WNT10B: A locus increasing risk of brachygnathia inferior in Brown Swiss cattle.

Shortening of the mandible (brachygnathia inferior) is a congenital, often inherited and variably expressed craniofacial anomaly in domestic animals including cattle. Brachygnathia inferior can lead to poorer animal health and welfare and reduced growth, which ultimately affects productivity. Within the course of the systematic conformation scoring, cases with a frequency of about 0.1% were observed in the Brown Swiss cattle population of Switzerland. In contrast, this anomaly is almost unknown in the Original Braunvieh population, representing the breed of origin. Because none of the individually examined 46 living offspring of our study cohort of 145 affected cows showed the trait, we can most likely exclude a monogenic-dominant mode of inheritance. We hypothesized that either a monogenic recessive or a complex mode of inheritance was underlying. Through a genome-wide association study of 145 cases and 509 controls with imputed 624k SNP data, we identified a 4.5 Mb genomic region on bovine chromosome 5 significantly associated with this anomaly. This locus was fine-mapped using whole-genome sequencing data. A run of homozygosity analysis revealed a critical interval of 430 kb. A breed specific frameshift duplication in WNT10B (rs525007739; c.910dupC; p.Arg304ProfsTer14) located in this genomic region was found to be associated with a 21.5-fold increased risk of brachygnathia inferior in homozygous carriers. Consequently, we present for the first time a genetic locus associated with this well-known anomaly in cattle, which allows DNA-based selection of Brown Swiss animals at decreased risk for mandibular shortening. In addition, this study represents the first large animal model of a WNT10B-related inherited developmental disorder in a mammalian species.

Genomic regions underlying positive selection in local, Alpine cattle breeds.

We used genome-wide SNP data from 18 local cattle breeds from six countries of the Alpine region to characterize population structure and identify genomic regions underlying positive selection. The geographically close breeds Evolèner, Eringer, Valdostana Pezzata Nera, and Valdostana Castana were found to differ from all other Alpine breeds. In addition, three breeds, Simmental, and Original Braunvieh from Switzerland and Pinzgauer from Austria built three separate clusters. Of the 18 breeds studied, the intra-alpine Swiss breed Evolèner had the highest average inbreeding based on runs of homozygosity (FROH ) and the highest average genomic relationship within the breed. In contrast, Slovenian Cika cattle had the lowest average genomic inbreeding and the lowest average genomic relationship within the breed. We found selection signatures on chromosome 6 near known genes such as KIT and LCORL explaining variation in coat color and body size in cattle. The most prominent selection signatures were similar regardless of marker density and the breeds in the data set. In addition, using available high-density SNP data from 14 of the breeds we identified 47 genome regions as ROH islands. The proportion of homozygous animals was higher in all studied animals of local breeds than in Holstein and Brown Swiss cattle, the two most important commercial breeds in the Alpine region. We report ROH islands near genes related to thermoregulation, coat color, production, and stature. The results of this study serve as a basis for the search for causal variants underlying adaptation to the alpine environment and other specific characteristics selected during the evolution of local Alpine cattle breeds.

Identification of two new recessive MC1R alleles in red-coloured Evolèner cattle and other breeds.

Sequence variations in the melanocortin-1 receptor (MC1R) gene are associated with melanism in different animal species. Six functionally relevant alleles have been described in cattle to date. In a hypothesis-free approach we performed a genome-wide allelic association study with black, red and wild-coloured cattle of three Alpine cattle breeds (Eringer, Evolèner and Valdostana), revealing a single significant association signal close to the MC1R gene. We searched for candidate causative variants by sequencing the entire coding sequence and identified two novel protein-changing variants. We propose designating the mutant alleles at MC1R:c.424C>T as ev1 and at MC1R:c.263G>A as ev2 . Both affect conserved amino acid residues in functionally important transmembrane domains (p.Arg142Cys and p.Ser88Asn). Both alleles segregate predominantly in the Swiss Evolèner breed. They occur in other European cattle breeds such as Abondance and Rotes Höhenvieh as well. We observed almost perfect association between the MC1R genotypes and the coat colour phenotype in a cohort of 513 black, red and wild-coloured cattle. Animals carrying two copies of MC1R loss-of-function alleles or that were compound heterozygous for e, ev1 , or ev2 have a red to dark red (chestnut-like red) coat colour. These findings expand the spectrum of causal MC1R variants causing recessive red in cattle.

Runs of homozygosity in Swiss goats reveal genetic changes associated with domestication and modern selection.

BACKGROUND: The domestication of goat (Capra hircus) started 11,000 years ago in the fertile crescent. Breed formation in the nineteenth century, establishment of herd books, and selection for specific traits resulted in 10 modern goat breeds in Switzerland. We analyzed whole-genome sequencing (WGS) data from 217 modern goats and nine wild Bezoar goats (Capra aegagrus). After quality control, 27,728,288 biallelic single nucleotide variants (SNVs) were used for the identification of runs of homozygosity (ROH) and the detection of ROH islands. RESULTS: Across the 226 caprine genomes from 11 populations, we detected 344 ROH islands that harbor 1220 annotated genes. We compared the ROH islands between the modern breeds and the Bezoar goats. As a proof of principle, we confirmed a signature of selection, which contains the ASIP gene that controls several breed-specific coat color patterns. In two other ROH islands, we identified two missense variants, STC1:p.Lys139Arg and TSHR:p.Ala239Thr, which might represent causative functional variants for domestication signatures. CONCLUSIONS: We have shown that the information from ROH islands using WGS data is suitable for the analysis of signatures of selection and allowed the detection of protein coding variants that may have conferred beneficial phenotypes during goat domestication. We hypothesize that the TSHR:p.Ala239Thr variant may have played a role in changing the seasonality of reproduction in modern domesticated goats. The exact functional significance of the STC1:p.Lys139Arg variant remains unclear and requires further investigation. Nonetheless, STC1 might represent a new domestication gene affecting relevant traits such as body size and/or milk yield in goats.

Short communication: Genomic prediction using imputed whole-genome sequence variants in Brown Swiss Cattle.

The accuracy of genomic prediction determines response to selection. It has been hypothesized that accuracy of genomic breeding values can be increased by a higher density of variants. We used imputed whole-genome sequence data and various single nucleotide polymorphism (SNP) selection criteria to estimate genomic breeding values in Brown Swiss cattle. The extreme scenarios were 50K SNP chip data and whole-genome sequence data with intermediate scenarios using linkage disequilibrium-pruned whole-genome sequence variants, only variants predicted to be missense, or the top 50K variants from genome-wide association studies. We estimated genomic breeding values for 3 traits (somatic cell score, nonreturn rate in heifers, and stature) and found differences in accuracy levels between traits. However, among different SNP sets, accuracy was very similar. In our analyses, sequence data led to a marginal increase in accuracy for 1 trait and was lower than 50K for the other traits. We concluded that the inclusion of imputed whole-genome sequence data does not lead to increased accuracy of genomic prediction with the methods.

Highly accurate sequence imputation enables precise QTL mapping in Brown Swiss cattle.

BACKGROUND: Within the last few years a large amount of genomic information has become available in cattle. Densities of genomic information vary from a few thousand variants up to whole genome sequence information. In order to combine genomic information from different sources and infer genotypes for a common set of variants, genotype imputation is required. RESULTS: In this study we evaluated the accuracy of imputation from high density chips to whole genome sequence data in Brown Swiss cattle. Using four popular imputation programs (Beagle, FImpute, Impute2, Minimac) and various compositions of reference panels, the accuracy of the imputed sequence variant genotypes was high and differences between the programs and scenarios were small. We imputed sequence variant genotypes for more than 1600 Brown Swiss bulls and performed genome-wide association studies for milk fat percentage at two stages of lactation. We found one and three quantitative trait loci for early and late lactation fat content, respectively. Known causal variants that were imputed from the sequenced reference panel were among the most significantly associated variants of the genome-wide association study. CONCLUSIONS: Our study demonstrates that whole-genome sequence information can be imputed at high accuracy in cattle populations. Using imputed sequence variant genotypes in genome-wide association studies may facilitate causal variant detection.

Population structure and genomic inbreeding in nine Swiss dairy cattle populations.

BACKGROUND: Domestication, breed formation and intensive selection have resulted in divergent cattle breeds that likely exhibit their own genomic signatures. In this study, we used genotypes from 27,612 autosomal single nucleotide polymorphisms to characterize population structure based on 9214 sires representing nine Swiss dairy cattle populations: Brown Swiss (BS), Braunvieh (BV), Original Braunvieh (OB), Holstein (HO), Red Holstein (RH), Swiss Fleckvieh (SF), Simmental (SI), Eringer (ER) and Evolèner (EV). Genomic inbreeding (F ROH) and signatures of selection were determined by calculating runs of homozygosity (ROH). The results build the basis for a better understanding of the genetic development of Swiss dairy cattle populations and highlight differences between the original populations (i.e. OB, SI, ER and EV) and those that have become more popular in Switzerland as currently reflected by their larger populations (i.e. BS, BV, HO, RH and SF). RESULTS: The levels of genetic diversity were highest and lowest in the SF and BS breeds, respectively. Based on F ST values, we conclude that, among all pairwise comparisons, BS and HO (0.156) differ more than the other pairs of populations. The original Swiss cattle populations OB, SI, ER, and EV are clearly genetically separated from the Swiss cattle populations that are now more common and represented by larger numbers of cows. Mean levels of F ROH ranged from 0.027 (ER) to 0.091 (BS). Three of the original Swiss cattle populations, ER (F ROH: 0.027), OB (F ROH: 0.029), and SI (F ROH: 0.039), showed low levels of genomic inbreeding, whereas it was much higher in EV (F ROH: 0.074). Private signatures of selection for the original Swiss cattle populations are reported for BTA4, 5, 11 and 26. CONCLUSIONS: The low levels of genomic inbreeding observed in the original Swiss cattle populations ER, OB and SI compared to the other breeds are explained by a lesser use of artificial insemination and greater use of natural service. Natural service results in more sires having progeny at each generation and thus this breeding practice is likely the major reason for the remarkable levels of genetic diversity retained within these populations. The fact that the EV population is regionally restricted and its small census size of herd-book cows explain its high level of genomic inbreeding.

Genome-wide association studies of fertility and calving traits in Brown Swiss cattle using imputed whole-genome sequences.

BACKGROUND: The detection of quantitative trait loci has accelerated with recent developments in genomics. The introduction of genomic selection in combination with sequencing efforts has made a large amount of genotypic data available. Functional traits such as fertility and calving traits have been included in routine genomic estimation of breeding values making large quantities of phenotypic data available for these traits. This data was used to investigate the genetics underlying fertility and calving traits and to identify potentially causative genomic regions and variants. We performed genome-wide association studies for 13 functional traits related to female fertility as well as for direct and maternal calving ease based on imputed whole-genome sequences. Deregressed breeding values from ~1000-5000 bulls per trait were used to test for associations with approximately 10 million imputed sequence SNPs. RESULTS: We identified a QTL on BTA17 associated with non-return rate at 56 days and with interval from first to last insemination. We found two significantly associated non-synonymous SNPs within this QTL region. Two more QTL for fertility traits were identified on BTA25 and 29. A single QTL was identified for maternal calving traits on BTA13 whereas three QTL on BTA19, 21 and 25 were identified for direct calving traits. The QTL on BTA19 co-localizes with the reported BH2 haplotype. The QTL on BTA25 is concordant for fertility and calving traits and co-localizes with a QTL previously reported to influence stature and related traits in Brown Swiss dairy cattle. CONCLUSION: The detection of QTL and their causative variants remains challenging. Combining comprehensive phenotypic data with imputed whole genome sequences seems promising. We present a QTL on BTA17 for female fertility in dairy cattle with two significantly associated non-synonymous SNPs, along with five additional QTL for fertility traits and calving traits. For all of these we fine mapped the regions and suggest candidate genes and candidate variants.

A structural variant in the 5'-flanking region of the TWIST2 gene affects melanocyte development in belted cattle.

Belted cattle have a circular belt of unpigmented hair and skin around their midsection. The belt is inherited as a monogenic autosomal dominant trait. We mapped the causative variant to a 37 kb segment on bovine chromosome 3. Whole genome sequence data of 2 belted and 130 control cattle yielded only one private genetic variant in the critical interval in the two belted animals. The belt-associated variant was a copy number variant (CNV) involving the quadruplication of a 6 kb non-coding sequence located approximately 16 kb upstream of the TWIST2 gene. Increased copy numbers at this CNV were strongly associated with the belt phenotype in a cohort of 333 cases and 1322 controls. We hypothesized that the CNV causes aberrant expression of TWIST2 during neural crest development, which might negatively affect melanoblasts. Functional studies showed that ectopic expression of bovine TWIST2 in neural crest in transgenic zebrafish led to a decrease in melanocyte numbers. Our results thus implicate an unsuspected involvement of TWIST2 in regulating pigmentation and reveal a non-coding CNV underlying a captivating Mendelian character.

Identification of key contributors in complex population structures.

Evaluating the genetic contribution of individuals to population structure is essential to select informative individuals for genome sequencing, genotype imputation and to ascertain complex population structures. Existing methods for the selection of informative individuals for genomic imputation solely focus on the identification of key ancestors, which can lead to a loss of phasing accuracy of the reference population. Currently many methods are independently applied to investigate complex population structures. Based on the Eigenvalue Decomposition (EVD) of a genomic relationship matrix we describe a novel approach to evaluate the genetic contribution of individuals to population structure. We combined the identification of key contributors with model-based clustering and population network visualization into an integrated three-step approach, which allows identification of high-resolution population structures and substructures around such key contributors. The approach was applied and validated in four disparate datasets including a simulated population (5,100 individuals and 10,000 SNPs), a highly structured experimental sheep population (1,421 individuals and 44,693 SNPs) and two large complex pedigree populations namely horse (1,077 individuals and 38,124 SNPs) and cattle (2,457 individuals and 45,765 SNPs). In the simulated and experimental sheep dataset, our method, which is unsupervised, successfully identified all known key contributors. Applying our three-step approach to the horse and cattle populations, we observed high-resolution population substructures including the absence of obvious important key contributors. Furthermore, we show that compared to commonly applied strategies to select informative individuals for genotype imputation including the computation of marginal gene contributions (Pedig) and the optimization of genetic relatedness (Rel), the selection of key contributors provided the highest phasing accuracies within the selected reference populations. The presented approach opens new perspectives in the characterization and informed management of populations in general, and in areas such as conservation genetics and selective animal breeding in particular, where assessing the genetic contribution of influential and admixed individuals is crucial for research and management applications. As such, this method provides a valuable complement to common applied tools to visualize complex population structures and to select individuals for re-sequencing.

A Non-Synonymous HMGA2 Variant Decreases Height in Shetland Ponies and Other Small Horses.

The identification of quantitative trait loci (QTL) such as height and their underlying causative variants is still challenging and often requires large sample sizes. In humans hundreds of loci with small effects control the heritable portion of height variability. In domestic animals, typically only a few loci with comparatively large effects explain a major fraction of the heritability. We investigated height at withers in Shetland ponies and mapped a QTL to ECA 6 by genome-wide association (GWAS) using a small cohort of only 48 animals and the Illumina equine SNP70 BeadChip. Fine-mapping revealed a shared haplotype block of 793 kb in small Shetland ponies. The HMGA2 gene, known to be associated with height in horses and many other species, was located in the associated haplotype. After closing a gap in the equine reference genome we identified a non-synonymous variant in the first exon of HMGA2 in small Shetland ponies. The variant was predicted to affect the functionally important first AT-hook DNA binding domain of the HMGA2 protein (c.83G>A; p.G28E). We assessed the functional impact and found impaired DNA binding of a peptide with the mutant sequence in an electrophoretic mobility shift assay. This suggests that the HMGA2 variant also affects DNA binding in vivo and thus leads to reduced growth and a smaller stature in Shetland ponies. The identified HMGA2 variant also segregates in several other pony breeds but was not found in regular-sized horse breeds. We therefore conclude that we identified a quantitative trait nucleotide for height in horses.

Evaluation of variant identification methods for whole genome sequencing data in dairy cattle.

BACKGROUND: Advances in human genomics have allowed unprecedented productivity in terms of algorithms, software, and literature available for translating raw next-generation sequence data into high-quality information. The challenges of variant identification in organisms with lower quality reference genomes are less well documented. We explored the consequences of commonly recommended preparatory steps and the effects of single and multi sample variant identification methods using four publicly available software applications (Platypus, HaplotypeCaller, Samtools and UnifiedGenotyper) on whole genome sequence data of 65 key ancestors of Swiss dairy cattle populations. Accuracy of calling next-generation sequence variants was assessed by comparison to the same loci from medium and high-density single nucleotide variant (SNV) arrays. RESULTS: The total number of SNVs identified varied by software and method, with single (multi) sample results ranging from 17.7 to 22.0 (16.9 to 22.0) million variants. Computing time varied considerably between software. Preparatory realignment of insertions and deletions and subsequent base quality score recalibration had only minor effects on the number and quality of SNVs identified by different software, but increased computing time considerably. Average concordance for single (multi) sample results with high-density chip data was 58.3% (87.0%) and average genotype concordance in correctly identified SNVs was 99.2% (99.2%) across software. The average quality of SNVs identified, measured as the ratio of transitions to transversions, was higher using single sample methods than multi sample methods. A consensus approach using results of different software generally provided the highest variant quality in terms of transition/transversion ratio. CONCLUSIONS: Our findings serve as a reference for variant identification pipeline development in non-human organisms and help assess the implication of preparatory steps in next-generation sequencing pipelines for organisms with incomplete reference genomes (pipeline code is included). Benchmarking this information should prove particularly useful in processing next-generation sequencing data for use in genome-wide association studies and genomic selection.

Congenital hepatic fibrosis in the Franches-Montagnes horse is associated with the polycystic kidney and hepatic disease 1 (PKHD1) gene.

Congenital hepatic fibrosis has been described as a lethal disease with monogenic autosomal recessive inheritance in the Swiss Franches-Montagnes horse breed. We performed a genome-wide association study with 5 cases and 12 controls and detected an association on chromosome 20. Subsequent homozygosity mapping defined a critical interval of 952 kb harboring 10 annotated genes and loci including the polycystic kidney and hepatic disease 1 (autosomal recessive) gene (PKHD1). PKHD1 represents an excellent functional candidate as variants in this gene were identified in human patients with autosomal recessive polycystic kidney and hepatic disease (ARPKD) as well as several mouse and rat mutants. Whereas most pathogenic PKHD1 variants lead to polycystic defects in kidney and liver, a small subset of the human ARPKD patients have only liver symptoms, similar to our horses with congenital hepatic fibrosis. The PKHD1 gene is one of the largest genes in the genome with multiple alternative transcripts that have not yet been fully characterized. We sequenced the genomes of an affected foal and 46 control horses to establish a comprehensive list of variants in the critical interval. We identified two missense variants in the PKHD1 gene which were strongly, but not perfectly associated with congenital hepatic fibrosis. We speculate that reduced penetrance and/or potential epistatic interactions with hypothetical modifier genes may explain the imperfect association of the detected PKHD1 variants. Our data thus indicate that horses with congenital hepatic fibrosis represent an interesting large animal model for the liver-restricted subtype of human ARPKD.

A chromosomal region on ECA13 is associated with maxillary prognathism in horses.

Hereditary variations in head morphology and head malformations are known in many species. The most common variation encountered in horses is maxillary prognathism. Prognathism and brachygnathism are syndromes of the upper and lower jaw, respectively. The resulting malocclusion can negatively affect teeth wear, and is considered a non-desirable trait in breeding programs. We performed a case-control analysis for maxillary prognathism in horses using 96 cases and 763 controls. All horses had been previously genotyped with a commercially available 50 k SNP array. We analyzed the data with a mixed-model considering the genomic relationships in order to account for population stratification. Two SNPs within a region on the distal end of chromosome ECA 13 reached the Bonferroni corrected genome-wide significance level. There is no known prognathism candidate gene located within this region. Therefore, our findings in the horse offer the possibility of identifying a novel gene involved in the complex genetics of prognathism that might also be relevant for humans and other livestock species.

Accumulating mutations in series of haplotypes at the KIT and MITF loci are major determinants of white markings in Franches-Montagnes horses.

Coat color and pattern variations in domestic animals are frequently inherited as simple monogenic traits, but a number are known to have a complex genetic basis. While the analysis of complex trait data remains a challenge in all species, we can use the reduced haplotypic diversity in domestic animal populations to gain insight into the genomic interactions underlying complex phenotypes. White face and leg markings are examples of complex traits in horses where little is known of the underlying genetics. In this study, Franches-Montagnes (FM) horses were scored for the occurrence of white facial and leg markings using a standardized scoring system. A genome-wide association study (GWAS) was performed for several white patterning traits in 1,077 FM horses. Seven quantitative trait loci (QTL) affecting the white marking score with p-values p≤10(-4) were identified. Three loci, MC1R and the known white spotting genes, KIT and MITF, were identified as the major loci underlying the extent of white patterning in this breed. Together, the seven loci explain 54% of the genetic variance in total white marking score, while MITF and KIT alone account for 26%. Although MITF and KIT are the major loci controlling white patterning, their influence varies according to the basic coat color of the horse and the specific body location of the white patterning. Fine mapping across the MITF and KIT loci was used to characterize haplotypes present. Phylogenetic relationships among haplotypes were calculated to assess their selective and evolutionary influences on the extent of white patterning. This novel approach shows that KIT and MITF act in an additive manner and that accumulating mutations at these loci progressively increase the extent of white markings.

A genome-wide association study reveals loci influencing height and other conformation traits in horses.

The molecular analysis of genes influencing human height has been notoriously difficult. Genome-wide association studies (GWAS) for height in humans based on tens of thousands to hundreds of thousands of samples so far revealed ∼200 loci for human height explaining only 20% of the heritability. In domestic animals isolated populations with a greatly reduced genetic heterogeneity facilitate a more efficient analysis of complex traits. We performed a genome-wide association study on 1,077 Franches-Montagnes (FM) horses using ∼40,000 SNPs. Our study revealed two QTL for height at withers on chromosomes 3 and 9. The association signal on chromosome 3 is close to the LCORL/NCAPG genes. The association signal on chromosome 9 is close to the ZFAT gene. Both loci have already been shown to influence height in humans. Interestingly, there are very large intergenic regions at the association signals. The two detected QTL together explain ∼18.2% of the heritable variation of height in horses. However, another large fraction of the variance for height in horses results from ECA 1 (11.0%), although the association analysis did not reveal significantly associated SNPs on this chromosome. The QTL region on ECA 3 associated with height at withers was also significantly associated with wither height, conformation of legs, ventral border of mandible, correctness of gaits, and expression of the head. The region on ECA 9 associated with height at withers was also associated with wither height, length of croup and length of back. In addition to these two QTL regions on ECA 3 and ECA 9 we detected another QTL on ECA 6 for correctness of gaits. Our study highlights the value of domestic animal populations for the genetic analysis of complex traits.