Mining And Identification Of Salt Tolerance Candidate Genes In Rice Seedling Stage By Genome-wide Association Analysis

Rice(Oryza sativa L)is a salt sensitive plant,especially in the early stage of vegetative growth,when the soluble salt concentration in the soil reaches 0.3%,it shows symptoms of damage.Therefore,it is of great significance to excavate and utilize excellent salt tolerance genes to breed salt tolerance rice varieties.In this study,based on 150 core germplasms constructed,a high-density SNP library was obtained by SLAF-seq sequencing,genome-wide association analysis was performed on 8 seedling stages and salt tolerance-related traits,and QTLs related to salt tolerance were located and screened,and identification of salt tolerance candidate genes at seedling stage,providing an excellent gene source for rice salt tolerance research and molecular breeding.The construction of salt tolerance candidate gene knockout mutants provides basic materials for revealing salt tolerance candidate genes and regulating the molecular mechanism of salt tolerance.The results of the study are as follows:1.A total of 831 significant SNPs were detected by genome-wide association analysis(P≤0.0001),including 42 lead SNPs,which were unequally distributed on 12 chromosomes.According to the LD decay distance,65 QTLs were identified,and compared with the previously reported salt tolerance QTLs,17 QTLs were found to overlap or be similar to the reported QTLs(≤500kbp).Compared with the QTLs or SNPs loci of rice cold tolerance and aluminum tolerance located in this group in the previous stage,it was found that 10 QTLs overlapped or were close to the QTLs and SNPs loci of cold tolerance or aluminum tolerance(≤500kb).23 known salt tolerance genes were detected around 65 QTLs(≤500 kbp).In addition to this,48 new salt tolerance QTLs were identified,which are all newly discovered and await further study.2.According to the positions of 65 QTLs,13 QTLs overlapped,thus 6 co-localized QTLs were identified.The genes in the range of SNPs ± 250 kb with the smallest P value in each co-localized QTL were retrieved,and multiple candidate genes for salt tolerance were screened through gene annotation information.Combined with the correlation analysis between haplotypes and salt tolerance phenotypic traits,11 were initially screened.Salt tolerance candidate genes.3.Gene expression analysis of the screened 11 salt tolerance candidate genes in two extreme salt tolerance materials(salt tolerance S125 and salt sensitive S87)showed that the expression levels of 11 salt tolerance candidate genes were significantly different in salt resistance and salt tolerance materials.4.The sequences of the above candidate genes in the two extreme materials were further compared with the Nipponbare sequences,and the results showed that 9 candidate genes of salt tolerance had site mutations in salt tolerance and salt sensitive materials.Expression profiling analysis also showed that these candidate genes were all related to abiotic stress,and it was finally determined that these 9 candidate genes may be involved in the regulation of salt tolerance in rice seedling stage.5.Using CRISPR-Cas9 gene editing technology and Agrobacterium-mediated rice callus,knockout mutants of 5 genes were constructed,which laid the foundation for further analysis of the molecular mechanism of these salt tolerance candidate genes regulating rice salt tolerance.