Genome-wide Association Analysis Of Cotton Resistance To Verticillium Wilt And Functional Study Of TIR-NBS-LRR Resistance Gene GhTNL1

The main pathogen of cotton Verticillium wilt in China is Verticillium dahliae.The occurrence of Verticillium wilt seriously affects the yield and quality of cotton.At present,disease-resistant varieties with excellent breeding are the most effective measures for controlling Verticillium wilt.Genome-wide association analysis(GWAS)research strategy is conducive to the full use of historical reorganization events in natural populations.Population genetic variation is abundant,and there is a higher resolution for the location of resistance loci.The completion of the genome sequencing of Gossypium hirsutum TM-1 provided strong support for GWAS analysis and gene cloning.In this study,299 elite cotton germplasm resources were used to construct G.hirsutum association populations resistance to Verticillium wilt.SLAF sequencing yielded 85,630 high-quality SNPs covering 26 chromosomes.The evaluation of kinship among individuals in the population revealed that the population was rich in genetic variation,and the individuals had relatively distant genetic relationships,with less interference to the GWAS.Principal component analysis and phylogenetic analysis showed that the population can be mainly divided into two groups.Group I mainly come from the Yangtze river region,and Group II mainly comes from the Yellow river region and the Northwest region.Although there are also different degrees of individual infiltration in these two groups,there was a certain correlation between the grouping and geographical origin of individuals.The phenotypic data in different environments were association analysis with genotype data and a total of 17 SNPs that were significantly associated with V.dahliae responses were identified.The peak SNPs on the chromosomes A04,A02 and D05 overlap with the reported QTLs and associate on the A10 chromosome to obtain a stable resistance locus VwRL3 in three environments.SNPs validity analysis showed that the varieties with favorable SNP genotypes were significantly more resistant,and the more number of SNPs was,the stronger resistance was,indicating that VW resistance had significant polymerization.The haplotype block map analysis identified a 372 kb candidate interval,which is a disease-resistant gene-rich region containing 22 candidate genes.The silencing analysis of the candidate genes by VIGS in the VwRL3 locus revealed that only the GhTNL1 gene silenced plants showed severe VW symptoms after inoculated with Verticillium dahliae.Analysis of GhTNL1 gene expression patterns in the resistant and susceptible materials induced by V.dahliae showed that the gene was strongly expressed at 6-24 h,which was consistent with V.dahliae's critical period of infection at 6-24 h.Phenotypic identification of Arabidopsis after inoculation with V.dahliae showed that compared with Col-0 and tnl1,OE1 and EC1 significantly reduced the yellowing and wilting of leaves,and the slow growth was partially recovered,indicating that the GhTNL1 gene was able to increase resistance of Arabidopsis thaliana to V.dahliae.Analysis revealed that the GhTNL1 gene contains a typical NB-ARC domain and LRRs repeat motif,it is predicted to contain two nuclear localization signals.Subcellular localization results show that both nuclear localization signals have localization activity.qRT-PCR analysis showed that GhTNL1 gene exerted its resistance function through the jasmonic acid pathway.Compared with Col-0 and tnl1,ROS accumulation and PR genes expression were significantly increased in OE1and EC1.Analysis of GhTNL1 polymorphisms in disease-resistant and susceptible materials revealed amino acid G/R mutations at position 225st,which is within the P-loop conserved motif of the NB-ARC domain.In order to verify the role of this mutation site,GhTNL1~R and GhTNL1~S were transiently transformed in Nicotiana benthamiana's leaves,and it was found that GhTNL1~S induced ROS accumulation and PR gene expression was significantly reduced.Therefore,it is speculated that this locus variation is one of the reasons leading to the differentiation of resistance and susceptibility to Verticillium wilt in different varieties of G.hirsutum.