鸡保种过程中繁殖性状近交衰退效应的遗传调控机制研究

The projective of this study is to investigate the inbreeding depression genetic regulatory mechanism of chicken reproductive traits in conservation. Lanshan chicken conservation populations, established by the family equally matching method, will be selected as the materials. The genomics will be applied to investigate it based on gene expression and epigenetic modification. The RAD-sequencing method will be used to identify SNP markers in the eleventh generations. By incorporation of SNP information and genealogy records, individual inbreeding coefficients and relationship coefficients among individuals will be measured accurately. Individuals with different inbreeding degrees will be selected to set up two populations, the high-inbreeding group and non-inbreeding group. After evaluating the inbreeding effects on reproduction performance in the two F1 populations by analysis of Pearson correlation coefficients and inbreeding depression coefficients, RNA-sequencing and MeDIP-Sequencing methods will be used to analyze the different expressed genes and DNA methylation levels of the HPG axis tissues between individuals with low performance in the high-inbreeding F1 group and individuals with normal performance in the non-inbreeding F1 group. To identify genes, pathways and methylation patterns related to production depression, and to investigate the links between inbreeding, epigenetic process and gene expression and inbreeding depression.

本项目研究目的是以国家级地方鸡种基因库保存的狼山鸡为素材，探讨保种过程中繁殖性状近交衰退效应的遗传调控机制。拟从基因表达和表观修饰两个研究基点，利用简化基因组RAD-seq测序技术鉴定保种群第11世代个体SNP标记，结合系谱信息精确度量个体（间）近交系数和亲缘关系系数，选配个体组建高近交系和非近交系两个组。通过分析Pearson相关系数和近交衰退系数，评价近交对F1后代群体繁殖性状的效应。利用转录组RNA-seq和甲基化组MeDIP-Seq测序技术，分析高近交系中繁殖性能显著下降个体与非近交系中正常表型个体性腺轴组织基因表达差异和DNA甲基化差异，分析差异基因和富集信号通路，探明近交、基因表达和DNA甲基化与繁殖性状近交衰退之间的关联。

近交衰退的分子机理是进化生物学、物种资源保护等领域的研究重点，针对有限规模鸡遗传资源活体保种群，繁殖性状的近交衰退会对群体的世代延续产生显著影响。本研究利用分子标记信息结合系谱信息选配个体，组建狼山鸡高、低近交组，评价繁殖性状的近交效应；利用RNA-seq和BS-seq测序技术，检测高、低近交组个体性腺轴组织基因表达差异和DNA甲基化差异，探索与繁殖性状近交衰退相关的基因及调控通路。研究结果：（1）组建了狼山鸡高、低近交组，高近交组中6个家系的平均分子近交系数均大于0.14，低近交组的平均分子近交系数小于0.04；高近交组狼山鸡开产日龄和300日龄产蛋数两个性状发生了显著衰退（P<0.05），而开产体重和开产蛋重的近交衰退未达到显著水平（P＞0.05）。（2）在卵巢中发现131个差异基因、34个差异lncRNA，差异基因中包括CPEB1、TDRD9、MOV10L1和PIWIL等繁殖功能相关基因，还包括MT4L、CCR6、BRSK2、VTN等免疫相关基因。GO和KEGG分析表明，差异基因富集在GnRH信号通路、卵母细胞成熟、性腺发育和叶酸的生物合成等生物学通路。在下丘脑中发现14个差异基因、27个差异lncRNA，差异基因主要与代谢相关，推测近交可能影响了狼山鸡的基础代谢，尤其是脂肪代谢，进而间接影响了其繁殖性能，导致高近交组繁殖性能的下降。（3）在下丘脑和卵巢中分别鉴定出5948 个和 4593个差异甲基化区域DMR，大量差异甲基化区域参与繁殖相关的神经内分泌调节和生殖细胞发育，推测其在调节狼山鸡繁殖近交衰退中发挥重要作用。通过整合分析转录组和DNA甲基化数据，推测SRD5A1和CDC27基因中的两个差异甲基化区域（DMR）可能是狼山鸡繁殖性能近交衰退的生物标记。研究初步阐明了鸡繁殖性能近交衰退的调控机制，为物种资源保护提供科学依据。