Genome DNA Methylation And Transcriptome Difference Analysis Of Female,Male And Neomale Gonad Of The Large Yellow Croaker

The sex determination mechanism of the large yellow croaker is still unclear.Finding out this mechanism will contribute to the development of large yellow croaker reproduction and sexual control breeding.Whole genome DNA methylation sequencing(WGBS)was adopted and transcriptome sequencing(RNA-seq)to perform genome-wide DNA methylation and transcriptome analyses on three different gonads(male,female,neomale)of the large yellow croaker in the study.Comprehensive investigation of the changes in DNA methylation patterns,the changes of gene expression levels and the regulatory mechanisms of DNA methylation on sex determination and differentiation-related gene expression in the whole genome of the different gonads of large yellow croaker provides a reference basis for revealing the genetic mechanism of the sex determination of the large yellow croaker.The main findings are as follows:(1).Through principal component analysis(PCA)and hierarchical clustering(ward)of the sample,we found that male and neomale samples had similar methylation patterns,and they both differed-from females in methylation genome widely.(2).In total,28072,95255 and 2161 differentially methylated regions(DMRs)were detected in the three groups of female and male(F-M),female and neomale(F-NM)and male and neomale(M-NM).The results suggested high concordance in DNA methylation patterns between M and NM,the difference in methylation between female and neomale gonads is the most significant.(3).Compared with males and neomale,female gonads had much lower levels of methylation,while males and neomales have similar level of methylation.(4).We identified 3,071,3,268,and 753 DMGs between F-M,F-NM and M-NM.DMGs between F-M that are up-methylated in testis was significantly enriched into 2 signaling pathways related to gonadal development,including nuclear receptors and focal adhesion.Moreover,DMGs of F-NM that are up-methylated in testis was significantly enriched into 4 signaling pathways related to gonadal development,including nuclear receptors,gap junction,focal adhesion,cytokines and growth factors.However,there were no enrichment results for ovarian hypermethylation genes of F-M and F-NM and DMGs in M-NM.(5).Based on analyses of the transcriptomic data,10,442,10,116 and 6,000 genes were differentially expressed between F-M,F-NM and M-NM,respectively(|Log2FC|>1,FDR<0.05).Among them,23 genes that were differentially expressed between ovaries and male testes,24 genes that were differentially expressed between ovaries and neomale testes,and 15 genes that were differentially expressed between M testes and NM testes.(6).There were sex-determining genes such as DMRT1,AMH,FST,ZP3 and CYP19B in F-M,F-NM,and M-NM that expression negatively correlated with methylated CpG number.The bisulfite sequencing(BSP)technique was used to verify the methylation level of the DMRT1 promoter region.It was found that the methylation level of females was higher than that of males,which was consistent with the sequencing results,suggesting that the difference in the expression of DMRT1 gene in different gonads of the large yellow croaker was largely affected by the difference in DNA methylation.