Epigenetic Analysis Of Non-coding RNA In Crassostrea Gigas With High Glycogen Content

Glycogen content is a quantitative trait.There are differences between different individuals in oysters due to environmental and genetic factors,including food,season,temperature,and salinity.Pacific oysters(Crassostrea gigas)are among the most vital aquaculture species worldwide.We used the gonad tissue of C.gigas as the research object,and designed two comparison groups,which are two siblings with high glycogen(GH)and low glycogen(GL)content in the same area of the Yellow Sea,Rizhao,China(RZ).And the same high-glycogen content sibling family produced two different trait oysters with high and low glycogen content in Rizhao and Kongtong Dao area(KTD),Yantai,respectively.In the study,contents of glycogen,amino acid,and fatty acid were compared and analyzed;RNA-seq and small RNA-seq technologies were used for transcriptome sequencing and functional enrichment analysis of differentially expressed RNA were used by GO and KEGG pathway.The main findings are as follows:1)Analysis of glycogen content in RZGH vs RZGL and RZGH vs KTDGL found that the glycogen content in the RZGH group was significantly higher than that in the RZGL and KTDGL groups(p < 0.05).In the RZGH group,the content of 10 glucogenic amino acids was significantly higher than that in the RZGL group(p < 0.05);in the KTDGL group,the content of 11 glucogenic amino acids was significantly higher than in the RZGH group(p < 0.05).In the RZGH vs KTDGL group,there were significant differences in the content of 13 fatty acid components(p < 0.05).2)A total of about 886M(million)high-quality double-end original reads with a read length of 150 nt were obtained in RNA-seq,of which about 76.66% could be compared to the C.gigas reference genome.A total of 2463 differential mRNAs,547 differential lncRNAs,and 84 differential circ RNAs were identified in the RZGH vs RZGL group;a total of 2084 differential mRNAs,1080 differential lncRNAs,and 34 differential circ RNAs were identified in the RZGH vs KTDGL group.Further analysis of lncRNAtranscripts showed that intergenic lncRNA(linc RNA)accounted for 29.0%,intronic lncRNA accounted for 62.1%,and anti-sense lncRNA accounted for 8.9%.Compared with mRNA,lncRNA has the characteristics of less exon number,shorter transcript length,and lower expression level.The transcription length of circ RNA in the gonad of C.gigas was identified to have a wide range of transcription lengths,the shortest being153 nt and the longest being 60025 nt,most of which are derived from large exons.3)In the RZGH vs RZGL group,functional enrichment analysis of differentially expressed mRNAs and lncRNA target genes were mainly involved in carbohydrate metabolism and glucogenic amino acids related pathways,and 20 related mRNAs were found,such as glucose-6-phosphatase 2(G6PC2),UTP-glucose-1-phosphate uridylyltransferase(UGP2),pyruvate kinase M1/M2(PKM),pyruvate dehydrogenase E1 component subunit alpha(PDHA),isocitrate dehydrogenase [NADP]cytoplasmic(IDH1),etc;A total of 395 lncRNA-mRNA interactions were found in cis role and 2020 lncRNA-mRNA interactions were found in trans role and 6 differential lncRNAs found to target glucose metabolism-related genes;Functional enrichment of circ RNA host genes was discovered,f differentially expressed novel-circ-0001838 was transcribed from inositol hexakisphosphate kinase 1(IHPK1).In the RZGH vs KTDGL group,differential mRNA analysis revealed that 29 genes were annotated to the calcium signaling pathway and 17 genes were annotated to the insulin signaling pathway,including calmodulin(CAM),tyrosine protein phosphatase(PTP),and insulin-like peptides receptor(INSR),and other genes;A total of 471 lncRNA-mRNA interactions were found in cis role and 1327 lncRNA-mRNA interactions were found in trans role,and functional enrichment analysis found that they were also related to insulin-like synthesis.4)About 79% of small RNA-seq were compared to the Danio rerio reference genome.A total of 39 mature mi RNAs and 75 pre-mi RNAs were identified and expressed in the C.gigas.In addition,101 new mi RNA matures and 103 pre-mi RNAs were identified.The results of the first base preference analysis showed that the first base was mostly U.6 and 7 differentially expressed mi RNAs were screened in the RZGH vs RZGL and RZGH vs KTDGL groups,respectively.The target genes were predicted using mi Randa software.After the combined analysis of the predicted target genes and differentiallyexpressed mRNAs,it was found that there were 1815 and 1077 target genes in the RZGH vs RZGL and RZGH vs KTDGL groups,respectively.At the same time,it was found that most mi RNAs are predicted to regulate hundreds or even thousands of target genes at the same time,and target genes may also be regulated by multiple mi RNAs simultaneously.5)Construction of differentially expressed lncRNA/circ RNA-mi RNA-mRNA regulatory networks.Functional enrichment analysis of differentially expressed mRNAs involved in it.In the RZGH vs RZGL group,novel-circ-0002559,novel-circ-0003404,novel-circ-0000158,and up-regulated novel-mi R-154 and down-regulated IDH1 genes were found to form a regulatory network.Novel-mi R-225,novel-mi R-154 and novel-mi R-91 targeted the regulation of β-glucosidase genes and formed a regulatory network with four down-regulated circ RNA genes novel-circ-0000166 and novel-circ-0002559;In the RZGH vs KTDGL group,282 up-regulated lncRNAs and one up-novel-circ-0000916 and novel-mi R-78 were found to target the INSR gene,and172 down-regulated lncRNAs and novel-mi R-154,novel-mi R-225 and PTP genes could form regulatory networks.This study speculates that non-coding RNA of C.gigas plays a role in regulating glycogen content.Analysis of RZGH vs RZGL groups in different families in the same sea area found that non-coding RNA can regulate the expression of genes directly or indirectly related to glycogen content,and it also can indirectly regulate the glycogen content of oysters by affecting the metabolism of amino acids and fatty acids.There are different breeding environments such as water temperature,salinity,and bait abundance in different seas.Oysters of the RZGH vs KTDGL experimental group are two phenotypes of high and low glycogen content produced by the same family in different seas.Studies have speculated that environmental factors may affect the regulation of genes by non-coding RNA in oysters,thereby further affecting glycogen content,and the regulatory mechanism remains to be further studied.