Whole-genome scan for guttural pouch tympany in Arabian and German warmblood horses

Equine guttural pouch tympany (GPT) is a hereditary disease in foals of several breeds, including thoroughbreds, Arabian, Quarter and warmblood horses. We performed a whole-genome scan for GPT in 143 horses from five Arabian and five German warmblood families and genotyped 257 microsatellites. Chromosome-wide significant linkage was detected on ECA2 and ECA15 using multipoint non-parametric linkage analyses. Analyses stratified by sex revealed chromosome-wide significant linkage on ECA2 for fillies and chromosome-wide significant linkage on ECA15 for colts. For Arabian colts, the quantitative trait locus (QTL) on ECA15 was genome-wide significant. Haplotypes including two to four microsatellites within the QTL on ECA2 and 15 in fillies and colts, respectively, were significantly associated with GPT for both breeds. Thus, our analysis indicated sex-specific QTL, a fact which is in agreement with a two- to fourfold higher incidence of GPT in females. This is the first report of QTL for equine GPT and a first step towards identifying genes responsible for GPT.     

Refinement of a quantitative gene locus on equine chromosome 16 responsible for osteochondrosis in Hanoverian warmblood horses

Osteochondrosis (OC) is an inherited developmental disease in young horses most frequently observed in thoroughbreds, trotters, warmblood and coldblood horses. Quantitative trait loci (QTL) for equine OC have been identified in Hanoverian warmblood horses employing a whole genome scan with microsatellites. A QTL on ECA16 reached the genome-wide significance level for hock osteochondrosis dissecans (OCD). The aim of this study was to refine this QTL on ECA16 using an extended marker set of 34 newly developed microsatellites and 15 single nucleotide polymorphisms (SNPs). We used the same 14 paternal half-sib groups as in the above-mentioned whole genome scan. The QTL for OCD in hock joints on ECA16 could be delimited at an interval between 17.60 and 45.18 Mb using multipoint non-parametric linkage analyses. In addition, six microsatellites and one SNP were significantly associated with hock OCD in the QTL region between 24.26 and 42.41 Mb. Furthermore, our analysis revealed a second QTL for fetlock OC between 6.55 and 24.26 Mb on ECA16. This report is a further step towards unravelling the genes underlying QTL for equine OC and towards the development of a marker test for OC in Hanoverian warmblood horses.     

A Genome-Wide Association Study Identifies Risk Loci to Equine Recurrent Uveitis in German Warmblood Horses

Equine recurrent uveitis (ERU) is a common eye disease affecting up to 3–15% of the horse population. A genome-wide association study (GWAS) using the Illumina equine SNP50 bead chip was performed to identify loci conferring risk to ERU. The sample included a total of 144 German warmblood horses. A GWAS showed a significant single nucleotide polymorphism (SNP) on horse chromosome (ECA) 20 at 49.3 Mb, with IL-17A and IL-17F being the closest genes. This locus explained a fraction of 23% of the phenotypic variance for ERU. A GWAS taking into account the severity of ERU, revealed a SNP on ECA18 nearby to the crystalline gene cluster CRYGA-CRYGF. For both genomic regions on ECA18 and 20, significantly associated haplotypes containing the genome-wide significant SNPs could be demonstrated. In conclusion, our results are indicative for a genetic component regulating the possible critical role of IL-17A and IL-17F in the pathogenesis of ERU. The associated SNP on ECA18 may be indicative for cataract formation in the course of ERU.     

Refinement of quantitative trait loci on equine chromosome 10 for radiological signs of navicular disease in Hanoverian warmblood horses

Navicular disease is characterized by a progressive degenerative alteration of the equine podotrochlea. In this study, we refined a previously identified quantitative trait locus (QTL) on horse chromosome 10 for the abnormal development of canales sesamoidales (DCS) of the navicular bone in Hanoverian warmblood horses. Genotyping was done in 192 Hanoverian warmblood horses from 17 paternal half-sib groups. The whole marker set comprised 45 markers including seven newly developed microsatellites and 13 single nucleotide polymorphisms (SNPs) within positional candidate genes. Chromosome-wide significant QTL were confirmed and refined for DCS on horse chromosome (ECA) 10 at 0.16-2.70 Mb and at 14.45-36.37 Mb. Nine microsatellites and three SNP markers reached the highest multipoint Zmeans and LOD scores at 19.34-20.38 Mb and at 23.17-30.73 Mb with genome-wide error probabilities of P<0.05. In addition, a significant association of a SNP within VSTM1 and a significant haplotype-trait association within IRF3 could be shown. These results support a possible role of the candidate genes VSTM1 and IRF3 within the QTL on ECA10 for DCS. This study is a further step towards the identification of the genes responsible for navicular disease in Hanoverian warmblood horses.     

Refinement of a quantitative trait locus on equine chromosome 5 responsible for fetlock osteochondrosis in Hanoverian warmblood horses

In this report, we provide 29 new informative microsatellites distributed over a region of 21 Mb on horse chromosome (ECA) 5 and refine a quantitative trait locus (QTL) for fetlock osteochondrosis dissecans (OCD) to a genome-wide significant interval between 78.03 and 90.23 Mb on ECA5. Genotyping was performed in 211 Hanoverian warmblood horses from 14 paternal half-sib groups. Within this OCD-QTL, collagen type XXIV alpha 1 was identified as a potential functional candidate gene for equine osteochondrosis. This report is a further step towards unravelling the genes that cause equine osteochondrosis.     

A genome-wide association study for quantitative trait loci of show-jumping in Hanoverian warmblood horses

Show-jumping is an economically important breeding goal in Hanoverian warmblood horses. The aim of this study was a genome-wide association study (GWAS) for quantitative trait loci (QTL) for show-jumping in Hanoverian warmblood horses, employing the Illumina equine SNP50 Beadchip. For our analyses, we genotyped 115 stallions of the National State stud of Lower Saxony. The show-jumping talent of a horse includes style and ability in free-jumping. To control spurious associations based on population stratification, two different mixed linear animal model (MLM) approaches were employed, besides linear models with fixed effects only and adaptive permutations for correcting multiple testing. Population stratification was explained best in the MLM considering Hanoverian, Thoroughbred, Trakehner and Holsteiner genes and the marker identity-by-state relationship matrix. We identified six QTL for show-jumping on horse chromosomes (ECA) 1, 8, 9 and 26 (-log(10) P-value >5) and further putative QTL with -log(10) P-values of 3-5 on ECA1, 3, 11, 17 and 21. Within six QTL regions, we identified human performance-related genes including PAPSS2 on ECA1, MYL2 on ECA8, TRHR on ECA9 and GABPA on ECA26 and within the putative QTL regions NRAP on ECA1, and TBX4 on ECA11. The results of our GWAS suggest that genes involved in muscle structure, development and metabolism are crucial for elite show-jumping performance. Further studies are required to validate these QTL in larger data sets and further horse populations.     

A genome-wide association study of osteochondritis dissecans in the Thoroughbred

Osteochondrosis is a developmental orthopaedic disease that occurs in horses, other livestock species, companion animal species, and humans. The principal aim of this study was to identify quantitative trait loci (QTL) associated with osteochondritis dissecans (OCD) in the Thoroughbred using a genome-wide association study. A secondary objective was to test the effect of previously identified QTL in the current population. Over 300 horses, classified as cases or controls according to clinical findings, were genotyped for the Illumina Equine SNP50 BeadChip. An animal model was first implemented in order to adjust each horse's phenotypic status for average relatedness among horses and other potentially confounding factors which were present in the data. The genome-wide association test was then conducted on the residuals from the animal model. A single SNP on chromosome 3 was found to be associated with OCD at a genome-wide level of significance, as determined by permutation. According to the current sequence annotation, the SNP is located in an intergenic region of the genome. The effects of 24 SNPs, representing QTL previously identified in a sample of Hanoverian Warmblood horses, were tested directly in the animal model. When fitted alongside the significant SNP on ECA3, two of these SNPs were found to be associated with OCD. Confirmation of the putative QTL identified on ECA3 requires validation in an independent sample. The results of this study suggest that a significant challenge faced by equine researchers is the generation of sufficiently large data sets to effectively study complex diseases such as osteochondrosis.     

A genome-wide scan for tying-up syndrome in Japanese Thoroughbreds

Tying-up syndrome, also known as recurrent exertional rhabdomyolysis in Thoroughbreds, is a common muscle disorder for racehorses. In this study, we performed a multipoint linkage analysis using LOKI based on the Bayesian Markov chain Monte Carlo method using 5 half-sib families (51 affected and 277 nonaffected horses in total), and a genome-wide association study (GWAS) using microsatellites (144 affected and 144 nonaffected horses) to map candidate regions for tying-up syndrome in Japanese Thoroughbreds. The linkage analysis identified one strong L-score (82.45) between the loci UCDEQ411 and COR058 (24.9-27.9 Mb) on ECA12. The GWAS identified two suggestive genomic regions on ECA12 (24.9-27.8 Mb) and ECA20 (29.3-33.5 Mb). Based on both results, the genomic region between UCDEQ411 and TKY499 (24.9-27.8 Mb) on ECA12 was the most significant and was considered as a candidate region for tying-up syndrome in Japanese Thoroughbreds.     

Fine mapping of a quantitative trait locus for osteochondrosis on horse chromosome 2

In this study, we refine a quantitative trait locus for equine osteochondrosis (OC) on horse chromosome (ECA) 2 to a genome-wide significant interval at 20.08-30.94 Mb. The marker set contained 27 newly developed microsatellites equidistantly distributed over ECA2 and 44 nucleotide polymorphisms, located in 16 positional candidate genes for OC. Genotyping was performed in 211 Hanoverian horses from 14 paternal half-sib groups. A NCDN-associated SNP and haplotype were significantly associated with OC in fetlock and/or hock joints. This study is a further step towards the identification of genes responsible for OC in horses.     

Genome-wide association analysis of osteochondrosis of the tibiotarsal joint in Norwegian Standardbred trotters

Osteochondrosis (OC), a disturbance in the process of endochondral ossification, is by far the most important equine developmental orthopaedic disease and is also common in other domestic animals and humans. The purpose of this study was to identify quantitative trait loci (QTL) associated with osteochondrosis dissecans (OCD) at the intermediate ridge of the distal tibia in Norwegian Standardbred (SB) using the Illumina Equine SNP50 BeadChip whole-genome single-nucleotide polymorphism (SNP) assay. Radiographic data and blood samples were obtained from 464 SB yearlings. Based on the radiographic examination, 162 horses were selected for genotyping; 80 of these were cases with an OCD at the intermediate ridge of the distal tibia, and 82 were controls without any developmental lesions in the joints examined. Genotyped horses descended from 22 sires, and the number of horses in each half-sib group ranged from 3 to 14. The population structure necessitated statistical correction for stratification. When conducting a case-control genome-wide association study (GWAS), mixed-model analyses displayed regions on chromosomes (Equus callabus chromosome - ECA) 5, 10, 27 and 28 that showed moderate evidence of association (P ≤ 5 × 10(-5); this P-value is uncorrected i.e. not adjusted for multiple comparisons) with OCD in the tibiotarsal joint. Two SNPs on ECA10 represent the most significant hits (uncorrected P=1.19 × 10(-5) in the mixed-model). In the basic association (chi-square) test, these SNPs achieved statistical significance with the Bonferroni correction (P=0.038) and were close in the permuted logistic regression test (P=0.054). Putative QTL on ECA 5, 10, 27 and 28 represent interesting areas for future research, validation studies and fine mapping of candidate regions. Results presented here represent the first GWAS of OC in horses using the recently released Illumina Equine SNP50 BeadChip.     

Fine mapping a quantitative trait locus on horse chromosome 2 associated with radiological signs of navicular disease in Hanoverian warmblood horses

Navicular disease or podotrochlosis is one of the main causes of progressive forelimb lameness in warmblood horses. The objective of this study was to refine a quantitative trait locus on horse chromosome 2 for radiological alterations in the contour of the navicular bone (RAC) in Hanoverian warmblood horses. Genotyping was performed in 192 Hanoverian warmblood horses from 17 paternal half-sib groups. The marker set was extended to 58 informative microsatellites including nine newly developed microsatellites. QTL for RAC could be delineated at 32.50–43.13 Mb and a further new QTL for RAC could be identified at 59.08–65.14 Mb. The markers ABGe342 and ABGe343 reached the highest multipoint Z _mean and LOD scores at 34.42 and 35.23 Mb with genome-wide error probabilities of P  = 0.013 and P  = 0.064. In addition, significant associations of markers and haplotypes within the QTL could be shown. The results support the location of the QTL on ECA2 associated with RAC. This work is a further step towards the development of a marker test for navicular disease in Hanoverian warmblood horses.     

Associations between candidate gene markers at a quantitative trait locus on equine chromosome 4 responsible for osteochondrosis dissecans in fetlock joints of South German Coldblood horses

A previously accomplished whole-genome scan for osteochondrosis (OC) and OC dissecans (OCD) in South German Coldblood horses using 250 microsatellite markers identified putative quantitative trait loci (QTL). A chromosome-wide significant QTL for fetlock OCD was found on Equus caballus chromosome (ECA) 4q at a relative position of 70.0-73.3 cM. The aim of this study was to analyze associations of single nucleotide polymorphisms (SNPs) in candidate genes for OC in this region. The association analysis included 32 affected and 64 unaffected horses. Three SNPs located in intron 8, intron 9, and 3'-untranslated region (UTR) of the acyloxyacyl hydrolase (AOAH) gene on ECA4q were significantly associated with OCD in fetlock joints. In order to control for systematic environmental and quantitative genetic effects, we employed a linear animal model. The association of the SNP (AJ543065:g.703A>G) in the 3'-UTR of exon 21 was confirmed in the animal model analysis and a significant additive genetic effect for fetlock OCD of 0.42 (P = 0.002) and a dominance effect of -0.32 (P = 0.03) was estimated. This is the first report on a marker in population-wide linkage disequilibrium with equine OCD in fetlock joints.     

Genome-wide search for microsatellite markers associated with radiologic alterations in the navicular bone of Hanoverian warmblood horses

The aim of this study was to identify quantitative trait loci (QTLs) for pathologic changes in the navicular bone in Hanoverian warmblood horses. Seventeen paternal half-sib groups comprising 192 individuals were analyzed in a whole-genome scan. These families included 144 progeny and grandchildren, which were randomly chosen from the Hanoverian warmblood. Three different traits were considered: deformed canales sesamoidales and radiographic changes in the contour and in the structure of the navicular bone. The genome scan included in total 214 highly polymorphic microsatellite markers. The putatively linked genomic regions on equine chromosomes (ECA) 2, 3, 10, and 15 were refined using 53 additional microsatellites. Chromosome-wide significant QTLs were located on five different equine chromosomes (ECA2, 3, 4, 10, and 26). Genome-wide significant QTLs were on ECA2 at 48 cM and on ECA10 from 45.5 to 49.8 cM. This study was a first step to get more insight into the molecular genetic determination of radiologic changes in the equine navicular bone. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Whole-genome scan identifies quantitative trait loci for chronic pastern dermatitis in German draft horses

Chronic pastern dermatitis (CPD), also known as chronic progressive lymphedema (CPL), is a skin disease that affects draft horses. This disease causes painful lower-leg swelling, nodule formation, and skin ulceration, interfering with movement. The aim of this whole-genome scan was to identify quantitative trait loci (QTL) for CPD in German draft horses. We recorded clinical data for CPD in 917 German draft horses and collected blood samples from these horses. Of these 917 horses, 31 paternal half-sib families comprising 378 horses from the breeds Rhenish German, Schleswig, Saxon-Thuringian, and South German were chosen for genotyping. Each half-sib family was constituted by only one draft horse breed. Genotyping was done for 318 polymorphic microsatellites evenly distributed on all equine autosomes and the X chromosome with a mean distance of 7.5 Mb. An across-breed multipoint linkage analysis revealed chromosome-wide significant QTL on horse chromosomes (ECA) 1, 9, 16, and 17. Analyses by breed confirmed the QTL on ECA1 in South German and the QTL on ECA9, 16, and 17 in Saxon-Thuringian draft horses. For the Rhenish German and Schleswig draft horses, additional QTL on ECA4 and 10 and for the South German draft horses an additional QTL on ECA7 were found. This is the first whole-genome scan for CPD in draft horses and it is an important step toward the identification of candidate genes.     

Detection of copy number variants in the horse genome and examination of their association with recurrent laryngeal neuropathy

We used the data from a recently performed genome‐wide association study using the Illumina Equine SNP50 beadchip for the detection of copy number variants (CNVs) and examined their association with recurrent laryngeal neuropathy (RLN), an important equine upper airway disease compromising performance. A total of 2797 CNVs were detected for 477 horses, covering 229 kb and seven SNPs on average. Overlapping CNVs were merged to define 478 CNV regions (CNVRs). CNVRs, particularly deletions, were shown to be significantly depleted in genes. Fifty‐two of the 67 common CNVRs (frequency ≥ 1%) were validated by association mapping, Mendelian inheritance, and/or Mendelian inconsistencies. None of the 67 common CNVRs were significantly associated with RLN when accounting for multiple testing. However, a duplication on chromosome 10 was detected in 10 cases (representing three breeds) and two unphenotyped parents but in none of the controls. The duplication was embedded in an 8‐Mb haplotype shared across breeds.     

Genome-wide association study among four horse breeds identifies a common haplotype associated with in vitro CD3+ T cell susceptibility/resistance to equine arteritis virus infection

Previously, we have shown that horses could be divided into susceptible and resistant groups based on an in vitro assay using dual-color flow cytometric analysis of CD3+ T cells infected with equine arteritis virus (EAV). Here, we demonstrate that the differences in in vitro susceptibility of equine CD3+ T lymphocytes to EAV infection have a genetic basis. To investigate the possible hereditary basis for this trait, we conducted a genome-wide association study (GWAS) to compare susceptible and resistant phenotypes. Testing of 267 DNA samples from four horse breeds that had a susceptible or a resistant CD3+ T lymphocyte phenotype using both Illumina Equine SNP50 BeadChip and Sequenom's MassARRAY system identified a common, genetically dominant haplotype associated with the susceptible phenotype in a region of equine chromosome 11 (ECA11), positions 49572804 to 49643932. The presence of a common haplotype indicates that the trait occurred in a common ancestor of all four breeds, suggesting that it may be segregated among other modern horse breeds. Biological pathway analysis revealed several cellular genes within this region of ECA11 encoding proteins associated with virus attachment and entry, cytoskeletal organization, and NF-κB pathways that may be associated with the trait responsible for the in vitro susceptibility/resistance of CD3+ T lymphocytes to EAV infection. The data presented in this study demonstrated a strong association of genetic markers with the trait, representing de facto proof that the trait is under genetic control. To our knowledge, this is the first GWAS of an equine infectious disease and the first GWAS of equine viral arteritis.     

Replication and fine-mapping of a QTL for recurrent airway obstruction in European Warmblood horses

Recurrent airway obstruction (RAO), or ‘heaves’, is a common performance‐limiting allergic respiratory disease of mature horses. It is related to sensitization and exposure to mouldy hay and has a familial basis with a complex mode of inheritance. In a previous study, we detected a QTL for RAO on ECA 13 in a half‐sib family of European Warmblood horses. In this study, we genotyped additional markers in the family and narrowed the QTL down to about 1.5 Mb (23.7–25.2 Mb). We detected the strongest association with SNP BIEC2‐224511 (24 309 405 bp). We also obtained SNP genotypes in an independent cohort of 646 unrelated Warmblood horses. There was no genome‐wide significant association with RAO in these unrelated horses. However, we performed a genotypic association study of the SNPs on ECA 13 in these unrelated horses, and the SNP BIEC2‐224511 also showed the strongest association with RAO in the unrelated horses (p_raw = 0.00037). The T allele at this SNP was associated with RAO both in the family and the unrelated horses. Thus, the association study in the unrelated animals provides independent support for the previously detected QTL. The association study allows further narrowing of the QTL interval to about 0.5 Mb (24.0–24.5 Mb). We sequenced the coding regions of the genes in the critical region but did not find any associated coding variants. Therefore, the causative variant underlying this QTL is likely to be a regulatory mutation.     

Identification of the myostatin locus (MSTN) as having a major effect on optimum racing distance in the Thoroughbred horse in the USA

One hundred and eighty-nine Thoroughbred horses that had won Graded Stakes races in North America were genotyped with the Illumina Equine SNP50 bead chip. Association tests using PLINK to determine whether any SNPs were associated with optimum racing distance (7 furlongs and under compared to 8-10 furlongs) identified a locus on ECA18 that was statistically significant (-log 10 EMP2=1.63) at the genome-wide level following permutation analysis (10,000 permutations). Bioinformatic analysis revealed that the two ECA18 SNPs with the highest statistical significance spanned the MSTN (myostatin) locus. Mutations in myostatin in several mammalian species have been associated with increased muscling, with a preferential increase in fast glycolytic type IIB fibres, which would increase power potential. Thoroughbred horses that race over sprint distances, which are 5-7 furlongs, are often characterized by impressive hind quarter musculature, strongly suggesting that the association observed between the ECA18 SNPs and optimum race distance is mediated through MSTN.     

Identification of a mutation associated with fatal Foal Immunodeficiency Syndrome in the Fell and Dales pony

The Fell and Dales are rare native UK pony breeds at risk due to falling numbers, in-breeding, and inherited disease. Specifically, the lethal Mendelian recessive disease Foal Immunodeficiency Syndrome (FIS), which manifests as B-lymphocyte immunodeficiency and progressive anemia, is a substantial threat. A significant percentage (～10%) of the Fell ponies born each year dies from FIS, compromising the long-term survival of this breed. Moreover, the likely spread of FIS into other breeds is of major concern. Indeed, FIS was identified in the Dales pony, a related breed, during the course of this work. Using a stepwise approach comprising linkage and homozygosity mapping followed by haplotype analysis, we mapped the mutation using 14 FIS-affected, 17 obligate carriers, and 10 adults of unknown carrier status to a ～1 Mb region (29.8 - 30.8 Mb) on chromosome (ECA) 26. A subsequent genome-wide association study identified two SNPs on ECA26 that showed genome-wide significance after Bonferroni correction for multiple testing: BIEC2-692674 at 29.804 Mb and BIEC2-693138 at 32.19 Mb. The associated region spanned 2.6 Mb from ～29.6 Mb to 32.2 Mb on ECA26. Re-sequencing of this region identified a mutation in the sodium/myo-inositol cotransporter gene (SLC5A3); this causes a P446L substitution in the protein. This gene plays a crucial role in the regulatory response to osmotic stress that is essential in many tissues including lymphoid tissues and during early embryonic development. We propose that the amino acid substitution we identify here alters the function of SLC5A3, leading to erythropoiesis failure and compromise of the immune system. FIS is of significant biological interest as it is unique and is caused by a gene not previously associated with a mammalian disease. Having identified the associated gene, we are now able to eradicate FIS from equine populations by informed selective breeding.