Comparative Analysis Of Whole-genome DNA Methylation Between Cytoplasmic-nuclear Male Sterile Line And Its Maintainer And Functional Study Of Differentially Methylated Genes In Soybean(Glycine Max(L.)Merr.)

Cytoplasmic-nuclear male sterility(CMS)is known to be a common phenomenon in higher plant,which plays an important role in crop heterosis utilization.Although extensive researches at home and abroad have been conducted on soybean CMS included genetics,cytology,transcriptomics and proteomics etc.,the epigenetic regulation of soybean CMS remains poorly understood.DNA methylation,as the main epigenetic modification means,has attracted more and more attention on the regulation mechanism of gene expression,and became an important method to study the mechanism of male sterility in plants.Therefore,in this study,the genome-wide DNA methylation profiles of soybean cytoplasmic male sterile line and its maintainer line were compared and analyzed,and some key differentially methylated genes were put forward for further functional study.The main results were as follows:1.Soybean CMS lines and their maintainer lines were selected as experimental materials in this study.The DNA methylation level of the flower bud at different developmental stages was analyzed using a methylation sensitive polymorphism method.The results indicated that,from the pollen mother cell stage to the binuclear pollen stage,the DNA methylation level in the maintainer lines had gradually increased.However,the DNA methylation level of the CMS lines had increased firstly and then decreased,reaching a peak value during the period of the uninucleate microspore to early binuclear pollen stage.The result of the cytological observation in our laboratory indicated that the microspore of the CMS lines also began to abort at the uninucleate microspore stage.Therefore,it was speculated that the change of the DNA methylation level might be related to the cytoplasmic male sterility of the soybean.2.The genome-wide DNA methylation profiles of the soybean CMS line NJCMS5A and its maintainer line NJCMS5B were compared and analyzed by whole-genome bisulfite sequencing(WGBS).In total,3,527 differentially methylated regions(DMRs),and 485 differentially methylated genes(DMGs),were identified.These included 353 highly credible methylated genes,56 genes encoding unknown proteins,and 76 new genes without functional annotation.In comparison with the maintainer line NJCMS5B,there were 281 hypo-methylated genes,88 hyper-methylated genes,and 116 unstable-DMGs identified in NJCMS5A.Finally,11 key DMGs involved in pollen and flower development were identified as being possibly related to the soybean CMS by referring to GO annotation and KEGG metabolic pathway analysis,as well as relevant literature report.In addition,25 DMRs were performed by bisulfite treatment,and the result showed that the methylation pattern of 21 DMRs were consistent with the result of WGBS,indicating that the sequencing result was good.qRT-RCR result of 9 DMGs showed that 8 of 9 DMGs had a negative correlation between gene expression and their DNA methylation level.3.In order to further reveal the relationship between DNA methylation and soybean CMS,three key DMGs were selected for cloning and functional studies in this study.These DMGs were cloned from NJCMS5A and named as GmGAUT1,GmFBH4 and GmACS2,respectively.The result of qRT-PCR showed that they were up-regulated in the flower bud of NJCMS5A,which had a negatively correlation with their hypo methylation level.The result of subcellular localization showed that GmGAUT1 might be located in the Golgi body and its membrane structure,GmFBH4 was located in the nucleolus,and GmACS2 was located in the cytoplasm.Functional studies were conducted through genetic transformation of Arabidopsis.As results,early flowering,decreased plant height,increased branch number,shorted silique length,decreased seed number,and pollen semi-sterility etc.appeared in GmGAUT1 overexpressed Arabidopsis.Overexpression of GmFBH4 showed delayed flowering,decreased silique number and seed number etc.in Arabidopsis.Overexpression of GmACS2 showed delayed flowering,decrescent flower,decreased silique number,early silique maturity,increased ethylene content etc.in Arabidopsis.Based on the above results,it was speculated that GmGAUT1,GmFBH4 and GmACS2 might parcipate in fertility regulation of soybean CMS.