Mining And Functional Identification Of Verticillium Wilt Resistance Genes Of In Upland Cotton

Cotton is an important economic crop in the world,and Verticillium wilt is a soil-borne fungal disease,commonly known as "the cancer of cotton",which causes a serious reduction in cotton yield and fiber quality.However,the genetic mechanism of Verticillium wilt is still unclear,and there are few Verticillium wilt resistance genes that can be applied to production.In this study,314 upland cotton resource materials were used as experimental materials,and the highly pathogenic Verticillium dahliae Vd592 was used as the test strain.Firstly,the phenotypic data of 11 environments were obtained based on the phenotypic identification results of 6 field environments,the phenotypic identification results of 4 greenhouse environments,and the best unbiased estimate(BLUP),Screening upland cotton resource materials with resistance to Verticillium wilt;Secondly,through the combined analysis of high-throughput genome-wide association analysis and RNA-seq technology,new main loci of resistance to Verticillium wilt in upland cotton were quickly and accurately discovered,and the disease resistance was screened out,the candidate range of genes;Finally,the candidate genes involved in Verticillium wilt resistance were selected,and the selected candidate genes were functionally identified by gene silencing technology,and the overexpression of Arabidopsis was verified at the level of transgene identification.The main results are as follows:1.Verticillium wilt resistance identification and excellent germplasm screening of upland cotton core germplasm(1)Statistical analysis on the correlation of 11 traits at 2 points in 3 years.The results showed that the relative disease index was significantly negatively correlated with plant height,number of effective bolls,and lint;the relative disease index was positively correlated with growth period;the correlation coefficient is 0.10,and the correlation is not significant;(2)From the perspective of several traits related to Verticillium wilt resistance,the core germplasm resources tested have rich genetic variation,which is conducive to the development of phenotypic traits.The excavation of excellent gene loci also shows that the phenotypic traits are greatly affected by the environment;(3)The disease index is calculated according to the different breeding periods of the varieties,and the results showed that the varieties bred in modern times are better than those bred in the middle and early stages;(4)There are differences in the resistance of materials from different geographical sources,especially the resistance of the varieties bred in the Yellow River in my country is generally better than that of other cotton planting areas;(5)The resistance analysis of different materials found that the Among the varieties without immunity,there is one high-resistant variety,and 47 resistant varieties were screened,accounting for 14.90% of the total tested materials;(6)A total of 39 materials from abroad showed disease resistance and susceptibility.Among them,17 were resistant to disease,accounting for 43.5%,and 22 were susceptible,accounting for 56.4%,the results showed that the resistance level of materials taken from abroad is lower.2.Genome-wide association analysis to identify resistance genes of cotton against Verticillium wilt(1)A total of 122,832 SNPs were identified by genome-wide association analysis,with a density of3.23 SNPs per kilobase,and the number of variants was approximately 3-104.The Manhattan results showed that there are significant correlation signals in chromosomes A02,A05,A06,A10,A11,D05,D07,D10,D11 and D12,and Chr A11 can be repeatedly detected in the three classical models of GLM,MLM and CMLM,indicating that A11(A11＿113830848-117065952)is a major correlation signal,containing18.7 % of correlated SNPs.(2)A total of 382 genes and 152 genes were screened in the interval A11＿117249761-119855924 and A11＿113830848-117065952,and 2 significant loci related to cotton resistance to Verticillium wilt were obtained,in 119011344 and 116221642,both located in A11 on the chromosome.(3)Through GO enrichment and KEGG pathway enrichment,89 genes involved in the interaction between plants and pathogens were initially selected,and 37 genes were compared with the transcriptome expression data.There was differential expression in root tissue,and showed a trend of high expression.After further verification,four genes highly related to cotton resistance to Verticillium wilt were obtained for gene cloning.From the differential expression analysis of genes,GhA11G373600 and GhA11G352200 were initially selected as candidate genes and named as GhNFYB8 and GhDUSP12,respectively.3.Functional identification of GhNFYB8 and GhDUSP12 in cottonFirstly,fluorescence quantitative differential expression experiments of GhNFYB8 and GhDUSP12 were performed in the root tissues of Zhongzhimian 2 and Xinluzao 36 at 0h,2h,12 h,36h,48 h,72h,and120h(sampling after inoculation).The results showed that among the disease-resistant materials,Zhongzhimian 2 showed a trend of first rising and then falling,and reached the maximum value at 48 h,and then showed a downward trend.The VIGS function verification of GhNFYB8 and GhDUSP12 was carried out,and it was found that the leaves of the plants wilted,turned yellow or even fell off after silencing the target gene,while the empty control plants grew relatively healthy.Quantitative and recovery experiments also verified that Zhongzhimian 2 was more sensitive to Verticillium dahliae after the target gene was not involved in expression.These two genes are involved in the immune process of plants against Verticillium dahliae infection.4.Functional identification of GhNFYB8 and GhDUSP12 in ArabidopsisGhNFYB8 and GhDUSP12 were overexpressed in Arabidopsis,and T2 generation positive seeds were obtained by phenotypic identification of their T2 generation plants in vegetative soil.