| Salt stress has serious impact on plant growth and crop production.Soybean is a moderately salt-sensitive crop,its growth,development,nodulation andnitrogen fixation are also seriously affected by salt stress resulting in great yield reduction.However,the mechanism of how soybean plant responds to salt stress remains largely unknown.To address these questions,this study intended to identify the key genes that are responsible for salt tolerance of soybean using forward and reverse genetics approaches.On the one hand,bioinfomatic and functional analyses were performed to identify the soybean homolog gene of Salt Overly Sensitive 2(AtSOS2),a key component of SOS signal pathway in Arabidopsis thaliana.As a protein kinase,SOS2 not only participates in salt response,but also mediates the crosstalk between salt tolerance signaling pathway and the other signaling pathways.On the other hand,a genome wide association analysis(GWAS) was conducted to mine the salt tolerant genes in soybean germplasms.Firstly,two SOS2 homologous genes(Glyma.17G113700 and Glyma.13G166100)were identified in soybean using AtSOS2 as a reference gene.The protein similarity and the phylogenetic analyses results showed that Glyma.17G113700 is the closest homologous to AtSOS2 and was named GmSOS2 a,while Glyma.13G166100 was named GmSOS2 b.Quantitative RT-PCR showed that GmSOS2 a and GmSOS2 b were mainly expressed in leaves and nodules,respectively.Both GmSOS2 a and GmSOS2b were induced by Na Cl and rhizobia,suggesting that they may be functionally diversed.Furthermore,knocking out GmSOS2b using a Cas9/CRISPR editing technology in hair root resulted in significantly reduced tolerance of the mutated roots compared with the empty vector control,but plant growth and nodule development were not apparently affected.Further analysis of the gmsos2b mutant plants showed that the mutant was more sensitive to salt stress than wild type with shorter and smaller root system and retarded development of root nodules.These results indicate that GmSOS2b might specifically mediate root and nodule plastic development in soybean under salt stress.Secondly,648 soybean varieties collected from all over the world were phenotypically analyzed with traits including plant height,root length,number of nodules,proportions of plant height,root length reduction,nodule number and salt tolerance.The genome-wide association study(GWAS) for the soybean salt tolerance was perfomed.Some significant SNP loci associated with plant salt tolerance were obtained on chromosomes 3,9 and 19.The significant SNP locus associated with plant height under salt stress were found on chromosomes 12,13,18 and 19.Then the candidate genes were determined using 100 kb as the LD attenuation distance.Within these loci,the candidate genes that possibly regulate plant height under salt stress and whole plant salt tolerance were obtained.In addition,we proved that the phenotype of the first pair of true leaves of young seedlings can be used as crucial and reliable criteria for evalulating the salt tolerance of plants.Unfortunately,the genetic loci associated with number of nodules with or without salt stress were not identified,suggesting that nodule number control is a more complex process and is not suitable for screening for key regulatory genes by genome-wide association study at the lab conditions.Taken together,this study has functionally characterized GmSOS2b as a gene that regulates the salt tolerance in soybean,and identified the candidate genes associated with salt toleranceof of soybean.These findings have laid a solid foundation for further understanding of the regulatory mechanisms underly salt response and tolerance in soybean. |
