DNA Methylation Inheritance Patterns And Imprinting And Expression Patterns Of Major Imprinted Genes In Horse,Donkey And Their Hybrids

DNA methylation and genomic imprinting is critical for growth,development,and especially normal embryonic development in mammals.Therefore,the research on mammalian DNA methylation patterns and imprinted gene expression patterns has always been a research hotspot.Although humans have so far gained a certain understanding of mammalian genome methylation and imprinted gene functions,expression regulation mechanisms,and inheritance patterns through rodent-based models,there are few studies on the rare hybrid species in mammals regarding the genomic methylation and imprinted genes.Because hybrid species generally have two sets of unequal gene sequences and different numbers of chromosomes,we believe that revealing the genomic methylation and imprinting patterns in hybrid species can help researchers more deeply to find the core mechanism underlying genomic methylation and imprinting.Therefore,in this study,we used the hybrid model between equine species horse and donkey to explore the pattern of genome methylation and imprinting in the case of hybrid mammalian species.In order to achieve the research goal,this study first used a simple PCR test to establish a system to identify the species and sex of horses,donkeys,mules and hinnies to ensure the accuracy of sampling;then horse,donkey,mule and hinny fetuses and caul,horse sperm,donkey sperm,adult equine tissues(viscera,brain and muscle)were used as the research objects,and whole-genome bisulfite sequencing was used to preliminarily analyze genomewide methylation patterns and inheritance patterns of equine embryonic fetuses and caul;after that,pyrosequencing and absolute quantitative PCR were used to analyze the imprinting and expression patterns of 10 major mammalian imprinted genes in equine adult tissues;at last we used,IGF2 R as an example to establish the regulation pattern of imprinting and expression in the equine hybrid species model,its association with imprinted DNA methylation and non-imprinted DNA methylation,and the association between its expression and phenotype.The main results we obtained from the above research were:1.Based on the sequence differences of CNGB3 gene and mitochondrial gene D-loop region between horse and donkey and the homology of SRY gene sequence,we established a method to simultaneously identify the species and sex of horse,donkey,mule and hinny by simple PCR.2.The fetuses and caul of horses,donkeys,mules,and hinnies were mapped for the first time by high-throughput sequencing of whole genome methylation of fetuses and caul of horses,donkeys,mules,and hinnies,and sperm of horses and donkeys.Genome-wide methylation maps of caul and preliminary analysis of their genome-wide DNA methylation distribution patterns revealed that DNA methylation in the equine hybrid model followed a cis-inherited trend and DMRs between horse,donkey fetuses and caul were identified and analyzed by GO and KEGG analysis,indicating that these DMRs are mainly related to RNA transcription,centrosome and metal ion binding function and metabolic pathways signaling pathways.These results lay a foundation for further study on the epigenetic regulation of DNA methylation and the development of fetuses and fetal membranes in equines.3.Parental expression of 10 mammalian imprinted genes including H19,IGF2 R,NAP1L5,MEST,DLK1,NNAT,RTL1,DGAT1,PHLDA2 and TSSC4 were identified in horse,donkey,mule and hinny heart,liver,spleen,lung,kidney,brain and muscle tissue and the results showed that well identified major mammalian imprinting genes IGF2 R and H19 were still stable in equine hybrid species models;NAP1L5 was prone to tissue-specific imprinting;the imprinting of MEST,DLK1,NNAT,RTL1 basically maintained,but accompanied by partial imprinting disorder;DGAT1,PHLDA2 and TSSC4 did not have imprinting expression characteristics in the equine hybrid species model.4.In the in-depth study of the imprinted gene IGF2 R,we found that,1)Although the sequence of equine IGF2 R intron 2 Cp G island was a maternal allelespecific methylation imprint,it had nothing to do with the regulation of IGF2 R imprint expression;2)There were three Cp G islands in the IGF2 R promoter region of horse,donkey and mule and hinny,and none of them contained allele-specific methylation regions.Cp G island1 and Cp G island 3 belonged to hypermethylated regions,and there was an insignificant difference in methylation between horses and donkeys.Horses were hypermethylated in the Cp G island 2 region,while donkeys showed tissue-specific methylation patterns.The DNA methylation status of hybrid species were inherited in cis-pattern,no matter the breeding pattern.3)The proliferation rate of hinny fetal fibroblasts was much lower than that of horse,donkey and mule fetal fibroblasts.Hinny fetal fibroblasts had a higher proportion of cells in the G0/G1 phase,and this phenomenon was caused by non-imprinted abnormality induced low expression of IGF2 R.