A Single Nucleotide Polymorphism In An R2R3 MYB Transcription Factor Gene Triggers Male Sterility In Soybean Ms6 (Ames1)

The exploitation of heterosis is one of the effective methods to boost crop yield,and the male sterile line can omit tedious artificial emasculation steps,which offers an important way to heterosis in soybean.In recent years,the Seed Production Technology（SPT）based on genic male sterility gene has developed rapidly in maize and rice,providing a new direction for application of genic male sterility in crop heterosis.The cloning of genic male sterility gene can provide theoretical support for the establishment of SPT,and it also helps us analyze the molecular mechanism of plant anther development.In this study,ms6 is a genic male sterility mutant,the previous studies have located ms6in the 3.7Mb interval between Satt030 and Satt149 on chromosome 13.Therefore,based on the previous research,this study shows that Glyma.13g066600 is a candidate gene of ms6through high-throughput sequencing combined with bioinformatics analysis.Glyma.13g066600 encodes a TDF1（Tapetal Development and Function 1）protein,and it has a homologous protein with 95%similarity,encoded by Glyma.19g017900 in soybean.We named Glyma.13g066600 as GmTDF1a,and named Glyma.19g017900 as GmTDF1b.Then we analyzed the function of GmTDF1a through genetic transformation of Arabidopsis thaliana experiments,real-time quantitative PCR,and subcellular localization.The results are as follows:（1）The phenotype and cytology observation of the mutant.Compared with wild type（WT）,ms6 developed normally during the vegetative growth stage,and the anthers shrink during the reproductive growth stage.Through cytological observation,we found that in the tetrad stage,both the anthers of WT and ms6 had 5 layers anther wall,but the parietal layer and tapetum of ms6 were abnormally enlarged and vacuolated.In the late stage of microspore development,the anther wall of WT and ms6 had four layers,but the epidermis was smaller and the parietal layer was abnormally vacuolated and swollen in ms6.（2）Screening candidate genes of ms6.Through high-throughput sequencing combined with bioinformatics analysis,we found the candidate gene Glyma.13g066600,which encodes an R2R3 MYB transcription factor.There is an SNP in the Glyma.13g066600 gene in ms6,leading to the 76^the Leucine to Histidine.The leucine at this position is a highly conserved amino acid residue.The protein,encoded by Glyma.13g066600,had 95%similarity with the other protein encoded by Glyma.19g017900 in soybean.Phylogenetic analysis showed that proteins encoded by Glyma.13g066600 and Glyma.19g017900 were TDF1 homologous proteins,therefore,we named Glyma.13g066600 gene as GmTDF1a,and named its homologous gene Glyma.19g017900 as GmTDF1b.（3）Genetic transformation of Arabidopsis thaliana experiments.Verifying the function of GmTDF1a and GmTDF1b in attdf1 heterozygote（+/tdf1）.The results showed that GmTDF1a and GmTDF1b driven by native promoter At TDF1p could both rescue the sterile phenotype of attdf1（tdf1/tdf1）,indicating that both GmTDF1a and GmTDF1b had the function of TDF1.In ms6,the 76th leucine of GmTDF1a was replaced by histidine(GmTDF1a^L76H),while GmTDF1a^L76H could not rescue the sterile phenotype of attdf1（tdf1/tdf1）,indicating that GmTDF1a^L76H was a functional loss protein.These results showed that the function of TDF1 protein was conserved,and that male sterility of ms6 was caused by the substitution of leucine 76th of GmTDF1a into histidine.（4）Quantitative Real-Time PCR（qRT-PCR）.The expression levels of GmTDF1a and GmTDF1b were detected in roots,stems,leaves,flowers,calyx,petals,pistils,and seeds.The results showed that GmTDF1a has high expression in flowers,but the expression levels were low in calyx,petals,pistils.Indicating that GmTDF1a was an anther-specific gene.However,the expression level of GmTDF1b was very low in all tissues,which further indicated that GmTDF1a was the major functional TDF1 in soybean.（5）Subcellular localization experiment.The GmTDF1a-GFP、GmTDF1a^L76H-GFP and GmTDF1b-GFP fusion proteins were expressed by 35S promoter in tobacco.We found that GmTDF1a、GmTDF1b and GmTDF1a^L76H were all localized in the nucleus,indicating that the 76th leucine was replaced by histidine of GmTDF1a not affect its subcellular location.