Animal and Bioscience Research Department, Animal and Grassland Research and Innovation Centre, Teagasc, Athenry, Galway, Ireland.
BMC Genomics. 2012 Jan 11;13:16. doi: 10.1186/1471-2164-13-16.
The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility.
In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom® MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total).
The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving interval plausibly harbouring causative variants contributing to heritable variation. To our knowledge, this is the first report describing sequencing of targeted genomic regions in any livestock species using groups with divergent phenotypes for an economically important trait.
生长轴在动物产后生长、发育和繁殖中的核心作用已得到充分证实。因此，鉴定影响该轴定量性状的遗传变异是一个有吸引力的目标。然而，要鉴定复杂性状的遗传变异，需要大量的样本数量，尽管技术正在迅速发展，但高通量测序大量完整个体基因组仍然过于昂贵。因此，本研究采用 pooled DNA 方法结合靶向富集和高通量测序，旨在鉴定 83 个生长轴候选基因中的多态性，并估计 150 头荷斯坦-弗里生奶牛的等位基因频率差异，这些奶牛分为两组，一组是繁殖力遗传优势高的牛，另一组是繁殖力遗传优势低的牛。
在对 83 个候选基因进行重测序的过程中，共鉴定出 4135 个 SNPs 和 893 个插入缺失。19%（n=952）的变异位于 5'和 3'UTR 内。72%（n=3612）为内含子，9%（n=464）为外显子，包括 65 个插入缺失和 236 个导致非同义取代（NSS）的 SNPs。在低繁殖力和高繁殖力两组之间，观察到 720 个 SNPs（58 个 NSS）的平均等位基因频率差异有统计学意义（P < 0.01）。通过对 150 头个体动物进行基因分型（Sequenom® MassARRAY），确定了其中 43 个 SNPs 的等位基因频率。在两个pool 中，两种方法对 43 个 SNP 的任何一个都没有观察到显著差异（P>0.1）（总共 86 次测试）。
本研究的结果支持以前使用 DNA 样本pooling 和高通量测序作为发现多态性和估计等位基因频率的可行策略的发现。使用这种方法，我们已经描述了生长轴及其相关途径基因中的遗传变异，这些基因对哺乳动物产后生长和发育以及随后的泌乳和繁殖至关重要。我们已经鉴定出大量在不同群体之间显著不同频率分离的变异，这些群体在产犊间隔方面存在差异，可能含有导致可遗传变异的致病变异。据我们所知，这是首次描述在任何家畜物种中使用具有重要经济性状差异表型的群体对靶向基因组区域进行测序。
