| Crassostrea gigas is an important biological resource among the major economic shellfish in China.China has been artificially breeding oysters for a long time,and some progress has been made in the breeding of black-shelled oyster.However,the biological mechanism of the difference between the black shell and white shell of the oyster has not yet been clarified.The preliminary work of our group found that the long oyster is consistent with other reported organisms affected by methylation epigenetic modifications.In this study,whole genome bisulfite sequencing(WGBS)and simplified genome methylation sequencing(Methyl RAD-seq)were combined with fluorescence PCR to analyze the changes in transcript expression of differentially methylated genes to identify candidate genes regulating different shell color formation mechanisms in the Crassostrea gigas.The studied the distribution of DNA methylation in the whole genomic region of the left and right mantle around the different shell colors of oyster,the functional annotation of the genes involved in the biological mechanism of oysters with different degrees of DNA methylation,the changes in the methylation status of single base precision of the full sib family and the changes in the expression level of genes affected by methylation to explore the formation mechanism of different shell colors of oysters and provide the theoretical basis for scientific breeding of oysters,the construction of different production systems and the expansion of market scale.WGBS,Methyl RAD-seq and q PCR transcript expression level verification were as follows.Six samples of the left and right mantle of oysters with black,white and yellow shells were sequenced by WGBS,and a total of 25.9G DNA sequence data were obtained.A total of 500 differentially methylated genes were screened by enrichment analysis among the black-shell,yellow-shell,and white-shell oysters.The enrichment analysis of differential methylation genes between black-shelled and yellow-shelled screened 2669 genes common to BL＿vs＿YL and BR＿vs＿YR.By enrichment analysis of differentially methylated genes between black-shelled and white-shelled,2752 common differential genes between BL＿vs＿WL and BR＿vs＿WR were screened.The enrichment analysis of differential methylation genes between yellow-shelled and white-shell screened 2801 genes in common between YL＿vs＿WL and YR＿vs＿WR.In addition,219 differential genes were screened in the differential methylation gene enrichment analysis of the left and right mantle of the same shell color in the black-shelled oysters.Similar methods,in the left and right mantle of yellow-shelled oyster,197 genes were screened.other in the left and right coat membranes of white oyster,200 differential genes were screened.A total of 152 M Raw reads were obtained from five black-shelled and five white-shelled oyster of the full sib family.999 differentially methylated genes were screened in five black-shelled and five white-shelled oysters of the full sib family samples,of which 519 genes were more highly methylated and 480 genes were less methylated in the black-shell sample group than in the white-shell sample group.The GO enrichment analysis of these differentially methylated loci and differentially methylated genes,respectively,revealed that the occurrence of differentially methylated loci and genes were mainly associated with several physiological processes such as receptor-mediated endocytosis,thiamin metabolism,stress-activated MAPK cascade,cellular senescence,spliceosome complex assembly,protointestinal embryo formation,DNA integration,and many other different molecular functions such as cation transmembrane transporter protein activity,hydrolase activity,calmodulin binding,carbohydrate binding,receptor binding,and protein homodimerization activity.The results of KEGG Pathway enrichment analysis showed that differential methylation levels and differential genes could be enriched to various Pathways such as taurine and hypotaurine metabolic pathway,Notch signaling pathway,dorsoventral axis formation,spliceosome,glycosphingolipid biosynthesis amino acid degradation,Notch signaling pathway,,AMPK signaling pathway,riboflavin metabolism,ABC transporter protein,AMPK signaling pathway,methane metabolism,Fox O signaling pathway,propionate metabolism,niacin and nicotinamide metabolism.The above results indicate that there are different DNA methylation regulatory mechanisms in black and white shell oyster and these DNA methylation regulatory mechanisms affect the activities of various enzymes and ion channel activities in black and white oyster,and a variety of biological processes including various metabolic pathways,hormone signaling pathways,defense responses,developmental processes,protein degradation,and DNA replication in Crassostrea gigas are also regulated by DNA level methylation.Finally,12 differentially methylated genes with the highest confidence were screened in this study,and the expression of these genes was examined for changes at the transcriptional level by q RT-PCR.The results showed that LOC105345749 and LOC105336214 were significantly up-regulated in black-shelled compared to white-shelled oyster,while LOC105347929,LOC105325380,LOC105332054 and LOC109618848 were significantly less expressed at the transcriptional level in black-shelled than in white-shelled oyster. |
