鉴别仔猪抗大肠杆菌F4ac侵染的MUC13基因因果突变

Enterotoxigenic Escherichia coli (ETEC) F4ac is a major determinant of diarrhea in neonatal and young pigs. We previously identified the F4ac receptor gene,MUC13,by a series of genetics technologies including whole-genome linkage mapping, recombination breakpoint analysis and association studies in a collection of diverse outbred populations. But the causative mutation has not been identified. It was inferred that the mutation might lie in ～10 Kb-long, GC-rich tandem repeat area in the coding region of the MUC13 gene. On the basis of these findings, we would sequence the tandem repeat area in the MUC13 gene by using single molecule sequencing and Sanger capillary sequencing. Then the identified polymorphisms would be genotyped on 292 outbred pigs representing 12 Chinese native pig breeds and 3 Western pig breeds. Association of MUC13 polymorphisms with susceptibility/resistance to ETEC F4ac will be performed to determine the causative mutation, which would be further validated by other pig population and functional analyses. The expected results of this study will provide novel insights into the molecular mechanism of ETEC F4ac disease resistance in pigs.

大肠杆菌F4ac是侵染新生仔猪引发腹泻的主要病原菌。申请人及所在课题组前期通过全基因组连锁定位分析、目的区域的重组断点事件分析和远缘群体高通量SNP标记的关联性分析等严谨的遗传学分析手段，确定了仔猪F4ac腹泻易感基因为MUC13基因，但至今尚未鉴别到因果突变，最大障碍在于该基因编码区存在一个尚未解析的约10 Kb富含GC 的高度串联重复区域。本项目拟在此基础上，借鉴近期高GC含量串联重复区变异位点研究的成功案例，采用PacBio单分子实时测序技术并结合Sanger测序特殊拼接技术对MUC13 基因10 Kb串联重复编码区开展比较测序分析，将鉴别到的变异位点，在已有F4ac黏附表型的15 个中、西方猪种远缘群体292 个仔猪个体中开展多态性检测，通过与黏附表型进行关联性分析，并经大样本验证及功能分析，最终确定因果突变位点，并探究其遗传起源，进而全面揭示猪F4ac侵染腹泻的抗病分子遗传机理。

仔猪腹泻给养猪业带来巨大经济损失，F4ac大肠杆菌是引发仔猪腹泻的主要病原菌，课题组前期通过全基因组连锁定位分析、目的区域的重组断点事件分析和远缘群体高通量SNP标记的关联性分析等严谨的遗传学分析手段，初步确定了仔猪F4ac腹泻易感基因为MUC13基因，本项目在前期工作基础上采用体外细胞黏附功能验证分析、小肠上皮细胞蛋白表达差异分析以及ITGB5基因的比较测序与关联分析进一步证实MUC13基因为目的基因；同时针对MUC13基因核心区域采用比较测序法结合关联分析、功能验证分析，发现了1个能准确鉴别易感个体和抗性个体的SNP位点（g319699192 G>A），准确率>97%；利用鉴别到的MUC13基因g319699192 G>A突变位点开展抗腹泻专门化新品系选育，结合遗传评估分析，成功培育出表型有鲜明特色并在生产上有重大价值、性能优良、遗传稳定且适合我国目前商业肉猪生产主流模式的瘦肉型抗病猪专门化新品系。本实验结果丰富了人们对仔猪腹泻分子遗传机理的认识，同时为实践生长中培育抗腹泻猪新品系提供理论依据。