混合、渗入和选择促进地方牛品种对特殊环境遗传适应的分子机制

China is one of the most rich countries in the world for animal genetic resources. Under long-term selection, Chinese indigenous cattle breeds have evolved genetic adaptations to various environmental conditions in the vast geographical region of China, including special environments, such as plateau hypoxia, arid, cold, hot and humid regions. However, the underlying physiological and molecular mechanism as well as genetic variation that is responsible for adaptation to stressful environments is not well understood. Our previous findings suggest that admixture and introgression occur widely in most of the Chinese native cattle breeds. Furthermore, selection during the process of domestication and subsequent breed formation has left detectable molecular signatures within the bovine genome. Therefore, we put forward the following hypothesis: "Admixture, introgression and selection play important roles in cattle domestication, breed formation and genetic adaptation to local extreme environments". To this end, first, we plan to investigate systematically the characteristics of genetic variations, population structure and history of breed formation in native cattle breeds via combinating bovine high-density SNP chips and whole-genome sequencing with the population statistics, such as PCA, Admixture, Treemix, Threepop and Fourpop-ratio; further, we will assemble a genomic dataset which represents the most comprehensive breed sampling of bovids and the largest cattle population in China. Using this dataset, we are able to accurately address a more detailed description of the population structure of domesticated cattle worldwide, specially in China. Second, we are going to identify the positive and balancing selective signatures, and harbour genes as well as genetic variations using the statistics including Fst, FLK, hapFLK, iHS, EXPHH and self-developed SelMafSweep methods. Third, we will confirm their functions of a few vital genes and genetic variants using the mixed-ancestry-corrected GEMMA method, and systematic RNAi, over-expression experiments, etc. Consequently, analyses of population structure and genomic diversity within and between breeds of cattle can be used to identify candidate genomic regions which provide valuable insights into the biology, origin, domestication and evolution of populations. Finally, we will focus on resolving the molecular mechanism for genetic adapations to special environments and clarifying their potential influences of admixture, introgression and selection on genetic adapation. These findings can give insights into theory and strategy for the conservation and application of genetic resources, and provide important ways contribute to better taking advantage of molecular breeding program to improve the production potential in cattle.

我国地方牛品种资源丰富，经长期选择，已很好地适应当地环境，尤其是高原低氧、干旱、高温高湿、寒冷等特殊环境。我们前期研究发现，我国地方牛品种普遍存在基因混合和渗入现象。为此，我们提出假设：“混合、渗入和选择在地方牛驯化、品种形成以及对当地极端环境的遗传适应中发挥了重要作用”。本研究拟采用高密度SNP芯片和全基因组重测序技术，联合PCA、Admixture、Treemix、f3、f4-ratio等系统解析地方牛品种的遗传变异特性、群体结构和形成历史；利用Fst、FLK、hapFLK、iHS、EXPHH、自主开发的SelMafSweep鉴定正选择和平衡选择信号；利用混合祖先校正的GEMMA、RNAi等验证关键基因和遗传变异的功能；揭示混合、渗入和选择在牛对特殊环境适应中的潜在作用及其分子遗传机制，为遗传资源保存和开发提供新的理论和策略，为人们更好地利用分子育种手段提高牛的生产潜能提供途径。

我国地方牛遗传资源丰富，并能很好地适应当地环境，包括高原低氧、干旱潮湿、冷热等极端环境。为了更好地了解中国地方牛的起源、混合对种群结构的影响程度，以及中国地方牛适应高海拔极端环境的机理，本研究利用42个牛品种的高密度SNP基因型数据，联合PCA、Admixture、Treemix、f3、f4-ratio等方法系统解析我国地方牛品种的遗传变异特性、群体结构和品种形成历史；利用FLK、hapFLK、Fst、XPEHH等方法鉴定和验证与高海拔适应相关的阳性选择信号、关键基因和遗传变异；使用配对的IBD方法和ILS分析牛品种间基因渗入情况；联合免疫组织化学、实时定量PCR、CRISPR/Cas9、双荧光素酶报告载体系统验证筛选到的高海拔适应关键候选基因和遗传变异的功能。我们发现中国瘤牛基因组有牦牛（Bos grunniens）和爪哇野牛（Bos javanicus）的基因渗入，导致了中国瘤牛与印度瘤牛的遗传分化，并且大部分中国地方牛品种存在渗入、混合和迁移现象。在中国普通牛品种形成之前，发生了高海拔地区牦牛的等位基因向中国普通牛的渗入的现象，随后经过选择，适应了高海拔环境。除了选择之外，与当地牛群体的混合、渗入也促进了高海拔适应。高海拔（>1800m）与低海拔(<1500m)牛品种的选择信号分析揭示几个候选基因（ACSS2, ALDOC, EPAS1, EGLN1, NUCB2）和信号通路可能与低氧适应有关。免疫组织化学、实时定量和CRISPR/cas9介导的ACSS2基因敲除实验分析表明在肝脏中上调ACSS2基因的表达可通过低氧诱导因子通路促进细胞对低氧的适应。本研究阐明了混合、渗入和选择在我国地方牛品种适应极端环境机制中发挥重要作用，鉴定到了牛基因组上与高海拔适应相关的候选基因和遗传变异，为高原低氧特色牛品种的培育和筛选等分子育种工作奠定重要基础，对牛遗传资源多样性保护和评价提供重要的理论和策略。另外对于人类高原医学也具有重要的借鉴意义。