解析猪12号染色体影响产仔数QTL的分子机理

Litter size affects economic benefits of pig industry directly. There are important theoretical and practical values to understand the molecular basis of litter size variation. Through long-term observation, an obvious separation of litter size within Erhualian pig breed was noted. A significant difference of corpora lutea numbers between high-yielding and low-yielding population was also observed. By a whole genome-wide scan, we identified a significant QTL for little size within Erhualian pig breed on SSC12 and individuals with dominant genotype of the most significant locus of this QTL region was higher than that with inferior genotype by 1.72 piglet. Moreover，many positional and functional candidate genes for ovulation rate or reproduction traits were detected within this relative large QTL region, but the causative gene is unknown. Based on the above background, this project was designed to fine map the QTL by increase marker density and population size firstly. Subsequently, combining with our gene expression profile of ovary from both high-yielding and low-yielding Erhualian sows, genes located in the refined QTL regions, expressed significantly different or related to reproductive physiological or biochemical pathways will be selected as candidate causal genes. Then verification analysis will be conducted at RNA and protein levels for the causal genes of the QTL. Finally, the causal mutations of the QTL will be detected by causal gene re-sequencing, conservation and functional prediction analysis, preliminary functional analysis combined with association analysis with little size in large population. Our results will be crucial for understanding the molecular basis of the QTL for litter size variation on pig chromosome 12 and will provide the new molecular markers to improve the litter size in pig breeding.

产仔数高低直接影响养猪效益，解析产仔数变异遗传机理具有重要理论和应用价值。申请人前期发现二花脸猪品种内产仔数分离明显，高、低产组产仔数和排卵数差异极显著；通过全基因组扫描在12号染色体鉴别到显著影响二花脸猪产仔数变异的QTL，QTL区域最显著标记优势基因型纯合子比劣势基因型纯合子总产仔数多1.72头，但QTL置信区间内存在多个与排卵数或繁殖相关的功能基因，真正的因果基因未知。本项目拟在此基础上，通过增加QTL区域标记密度和样本量精细定位QTL，整合申请人已有的卵泡发育关键时期高、低产二花脸猪卵巢基因差异表达结果，鉴别QTL因果基因，并进行RNA、蛋白表达差异验证；通过对因果基因的深度重测序、突变位点体外细胞初步功能分析和大群体产仔数关联性验证，最终鉴别因果基因及突变位点，由此揭示猪12号染色体影响产仔数QTL的分子机理，并为猪产仔性能遗传改良提供新的分子标记。

产仔数是猪最重要的经济性状之一，解析产仔数变异遗传机理具有重要理论和应用价值。申请人前期发现二花脸猪品种内产仔数分离明显，高、低产组排卵数差异极显著，通过极端高、低产二花脸猪全基因组扫描分析，在12号染色体鉴别到显著影响二花脸猪产仔数变异的QTL，但QTL置信区间内存在多个繁殖相关的功能基因，因果基因未知。本项目通过在QTL置信区间内增加标记密度（1个SNP/8Kb），增加标记分型群体(334头二花脸猪和269头梅山猪)，基于单点和单倍型关联性分析方法成功精细定位该QTL区间，区间内拥有TUBD1、RPS6KB1、RNFT1和MED13等基因。通过定量和蛋白分析，发现区间内仅有RPS6KB1基因在二花脸高、低产组卵巢中存在转录水平和蛋白表达差异，免疫组化分析发现该基因在卵巢的主要表达部位为卵泡内膜细胞，且该基因已被发现与卵泡发育及成熟有关，因此RPS6KB1基因是该QTL的候选因果基因。利用极端高、低产二花脸猪对RPS6KB1基因进行重测序，鉴别所有SNP位点，结果发现基因编码区没有SNP位点，基因调控区存在多个SNP位点。通过对调控区SNP位点功能预测分析、与二花脸猪产仔数关联性分析，发现RPS6KB1基因3’UTR的g.37433196位点与二花脸猪产仔数显著关联，且功能预测发现该位点突变可影响RPS6KB1基因与ssc-miR-7135-5P的结合效率，是重要的候选因果位点。通过体外双荧光素酶试验分析，发现g.37433196位点由G突变为C影响了该位点与ssc-miR-7135-5P结合效率，调节了RPS6KB1基因的表达。通过g.37433196位点与苏太猪、梅山猪、沙乌头猪、大白猪产仔数关联性验证分析发现，g.37433196位点与梅山猪、大白猪显著关联，与苏太和沙乌头猪有关联趋势，可初步证明g.37433196位点为该QTL上的候选因果位点。综上所述，本研究通过QTL精细定位，候选因果基因RNA、蛋白、免疫组化分析，候选因果位点功能预测、体外细胞初步验证和大群体产仔数关联性验证分析，成功鉴别到12号染色体影响二花脸猪产仔数变异的QTL的候选因果基因位点：RPS6KB1基因3’UTR的g.37433196位点。本研究初步揭示了猪12号染色体影响产仔数QTL的分子机理，并为猪产仔性能遗传改良提供了新的重要分子标记。