Schrimpf Rahel, Gottschalk Maren, Metzger Julia, Martinsson Gunilla, Sieme Harald, Distl Ottmar
Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, 30559, Hannover, Germany.
State Stud Celle of Lower Saxony, Spörckenstraße 10, 29221, Celle, Germany.
BMC Genomics. 2016 Apr 14;17:288. doi: 10.1186/s12864-016-2608-3.
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.
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).
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.
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.
由于马人工授精的增加，种公马的繁殖力是一个具有经济重要性的性状。全基因组序列数据的可用性有助于识别影响种公马繁殖力的罕见高影响变异。我们研究的目的是对从11匹马的下一代测序（NGS）数据中检索到的罕见高影响变异进行基因分型，以揭示大量种公马样本中的有害遗传变异。
使用基因本体（GO）术语和公共数据库的搜索结果，获得预测参与雄性生殖调控的人类和小鼠基因的综合列表。在7匹种公马和4匹母马的全基因组序列数据中搜索相应的马直系同源基因，并使用SnpEFF、SIFT和Polyphen 2软件筛选高影响遗传变异。使用KASP基因分型检测或PCR-RFLP对19个品种的337匹育种种公马中所有缺失纯合突变基因型的遗传变异进行基因分型。采用混合线性模型分析与每个发情期受胎率父本成分的去回归估计育种值（EBV-PAT）进行关联分析。
我们在1194个人类和小鼠基因中筛选了11匹马全基因组的下一代测序数据中的马遗传变异，这些基因参与雄性生育，并通过共同的基因本体（GO）与雄性生殖过程相关联。对蛋白质结构有高影响的变异进行筛选，并通过SIFT和Polyphen 2进行验证。仅当在包含11匹马的检测样本中缺失纯合突变基因型时，才对这些遗传变异进行后续研究。经过此筛选过程，留下了17个单核苷酸多态性（SNP）。使用KASP基因分型检测或PCR-RFLP对19个品种的337匹育种种公马中的这些SNP进行基因分型。对216匹汉诺威种公马的关联分析显示，NOTCH1基因中的剪接位点破坏变异g.37455302G>A与每个发情期受胎率父本成分的去回归估计育种值（EBV-PAT）显著相关。在所有337匹育种种公马的验证样本中，CFTR、OVGP1、FBXO43、TSSK6、PKD1、FOXP1、TCP11、SPATA31E1和NOTCH1基因（g.37453246G>C）中的9个高影响变异明显不存在纯合突变基因型。因此，这些变异被认为是种公马繁殖力的潜在有害因素。
总之，本研究揭示了17个对蛋白质结构具有预测高破坏作用且缺失纯合突变基因型的遗传变异。g.37455302G>A NOTCH1变异被确定为汉诺威种公马中一个显著的种公马繁殖力位点，并且在包括19个马品种的样本中验证了另外9个缺失纯合突变基因型的候选繁殖力位点。据我们所知，这是首次在马中使用下一代测序数据来揭示种公马繁殖力的强候选因素的研究。
