Identification Of Salt-tolerant Germplasm And Candidate Genes In Barley

Soil salinization is one of major abiotic stresses that limits global crop production,posing a long-term threat to food safety and restricting the development of agricultural modernization.Recently,President Jinping Xi emphasized that we must update our concept of breeding from the repairment of saline-alkali lands to the adaption of saline-alkali lands with breeding more salt-tolerant crops.Therefore,it is of great significance to clarify the mechanism of crop salt tolerance and develop the crops with higher salt tolerance for the utilization of salty land and food safety.In this study,worldwide barley（Hordeum vulgare L.）mini-core germplasm and a transformation cultivar Golden Promise（GP）were used as plant materials for the determination of seedling-stage physiological traits（fresh weight,dry weight and ion content of shoot and root）in response to salt stress.Genome-wide association analysis（GWAS）and transcriptome analysis were used to identify salt-tolerant candidate genes for ion homeostasis in barley roots.On the other hand,we constructed a double haploid（DH）population by using a salt-tolerant genotype XZ26 from Tibetan wild barley accessions and a cultivar Zhejiang University No.9（ZU9）from Zhejiang and Jiangsu province to investigate their response to salt stress including physiological growth index and ion content.Furthermore,QTL mapping and transcriptome analysis were used to identify genetic loci and candidate genes.The main results are as follows:1.Integration analysis of GWAS and transcriptome for the identification of salt-tolerant genes in barley rootsWe evaluated the difference of salt tolerance among 100 global barley core germplasm at seedling stage,which showed rich genetic variations in salt tolerance.There were three genotypes identified as salt-tolerant lines（GB37,GB19 and GB14）and three were salt sensitive lines（GB44,GB90 and GB39）.After salt treatment,the salt-tolerant lines showed higher whole-plant and root dry matter weight and root Na⁺accumulation,but with significantly lower whole-plant and shoot Na⁺accumulation of salt-sensitive lines than those of salt-tolerant lines.GWAS was performed on root relative dry weight and Na⁺content of 100 barley core germplasm planted in growth chamber and net house,and identified a total of 215 candidate genes.Transcriptome analysis of GP in response to salt stress identified a total of 1,904 up-regulated and 1,313 down-regulated genes,which were mainly enriched in"phenylpropyl biosynthesis","glutathione metabolism","plant hormone signal transduction"and other metabolic pathways.Integration analysis of GWAS and transcriptome identified39 candidate genes,including CML,LRR-KISS and CYPs,which may play important roles in the response and defense against salt stress for barley seedlings.2.Integration analysis of QTL mapping and transcriptome for the identification of salt-tolerant genes in barleyWe compared the salt tolerance between XZ26 and ZU9,which was used as parent materials for DH population.The results showed that XZ26 had stronger salt tolerance than ZU9,with significantly higher shoot dry weight and lower Na⁺concentration than that of ZU9.A total of 94 DH lines were randomly selected and used for salt treatment.QTL mapping was performed using Map QTL software on the relative dry weight and ion content of shoot after salt treatment.We identified an interval 278.7 Mb-299.7 Mb located on 6H chromosome associated with relative dry weight explaining 23.5%total variation with 67 candidate genes,and an interval 8.1Mb-9.2 Mb located on 1H chromosome associated with Na⁺,Fe²⁺and Ca²⁺content covering 20.5%total variation with 37 candidate genes,respectively.Transcriptome analysis of XZ26 in response to salt stress identified a total of 1,669 up-regulated genes and 2,086 down-regulated genes,which were mainly enriched in metabolic pathways such as"plant hormone signal transduction"and"phenylpropanoid biosynthesis".At last,integration analysis of QTL mapping and transcriptome identified 20 candidate genes,including PPR and ZF,which might be involved in the response and defense against salt stress in barley.In conclusion,this study investigated the genetic diversity of salt tolerance among barley core germplasm and finally identified 3 distinct salt-tolerant and sensitive germplasms,as well as 39 genes associated with ion balance by GWAS and transcriptome analysis.Meanwhile,we also analyzed the physiological and element content differences of DH population under salt stress,and identified 20 salt-tolerant candidate genes by QTL mapping and transcriptome analysis.This research further enriched the theory of salt tolerance in barley,and provided elite germplasm and gene resources for crop genetic improvement of salt tolerance.