| Soil salinity/alkalinity is one of the most serious stresses threatening wheat productivity,especially,with rapid urbanization on previously prime agricultural land.Many studies attempted to address the food security issues by increasing the yield in high nutrition and optimal field.However,few studies provided methods for breeding salt-tolerant wheat varieties.Therefor,a collection of 307 representives from over 5,000wheat accessions,was uesd for salt tolerance screening experiments,and we identified a number of candidate genes associated with salt tolerance using GWAS and RNA-seq based on the lastest common wheat genome.The results are as followed:?1?We proposed a new method for accurate evaluation of salt tolerance by calculating the salt tolerance index??.Suppose,the environmental salinity is in the range of 0 to 1standard salinity,with other environmental conditions unchanged,and the variety's germination rate,survival rate or reproduction rate change curve just be determined by the cultivar's salt tolerance ability.And integral method was used to calculate the constant proportional by eliminating the influence of the salinity factor.?2?The of 307 wheat cultivars was evaluated,and the salt tolerance of 307 wheat cultivars was identified by using method established.The results show:the of 307accessions ranged from 0.1257 to 0.9522.And the frequency distribution is normal,which is consistent with the model assumptions of GWAS.At the same time,the results confirmed that there is a defect in selecting a single salt concentration for salt tolerance identification.?3?The genotype data of 307 wheat cultivars was analyzed for population structure,linkage disequilibrium?LD?analysis,and GWAS analysis.The results showed that 307materials can be divided into 7 subgroups.The LD attenuation distance of the whole wheat genome is 3.2 Mb,and the LD attenuation distances of subgenomes A,B and D are 2.7 Mb,4.8 Mb and 2.0 Mb,respectively.The 117 significant loci(P<1×10-5)related to salt tolerance of wheat at bud stage are mainly clustered?on chromosomes 1A,3B and 6B,and the number of candidate sites on the three chromosomes is 73 and 15 and 20,explaining the phenotypic variation of 8.92%,5.23%and 1.98%,respectively.The three chromosome positioning intervals contain 53 genes,including transcription factors,calmodulin,antioxidant enzymes,and osmotic regulating substance synthase.Among them,35 genes are up-or down-regulated under salt stress.?4?Wheat cultivars Xiaoyan 22?salt tolerance?and Bima 4?salt sensitive?were used for RNA-seq analysis in this study.The seedlings treated with water?control?and artificial seawater solution?salt treatment?were harvested at 0,1,3,6,12 and 24 hours and were extracted for RNA-seq.After analysis of the RNA-seq data,617 differentially expressed genes?DEGs?were identified.Gene Ontology?Go?and Kyoto Encyclopedia of Genes and Genomes?KEGG?analyses revealed that the differential genes were enriched in ABA signaling pathway and hormone signaling pathway.?5?In a search for the genes'names associated with salt tolerance and their frequencies in the literatures in recent ten years,56 gene with the most frequencies were listed.Then,the protein sequences of these 56 genes from NCBI database,and the 501 protein sequences from Swiss-prot database were downloaded.Homologous comparison of these sequences based on wheat reference genome IWGSC Ref Seqv1.0 protein sequences,4,620 wheat candidate genes associated with salt tolerance were identified.Then 35 members of Ta Sn RK2 gene family were recognized,and the Ta Sn RK2.29 gene was selected as an important candidate gene for further molecular function experiments.This study provided a method for accurate evaluation of salt tolerance and identification of the corresponding genes by GWAS and RNA-seq.All of above results provided insights into understanding the molecular mechanism of salt tolerance and novel QTL/genes resources for wheat improvement of salt tolerance. |
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