基于基因组目标区域重测序技术鉴定影响奶牛产奶性状的功能突变

The identification of genetic variants affecting milk production traits is very crucial for molecular breeding in dairy cattle. So far, genome wide association study (GWAS) has been widely accepted as a primary approach for gene finding relevant to complex traits and achieved huge success in identifying genes. The potential limitation of GWAS is the specific genes and causal variants underlying these traits have not yet been clearly defined because most of detected SNPs are mostly not the causal genes due to the potential linkage disequilibrium (LD) between the causal variant and genotyped markers. This concern may be overcome with the recent advances in next generation sequencing technology, which makes it possible to comprehensively catalog genetic variation in population samples and creates a foundation for understanding complex traits. In current project, by using target-enrichment strategy coupled with high-throughput next generation sequencing of illumina Solex platform, we will resequence genomic regions associated with milk production traits in 60 unrelated sire individuals. These target regions of the bovine genome were determined according to the findings from our initial GWAS. Among all common as well as rare variants detected in the sequencing target regions, we further perform bioinformatics and comparative genomics analyses to sift out important candidate variants. Subsequently, these chosen mutations are detected through follow-up association analyses in another experimental population to finally discover functional mutations affecting milk production performance. The aim of this study is to reveal the genetic basis of milk traits and enhance our understanding of the mechanisms of mutations underlying quantitative phenotypes. It can be expected that findings of this study will offer gene resources and further lay a foundation for molecular breeding in dairy cattle in the future.

挖掘产奶性状功能基因历来是奶牛分子育种的关键。全基因组关联分析（GWAS）是当前基因检测的主流方法，但由于GWAS检测的SNP大多与潜在功能突变处于连锁不平衡，真正发现的功能基因极为有限。基于第二代测序的基因组目标区域捕获技术，为深入分析GWAS结果、检测功能基因突变提供了新的策略。本研究基于团队前期开展的中国荷斯坦奶牛GWAS研究结果，选择与产奶性状显著相关的候选基因组区段，对60头公牛实施基因组目标区域捕获、富集，并采用Illumina高通量测序平台进行重测序，从而挖掘目标区段的所有遗传变异和新基因，构建产奶性状相关基因组热点区域的全新遗传标记图谱。同时利用生物信息学和比较基因组学方法筛选重要功能候选突变，并利用新的资源群体进行关联分析，检测影响产奶性状的功能突变位点。本项目的开展对揭示奶牛产奶性能遗传基础及关键变异作用的分子机理具有重要意义，为我国奶牛分子育种提供科学依据和基因资源。

挖掘奶牛产奶性状功能基因是奶牛分子育种研究的重要内容。本课题组前期利用Illumina牛54K全基因组SNP芯片对我国荷斯坦奶牛群体进行了产奶性状的全基因组关联分析研究（GWAS）。本项目基于前期GWAS结果，对6.6Mb的显著染色体区段，利用目标区域捕获测序技术和重测序，进一步对影响产奶性状的功能基因进行精细定位。通过生物信息学分析，筛选了200个位于重要候选基因功能区（外显子，启动子和UTR区）的SNP在30个公牛家系的734头中国荷斯坦母牛群体中进行关联分析。结果显示40个SNP与产奶性状关联显著，分布在33个基因中。在此基础上，我们使用基因型填充的方法将资源群体扩大到2634头母牛再次进行关联分析，结果共检测到53个重要候选基因，其中有26个与前期GWAS结果一致。为进一步验证这些关联分析显著基因，对其中22个基因组序列较为明确的基因在4头泌乳时期奶牛的8个不同组织中进行表达分析研究。结果显示，15个基因在乳腺组织呈现特异性的高表达，说明这些基因很可能在奶牛泌乳时期起到重要作用。随后，我们利用RNA-seq技术，对高、低产奶牛不同泌乳时期牛奶组织进行分析，发现了15个重要差异表达基因，并集中在乳腺组织发育和脂肪酸代谢两个重要通路。通过上述研究结果我们筛选了GPIHBP1、EEF1D等一批重要候选基因开展了进一步的功能验证。结果发现GPIHBP1存在一种新的可变剪切体形式(GPIHBP1-X5)。同时发现，EEF1D基因在不同组织和不同泌乳时期的表达都受到其启动子区的一个CpG岛甲基化水平的调控，说明表观遗传学可能对产奶性状发挥了重要作用。本研究首次利用目标区域捕获测序策略进行产奶性状基因定位，并取得了良好效果，研究结果为我国奶牛分子育种提供了重要信息，同时为后续深入开展奶牛功能基因验证工作奠定了坚实基础。