| When cotton is subjected to drought stress in different growth periods of different environments,its drought resistance mechanisms are different.Based on the complexity of cotton drought resistance,researchers have carried out extensive and in-depth research on the drought response mechanisms of cotton at different levels.The QTL for drought resistance traits and the mining of related candidate genes are thought to play an important role in this process.In this study,Xinluzhong 36 with weak drought resistance of Gossypium hirsutum and Acala1517-08 with strong drought resistance of Gossypium hirsutum were used as parents,and a recombinant inbred line(RIL)population containing 240 progeny was constructed.Stress treatment,by measuring the field phenotype of 3 environmental points for two years,comprehensive evaluation of population drought resistance and screening of key drought resistance traits,using BSA-seq to map the drought resistance QTL of Upland cotton,through the combination of inter-parental genotyping and RNA-seq mines potential candidate genes and performs functional verification.Based on the relative change rate after drought stress,through dimensionality reduction analysis,plant height(PH),single boll weight(SBW)and transpiration rate(Tr)were obtained as the key drought resistance traits of Gossypium hirsutum.According to these three key traits,RIL was performed.For the screening of population extreme materials,3 groups of 28,28 and 23 separate drought-resistant and drought-sensitive mixed pools were constructed.BSA-seq was used to obtain QTL segments related to drought resistance,and 5 candidate genes for drought resistance were mined and their functions were verified.To provide genetic resources for molecular directional breeding of cotton drought resistance.The main findings are as follows:(1)Artificial drought stress was applied to the parental and RIL populations at the flowering and boll stage,and their agronomic,photosynthetic,biomass and yield traits were determined.Based on the field phenotype data of three environmental points in two years,the drought resistance of the population materials was comprehensively evaluated by the comprehensive drought resistance metric value(D),the drought resistance membership function value(DM),the relative change rate of each trait(V)and other drought resistance indicators.It was found that D There was a very significant negative correlation between the value and V,which could be used as an index to evaluate the drought resistance of the population.The drought resistance of the population materials was sorted and divided into 5 categories by D-value cluster analysis.At the same time,using correlation,principal component,linear regression and other analysis methods,6samples with stable performance and large contribution rate were screened at each environmental point.Key traits of drought resistance,yield per plant(YPP),transpiration rate(Tr),boll weight(SBW),plant height(PH),aboveground dry matter accumulation(OP),root shoot ratio(RSR).Combined with the analysis of variance among the traits of the four environmental points of the parents,the key traits with high phenotypic variation interpretation rates and significant differences among the parents were determined,and the plant height(PH),single boll weight(SBW),and transpiration rate were finally determined.(Tr)as a key trait for drought resistance in Gossypium hirsutum..(2)Using phenotypic data from 3 environmental points,the extreme materials with stable performance at multiple environmental points were selected to construct 3 sets of BSA-seq mixed pool sequencing based on the intersection of ED and SNP-index algorithms,and one drought resistance QTL associated with PH traits was identified on chromosome A13,which was 3.2 Mb in size and contained 123 genes.A total of 2 drought tolerance QTLs with a size of 6.74 Mb and containing 188 genes were identified in the mixed pool of tolerance constructed in SBW,which were located on chromosome A03 and A10.A total of 2 drought resistance QTLs with a size of1.15 Mb and containing 97 genes were localized in the hybrid pool constructed by Tr,which were located on chromosomes A07 and D07.Finally,based on the localization results of PH,SBW and Tr traits,a total of 6.27 Mb sized drought resistance QTL intervals containing 248 genes were obtained on chromosomes A07,A10 and A13.(3)Based on the SNP information between the parents,248 genes in the candidate interval were typed,and 76 genes were found to be non-synonymous mutation genes between the parent Acala1517-08 and Xinluzhong 36.Combined with the transcriptome data of drought resistance,the expression levels of 19 candidate genes in the 76 candidate genes in the five time periods of 6h,12 h,24 h,48 h and 72 h under drought stress were significant compared with 0 h.difference.q RT-PCR was used to analyze the expression levels of 19 candidate genes in the population parents,drought-resistant extreme materials of Gossypium hirsutum and tissue-specific,and finally 5 genes(Gh NCED1,Gh F6’H1,Gh PER55,Gh3AT1,Gh GAPA2)were screened as upland cotton The candidate genes for drought resistance were also analyzed by bioinformatics,which laid the foundation for the preliminary identification of the response functions of these five genes to the drought process.(4)Five candidate genes were cloned and VIGS silencing vector and overexpression vector were constructed respectively.After the candidate gene was silenced,drought treatment was performed to observe its phenotype and its physiological indicators were determined.Combined with the drought resistance analysis of overexpressed Arabidopsis(OE),wild type Arabidopsis(WT)and Arabidopsis deletion mutants,it was found that,After drought stress,the MDA content of the gene Gh GAPA2 silenced plants increased significantly,and the Pro content decreased significantly.Compared with WT,the whole plant of OE-Gh GAPA2 Arabidopsis thaliana did not show obvious wilting,and the Arabidopsis deletion mutant of Gh GAPA2 showed obvious wilting.This is consistent with the high expression of q RT-PCR in drought-resistant parents and extreme materials,indicating that the Gh GAPA2 gene is a positive regulator in response to drought stress.The MDA content of p TRV2-Gh3AT1 and p TRV2-Gh NCED1 plants did not increase significantly,but the content of Pro and chlorophyll increased significantly compared with CK treatment.At the same time,OE-Gh3AT1 and OE-Gh NCED1 Arabidopsis plants showed severe leaf wilting and stem wilting.Severe culm dryness and plant death,it is speculated that the above genes may be negatively regulated genes.The MDA content of the candidate gene Gh PER55 silenced plants was significantly increased,and the Pro and chlorophyll contents were extremely significantly increased.Compared with the WT,the OE-Gh PER55 Arabidopsis plants had no significant change.When verifying its function,the phenotype and resistance were inconsistent.The mechanism of action Further experimental verification is required.MDA content of Gh F6’H1silenced plants did not increase significantly,but Pro and chlorophyll content increased slightly,and OE-Gh F6’H1 Arabidopsis thaliana plants showed a small amount of wilting.This gene may be involved in the negative regulation of drought resistance. |
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