Mechanism Of GmDME-Mediated Regulation Of Soybean Seed Size

Soybean is an important oil crop and high-protein feed crop with strategic significance for China’s food security and economic growth.In recent years,with the increasing demand for soybeans and the severe dependence on imports,it is urgent to increase soybean yield and improve China’s self-sufficiency in soybean production.Yield is a quantitative trait regulated by multiple genes and affected by environmental factors.Over the years,breeders have made breakthroughs in deciphering the molecular mechanisms of yield-related genes in the three major crops through means such as the "green revolution" and heterosis utilization,using techniques such as QTL analysis,whole-genome association analysis,and positional cloning.However,the corresponding yield-increasing strategies are not applicable to molecular breeding of soybeans due to their different biological characteristics from the three major crops.The slow progress in the study of molecular mechanisms related to soybean yield has greatly constrained related breeding work,leading to a serious bottleneck in increasing soybean yield.Therefore,deciphering the molecular mechanism of soybean yield regulation,innovating yield breeding methods,and obtaining high-yielding soybean varieties are urgently needed to fundamentally solve the problem of soybean yield.Research has found that epigenetic modifications play an important role in regulating plant growth,development,and response to environmental stress.DNA methyltransferases and DNA demethylases play important regulatory roles in the seed development process of rice,Arabidopsis,and maize,and important genes involved in soybean seed development are located in regions of low methylation.In previous studies,the project team used CRISPR/Cas9 technology and the small,densely-packed variety "Dongnong 50" as the recipient material to obtain gmdme mutants.These mutants exhibited larger seeds,increased hundred-grain weight,and increased yield.To further explore the important role of DNA demethylase Gm DME in active demethylation during soybean seed development,this study used high-throughput sequencing,microscopic analysis,molecular biology,and epigenetics research techniques to analyze the regulatory mechanism of DNA demethylation on soybean seed size.The study provides a theoretical basis for elucidating the epigenetic regulatory mechanism of important agronomic traits genes,and has important significance for innovative epigenetic breeding methods for precise improvement of soybean yield and the creation of breakthrough soybean germplasm resources.The main results are as follows:(1)Transcriptome analysis of two groups of soybean varieties with different seed sizes revealed that Gm DME was significantly less expressed in the large-seed variety than in the small-seed variety.Transcription factors and embryo development-related genes were also enriched.Whole-genome methylation sequencing analysis showed that CHH type DNA methylation was significantly higher in the whole-genome transposon region(TE)than in the small-seed group in the large-seed population;the number of differentially methylated regions(CHH-DMR)of CHH type was relatively high.(2)The gmdme mutant seed cells were significantly larger,resulting in larger seeds,but the number of seed cells did not change significantly.gmdme mutant single plant yield increased significantly,but the strain type,oil content and protein content did not change significantly.(3)In the mutant gmdme-3,the CHH type had significantly higher DNA methylation levels in the transposon region.Analysis of differentially methylated regions(DMRs)revealed a significantly higher number of CHH-DMRs than CG-and CHG-DMRs.combined with transcriptome data,elevated CHH methylation in the promoter regions and decreased gene expression levels were found for some abscisic acid(ABA)pathway genes,transcription factors and ubiquitination-related genes.Sequence analysis of the promoter regions of these genes showed that all contained transposons enriched in AT sequences.The results suggest that the gmdme-3 mutant and regulates the expression of seed size-related genes by enhancing the CHH methylation level of transposons,which in turn affects seed size.(4)The heritability of Gm DME-mediated seed size variation was investigated by seed size,methylation level and gene expression level using the progeny of chromosome fragment substitution lines(CSSL)with Suinong 14 and ZYD00006 as parents.The results showed that the expression level of Gm DME was significantly lower in Suinong 14(large seed)than in ZYD00006(small seed).In the CSSL progeny population,the expression level of Gm DME was also significantly lower in the large-seed progeny than in the small-seed progeny.the expression level of Gm DME was significantly negatively correlated with seed size.In parental Suinong 14,the promoter regions of SAMBA and Go LS1 had higher methylation levels,and the expression levels of both genes were also lower than in parental ZYD00006.The effect of Gm DME on soybean seed size was stably inherited.The above findings reveal that the DNA demethylation enzyme Gm DME in soybean negatively regulates soybean seed size by targeting AT-rich transposons and affecting the level of CHH-type DNA methylation in the transposon region,which in turn regulates the expression of ABA-responsive genes,transcription factors and ubiquitination pathway genes.