高寒低氧胁迫下藏绵羊基因组内大片段获得与缺失变异发掘及功能分析

The presence/absence variants (PAVs) are a major source of genome structure variation, and often affect a larger fraction of the genome than those attributable to single nucleotide polymorphism (SNPs), insertions and deletions (InDels, normally<10bp). PAVs are widespread in the human and animal genome and may have a significant role in human genetic diseases, animal phenotypic traits, and adaptation to adverse environmental conditions. Tibetan sheep is one of the domestic animals that can thrive in the harsh environment of the Qinghai-Tibetan Plateau in China. Tibetan sheep provides essential resources such as meat, fibre and milk products allowing the communities of Tibetans to survive well at high altitudes. However, the origin, formation mechanism and function of PAVs in Tibetan sheep genome are still unclear. To gain full insight into the potential role of PAVs to adaptation of Tibetan sheep to high altitude, the genome of Tibetan sheep and Hu sheep (low-altitude sheep breed as control group) will be resequenced. The type, number, the distribution frequency of PAVs in natural populations, location of the breakpoints and genetic diversity of PAVs in Tibetan sheep will be identified and verified by PCR and Southern hybridization. The mechanism of the occurrence and diversity of PAVs on the adaptation of Tibetan sheep to high altitude will be determined. This information will provide scientific support for enriching the basic theory of molecular ecology of ruminants in extreme environments, excavating Tibetan elite germplasm resources, molecular breeding, and functional genomics.

获得与缺失变异（PAVs）是一种重要的基因组结构变异，对人类多种遗传疾病、动物表型变异及逆境胁迫适应性起到关键作用。然而，藏绵羊适应高寒低氧胁迫的基因组内大片段获得与缺失变异（lsPAVs）的形成机理和功能尚不清楚。本项目采用高通量测序技术和生物信息分析方法，拟对高海拔藏绵羊和低海拔湖羊进行基因组重测序，识别高海拔藏绵羊基因组中lsPAVs的类别、数目和分布；通过PCR和Southern杂交对lsPAVs进行验证，解析lsPAVs在自然群体中分布频率，精确定位lsPAVs发生的断点，剖析其生物学功能，阐释lsPAVs的低氧适应性形成机制，为丰富极端环境下反刍动物分子生态适应性基础理论，发掘藏绵羊优良种质资源和分子育种及功能基因组学研究提供科学支撑。

本项目利用高通量第三代PacBio、第二代Illumina和BGI联合测序技术平台及多组学生物信息分析方法，通过研究藏绵羊低氧适应性lncRNA和mRNA、功能基因及基因组内大片段获得与缺失变异发掘及功能分析，揭示了藏绵羊对高寒低氧环境胁迫下的独特适应性遗传机制。研究主要发现：（1）藏绵羊对高寒低氧的适应性并不是从增加血红蛋白浓度来实现的，可能是提高了血红蛋白输送氧气的能力和效率，增加了肺通气量和肺血流量，并通过内源NO产量高低来调节肺部血管系统以适应高寒低氧的逆境胁迫性环境，形成有效摄取、输送和利用氧的结构特征和生理机制，而且通过对细胞代谢和抗低氧因子诱导从分子水平上来适应高原低氧环境；（2）筛选出了高寒低氧适应性相关的33个差异表达基因、16个lncRNA及其35个靶基因，表达基因的lncRNA靶基因和22个特异候选基因主要调控绑定、细胞过程、免疫系统等过程的信号通路的变化上，另外，上调lncRNA与信号转导、DNA结合蛋白、血红蛋白结合、能量代谢、免疫功能相关；（3）鉴定得到了37基因与高原低氧适应性和环境因素相关，剖析了27号性染色体上6个关键候选基因的INDEL变异，发现一个INDEL位于CSDE1直系同源物HAG1基因的5'UTR区，HAG1的INDEL与APS的变化高度相关；（4）精准组装和注释了湖羊、贵德黑裘皮羊和霍巴藏绵羊基因组，挖掘了藏绵羊基因组中与低氧适应性关联的lsPAVs类别、数目和分布，精确定位lsPAVs发生的断点，主要与细胞因子、淋巴因子、激素、能量代谢、氧传输、响应缺氧、DNA修复酶等适应性相关基因和信号通路变化相关；（5）首次重大的发现是湖羊基因组chr6:72984208～88918197和霍巴藏绵羊基因组chr6:72941190～88051854有20M片段的1个大的到位，涉及最重要的基因是UGT家族，UGT基因家族与高寒低氧适应性高度相关。