Drought Tolerance Evaluation Of Tartary Buckwheat Germplasm Resources And Discovery And Functional Analysis Of Associated Genes

In rencent years,with the improvement of people’s living strandard,people pay more and more attention to the health problem.As a small grain crop in China,Tartary buckwheat has been paid more attention because of its high nutrition value and medicinal value.Tartary buckwheat is mainly distributed in arid and semi-arid highland mountainous area in China,which is vulnerable to drought stress.So,it is significant for cultivation of high drought resistance tartary buchwheat to scree elite tartary buckwheat germplasms with drought tolerance and excavate drought related genes.In this study,20% polyethylene glycol(PEG6000)was used to simulate drought stress,the germination rate,germination energy,germination index and germination length of290 tartary buckwheat germplasm were measured.The relative value of each index was taken as evaluation index of drought resistance.The drought resistance of tartary buckwheat were evaluated with subordinate function method and classified by classifications groups step by step.The SNPs and candidate genes related to drought resistance were detected by genome-wide association analysis.The function of selected candidate genes were validated.The results are as follows:1.The germination rate,germination energy,germination index and germination length of tartary buckwheat germplasms were inhibited under 20% PEG stress;the 290 tartary buckwheat germplasms were divided into 5 groups using classifications groups step by step: Hightly resistant(HR)41,Resistant(R)37,Moderately resistant(M)125,Susceptible(S)38,and Hightly susceptible(HS)49.2.15 SNP associated with drought resistance at tartary buckwheat germination stage and 295 genes with functional annotation were screened through GWAS.According to gene expression and gene function annotation,four genes Ft2OGDD1,Ft AAD1,Ft ERF68 and Ft ERF99 were selected for further study.3.The results of haplotype analysis showed that there were 2、1、2 SNP on the promoter of Ft ERF68、Ft2OGDD1 and Ft AAD1 were significantly correlated with drought resistance of tartary buckwheat at germination stage.The expression level analysis of candidate genes in different materials showed that the expression level of Ft ERF68 was positively correlated with drought resistance of tartary buckwheat at germination stage and Ft2OGDD1 and Ft AAD1 were on the contrary.4.Sequence analysis showed that the full-length coding sequence of Ft ERF68 was447 bp encoding for 148 amino acids residues and Ft ERF99 was 807 bp encoding for268 amino acids residues.The expression of Ft ERF68 and Ft ERF99 in tartary buckwheat were induced by PEG and Na Cl treatment and overexpression of Ft ERF68 or Ft ERF99 in Arabidopsis significantly enhance drought and salt tolerance of transgenic Arabidopsis plants.5.Sequence analysis showed that the full-length coding sequence of Ft2OGDD1 was 285 bp encoding for 94 amino acids residues and Ft AAD1 was 1194 bp encoding for 397 amino acids residues.The expression of Ft2OGDD1 and Ft AAD1 in tartary buckwheat was reduced by PEG and Na Cl treatment and overexpression of Ft2OGDD1 or Ft AAD1 in Arabidopsis significantly reduce drought and salt tolerance of transgenic Arabidopsis plants.In this study,41 hightly resistant drought and 37 resistant drought materials were selected by evaluating the drought resistance of 290 tartary buckwheat germplasm resources;15 SNPs corresponding to drought tolerance were detected through GWAS;functional verification of transgenic Arabidopsis showed that four genes Ft ERF68、Ft ERF99、Ft2OGDD1 and Ft AAD1 played a role in drought and salt stress adaptation of tartary buckwheat.These results provide some reference for tartary buckwheat breeding in the future.