity analysis. These private signatures underline the genetic uniqueness of the Valais Blacknose sheep breed. In conclusion, we identified differences in the genetic make‐up of Swiss sheep breeds, and we present relevant candidate genes responsible for breed differentiation in locally adapted breeds.     

Genetic diversity and investigation of polledness in divergent goat populations using 52 088 SNPs

The recent availability of a genome‐wide SNP array for the goat genome dramatically increases the power to investigate aspects of genetic diversity and to conduct genome‐wide association studies in this important domestic species. We collected and analysed genotypes from 52 088 SNPs in Boer, Cashmere and Rangeland goats that had both polled and horned individuals. Principal components analysis revealed a clear genetic division between animals for each population, and model‐based clustering successfully detected evidence of admixture that matched aspects of their recorded history. For example, shared co‐ancestry was detected, suggesting Boer goats have been introgressed into the Rangeland population. Further, allele frequency data successfully tracked the altered genetic profile that has taken place after 40 years of breeding Australian Cashmere goats using the Rangeland animals as the founding population. Genome‐wide association mapping of the POLL locus revealed a strong signal on goat chromosome 1. The 769‐kb critical interval contained the polled intersex syndrome locus, confirming the genetic basis in non‐European animals is the same as identified previously in Saanen goats. Interestingly, analysis of the haplotypes carried by a small set of sex‐reversed animals, known to be associated with polledness, revealed some animals carried the wild‐type chromosome associated with the presence of horns. This suggests a more complex basis for the relationship between polledness and the intersex condition than initially thought while validating the application of the goat SNP50 BeadChip for fine‐mapping traits in goat.     

Genome‐wide association study to identify genomic regions affecting prolificacy in Lori‐Bakhtiari sheep

Several causative mutations in candidate genes affecting prolificacy have been detected in various sheep breeds. A genome‐wide association study was performed on estimated breeding values for litter size in Lori‐Bakhtiari sheep. Prolific ewes with twinning records and others with only singleton records were genotyped using the medium‐density Illumina Ovine SNP50 array. Four single nucleotide polymorphisms (SNPs) associated with litter size were identified on chromosomes 3, 6 and 22. The region on sheep chromosome 3 between 75 739 167 and 75 745 152 bp included two significant SNPs (s52383.1 and OAR3_80038014_X.1) in high linkage disequilibrium with each other. The region that surrounds these SNPs contains a novel putative candidate gene: luteinizing hormone/choriogonadotropin receptor (LHCGR), known to be involved in ovarian steroidogenesis and organism‐specific biosystem pathways in sheep. Known prolificacy genes BMPR1B, BMP15 and GDF9 were not associated with litter size in Lori‐Bakhtiari sheep, suggesting that other biological mechanisms could be responsible for the trait's variation in this breed.     

Bovine polledness--an autosomal dominant trait with allelic heterogeneity

The persistent horns are an important trait of speciation for the family Bovidae with complex morphogenesis taking place briefly after birth. The polledness is highly favourable in modern cattle breeding systems but serious animal welfare issues urge for a solution in the production of hornless cattle other than dehorning. Although the dominant inhibition of horn morphogenesis was discovered more than 70 years ago, and the causative mutation was mapped almost 20 years ago, its molecular nature remained unknown. Here, we report allelic heterogeneity of the POLLED locus. First, we mapped the POLLED locus to a ～381-kb interval in a multi-breed case-control design. Targeted re-sequencing of an enlarged candidate interval (547 kb) in 16 sires with known POLLED genotype did not detect a common allele associated with polled status. In eight sires of Alpine and Scottish origin (four polled versus four horned), we identified a single candidate mutation, a complex 202 bp insertion-deletion event that showed perfect association to the polled phenotype in various European cattle breeds, except Holstein-Friesian. The analysis of the same candidate interval in eight Holsteins identified five candidate variants which segregate as a 260 kb haplotype also perfectly associated with the POLLED gene without recombination or interference with the 202 bp insertion-deletion. We further identified bulls which are progeny tested as homozygous polled but bearing both, 202 bp insertion-deletion and Friesian haplotype. The distribution of genotypes of the two putative POLLED alleles in large semi-random sample (1,261 animals) supports the hypothesis of two independent mutations.     

Design of a low‐density SNP chip for the main Australian sheep breeds and its effect on imputation and genomic prediction accuracy

Genotyping sheep for genome‐wide SNPs at lower density and imputing to a higher density would enable cost‐effective implementation of genomic selection, provided imputation was accurate enough. Here, we describe the design of a low‐density (12k) SNP chip and evaluate the accuracy of imputation from the 12k SNP genotypes to 50k SNP genotypes in the major Australian sheep breeds. In addition, the impact of imperfect imputation on genomic predictions was evaluated by comparing the accuracy of genomic predictions for 15 novel meat traits including carcass and meat quality and omega fatty acid traits in sheep, from 12k SNP genotypes, imputed 50k SNP genotypes and real 50k SNP genotypes. The 12k chip design included 12 223 SNPs with a high minor allele frequency that were selected with intermarker spacing of 50–475 kb. SNPs for parentage and horned or polled tests also were represented. Chromosome ends were enriched with SNPs to reduce edge effects on imputation. The imputation performance of the 12k SNP chip was evaluated using 50k SNP genotypes of 4642 animals from six breeds in three different scenarios: (1) within breed, (2) single breed from multibreed reference and (3) multibreed from a single‐breed reference. The highest imputation accuracies were found with scenario 2, whereas scenario 3 was the worst, as expected. Using scenario 2, the average imputation accuracy in Border Leicester, Polled Dorset, Merino, White Suffolk and crosses was 0.95, 0.95, 0.92, 0.91 and 0.93 respectively. Imputation scenario 2 was used to impute 50k genotypes for 10 396 animals with novel meat trait phenotypes to compare genomic prediction accuracy using genomic best linear unbiased prediction (GBLUP) with real and imputed 50k genotypes. The weighted mean imputation accuracy achieved was 0.92. The average accuracy of genomic estimated breeding values (GEBVs) based on only 12k data was 0.08 across traits and breeds, but accuracies varied widely. The mean GBLUP accuracies with imputed 50k data more than doubled to 0.21. Accuracies of genomic prediction were very similar for imputed and real 50k genotypes. There was no apparent impact on accuracy of GEBVs as a result of using imputed rather than real 50k genotypes, provided imputation accuracy was >90%.     

Genome‐wide association analysis identifies the genetic basis of fat deposition in the tails of sheep (Ovis aries)

Fat‐tailed sheep (Ovis aries) can survive in harsh environments and satisfy human's intake of dietary fat. However, the animals require more feed, which increases the cost of farming. Thus, most farmers currently prefer thin‐tailed, short‐tailed or docked sheep. To date, the molecular mechanism of the formation of fat tails in sheep has not been completely elucidated. Here, we conducted a genome‐wide association study using phenotypes and genotypes (the Ovine Infinium HD SNP BeadChip genotype data) of two breeds of contrasting tail types (78 Small‐tailed and 78 Large‐tailed Han sheep breeds) to identify functional genes and variants associated with fat deposition. We identified four significantly (rs416433540, rs409848439, rs408118325 and rs402128848) and three approximately associated autosomal SNPs (rs401248376, rs402445895 and rs416201901). Gene annotation indicated that the surrounding genes (CREB1, STEAP4, CTBP1 and RIP140, also known as NRIP1) function in lipid storage or fat cell regulation. Furthermore, through an X‐chromosome‐wide association analysis, we detected significantly associated SNPs in the OARX: 88–89 Mb region, which could be a strong candidate genomic region for fat deposition in tails of sheep. Our results represent a new genomic resource for sheep genetics and breeding. In addition, the findings provide novel insights into genetic mechanisms of fat deposition in the tail of sheep and other mammals.     

Genome‐wide detection of signatures of selection in Korean Hanwoo cattle

The Korean Hanwoo cattle have been intensively selected for production traits, especially high intramuscular fat content. It is believed that ancient crossings between different breeds contributed to forming the Hanwoo, but little is known about the genomic differences and similarities between other cattle breeds and the Hanwoo. In this work, cattle breeds were grouped by origin into four types and used for comparisons: the Europeans (represented by six breeds), zebu (Nelore), African taurine (N'Dama) and Hanwoo. All animals had genotypes for around 680 000 SNPs after quality control of genotypes. Average heterozygosity was lower in Nelore and N'Dama (0.22 and 0.21 respectively) than in Europeans (0.26–0.31, with Shorthorn as outlier at 0.24) and Hanwoo (0.29). Pairwise F_ST analyses demonstrated that Hanwoo are more related to European cattle than to Nelore, with N'Dama in an intermediate position. This finding was corroborated by principal components and unsupervised hierarchical clustering. Using genome‐wide smoothed F_ST, 55 genomic regions potentially under positive selection in Hanwoo were identified. Among these, 29 were regions also detected in previous studies. Twenty‐four regions were exclusive to Hanwoo, and a number of other regions were shared with one or two of the other groups. These regions overlap a number of genes that are related to immune, reproduction and fatty acid metabolism pathways. Further analyses are needed to better characterize the ancestry of the Hanwoo cattle and to define the genes responsible to the identified selection peaks.     

Genome‐wide population structure and admixture analysis reveals weak differentiation among Ugandan goat breeds

Uganda has a large population of goats, predominantly from indigenous breeds reared in diverse production systems, whose existence is threatened by crossbreeding with exotic Boer goats. Knowledge about the genetic characteristics and relationships among these Ugandan goat breeds and the potential admixture with Boer goats is still limited. Using a medium‐density single nucleotide polymorphism (SNP) panel, we assessed the genetic diversity, population structure and admixture in six goat breeds in Uganda: Boer, Karamojong, Kigezi, Mubende, Small East African and Sebei. All the animals had genotypes for about 46 105 SNPs after quality control. We found high proportions of polymorphic SNPs ranging from 0.885 (Kigezi) to 0.928 (Sebei). The overall mean observed (H_O) and expected (H_E) heterozygosity across breeds was 0.355 ± 0.147 and 0.384 ± 0.143 respectively. Principal components, genetic distances and admixture analyses revealed weak population sub‐structuring among the breeds. Principal components separated Kigezi and weakly Small East African from other indigenous goats. Sebei and Karamojong were tightly entangled together, whereas Mubende occupied a more central position with high admixture from all other local breeds. The Boer breed showed a unique cluster from the Ugandan indigenous goat breeds. The results reflect common ancestry but also some level of geographical differentiation. admixture and f₄ statistics revealed gene flow from Boer and varying levels of genetic admixture among the breeds. Generally, moderate to high levels of genetic variability were observed. Our findings provide useful insights into maintaining genetic diversity and designing appropriate breeding programs to exploit within‐breed diversity and heterozygote advantage in crossbreeding schemes.     

Y chromosome haplotype diversity of domestic sheep (Ovis aries) in northern Eurasia

Variation in two SNPs and one microsatellite on the Y chromosome was analyzed in a total of 663 rams representing 59 breeds from a large geographic range in northern Eurasia. SNPA‐oY1 showed the highest allele frequency (91.55%) across the breeds, whereas SNPG‐oY1 was present in only 56 samples. Combined genotypes established seven haplotypes (H4, H5, H6, H7, H8, H12 and H19). H6 dominated in northern Eurasia, and H8 showed the second‐highest frequency. H4, which had been earlier reported to be absent in European breeds, was detected in one European breed (Swiniarka), whereas H7, which had been previously identified to be unique to European breeds, was present in two Chinese breeds (Ninglang Black and Large‐tailed Han), one Buryatian (Transbaikal Finewool) and two Russian breeds (North Caucasus Mutton‐Wool and Kuibyshev). H12, which had been detected only in Turkish breeds, was also found in Chinese breeds in this work. An overall low level of haplotype diversity (median h = 0.1288) was observed across the breeds with relatively higher median values in breeds from the regions neighboring the Near Eastern domestication center of sheep. H6 is the dominant haplotype in northwestern and eastern China, in which the haplotype distribution could be explained by the historical translocations of the H4 and H8 Y chromosomes to China via the Mongol invasions followed by expansions to northwestern and eastern China. Our findings extend previous results of sheep Y chromosomal genetic variability and indicate probably recent paternal gene flows between sheep breeds from distinct major geographic regions.     

Genome-wide association mapping identifies the genetic basis of discrete and quantitative variation in sexual weaponry in a wild sheep population

Understanding the genetic architecture of phenotypic variation in natural populations is a fundamental goal of evolutionary genetics. Wild Soay sheep (Ovis aries) have an inherited polymorphism for horn morphology in both sexes, controlled by a single autosomal locus, Horns. The majority of males have large normal horns, but a small number have vestigial, deformed horns, known as scurs; females have either normal horns, scurs or no horns (polled). Given that scurred males and polled females have reduced fitness within each sex, it is counterintuitive that the polymorphism persists within the population. Therefore, identifying the genetic basis of horn type will provide a vital foundation for understanding why the different morphs are maintained in the face of natural selection. We conducted a genome-wide association study using ～36000 single nucleotide polymorphisms (SNPs) and determined the main candidate for Horns as RXFP2, an autosomal gene with a known involvement in determining primary sex characters in humans and mice. Evidence from additional SNPs in and around RXFP2 supports a new model of horn-type inheritance in Soay sheep, and for the first time, sheep with the same horn phenotype but different underlying genotypes can be identified. In addition, RXFP2 was shown to be an additive quantitative trait locus (QTL) for horn size in normal-horned males, accounting for up to 76% of additive genetic variation in this trait. This finding contrasts markedly from genome-wide association studies of quantitative traits in humans and some model species, where it is often observed that mapped loci only explain a modest proportion of the overall genetic variation.     

Nucleotide variability and haplotype heterogeneity at the porcine fat mass and obesity‐associated (FTO) gene

A large number of studies have confirmed that variants within the fat mass and obesity‐associated (FTO) gene are associated with higher obesity risk in humans. We and others have shown that FTO polymorphisms are associated with fat deposition and related traits in several pig populations, thus confirming the role of this gene in fatness across species. However, some differences observed in different pig populations may be derived, at least in part, from genetic heterogeneity at this locus. Here, we characterise the nucleotide variability and haplotype diversity of the porcine FTO gene in breeds having different predispositions to fat deposition traits. We resequenced 4749 bp of coding and non‐coding regions of the porcine FTO gene in 44 pigs of eight different breeds and identified 27 single nucleotide polymorphisms (SNPs) and four insertions/deletions. A positive Tajima's D‐value (P < 0.10) obtained in Italian Duroc pigs may be compatible with putative balancing selection. From the sequenced pig panel, 20 haplotypes were inferred, some of which clustered according to the breed of origin (Meishan and Italian Duroc). Genetic heterogeneity at this locus could complicate the dissection of the effects of this gene on fat deposition and production traits in pigs. This situation resembles, to some extent, what has been reported in humans, thus making the study of the porcine FTO gene variability especially interesting, as it could be used as a model to understand the complex and elusive role of this gene in human obesity.     

Genome‐wide association analysis for quantitative trait loci influencing Warner–Bratzler shear force in five taurine cattle breeds

We performed a genome‐wide association study for Warner–Bratzler shear force (WBSF), a measure of meat tenderness, by genotyping 3360 animals from five breeds with 54 790 BovineSNP50 and 96 putative single‐nucleotide polymorphisms (SNPs) within μ‐calpain [HUGO nomenclature calpain 1, (mu/I) large subunit; CAPN1] and calpastatin (CAST). Within‐ and across‐breed analyses estimated SNP allele substitution effects (ASEs) by genomic best linear unbiased prediction (GBLUP) and variance components by restricted maximum likelihood under an animal model incorporating a genomic relationship matrix. GBLUP estimates of ASEs from the across‐breed analysis were moderately correlated (0.31–0.66) with those from the individual within‐breed analyses, indicating that prediction equations for molecular estimates of breeding value developed from across‐breed analyses should be effective for genomic selection within breeds. We identified 79 genomic regions associated with WBSF in at least three breeds, but only eight were detected in all five breeds, suggesting that the within‐breed analyses were underpowered, that different quantitative trait loci (QTL) underlie variation between breeds or that the BovineSNP50 SNP density is insufficient to detect common QTL among breeds. In the across‐breed analysis, CAPN1 was followed by CAST as the most strongly associated WBSF QTL genome‐wide, and associations with both were detected in all five breeds. We show that none of the four commercialized CAST and CAPN1 SNP diagnostics are causal for associations with WBSF, and we putatively fine‐map the CAPN1 causal mutation to a 4581‐bp region. We estimate that variation in CAST and CAPN1 explains 1.02 and 1.85% of the phenotypic variation in WBSF respectively.     

Beta‐globin gene evolution in the ruminants: evidence for an ancient origin of sheep haplotype B

Domestic sheep (Ovis aries) can be divided into two groups with significantly different responses to hypoxic environments, determined by two allelic beta‐globin haplotypes. Haplotype A is very similar to the goat beta‐globin locus, whereas haplotype B has a deletion spanning four globin genes, including beta‐C globin, which encodes a globin with high oxygen affinity. We surveyed the beta‐globin locus using resequencing data from 70 domestic sheep from 42 worldwide breeds and three Ovis canadensis and two Ovis dalli individuals. Haplotype B has an allele frequency of 71.4% in O. aries and was homozygous (BB) in all five wild sheep. This shared ancestry indicates haplotype B is at least 2–3 million years old. Approximately 40 kb of the sequence flanking the ~37‐kb haplotype B deletion had unexpectedly low identity between haplotypes A and B. Phylogenetic analysis showed that the divergent region of sheep haplotype B is remarkably distinct from the beta‐globin loci in goat and cattle but still groups with the Ruminantia. We hypothesize that this divergent ~40‐kb region in haplotype B may be from an unknown ancestral ruminant and was maintained in the lineage to O. aries, but not other Bovidae, evolving independently of haplotype A. Alternatively, the ~40‐kb sequence in haplotype B was more recently acquired by an ancestor of sheep from an unknown non‐Bovidae ruminant, replacing part of haplotype A. Haplotype B has a lower nucleotide diversity than does haplotype A, suggesting a recent bottleneck, whereas the higher frequency of haplotype B suggests a subsequent spread through the global population of O. aries.