鸡繁殖性能近交衰退相关CpG岛差异甲基化基因的筛选

鸡繁殖性能近交衰退是地方鸡遗传资源活体保种过程中面临的重要问题之一，本研究旨在探讨全基因组CpG岛（CpG island，CGI）区DNA甲基化在鸡繁殖性能近交衰退中的作用。分别从狼山鸡高近交组和低近交组中各选取健康母鸡3只，即试验分2个组，每组3个重复，然后采用全基因组重亚硫酸盐测序（WGBS）技术，检测分析两组个体性腺轴组织（包括卵巢和下丘脑）全基因组DNA甲基化差异，筛选差异甲基化区域（DMRs），并对CpG岛区差异甲基化基因进行功能注释和富集分析。结果表明，狼山鸡高近交组和低近交组比较，其卵巢和下丘脑基因组整体甲基化水平均不存在显著差异（P>0.05）；高、低近交组间差异甲基化区域检测发现，下丘脑和卵巢中分别检测到5 948和4 593个差异甲基化区域，其中1 798和995个差异甲基化区域位于基因组CpG岛区，分别注释到1 020和552个基因；下丘脑中，这些CpG岛区差异甲基化基因显著富集在信号转导、神经系统发育、生殖系统发育和卵母细胞成熟调控等繁殖相关的GO条目，以及转化生长因子β信号通路、乙型肝炎、脂肪酸代谢、胰岛素信号通路等19条KEGG信号通路（P<0.05）；卵巢中，CpG岛区差异甲基化基因显著富集于12条信号通路（P<0.05），包括慢性骨髓白血病、流感A、精氨酸和脯氨酸代谢、粘着连接等，一些与卵子发育和性激素分泌相关的信号通路也被富集到，如黄体酮介导的卵母细胞成熟、卵母细胞减数分裂、GnRH信号通路、雌激素信号通路等，其中包含CDC27、ADCY8、AKT3等10个差异甲基化基因。因此，本研究在狼山鸡高、低近交组间检测到了大量差异甲基化区域，并发现大量差异甲基化基因与繁殖性状相关，推测这些基因CpG岛区DNA甲基化可能在狼山鸡繁殖性能近交衰退调控中发挥重要作用，研究结果为进一步深入探索鸡繁殖性能近交衰退调控机制奠定了基础，为物种资源保护和家禽育种工作提供了理论参考依据。

Screening of Genes with Differential Methylated CpG Island Related to Inbreeding Depression of Chicken Reproduction

Inbreeding depression of chicken reproduction is one of major concerns during preserving local chicken genetic resources. This study aimed to investigate the role of genome-wide CpG island (CGI) DNA methylation in inbreeding depression of chicken reproduction. Three healthy hens were selected from the strongly and weakly inbred chicken groups respectively, that is, two groups were set in the experiment with 3 replicates in each group. Whole-genome bisulfite sequencing (WGBS) technology was used to detect and analyze genome-wide methylation differences in gonad axis tissues (including ovary and hypothalamus) between the two groups, and to identify the differentially methylated regions (DMRs). Functional annotation and enrichment analysis of genes with DMRs in their CpG islands were performed. The results showed that no significant differences were found at the methylation levels of ovary and hypothalamus genome between the strongly and weakly inbred chickens (P>0.05). However, 5 948 and 4 593 DMRs were identified between the two groups in hypothalamus and ovary, respectively, of which 1 798 and 995 DMRs were located in CpG islands of the genome. These DMRs were annotated to 1 020 and 552 genes, respectively. In hypothalamus, the genes with DMRs in CpG islands were significantly enriched in GO terms related to reproduction(P<0.05), such as signal transduction, nervous system development, reproductive system development and oocyte maturation regulation. And a total of 19 KEGG pathways were significantly enriched(P<0.05), including TGF-beta signaling pathway, Hepatitis B, fatty acid metabolism and insulin signaling pathways, etc. In ovary, the differentially methylated genes were significantly enriched in 12 pathways(P<0.05), such as chronic myeloid leukemia, influenza A, arginine and proline metabolism and focal adhesion, etc. Particularly, several pathways associated with oocyte development and sex hormone secretion were also enriched, for example, progesterone mediated oocyte maturation, oocyte meiosis, GnRH signaling pathway and estrogen signaling pathways, etc, which included a total of 10 genes, such as CDC27, ADCY8 and AKT3, etc. Thus, a large number of differentially methylated regions were detected between the strongly and weakly inbred Langshan chickens, and a number of differentially methylated genes were found to be related to reproductive traits. It is inferred that the DNA methylation in CpG islands of these genes may play important roles in regulation of inbreeding depression of reproduction in Langshan chicken. The study results will provide a basis for further research on the regulation mechanism of inbreeding depression of chicken reproduction, and will supply a reference for conservation of species resource and future poultry breeding.