种间生殖隔离的机制至今仍困扰遗传学家。马驴杂交呈现出明显的杂种不育现象，这为从表观遗传学角度研究杂交不育提供了良好的动物模型。针对骡不育难题，我们前期绘制了马骡及驴骡家系24个个体的血液全基因组甲基化图谱，分析发现杂交部分改变了骡的甲基化表达模式，对上调或下调的基因进行通路构建，发现与移植排斥、移植物抗宿主病、抗原的加工与呈递等通路相关。据此我们推测杂交后代甲基化模式改变可引发基因表达谱模式异常，导致遗传不相容而致骡不育。为证实上述假设，拟对上述试验材料中骡睾丸和卵巢进行全基因组甲基化测序，同时对相应组织进行转录组测序研究；联合分析甲基化和转录组数据，鉴定骡基因组特有的甲基化模式及基因表达谱模式；通过BSP和RT-qPCR实验验证关键调控基因的甲基化水平和基因的表达情况；利用骡睾丸、卵巢组织及小鼠睾丸细胞系研究关键基因的甲基化调控机制。从甲基化表观修饰角度解析骡不育分子机制。

Interspecific hybridization leads to changes in phenotypes and genotypes of the progeny, with major implications for evolutionary biology, genetics, and breeding. Hybrids of the horse and donkey provide an ideal system for investigating hybrid sterility. Both mule (a female horse is mated to a male donkey) and hinny (a female ass mated to a male horse) are phenotypically different from either of the parent species. Sterility in mules and hinnies has been a subject of discussion for as many centuries. To investigate the mechanism of sterility of mule or hinny, many researchers approached the problem from different perspectives, and the mechanism was only partially revealed. Each of these hybrids is considered to be sterile due to the incompatibility of the maternal and paternal chromosomes or genes. However, the detailed mechanism of hybrid sterility remains unknown.. Since 2014, we began to implement donkey genome de novo. Last year, we generated and provided a genome-wide landscape of DNA methylations from 6 mules and 6 hinnies and 12 their parent species, respectively by MeDIP-seq. We successfully identified differential methylated regions through a comparative analysis of mule and hinny and their parents, indicating that Allograft rejection, Graft-versus-host disease and Antigen processing and presentation etc. pathway from mule and hinny showed different from the their parents. Furthermore, we found the relatively density of methylation of some imprinted gene were different between mule and hinny and their parents. These results indicate that DNA methylation may contribute to hybrid sterility by hybrid incompatibility. We propose a hypothetical model in which hybrid incompatibilities resulted from the difference between gene expression from horse and donkey by DNA methylation, could contribute to hybrid sterility and hybrid segregation distortion between reciprocal hybrids. . DNA methylation is a stably inherited epigenetic modification in eukaryotes. The regulation and characteristics of the DNA methylation still remain enigmatic, although the importance of it has been demonstrated in many biological processes such as gene expression regulation, genomic imprinting, X chromosome inactivation, maintenance of genomic stability by transposon silencing. . We sampled the testes or ovary tissues from 6 mules and 6 hinnies respectively. We will generate and provide a genome-wide landscape of DNA methylations respectively by MeDIP-seq and mRNA transcriptome respectively. Integration analysis will reveal a total of negatively correlated genes with methylation in the promoter and gene body regions, respectively. Furthermore, we will identify expression patterns of high-read genes that exhibit a negative correlation between methylation and expression from blood and testes and ovary of mule, hinny and their parents. To further investigate the biological processes and biological functions associated with the differentially expressed genes, we will perform GO analysis and KEGG pathway analysis. In addition, we will validate the MeDIP-seq results by BSP in some of the differentially methylated promoters. RT-qPCR will be performed to valid the differentially expression gene between the mule and hinny and their parents. Further, we will compare the above integration analysis results with X-linked gene expression, genomic imprinting and X chromosome inactivation in testes or ovary of mule and hinny, respectively. Finally, we will test the above integration analysis results by transfection into MLTC-1 cell lines by 5-Aza-dC. These results will provide valuable data to uncover the molecular basis underlying hybrid sterility. This comprehensive map may also provide a solid basis for exploring the epigenetic mechanisms of hybrid sterility.