title: Screening of whole genome sequences identified high-impact variants for stallion fertility.
abstract: 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.
authors.affiliation: Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, 30559, Hannover, Germany.
authors.forename: Rahel
authors.initials: R
authors.lastname: Schrimpf
authors.affiliation: Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, 30559, Hannover, Germany.
authors.forename: Maren
authors.initials: M
authors.lastname: Gottschalk
authors.affiliation: Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, 30559, Hannover, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Metzger
authors.affiliation: State Stud Celle of Lower Saxony, Spörckenstraße 10, 29221, Celle, Germany.
authors.forename: Gunilla
authors.initials: G
authors.lastname: Martinsson
authors.affiliation: Clinic for Horses, Unit for Reproduction Medicine, University of Veterinary Medicine Hannover, Bünteweg 15, 30559, Hannover, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Sieme
authors.affiliation: Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, 30559, Hannover, Germany. ottmar.distl@tiho-hannover.de.
authors.forename: Ottmar
authors.initials: O
authors.lastname: Distl
mesh_terms: D000818:Animals; D001947:Breeding; D019295:Computational Biology; D004252:DNA Mutational Analysis; D005260:Female; D005298:Fertility / Q000235:genetics*; D005838:Genotype; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing; D006720:Homozygote; D006736:Horses / Q000235:genetics*; D007248:Infertility, Male / Q000235:genetics; D007315:Insemination, Artificial; D008297:Male; D020641:Polymorphism, Single Nucleotide; D011247:Pregnancy; D011270:Pregnancy, Animal
keywords: High-impact variants; NOTCH1; Stallion fertility; Whole genome screening