Simultaneous testing of rule- and model-based approaches for runs of homozygosity detection opens up a window into genomic footprints of selection in pigs.

BACKGROUND: Past selection events left footprints in the genome of domestic animals, which can be traced back by stretches of homozygous genotypes, designated as runs of homozygosity (ROHs). The analysis of common ROH regions within groups or populations displaying potential signatures of selection requires high-quality SNP data as well as carefully adjusted ROH-defining parameters. In this study, we used a simultaneous testing of rule- and model-based approaches to perform strategic ROH calling in genomic data from different pig populations to detect genomic regions under selection for specific phenotypes. RESULTS: Our ROH analysis using a rule-based approach offered by PLINK, as well as a model-based approach run by RZooRoH demonstrated a high efficiency of both methods. It underlined the importance of providing a high-quality SNP set as input as well as adjusting parameters based on dataset and population for ROH calling. Particularly, ROHs ≤ 20 kb were called in a high frequency by both tools, but to some extent covered different gene sets in subsequent analysis of ROH regions common for investigated pig groups. Phenotype associated ROH analysis resulted in regions under potential selection characterizing heritage pig breeds, known to harbour a long-established breeding history. In particular, the selection focus on fitness-related traits was underlined by various ROHs harbouring disease resistance or tolerance-associated genes. Moreover, we identified potential selection signatures associated with ear morphology, which confirmed known candidate genes as well as uncovered a missense mutation in the ABCA6 gene potentially supporting ear cartilage formation. CONCLUSIONS: The results of this study highlight the strengths and unique features of rule- and model-based approaches as well as demonstrate their potential for ROH analysis in animal populations. We provide a workflow for ROH detection, evaluating the major steps from filtering for high-quality SNP sets to intersecting ROH regions. Formula-based estimations defining ROHs for rule-based method show its limits, particularly for efficient detection of smaller ROHs. Moreover, we emphasize the role of ROH detection for the identification of potential footprints of selection in pigs, displaying their breed-specific characteristics or favourable phenotypes.

De novo ZIC2 frameshift variant associated with frontonasal dysplasia in a Limousin calf.

BACKGROUND: Bovine frontonasal dysplasias like arhinencephaly, synophthalmia, cyclopia and anophthalmia are sporadic congenital facial malformations. In this study, computed tomography, necropsy, histopathological examinations and whole genome sequencing on an Illumina NextSeq500 were performed to characterize a stillborn Limousin calf with frontonasal dysplasia. In order to identify private genetic and structural variants, we screened whole genome sequencing data of the affected calf and unaffected relatives including parents, a maternal and paternal halfsibling. RESULTS: The stillborn calf exhibited severe craniofacial malformations. Nose and maxilla were absent, mandibles were upwardly curved and a median cleft palate was evident. Eyes, optic nerve and orbital cavities were not developed and the rudimentary orbita showed hypotelorism. A defect centrally in the front skull covered with a membrane extended into the intracranial cavity. Aprosencephaly affected telencephalic and diencephalic structures and cerebellum. In addition, a shortened tail was seen. Filtering whole genome sequencing data revealed a private frameshift variant within the candidate gene ZIC2 in the affected calf. This variant was heterozygous mutant in this case and homozygous wild type in parents, half-siblings and controls. CONCLUSIONS: We found a novel ZIC2 frameshift mutation in an aprosencephalic Limousin calf. The origin of this variant is most likely due to a de novo mutation in the germline of one parent or during very early embryonic development. To the authors' best knowledge, this is the first identified mutation in cattle associated with bovine frontonasal dysplasia.

A structural UGDH variant associated with standard Munchkin cats.

BACKGROUND: Munchkin cats were founded on a naturally occurring mutation segregating into long-legged and short-legged types. Short-legged cats showed disproportionate dwarfism (chondrodysplasia) in which all four legs are short and are referred as standard Munchkin cats. Long-legged animals are referred as non-standard Munchkin cats. A previous study using genome-wide single nucleotide polymorphisms (SNPs) for genome-wide association analysis identified a significantly associated region at 168-184 Mb on feline chromosome (FCA) B1. RESULTS: In this study, we validated the critical region on FCA B1 using a case-control study with 89 cats and 14 FCA B1-SNPs. A structural variant within UGDH (NC_018726.2:g.173294289_173297592delins108, Felis catus 8.0, equivalent to NC_018726.3:g.174882895_174886198delins108, Felis catus 9.0) on FCA B1 was perfectly associated with the phenotype of short-legged standard Munchkin cats. CONCLUSION: This UGDH structural variant very likely causes the chondrodysplastic (standard) phenotype in Munchkin cats. The lack of homozygous mutant phenotypes and reduced litter sizes in standard Munchkin cats suggest an autosomal recessive lethal trait in the homozygote state. We propose an autosomal dominant mode of inheritance for the chondrodysplastic condition in Munchkin cats.

Genetics of Equine Orthopedic Disease.

Orthopedic diseases are a common cause for limited exercise capacity in the horse. They often underlie genetic risk factors, which can affect bone, articular cartilage, tendons, ligaments, and adnexal structures among others. The genetic effects can directly interfere with tissue development and skeletal growth or can trigger degenerative or inflammatory processes. Many of these diseases of the locomotor system like osteochondrosis are complex and can be affected by multifactorial influences. For this reason, it is important for those performing diagnostic procedures to have a comprehensive knowledge of orthopedic diseases, their prevalence within breeds, and genetic background.

Genome data uncover four synergistic key regulators for extremely small body size in horses.

BACKGROUND: Miniature size in horses represents an extreme reduction of withers height that originated after domestication. In some breeds, it is a highly desired trait representing a breed- or subtype-specific feature. The genomic changes that emerged due to strong-targeted selection towards this distinct type remain unclear. RESULTS: Comparisons of whole-genome sequencing data from two Miniature Shetland ponies and one standard-sized Shetland pony, performed to elucidate genetic determinants for miniature size, revealed four synergistic variants, limiting withers height to 34.25 in. (87 cm). Runs of homozygosity regions were detected spanning these four variants in both the Miniature Shetland ponies and the standard-sized Shetland pony. They were shown to be characteristic of the Shetland pony breed, resulting in a miniature type under specific genotypic combinations. These four genetic variants explained 72% of the size variation among Shetland ponies and related breeds. The length of the homozygous regions indicate that they arose over 1000 years ago. In addition, a copy number variant was identified in DIAPH3 harboring a loss exclusively in ponies and donkeys and thus representing a potential height-associated variant. CONCLUSION: This study reveals main drivers for miniature size in horses identified in whole genome data and thus provides relevant candidate genes for extremely short stature in mammals.

Autozygosity islands and ROH patterns in Nellore lineages: evidence of selection for functionally important traits.

BACKGROUND: The aim of this study was to assess genome-wide autozygosity in a Nellore cattle population and to characterize ROH patterns and autozygosity islands that may have occurred due to selection within its lineages. It attempts also to compare estimates of inbreeding calculated from ROH (FROH), genomic relationship matrix (FGRM), and pedigree-based coefficient (FPED). RESULTS: The average number of ROH per animal was 55.15 ± 13.01 with an average size of 3.24 Mb. The Nellore genome is composed mostly by a high number of shorter segments accounting for 78% of all ROH, although the proportion of the genome covered by them was relatively small. The genome autozygosity proportion indicates moderate to high inbreeding levels for classical standards, with an average value of 7.15% (178.70 Mb). The average of FPED and FROH, and their correlations (- 0.05 to 0.26) were low. Estimates of correlation between FGRM-FPED was zero, while the correlation (- 0.01 to - 0.07) between FGRM-FROH decreased as a function of ROH length, except for FROH > 8Mb (- 0.03). Overall, inbreeding coefficients were not high for the genotyped animals. Autozygosity islands were evident across the genome (n = 62) and their genomic location did not largely differ within lineages. Enriched terms (p < 0.01) associated with defense response to bacteria (GO:0042742), immune complex reaction (GO:0045647), pregnancy-associated glycoproteins genes (GO:0030163), and organism growth (GO:0040014) were described within the autozygotic islands. CONCLUSIONS: Low FPED-FROH correlation estimates indicate that FPED is not the most suitable method for capturing ancient inbreeding when the pedigree does not extend back many generations and FROH should be used instead. Enriched terms (p < 0.01) suggest a strong selection for immune response. Non-overlapping islands within the lineages greatly explain the mechanism underlying selection for functionally important traits in Nellore cattle.

Association of Batai Virus Infection and Encephalitis in Harbor Seals, Germany, 2016.

We isolated Batai virus from the brain of a euthanized, 26-year-old, captive harbor seal with meningoencephalomyelitis in Germany. We provide evidence that this orthobunyavirus can naturally infect the central nervous system of a mammal. The full-genome sequence showed differences from a previously reported virus isolate from a mosquito in Germany.

A recessive lethal chondrodysplasia in a miniature zebu family results from an insertion affecting the chondroitin sulfat domain of aggrecan.

BACKGROUND: Congenital skeletal malformations represent a heterogeneous group of disorders affecting bone and cartilage development. In cattle, particular chondrodysplastic forms have been identified in several miniature breeds. In this study, a phenotypic characterization was performed of an affected Miniature Zebu calf using computed tomography, necropsy and histopathological examinations, whole genome sequencing of the case and its parents on an Illumina NextSeq 500 in 2 × 150 bp paired-end mode and validation using Sanger sequencing and a Kompetitive Allele Specific PCR assay. Samples from the family of an affected Miniature Zebu with bulldog syndrome including parents and siblings, 42 healthy Miniature Zebu not related with members of the herd and 88 individuals from eight different taurine cattle breeds were available for validation. RESULTS: A bulldog-like Miniature Zebu calf showing a large bulging head, a short and compressed body and extremely short and stocky limbs was delivered after a fetotomy. Computed tomography and necropsy revealed severe craniofacial abnormalities including a shortening of the ventral nasal conchae, a cleft hard palate, rotated limbs as well as malformed and fused vertebrae and ribs. Histopathologic examination showed a disorganization of the physeal cartilage with disorderly arranged chondrocytes in columns and a multifocal closed epiphyseal plate. Whole-genome sequencing of this malformed Miniature Zebu calf, its dam and sire and subsequent comparative sequence analysis revealed a one base pair insertion (ACAN:c.5686insC) located within the cartilage development gene aggrecan (ACAN) exclusively homozygous in the affected calf and heterozygous in its parents. This variant was predicted to cause a frameshift (p.Val1898fsTer9) and thus a truncation of the chondroitin sulfate domain as well as a loss of the C-terminal globular domain of ACAN. It perfectly co-segregated with the lethal bulldog syndrome in Miniature Zebus. CONCLUSIONS: We found a novel mutation in ACAN causing a recessive lethal chondrodysplasia in Miniature Zebu cattle. A diagnostic test for this mutation is now available for Miniature Zebu breeders preventing further cases of bulldog syndrome by targeted matings. To the authors' best knowledge, this is the first case of a Miniature Zebu associated with an ACAN mutation.

Whole genome sequencing identifies missense mutation in MTBP in Shar-Pei affected with Autoinflammatory Disease (SPAID).

BACKGROUND: Autoinflammatory diseases in dogs are characterized by complex disease processes with varying clinical signs. In Shar-Pei, signs of inflammation including fever and arthritis are known to be related with a breed-specific predisposition for Shar-Pei Autoinflammatory Disease (SPAID). RESULTS: Clinical and histopathological examinations of two severely SPAID-affected Shar-Pei revealed signs of inflammation including fever, arthritis, and perivascular and diffuse dermatitis in both dogs. A multifocal accumulation of amyloid in different organs was found in one SPAID-affected case. Whole genome sequencing resulted in 37 variants, which were homozygous mutant private mutations in SPAID-affected Shar-Pei. Nine SNVs with predicted damaging effects and three INDELs were further investigated in 102 Shar-Pei affected with SPAID, 62 unaffected Shar-Pei and 162 controls from 11 different dog breeds. The results showed the missense variant MTBP:g.19383758G > A in MTBP to be highly associated with SPAID in Shar-Pei. In the region of this gene a large ROH (runs of homozygosity) region could be detected exclusively in the two investigated SPAID-affected Shar-Pei compared to control dog breeds. No further SPAID-associated variant with predicted high or moderate effects could be found in genes identified in ROH regions. This MTBP variant was predicted to affect the MDN2-binding protein domain and consequently promote proinflammatory reactions. In the investigated group of Shar-Pei older than six years all dogs with the mutant genotype A/A were SPAID-affected whereas SPAID-unaffected dogs harbored the homozygous wildtype (G/G). Shar-Pei with a heterozygous genotype (G/A) were shown to have a 2.13-fold higher risk for disease development, which gave evidence for an incomplete dominant mode of inheritance. CONCLUSIONS: The results of this study give strong evidence for a variant in MTBP related with proinflammatory processes via MTBP-MDM2 pathway. Thus, these results enable a reliable detection of SPAID in Shar-Pei dogs.

Germline mutation within COL2A1 associated with lethal chondrodysplasia in a polled Holstein family.

BACKGROUND: The bulldog calf syndrome is a lethal form of the inherited congenital chondrodysplasias. Among the progeny of the polled Holstein bull Energy P cases of lethal chondrodysplasia were observed. Pedigrees of the cases and the frequency of 3/8 cases among the offspring of Energy P at our teaching and experimental farm Ruthe (LuFG Ruthe) supported the assumption of a germline mutation with a mosaic of normal and defective sperm. RESULTS: All three malformed calves were examined using necropsy, histopathology and computed tomography scanning. The phenotypic appearance of the affected calves was highly similar; they presented with severe disproportionate dwarfism and reduced body weight. The syndrome was characterized by brachygnathia superior, bilateral palatoschisis, shortening and compression of the body due to malformed vertebrae, in their size reduced and malformed ribs and reduced length of the long bones of the limbs. The bones had small irregular diaphyses and enlarged epiphyses. Whole genome sequencing of one bulldog calf, sperm of its sire Energy P and a normal progeny of Energy P identified a deleterious missense mutation (g.32476082G > A, c.2986G > A, ss2019324576) within COL2A1 on bovine chromosome (BTA) 5. Sanger sequencing confirmed the ss2019324576 variant in the affected calves and sperm of Energy P. This mutation is located within the collagen triple helix repeat and causes an exchange of glycine to serine (p.996G > S) in COL2A1. This private single nucleotide variant (SNV) was present as a gonadal mosaic in sperm of the bull. All affected calves were in a heterozygous state whereas normal half-siblings and all dams of the progeny from Energy P were missing this SNV. Validation in polled Holstein bulls and normal Holstein calves randomly sampled from several herds and from the LuFG Ruthe confirmed this SNV as private. CONCLUSIONS: The identified spontaneous missense mutation within COL2A1 is most likely the cause of lethal chondrodysplasia in the progeny of Energy P through a dominant negative effect. This example suggests that it would be beneficial to conduct whole genome sequencing of sperm from bulls widely used in artificial insemination in order to detect germline mosaicism.

Developing a 670k genotyping array to tag ~2M SNPs across 24 horse breeds.

BACKGROUND: To date, genome-scale analyses in the domestic horse have been limited by suboptimal single nucleotide polymorphism (SNP) density and uneven genomic coverage of the current SNP genotyping arrays. The recent availability of whole genome sequences has created the opportunity to develop a next generation, high-density equine SNP array. RESULTS: Using whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 million de novo discovered genetic variants. Leveraging genotype data from individuals with both whole genome sequence, and genotypes from lower-density, legacy SNP arrays, a subset of ~5 million high-quality, high-density array candidate SNPs were selected based on breed representation and uniform spacing across the genome. Considering probe design recommendations from a commercial vendor (Affymetrix, now Thermo Fisher Scientific) a set of ~2 million SNPs were selected for a next-generation high-density SNP chip (MNEc2M). Genotype data were generated using the MNEc2M array from a cohort of 332 horses from 20 breeds and a lower-density array, consisting of ~670 thousand SNPs (MNEc670k), was designed for genotype imputation. CONCLUSIONS: Here, we document the steps taken to design both the MNEc2M and MNEc670k arrays, report genomic and technical properties of these genotyping platforms, and demonstrate the imputation capabilities of these tools for the domestic horse.

New Avian Hepadnavirus in Palaeognathous Bird, Germany.

In 2015, we identified an avian hepatitis B virus associated with hepatitis in a group of captive elegant-crested tinamous (Eudromia elegans) in Germany. The full-length genome of this virus shares <76% sequence identity with other avihepadnaviruses. The virus may therefore be considered a new extant avian hepadnavirus.

Whole-genome sequencing reveals a potential causal mutation for dwarfism in the Miniature Shetland pony.

The Miniature Shetland pony represents a horse breed with an extremely small body size. Clinical examination of a dwarf Miniature Shetland pony revealed a lowered size at the withers, malformed skull and brachygnathia superior. Computed tomography (CT) showed a shortened maxilla and a cleft of the hard and soft palate which protruded into the nasal passage leading to breathing difficulties. Pathological examination confirmed these findings but did not reveal histopathological signs of premature ossification in limbs or cranial sutures. Whole-genome sequencing of this dwarf Miniature Shetland pony and comparative sequence analysis using 26 reference equids from NCBI Sequence Read Archive revealed three probably damaging missense variants which could be exclusively found in the affected foal. Validation of these three missense mutations in 159 control horses from different horse breeds and five donkeys revealed only the aggrecan (ACAN)-associated g.94370258G>C variant as homozygous wild-type in all control samples. The dwarf Miniature Shetland pony had the homozygous mutant genotype C/C of the ACAN:g.94370258G>C variant and the normal parents were heterozygous G/C. An unaffected full sib and 3/5 unaffected half-sibs were heterozygous G/C for the ACAN:g.94370258G>C variant. In summary, we could demonstrate a dwarf phenotype in a miniature pony breed perfectly associated with a missense mutation within the ACAN gene.

Variant detection and runs of homozygosity in next generation sequencing data elucidate the genetic background of Lundehund syndrome.

BACKGROUND: The Lundehund is a highly specialized breed characterized by a unique flexibility of the joints and polydactyly in all four limbs. The extremely small population size and high inbreeding has promoted a high frequency of diseased dogs affected by the Lundehund syndrome (LS), a severe gastro-enteropathic disease. RESULTS: Comprehensive analysis of bead chip and whole-genome sequencing data for LS in the Lundehund resulted in a genome-wide association signal on CFA 34 and LS-specific runs of homozygosity (ROH) in this region. Filtering analysis for variants with predicted high or moderate effects revealed a missense mutation in LEPREL1 1.2 Mb proximal to the region of the genome-wide association, which was shown to be significantly associated with LS. LS-affected Lundehund harbored the mutant LEPREL1:g.139212C>G genotype A/A whereas all controls of other breeds showed the C/C wild type. In addition, ROH analysis for the Lundehund indicated a high enrichment of genes in potential signatures of selection affecting protein activation and immunoregulatory processes like NOD1 potentially involved in LS breed disposition. CONCLUSIONS: Sequencing results for Lundehund specific traits reveal a potential causative mutation for LS in the neuropeptide operating gene LEPREL1 and suggests it as a precursor of the inflammatory process. Analyses of ROH regions give an insight into the genetic background of characteristic traits in the Lundehund that remain to be elucidated in the future.

Screening of whole genome sequences identified high-impact variants for stallion fertility.

BACKGROUND: Stallion fertility is an economically important trait due to the increase of artificial insemination in horses. The availability of whole genome sequence data facilitates identification of rare high-impact variants contributing to stallion fertility. The aim of our study was to genotype rare high-impact variants retrieved from next-generation sequencing (NGS)-data of 11 horses in order to unravel harmful genetic variants in large samples of stallions. METHODS: Gene ontology (GO) terms and search results from public databases were used to obtain a comprehensive list of human und mice genes predicted to participate in the regulation of male reproduction. The corresponding equine orthologous genes were searched in whole genome sequence data of seven stallions and four mares and filtered for high-impact genetic variants using SnpEFF, SIFT and Polyphen 2 software. All genetic variants with the missing homozygous mutant genotype were genotyped on 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. Mixed linear model analysis was employed for an association analysis with de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT). RESULTS: We screened next generation sequenced data of whole genomes from 11 horses for equine genetic variants in 1194 human and mice genes involved in male fertility and linked through common gene ontology (GO) with male reproductive processes. Variants were filtered for high-impact on protein structure and validated through SIFT and Polyphen 2. Only those genetic variants were followed up when the homozygote mutant genotype was missing in the detection sample comprising 11 horses. After this filtering process, 17 single nucleotide polymorphism (SNPs) were left. These SNPs were genotyped in 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. An association analysis in 216 Hanoverian stallions revealed a significant association of the splice-site disruption variant g.37455302G>A in NOTCH1 with the de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT). For 9 high-impact variants within the genes CFTR, OVGP1, FBXO43, TSSK6, PKD1, FOXP1, TCP11, SPATA31E1 and NOTCH1 (g.37453246G>C) absence of the homozygous mutant genotype in the validation sample of all 337 fertile stallions was obvious. Therefore, these variants were considered as potentially deleterious factors for stallion fertility. CONCLUSIONS: In conclusion, this study revealed 17 genetic variants with a predicted high damaging effect on protein structure and missing homozygous mutant genotype. The g.37455302G>A NOTCH1 variant was identified as a significant stallion fertility locus in Hanoverian stallions and further 9 candidate fertility loci with missing homozygous mutant genotypes were validated in a panel including 19 horse breeds. To our knowledge this is the first study in horses using next generation sequencing data to uncover strong candidate factors for stallion fertility.

Genome-wide association study for hereditary ataxia in the Parson Russell Terrier and DNA-testing for ataxia-associated mutations in the Parson and Jack Russell Terrier.

BACKGROUND: Spinocerebellar ataxia also referred to as hereditary ataxia comprises different forms of progressive neurodegenerative diseases. A complex mode of inheritance was most likely in Parson Russell Terriers (PRT) and in Jack Russell Terriers (JRT). Recently, the missense mutation KCNJ10:c.627C > G was shown to be associated with the spinocerebellar ataxia (SCA) in JRT and related Russell group of terriers, whereas the missense mutation CAPN1:c.344G > A was associated with late onset ataxia (LOA) in PRT. RESULTS: We performed a genome-wide association study (GWAS) in PRT including 15 cases and 29 controls and found a statistically strong signal in the genomic region on dog chromosome 38 (CFA38) where KCNJ10 is located. We tested the CAPN1:c.344G > A and KCNJ10:c.627C > G (Transcript XM_545752.4) mutations in a sample of 77 PRT and 9 JRT from Germany as well as further 179 controls from 20 different dog breeds. All cases and controls genotyped carried the wild-type for the CAPN1:c.344G > A mutation. Among the PRT, 17/77 (22.1 %) dogs were homozygous for the mutant KCNJ10 allele and 22/77 (28.6 %) dogs were heterozygous. Three cases of PRT had the homozygous KCNJ10 wild-type. In JRT, 1/3 cases did show the mutant KCNJ10 allele homozygous. Thus, we sequenced the KCNJ10 exons with their adjacent regions from 10 PRT and 3 JRT including the animals with imperfect co-segregation of the c.627C > G mutation. We identified a total of 45 genetic variants within KCNJ10. The most likely variant explaining the cases appeared a 1-bp-insertion in a C-stretch within exon 3 (KCNJ10:g.22141027insC). In silico analysis showed that this indel may influence the regulation of gene expression. CONCLUSIONS: In the present study, 16/21 cases of hereditary ataxia perfectly co-segregated with the KCNJ10:c.627C > G mutation. The CAPN1:c.344G > A mutation could not be validated and seems to be a rare variant in the samples screened. Screening KCNJ10 for further mutations did result in a genetic variant explaining 2 JRT cases but further 3 cases with a non-mutant homozygous c.627C > G genotype could not be resolved. Breeders have to be aware that DNA-testing for hereditary ataxia in PRT and JRT does not capture all cases of hereditary ataxia in these dog breeds. At least one further form of hereditary ataxia not yet resolved by a mutation may occur in PRT and JRT.

Runs of homozygosity reveal signatures of positive selection for reproduction traits in breed and non-breed horses.

BACKGROUND: Modern horses represent heterogeneous populations specifically selected for appearance and performance. Genomic regions under high selective pressure show characteristic runs of homozygosity (ROH) which represent a low genetic diversity. This study aims at detecting the number and functional distribution of ROHs in different horse populations using next generation sequencing data. METHODS: Next generation sequencing was performed for two Sorraia, one Dülmen Horse, one Arabian, one Saxon-Thuringian Heavy Warmblood, one Thoroughbred and four Hanoverian. After quality control reads were mapped to the reference genome EquCab2.70. ROH detection was performed using PLINK, version 1.07 for a trimmed dataset with 11,325,777 SNPs and a mean read depth of 12. Stretches with homozygous genotypes of >40 kb as well as >400 kb were defined as ROHs. SNPs within consensus ROHs were tested for neutrality. Functional classification was done for genes annotated within ROHs using PANTHER gene list analysis and functional variants were tested for their distribution among breed or non-breed groups. RESULTS: ROH detection was performed using whole genome sequences of ten horses of six populations representing various breed types and non-breed horses. In total, an average number of 3492 ROHs were detected in windows of a minimum of 50 consecutive homozygous SNPs and an average number of 292 ROHs in windows of 500 consecutive homozygous SNPs. Functional analyses of private ROHs in each horse revealed a high frequency of genes affecting cellular, metabolic, developmental, immune system and reproduction processes. In non-breed horses, 198 ROHs in 50-SNP windows and seven ROHs in 500-SNP windows showed an enrichment of genes involved in reproduction, embryonic development, energy metabolism, muscle and cardiac development whereas all seven breed horses revealed only three common ROHs in 50-SNP windows harboring the fertility-related gene YES1. In the Hanoverian, a total of 18 private ROHs could be shown to be located in the region of genes potentially involved in neurologic control, signaling, glycogen balance and reproduction. Comparative analysis of homozygous stretches common in all ten horses displayed three ROHs which were all located in the region of KITLG, the ligand of KIT known to be involved in melanogenesis, haematopoiesis and gametogenesis. CONCLUSIONS: The results of this study give a comprehensive insight into the frequency and number of ROHs in various horses and their potential influence on population diversity and selection pressures. Comparisons of breed and non-breed horses suggest a significant artificial as well as natural selection pressure on reproduction performance in all types of horse populations.

Next generation sequencing gives an insight into the characteristics of highly selected breeds versus non-breed horses in the course of domestication.

BACKGROUND: Domestication has shaped the horse and lead to a group of many different types. Some have been under strong human selection while others developed in close relationship with nature. The aim of our study was to perform next generation sequencing of breed and non-breed horses to provide an insight into genetic influences on selective forces. RESULTS: Whole genome sequencing of five horses of four different populations revealed 10,193,421 single nucleotide polymorphisms (SNPs) and 1,361,948 insertion/deletion polymorphisms (indels). In comparison to horse variant databases and previous reports, we were able to identify 3,394,883 novel SNPs and 868,525 novel indels. We analyzed the distribution of individual variants and found significant enrichment of private mutations in coding regions of genes involved in primary metabolic processes, anatomical structures, morphogenesis and cellular components in non-breed horses and in contrast to that private mutations in genes affecting cell communication, lipid metabolic process, neurological system process, muscle contraction, ion transport, developmental processes of the nervous system and ectoderm in breed horses. CONCLUSIONS: Our next generation sequencing data constitute an important first step for the characterization of non-breed in comparison to breed horses and provide a large number of novel variants for future analyses. Functional annotations suggest specific variants that could play a role for the characterization of breed or non-breed horses.

Analysis of copy number variants by three detection algorithms and their association with body size in horses.

BACKGROUND: Copy number variants (CNVs) have been shown to play an important role in genetic diversity of mammals and in the development of many complex phenotypic traits. The aim of this study was to perform a standard comparative evaluation of CNVs in horses using three different CNV detection programs and to identify genomic regions associated with body size in horses. RESULTS: Analysis was performed using the Illumina Equine SNP50 genotyping beadchip for 854 horses. CNVs were detected by three different algorithms, CNVPartition, PennCNV and QuantiSNP. Comparative analysis revealed 50 CNVs that affected 153 different genes mainly involved in sensory perception, signal transduction and cellular components. Genome-wide association analysis for body size showed highly significant deleted regions on ECA1, ECA8 and ECA9. Homologous regions to the detected CNVs on ECA1 and ECA9 have also been shown to be correlated with human height. CONCLUSIONS: Comparative analysis of CNV detection algorithms was useful to increase the specificity of CNV detection but had certain limitations dependent on the detection tool. GWAS revealed genome-wide associated CNVs for body size in horses.

Morphometric magnetic resonance imaging and genetic testing in cerebellar abiotrophy in Arabian horses.

BACKGROUND: Cerebellar abiotrophy (CA) is a rare but significant disease in Arabian horses caused by progressive death of the Purkinje cells resulting in cerebellar ataxia characterized by a typical head tremor, jerky head movements and lack of menace response. The specific role of magnetic resonance imaging (MRI) to support clinical diagnosis has been discussed. However, as yet MR imaging has only been described in one equine CA case. The role of MR morphometry in this regard is currently unknown. Due to the hereditary nature of the disease, genetic testing can support the diagnosis of CA. Therefore, the objective of this study was to perform MR morphometric analysis and genetic testing in four CA-affected Arabian horses and one German Riding Pony with purebred Arabian bloodlines in the third generation. RESULTS: CA was diagnosed pathohistologically in the five affected horses (2 months - 3 years) supported by clinical signs, necropsy, and genetic testing which confirmed the TOE1:g.2171G>A SNP genotype A/A in all CA-affected horses. On MR images morphometric analysis of the relative cerebellar size and relative cerebellar cerebrospinal fluid (CSF) space were compared to control images of 15 unaffected horses. It was demonstrated that in MR morphometric analyses, CA affected horses displayed a relatively smaller cerebellum compared to the entire brain mass than control animals (P = 0.0088). The relative cerebellar CSF space was larger in affected horses (P = 0.0017). Using a cut off value of 11.0% for relative cerebellar CSF space, the parameter differentiated between CA-affected horses and controls with a sensitivity of 100% and a specificity of 93.3%. CONCLUSIONS: In conclusion, morphometric MRI and genetic analysis could be helpful to support the diagnosis of CA in vivo.