Exploration Of Verticillium Wilt Resistance Genes And Functional Analysis Of Important Candidate Genes In Gossypium Hirsutum

Cotton Verticillium wilt(VW),one of the most devastating diseases in cotton production,reduces cotton quality and yield.It is of great significance for the genetic improvement of VW resistance in cotton to explore the resistance genes and analyze the resistance mechanism.In the previous study of our research team,a cotton core collection with 401 germplasm resources were resequenced up to 11.0-fold-coverage depth.In this study,we identified the VW resistance of 401 upland cotton germplasms across two V.dahliae strains including strong pathogenicity strain LX2-1 and medium pathogenicity strain Vd991.The SNPs significantly associated with VW resistance were identified via genome-wide association study.Based on the linkage disequilibrium decay of this population,the candidate genes were identified with a 300-kb region of the significant SNPs by combining transcriptome data induced by Verticillium dahliae.The transgenic Arabidopsis thaliana was used to study the effect of overexpressed candidate genes on VW resistance.In order to further study the function of candidate genes in cotton resistant to V.dahliae,the virus-induced gene silencing technique was used to inhibit the expression of cotton endogenous genes.At the same time,combining a comparative proteomic analysis by two-dimensional electrophoresis combined with MS/MS analysis in a previous study,the disease resistance mechanism of candidate genes GhnsLTPsA10 and GhGPA were preliminarily analyzed.The main results were as follows:1.The disease resistance identification showed that there were a few of resistant varieties in this 401-core collection,and 12 resistant varieties were screened out,and the disease index ranged from 21.67 to 25.00.A total of 253 varieties displayed tolerant,and the disease index ranged from 25.47 to 50.00.There was no variety with immunity and high VW resistance.VW resistance of the modern varieties(the average disease index was 43.75)was better than that of middle-stage varieties(48.60)and early-stage varieties(51.55).The resistance of the varieties from the Yellow River cotton-growing areas(the average disease index was 43.21)were better than those of other cotton-growing areas or geographic origins.2.We found 352 SNPs significantly associated with VW resistance via GW AS.There were 349 SNPs occurred within a region located at chromosome Dtl1(Dt11:61909252-62256064),containing 18 SNPs(core elite alleles)that were repeatedly detected and undescribed previously.Gene function analysis proved that the associated Dt11 region was a major genetic locus responsible for VW resistance.Of the causal genes associated with Dtll signal,27 candidate genes out of 60 associated genes showed differential expression upon V.dahliae inoculation.The 23 candidate genes were proved to be involved in VW resistance via expression,gene knockdown and/or overexpression experiments.The frequency of the core elite alleles(FEA)in modern varieties was 12.7%and significantly higher than that in early&middle varieties(4.4%),indicating that the FEAs increased during artificial selection breeding.The newly-developed cultivars,JND23 and JND24,have fixed these core elite alleles during breeding without yield penalty.3.We identified one locus on chromosome At 10 significantly associated with VW resistance,and within a 145.5 kb LD and found two nonspecific lipid transfer protein genes(named GhnsLTPsA10).Expression of GhnsLTPsA10 dramatically increased in root against V.dahliae,whereas significantly decreased in leaf under insect attack.We found that the nsLTPs content positively correlated with VW resistance.Furthermore,GhnsLTPsA10 played antagonistic roles in positively regulating VW and Fusarium wilt resistance in transgenic Arabidopsis and VIGS cotton under the control of 35S promoter.Combining transcriptomic,histological and physiological analyses,we discovered that GhnsLTPsA10 mediated phenylpropanoid metabolism,which further affected the balance of its downstream metabolic flux of flavonoid-and lignin-biosynthesis.Besides,GhnsLTPsA10 also contributed to maintain ROS in a relatively high homeostasis state in plants under fungus infection.4.A cotton G-protein α-subunit-encoding gene(GhGPA)was isolated from VW resistant Gossypium hirsutum(upland cotton)cv.ND601.Subcellular localization revealed GhGPA was located in the plasma membrane.GhGPA transcription was up-regulated under V.dahliae stress and higher in the tolerant cotton cultivars than in the susceptible.GhGPA showed high(85.0%)identity with Arabidopsis AT2G26300(AtGPA1),and AtGPA1 gpa1-4 mutants displayed susceptibility to V.dahliae.Ectopic expression of GhGPA successfully restored the resistance of Arabidopsis gpa1-4 mutants to Verticillium wilt and made them more resistant than the wild type.Overexpression of GhGPA in Arabidopsis markedly increased the resistance.Further analysis found that,GhGPA played a vital role in VW resistance by inducing SA and JA signaling pathways and regulating the production of reactive oxygen species.In summary,the VW resistance of 401 cotton core collection was determined in this study.A total of 12 resistant varieties were screened out,and can be applied to the genetic improvement of VW resistance.The genetic basis and elite alleles variation loci of VW resistance in the core germplasm were revealed.We identified a novel chromosome region in Dt11(D11:61909252—D11:62256064)was significantly associated with VW resistance via GWAS,containing 18 SNPs(core elite alleles)and 23 candidate gene that played an important role in cotton VW resistance.We found that our associated region in the Dt11 was not overlapped with the reported signal region associated with cotton other main agronomic traits,and the frequency of 18 elite alleles of modern varieties was 12.7%,significantly higher than that of early&middle varieties(4.4%).Further research on the function of important candidate genes and found that,GhnsLTPsA10 coordinated disease resistance by regulating the redirection of the metabolic flow of flavonoids and lignin biosynthesis pathways in the phenylpropane-metabolic pathway.Besides,GhnsLTPsA10 was able to inhibit the fungal growth by permeabilizing fungal membranes and changing the permeability of the cell membrane,and maintained the ROS in the plant at a relatively high level and positively regulated the disease resistance.GhGPA positively regulated cotton VW resistance by increasing PR gene expression and regulating SA and JA signaling pathways,H2O2 accumulation,and enzymatic activity.