Comparative Analysis Of DNA Methylation Between Cytoplasmic-nuclear Male Sterile Line NJCMS1A And Its Maintainer NJCMS1B,and Cloning Of Candidate Genes In Soybeans

The utilization of heterosis in crops is an effective way to improve yield.The study of cytoplasmic-nuclear male sterility has significant value for taking advantage of heterosis.Although many studies on cytoplasmic-nuclear male sterility of soybean have been conducted by a large number of scholars,the molecular mechanism of cytoplasmic-nuclear male sterility remains unknown.In addition,numerous studies on DNA methylation and male sterility in rice have been reported,but there have been no reports regarding soybeans.This study intends to perform a comparative analysis for the DNA methylation between cytoplasmic-nuclear male sterile line NJCMS1A and its maintainer NJCMS1B in soybeans,as well as the functional analysis of candidate genes.The main results are as follows:1.The whole-genome DNA methylation for cytoplasmic-nuclear male sterile line NJCMS1A and its maintainer NJCMS1B were detected using methylated DNA immunoprecipitation combined with high-throughput sequencing(MeDIP-seq)technology.The MeDIP-seq showed that the distribution of the clean reads was homogeneous,covering the majority of CpGs of the soybean genomes,and the clean reads were mainly located in the transposon elements(TEs),promoter,intron,etc.In addition,the analysis of the DNA methylation profiles revealed that the DNA methylation levels in and around the transcription start sites(TSS)and transcription termination sites(TTS)were the lowest,while the methylation levels dramatically increased when departing the TSS and TTS,and the methylation level in the gene body were generally higher than those in the 2 kb upstream of TSS and 2 kb downstream of TTS.The distribution of methylated peaks in the genomic components showed that a major portion of peaks were located in TEs,while the numbers of peaks in the 5'UTR,3'UTR and coding sequence(CDS)was relatively smaller.Furthermore,the long terminal repeat(LTR)and terminal inverted repeat(TIR)took the lead in terms of methylated TEs.A total of 178 differential methylated genes(DMGs)were identified,with 156 down-methylated and 22 up-methylated genes in NJCMS1A relative to NJCMS1B.The Gene Ontology(GO)analysis showed that 114 DMGs participated in one or more GO categorizations,in which four GO terms were significantly enriched,and the KEGG pathway analysis showed that 18 DMGs participated and were enriched in homologous recombination,purine metabolism,proteasome,non-homologous end-joining,pyrimidine metabolism,etc.Finally,eight differential methylated regions(DMRs)were selected to validate the reliability of the MeDIP-seq by means of bisulfite sequencing,and the DNA methylation pattern of six DMRs was in accordance with MeDIP-seq,which indicated that the results of MeDIP-seq were reliable.2.The two candidate genes GmGST 12 and GmIscU1-like were selected to perform gene cloning and expression analysis by processing the data of MeDIP-seq and previous RNA-seq data.The GmGST 12 and GmIscU1-like genes were cloned from the flower buds of NJCMS1A and NICMS1B,of which the open reading frame(ORF)reached up to 708 bp and 516 bp in length,respectively.The bioinformatic analysis indicated that GmGST 12 and GmIscU1-like coded a Glutathione S-transferases(GSTs)and an iron-sulfur cluster assemble protein,respectively,which contained two conserved domains of GST_N and SCOP,and a conserved domain of IscU like.The phylogenetic tree analysis showed that the respective homologies of GmGST 12 and GmIscU1-like of soybean with CcGST 12 of Capsicum chinense and MtISU1 of Medicago truncatula were the highest,and the identities of the amino acid sequences were 53%and 83.72%.The tissue expression analysis indicated that the expression level of GmGST 12 in the flower buds was significantly higher in NJCMS1A than NJCMS1B,and there was no difference in the roots,steams or leaves.In addition,the expression levels of GmIscUl-like in the roots,steams and flower buds were significantly higher in NJCMS1A than NJCMS1B,but significantly lower in the leaves of NJCMS1A.The results of the sub-cellular localization indicated that the GmGST 12 was located in the whole-cell.The plant over-expression vectors pC AMBI A3 3 01-GFP-GmGST 12 and pCAMBIA3301-GFP-GmIscU1-like were constructed,and the vectors were successfully transferred to the agrobacterium tumefaciens,thus laying the foundation for functional verification of transgenic research.