A Comparative Study On Genomic DNA Methylation Of Four Mongolian Sheep Breeds In My Country

Mongolian sheep is widely distributed in our country and has large differences in production performance.DNA methylation is one of the epigenetic modifications,and plays an important role in the biological process of growth and gender differentiation.However,few studies are reported about the DNA methylation of Mongolian sheep.In this study,we used four major Mongolian sheep(2 Ujimqin female sheep,2 Ujimqin male sheep,2 Tan female sheep,2 Small-tailed Han female sheep and 2 female Hu sheep,10 sheep in total)as the research materials,and applied MeDIP-seq as the research method to detect breed-specific methylation regions and phenotype related DNA methylation variation regions.And using MassARRAY,qPCR and Western Blot to test candidate gene's DNA methylation,mRNA expression and protein expression level.Meanwhile,the MeDIP-seq data was used to exploit reproduction related single nucleotide variations.The main results are showed as following.1.The Fst values of Ujimqin sheep,Tan sheep,Small-tailed Han sheep and Hu sheep are less than 0.05 in D-loop regions of mitochondria among 167 individuals,they are 0.00258(Hu vs.Tan),0.02145(Hu vs.UQ),0.03375(Hu vs.StH),-0.01169(Tan vs.UQ),0.03150(Tan vs.StH)and 0.03323(UQ vs.StH),indicating there is no genetic diversity among four subpopulations of Mongolian sheep,and small difference genetic distance among four breeds.It also indicates that all the samples used in our study are reliable.2.We obtained the first high quality DNA methylation landscape of Mongolian sheep by using MeDIP-seq technology.And we got 725,844,769 clean reads,then submitted to NCBI database(Access number is GSE62345).84.74%clean reads were mapped to ovis aries reference genome.All the Pearson correlation coefficient of biological replicates are above 0.94(all P are less than 2.2e-16),meanwhile,we used MassARRAY method to validate the MeDIP-seq data.And two relatively hypermethylated and hypomethylated regions were randomly selected.The results agreed with the MeDIP-seq data,again it indicated the accuracy of MeDIP-seq data.3.Depending on MeDIP-seq data and combining with results of diffReps and RPKM value of 300 bp window,we obtained 8068 UQ-specific different methylated regions,and mapped to 1037 genes;14387 Tan-specific different methylated regions,and mapped to 1645 genes;10195 StH-specific different methylated regions,and mapped to 1340 genes;7672 Hu-specific different methylated regions,and mapped to 969 genes.Then we annotated those genes by GO method,and found each breed's specific genes were annotated to Developmental Process(GO:0032502).4.There are 4972 different methylated regions(3144 Up-regulated and 1828 Down-regulated)between Ujimqin female and male sheep,and were mapped to 1348 genes.GO analysis shows most of genes are enriched in metabolic and hormone-related pathways,and we also obtained the GnRH signaling pathway.It indicates those sex-different methylated regions may participate in the regulations of gender-differentiation or reproduction.5.Modified body mass index result shows that there is significant difference among four breeds.UQ has highest MBMI value(84.78±7.94),significantly higher than the rest three breeds(P<1.0×10-7);there are no significant difference between StH(72.78±9.46)and Hu(73.19±9.97);Tan displays lowest MBMI value,significantly lower than three other breeds(P<1.0×10-7).All breed-specific different methylated regions enriched in Developmental Process(GO:0032502),which is a biological process associate with growth and development of cell,tissue and organ.And it potentially influences body size,then we defined the different methylated regions enriched in Developmental Process(GO:0032502)as body size variation related candidates.641 different methylated regions were identified,and annotated in 308 genes.6.Then,we tested the DNA methylation level,mRNA expression and protein expression patterns of BMPR1B,SMAD1,SMURF 1,AKT1 and TSC1 in Ujimqin,Tan and Samll-tailed Han sheep.The candidate regions show large difference among breeds,the fatter Ujimqin and Samll-tailed Han sheep have higher DNA methylation levels.Except AKTl mRNA expression is found significantly higher in Ujimqin compared to Tan and Small-tailed Han sheep,there are no significant difference of mRNA expression are observed in other four genes within breeds.However,there are six CpG sites'methylation level have significant correlation with their own mRNA expression.Unfortunately,there is no significant variation of protein expression within breeds.Only TSC1 was found significant correlation among CpG methylation,mRNA and protein expression.7.We used MeDIP-seq data to scan different methylated regions between high-fecundity sheep(Hu and Small-tailed Han sheep)and low-fecundity sheep(Ujimqin and Tan sheep).240 Up-regulated and 303 Down-regulated regions were obtained,and they were in 179 genes.Besides,we obtained 499 single nucleotide variations from our MeDIP-seq data.Among them,19 SNVs located at aseasonal QTL.There is one variation which is at 253426198 on chromosome 1 not only in aseasonal QTL,but also in different methylated regions,which may be a single-nuclotide mutations related to reproduction.Most important of all,we only detected FecB mutation in high fecundity sheep,which indicated our analysis pipeline was accurate.