Study On Methylation Difference Of Charybdis Japonica In Offshore Of China

Charybdis japonica is an important marine economy crab,belonging to the Decapoda,Poratunidae,Charybdis.At present,a series of studies on morphology,fishery biology,germplasm resources,and population genetics have been carried out,while epigenetics research has not been reported.Epigenetic modifications are inherited across generations and can produce specific responses to environmental stress.The pattern of stress was recorded in genetic modifications in the form of DNA methylation,etc.,producing adaptive,short-or long-term phenotypic plasticity of gene expression,increasing the potential to adapt to the environment,or buffering the organism from harmful environmental effects.Heat shock proteins（Hsps）play a key role in cellular processes and responses to environmental stresses.In particular,their roles in maintaining cell homeostasis,transport,and involvement in signal transduction processes directly affect crabs’ behavior,physiology,adaptive responses,and regulatory mechanisms.The Hsp70 and Hsp40 gene families are the most widely studied Hsps,which play important roles in the response of crabs to complex and changeable intertidal life,especially temperature fluctuations.In this study,25 female individuals from five northern and southern populations（Taizhou,Zhoushan,Lianyungang,Yantai,and Dalian）were selected under different water temperatures.The whole genome methylation sequencing was performed to obtain the differentially expressed methylated regions among populations and individuals,as well as the differentially methylated genes and their corresponding functional annotations and signals.A high level of assembly annotation information was obtained.Second,the whole genome identification and analysis of Hsp70 and Hsp40 gene families of C.japonica were performed by bioinformatics method.At the same time,the gene expression data obtained by transcriptome sequencing were jointly analyzed to obtain the differentially expressed genes between different populations and analyze the DNA methylation of the differentially expressed genes.The differences in Hsps gene methylation levels between different populations were compared,and the contribution of DNA methylation to temperature adaptation in different populations was analyzed.The results of this study elucidated the mechanism of temperature adaptation in different populations at the epigenetic regulation and transcriptome level.This study provides a preliminary theoretical reference for the involvement of Hsp70 and Hsp40 genes in the epigenetic difference of C japonica under the selective pressure of temperature and provides a basis for the epigenetic study of Decapods.The main research results are as follows:1.Whole genome methylation measurement of different populations of Charybdis japonicaIn this study,1474.58G of total raw data and 1386.04G of total valid data were obtained by combining bisulfite conversion method and next-generation sequencing technology,with an average sequencing depth of 41.25x.The average methylated CpG proportion（mCpG%）of all chromosomes in C.japonica genes was 21.57%,of which the methylated CHG and CHH proportions were 0.42%and 0.43%,respectively,and the average proportion of overall methylated cytosines was 3.23%.The methylation levels and distribution patterns of CHG,CHH and CpG were analyzed in 25 samples.The methylation levels of CHG and CHH were similar in general,and the methylation levels 2kb upstream of transcriptional start site（TSS）and 2kb downstream of transcription termination site（TES）were both lower.In the CpG methylation level,the methylation level near the TSS and downstream of the gene was maintained at hypermethylation,and the methylation level in the gene region fluctuated.Taking Haizhou Bay as the boundary and Lianyungang population as the reference group,we compared the differences between groups of Taizhou,Zhoushan,Yantai and Dalian,respectively.We found that there were differences in the number of differentially methylated regions（DMRs）between different populations,both hypermethylated and hypomethylated DMRs,and differences in hypermethylation and hypomethylation between different gene constructs in different populations.Functional enrichment in different populations of C.japonica also exists.Each group was mainly enriched in the signaling pathway of G-protein coupled receptor（GO:0007186）,structural components of the cuticle（GO:0042302）,synaptic tissue（GO:0050808）,endoplasmic reticulum（GO:0005783）,extracellular space（GO:0005615）,nuclease activity（GO:0004518）,negative regulation of transcription by RNA polymeraseⅡ（GO:0000122）,and other pathways.KEGG pathways of each group were mainly related to pyrimidine metabolism（ko240）,RNA polymerase（ko3020）,DNA replication（ko3030）,peroxisome（ko4146）,Glycerophospholipid metabolism（ko564）,PPAR signaling pathway（ko3320）,regulation of actin cytoskeleton（ko4810）,nucleotide excision repair（ko3420）,and phagosome pathway（ko4145）.In the RNA polymerase pathway,RPABC4,RPB12,RPABC5,and RPC8 genes were involved.In the pyrimidine metabolism pathway,ndk,NME,ENPP1₃,and CD203 genes were involved.Peroxisome pathway,PECR,PXMP2,and RMP22 genes were involved.In the DNA replication pathway,POLA2,POLE,PRI1,PRI2 and POLE4 genes were involved.2.Hsp70 gene family in Charybdis japonica:genome-wide Screening,molecular characterization,and combined analysis of gene expression and methylation dataIn this study,the Hsp70 gene family was identified from the complete genome sequence of C.japonica.The sequence characteristics,structure and phylogenetic analysis of 22 identified Hsp70 genes were performed.The 22 Hsp70 genes of C.japonica and the direct homologous genes of other species all fall into their own clade in the phylogenetic tree,and were classified into four subfamilies,of which 16 members were in the Hspa9 subfamily（hspa9-hspa9l.15）.There are four members of the Hspa8 subfamily（hspa8l.1-hspa81.4）,which may indicate crustacean-specific gene amplification.In addition,hsph1 belongs to the Hsphl subfamily and hspa5 belongs to the Hspa5 subfamily.The CDS length of Hsp70 gene was 1389-2769 bp,and the length of encoded protein was 462-922 amino acids（aa）.Selection pressure analysis showed that the Ka/Ks values of the five Hsp70 homologous gene pairs were all less than 1.0,indicating that the gene underwent highly purifying selection during evolution.A total of 5 differentially methylated genes were found in different north and south populations:hspa8l.1,hspa8l.2,hspa8l.3,hsphl,and hspa9,and 4 genes were differentially expressed in each population.Functional enrichment analysis mainly enriched 9 GO terms and 4 KEGG pathways.hspa8l.1 and hspa8l.2 were significantly down-regulated in Taizhou and Yantai populations.In Zhoushan and Dalian populations,only hspa8l.1 was significantly down-regulated.No other genes were significantly up-regulated in each population.3.Identification of the Hsp40 gene family of Charybdis japonica and conjoint analysis of gene expression and methylation dataIn this study,47 DNAJ genes were identified from the complete genome sequence of C.japonica,including 9 DNAJA subfamily members,8 DNAJB subfamily members,and 30 DNAJC subfamily members.The length of the coding sequence of DNAJ gene ranged from 357 to 2421 bp,and the length of the coding protein ranged from 118 to 806 aa.Selection pressure analysis showed that the Ka/Ks ratio of the expanded gene pair of DNAJ subfamily in C.japonica was less than 1.0,indicating that it underwent obvious negative selection.This study provides a good preliminary classification of the DNAJ gene family of C.japonica,which is an important step for functional identification and provides new information for the study of regulation mechanism and evolution of the C.japonica DNAJ gene family to environmental stress.DNAJC25,DNAJB11L.L and DNAJA1L were differentially methylated genes in the Hsp40 gene family.Functional enrichment analysis mainly enriched 7 GO terms and KEGG pathways were not enriched.Among the five populations,only three Hsp40 genes,DNAJB11L,DNAJC7L,and DNAJC2,were found to be differentially expressed.DNAJB11L was significantly down-regulated in all populations,DNAJC7L was significantly down-regulated in Taizhou and Zhoushan populations,and DNAJC2 was significantly down-regulated in Zhoushan populations.