| 【Object】Cotton is an important cash crop in China and one of the important textile raw materials.Improving the yield of cotton fiber quality and disease resistance is considered to be an important goal of breeding.However,these traits are complex quantitative traits controlled by minor-gene and influenced by external environment.Among them,Verticillium wilt,which can significantly reduce the yield and quality of cotton,is seriously harmful for cotton.Moreover,due to the rapid mutation rate of pathogen causing Verticillium wilt and its wide host range,and no effective control method in production,it is called the"cancer"of cotton.At present,the most effective way to control cotton Verticillium wilt is to breed and popularize resistant varieties.Since the 1950s,some upland cotton varieties with Verticillium wilt resistance have been bred by conventional breeding methods,but their resistance is limited.At present,the major genes and molecular mechanisms of resistance to Verticillium wilt in cotton are not clear.【Methods】In this study,eight varieties/lines selected by our research group in the cotton area of northern Xinjiang were used as parents to construct the multi-parent advanced generation intercross,63K SNP chip was used to mine population polymorphism SNP sites to identify Verticillium wilt resistance traits.Sixty-two SNP markers significantly associated with Verticillium wilt resistance were identified by GWAS analysis.Combined with the early transcriptome analysis of a pair of extremely different materials,the key candidate genes for resistance to Verticillium wilt were obtained and their functions and molecular mechanisms were preliminarily analyzed.【Results】1.Genome-wide association analysis of Verticillium wilt resistance in MAGIC population（1）Phenotypic analysis of Verticillium wilt resistance in 328 population showed that the disease index showed continuous variation in different environments with approximately normal distribution.The population was significantly influenced by the genotype,environment,the genotype and environmental interaction effect.The variation richness coefficient of Verticillium wilt was 12.3%～49.4%.The correlations among the environments were all highly significant,with correlation coefficients ranging from 0.35 to 0.78.（2）The MAGIC populations were divided into three subgroups.9,323 high quality SNPs were obtained by filtering the 63k chip.The average density of SNPs were 2.44 SNP/c M,an average polymorphism coefficient ranged from 1.02 to 4.22,and an average genetic diversity coefficient were0.29.（3）Genome-wide association analysis of cotton resistance to Verticillium wilt was performed using9,323 high quality SNPs.Threshold-log10P>3,62 SNPs sites were found to be significantly related to Verticillium wilt resistance in cotton.Moreover,the A11 and D11 chromosomes were identified with continuous distribution in the 2018-SHZ and BLUP environments.（4）The most significant SNP site i32100Gh（P-value:2.67E-04）on A11 chromosome and the 500Kb upstream and downstream of i07205Gh（P-value:2.16E-04）on D11 chromosome were used as the range of disease-resistance related candidate genes.Then were BLASTn in the genomic sequences of G.hirsutum＿TM-1＿WHU＿genome,extracted all the candidate genes from A11:11,224,619-12,224,619 and D11:15,955,857-16,955,857 region.Finally,37 genes were obtained from A11,and 62 genes were obtained from D11.2.Transcriptome analysis of cotton under Verticillium dahliae infection（1）The MAGIC line M138（resistant cultivar）and the MAGIC parent P2（susceptible cultivar）were selected for transcriptome analysis.Compared no V.dahliae infection with V.dahliae infection in M138 and P2 at all time-points,respectively.In M138,11,076 genes were differentially expressed.In P2,6,640 genes were differentially expressed.（2）WGCNA was performed on the 4,633 differentially expressed TFs,and sorted into 16 different modules based on clustering analysis of gene expression across all samples.Total of 654 transcription factors were aggregated in the MEblue module,which were highly expressed in M138 at 6 h and 12 h.KEGG pathway enrichment analysis showed that“Plant hormone signal transduction”,“Plant-pathogen interaction”,“MAPK signaling pathway”and“Glyoxylate and dicarboxylate metabolism”etc significant pathways were enriched in the gens of the’MEblue’modules.（3）According to heatmaps and characteristic gene expression patterns of the’MEblue’module,the Z-score method was performed to identify hub genes and co-expression networks indicated by high eigengene-based connectivities（Kme）.Based on expression pattern of V.dahliae infection,tissue expression analysis,interaction intensity and Kme values,we determined that Gh ERF91 was the key gene for resistance to Verticillium wilt in cotton.（4）Multiple sequence alignment between Gh ERF91 and its homologous genes from various plant species showed Gh ERF91 was highly similar to ERF91 proteins of other species.Gh ERF91 contained a highly conserved AP2/ERF domain with 58 amino acids.The expression of Gh ERF91 was induced 3 h after exogenous application of ETH.Silencing of Gh EFR91 increased susceptibility to V.dahliae in cotton.The transcription of Gh ERF1,Gh ERF6,Gh ACO1 and Gh PR1 were decreased in Gh ERF91-silenced cotton.Interestingly,after sprayed 0.5μg/m L ethephon 3 h before V.dahliae inoculation to TRV:00 and TRV:Gh ERF91 plants,the disease index and the rate of defoliation were significantly lower in ethephon treatment TRV:00 plants than without ethephon treatment.3.The key candidate genes for resistance to Verticillium wilt in cotton were extracted by combining GWAS and transcriptome（1）A total of 34 differentially expressed genes were identified in the candidate regions significantly associated with Verticillium wilt resistance combined GWAS analysis.The expression of Ghi＿A11G08556 and Ghi＿D11G08281 were higher in M138 than P2 and induced up-regulated by V.dahliae.Moreover,sequence analysis found that Ghi＿A11G08556 and Ghi＿D11G08281 were a pair of homologous genes with 98.02%nucleotide sequence similarity.However,the expression of Gh FBX92was higher in P2 than M138,and induced down-regulated by V.dahliae.The non-synonymous mutant SNP were found in three candidate genes between M138 and P2.（2）Inhibiting the expression of Ghi＿A11G08556,Ghi＿D11G08281 and Gh FBX92 in cotton by VIGS,showed that Ghi＿A11G08556 and Ghi＿D11G08281 commonly silenced to reduce the resistance to V.dahliae,and the specific silencing effect of single gene was not obvious.Silencing Gh FBX92improved resistance to V.dahliae.Moreover,inhibiting Gh FBX92 expression activated the expression of key genes in jasmonic acid signaling pathway.The disease rate of Arabidopsis homologous mutant fbx92showed that its homologous gene AT3G07870 was involved in defense against Verticillium wilt.Gh FBX92 overexpression induced downregulation of key genes in JA signaling pathway in Arabidopsis thaliana. |
