猪基因组中活性转座子的插入多态鉴定及重要经济性状分子标记筛选

The study of human, mice and plants confirmed the transposon insertion polymorphism (TIP) are new molecular marker with many advantages, and which represents a high application value in the research fields of gene fine mapping, genetic evolution, breed identification, but the research and development of pig genome TIP was rare in the past, which, to some extent, restricted the swine molecular breeding process. In this study, the mixing porcine genomic insert multiple transposon libraries were constructed, and then the transposon insertion site sequence data were obtained by targeted high-throughput sequencing. The population genetic analysis was further conducted to setup the pig genome-wide TIP database and to assess the application value of TIP in the research of pig evolution, breed formation. Then the TIP distribution in the genome was annotated to identify the TIPs inserted in or around functional genes. On this basis, the candidate TIPs associated with genes, which may affect important economic traits, were selected for phenotype association studies, and the strong correlation TIPs with phenotype were remained for further explore the molecular mechanisms of the gene activity changes due to the transposon insertion. This project is expected to get the pig genome TIP data, obtain some valuable TIPs in pig molecular breeding, publish 2-4 papers, and apply 1-2 patents. The research and development of pig TIPs will promote the development of pig functional genomics and molecular breeding process, deepen the understanding of the origin and evolution of swine, and the breed and strain formation.

人类、鼠和植物等研究证实：转座子插入多态（TIP）是具有诸多优点的新型分子标记，在基因精细定位、遗传进化、品种鉴定等研究中具有很高的应用价值，但目前猪基因组TIP研发不足，一定程度上制约了分子育种进程。本研究通过构建猪混合基因组转座子多重插入富集文库，进行靶向高通量测序，获得猪基因组转座子插入位点信息后进行群体遗传学分析，建立猪基因组TIP数据库，评估TIP对猪遗传进化和品种形成的影响。然后注释TIP在基因组上的分布特征，获得与功能基因相关联的候选TIP。在此基础上，筛选一些可能影响重要经济性状的TIP，进行表型关联研究，筛选与表型关联性较强的位点，探究转座子插入对关联基因活性影响的分子机制。项目预期能够建立猪全基因组TIP数据库，获得若干有育种价值的TIP，发表论文2-4篇，申请专利1-2项。TIP研发将推动猪功能基因学研究和分子育种的进程，加深对猪起源进化和品种形成的理解和认识。

转座组（基因组上所有转座子）是哺乳动物遗传变异的重要来源，目前以转座子插入多态为基础的分子标记在猪上的开发和应用非常有限。本项目对猪基因组中的转座成份进行重新挖掘、分类和进化动力学，转座活性验证，并对多态位点进行鉴定和分子标记开发，同时探究转座子对插入基因产生的影响。.反转录转座子是猪基因组的主要成分（37.13%），LINE、LTR和SINE分别占基因组的18.52%、7.56%和11.05%。其中L1分为四个家族，53个亚家族，L1D1-7为代表最年轻的亚家族。SINE分为三个家族，25个亚家族，SINEA1-3代表最年轻的亚家族。ERV分为十八个家族，ERV6A和ERV6B代表最年轻的亚家族。细胞实验证实年轻L1具有转座活性，L1D-5’UTR与ERV6-LTR具有正反向启动子活性。超过80%的蛋白编码基因和lncRNA基因含转座子插入，44.30%的蛋白编码基因和24.13%的lncRNA基因含年轻反转录转座子插入。PCR证实外显子区存在151个SINE插入多态位点。通过生信分析并经PCR验证表明GHR、IGF1、VRTN和KTN1基因及其侧翼区分别含有4个、1个、3个和5个由转座子插入引起的结构变异（>100bp）。其中VRTN基因中VRTN-sRTIP1位点在大白群体中存在丰富多态性，无插入个体的百公斤体重日龄显著低于有插入个体，KTN1基因中KTN1-intron5-SINE位点有SINE插入个体所产仔猪断奶窝重显著高于无插入个体。.鉴定到lncRNA基因内L1插入多态位点114个，ERV 107个，其中两个L1插入多态位点L1-lnc-49和L1-lnc-253-F1在大白猪群体中检测表明，L1-lnc-49位点有L1插入个体的校正背膘厚显著低于无插入个体，L1-lnc-253-F1位点有L1插入个体的繁殖性状同窝活仔数显著低于无插入个体。两个ERV插入多态位点lnc97和lnc66在大白猪群体中检测表明，lnc97位点含ERV插入的个体眼肌面积比无插入个体显著增加，而lnc66位点有ERV插入个体百公斤体重日龄显著高于无插入个体。