运用比较群体遗传学方法快速有效发掘与鉴定乌骨绵羊乌质性状致因基因及其分子标记

The Lanping black-boned sheep (Ovis aries), which is discovered and reported firstly by our team, is the first mammal with black traits around the world. In our previous studies, it has systematically revealed that the black traits of black-boned sheep are closely associated with the abnormal migration of melanoblast and biosynthesis and metabolism of melanin compared with normal sheep (Ovis aries). Moreover, we have preliminary identified some selective signals of black traits based on the genome scan by using Illumina 50K sheep SNP beadchip (all 15 heads) and whole genome re-sequencing (all 10 heads) technologies between the Lanping black-boned and Lanping normal sheep. However, the primary signals are weak and broad. As a result, further research is needed to confirm.. Therefore, in the present study, this project will re-sequence (30G/sample) the whole genome of the Lanping black-boned (40 heads) and Lanping normal (10 heads) sheep based on the theory and calculation of comparative populations genetics (MAF>0.05). The differential genome mutations including SNPs, Indel and CNV types between 50 Lanping black-boned sheep and 20 Lanping normal sheep will be compared by combining the above re-sequencing data at the first step. Then, the data of 80 universally normal sheep with sequencing depth >10x will be selected and downloaded from the free NCBI SRA database. By using the F-statistics (Fst), the locus-specific branch lengths (LSBL), the Z transformations of Hp (ZHp) and overlapping methodologies, the unique mutations of black-boned sheep will be retrieved at the second step. Then, the specific mutations in the coding and regulatory regions of black-boned sheep will be selected to analyze their role and function by using the cis-trans regulatory elements mutual experiment and the reporter gene assay, respectively. Furthermore, the gene frequency of mutations will be confirmed in the largely random populations from black-boned and normal sheep using the association analysis of genotype and melanin content. Therefore, the causative genes and molecular markers of black traits from the black-boned sheep will be quickly and effectively isolated, cloned and identified. Finally, our results will provide the significantly theoretical basis for the protection, development and utilization of the black-boned sheep in future.

兰坪乌骨绵羊是我们发现并报道的全球第一个具有乌质性状的哺乳动物。前期我们系统揭示其乌质性状与成黑色素细胞异向迁徙和黑色素合成代谢有关，并对乌骨和普通绵羊利用芯片（各15只）和重测序（各10只）技术开展扫描，发现了一些选择信号，但信号弱且宽泛。.据此，基于比较群体遗传学理论和推算（MAF>5%），本项目继续对40只乌骨和10只兰坪普通绵羊开展全基因组重测序（30G/样），系统检测比较50只乌骨和20只兰坪普通绵羊的SNPs、Indel和CNV；选择NCBI上80只普通绵羊数据（>10x），采用Fst、LSBL和ZHp等方法再次分析，获取乌骨绵羊特有变异，分析其编码区和调控区突变；预测相关调控因子并开展顺反式调控元件互作实验和报告基因分析；在这2个群体中大规模筛查，与黑色素含量进行关联分析，可望快速有效分离、克隆并鉴定出其乌质性状致因基因及其分子标记。为乌骨绵羊的保护、开发与利用提供理论依据。

为探索兰坪乌骨绵羊乌质性状形成的分子机理，本研究在前期系统阐明了兰坪乌骨绵羊乌质性状是由高含量的酪氨酸酶催化合成了显著多的总黑色素并沉积在不同部位造成的基础上，运用高通量的全基因组重测序技术。对兰坪乌骨绵羊（100只）和兰坪普通绵羊（50只）开展了大规模测序（原始数据量5369G，平均测序深度9.3×），结合野生羊（38只）和云南省外家养绵羊（386只）全基因组重测序数据（Clean 数据量 10818G，平均测序深度 9.7x），开展了系统分析，获得兰坪乌骨绵羊及兰坪普通绵羊SNP各24,335,298、22,558,944个；群体遗传结构分析表明兰坪绵羊与中国其他两个主要绵羊类群的遗传关系较远；兰坪乌骨绵羊从兰坪普通绵羊的一个支系中分化出来；二者虽具有相似背景，但已分化为两个不同的绵羊群体并且各自具有独特的基因组成，二者的祖先可能是亚洲摩弗伦野绵羊、欧洲摩弗伦野绵羊及东方盘羊，并在较近的时期受到自然选择后形成两个不同的群体。通过基于等位基因频率、选择压力分析及全基因关联分析鉴定到与乌质性状形成较相关的基因：SLC17A8 、TRPM3、ROBO2及WWOX，并且4个基因分别在乌质性状形成的3个生物学过程中发挥作用，TRPM3 可能与SLC17A8协同作用参与神经递质谷氨酸的释放，促进黑色素细胞的形成；ROBO2可能与黑色素细胞树突的延伸有关；WWOX的抑制表达会促进酪氨酸酶的表达及活性，从而影响黑色素的合成。同时对发掘的PDGFRL和SLC17A8基因开展了克隆及其关联分析。.依托本研究，指导毕业硕士研究生2人、博士研究生2人，2人晋升为副高职，发表学术论文9篇（含2篇SCI论文），参加国际和国内学术会议5次（含1次特邀报告），在CCTV上播放2次，研究成果受到党和国家领导人关注。