Genome-wide Association Analysis Of Fiber Quality And Yield And Functional Analysis Of Candidate Genes In Upland Cotton

Cotton is not only an important economic crop,but also an ideal species for studying the origin,evolution and domestication of polyploids.As one of the main cultivated varieties,the mechanism of fiber quality and yield of upland cotton has been the focus of research.Cotton fiber is the trichome developed from a single epidermal cell,which is almost composed of pure cellulose,is an ideal single cell model for studying cell elongation and cell wall modification.In this study,we estimated the natural populations and fiber-related F2 populations of 355 upland cotton accessions and collected genotype data?including genome-wide resequencing and simplified sequencing?,aimed to find SNPs associated with fiber yield and quality.The candidate genes were identified and verified.Transgenic cotton and transient expression of VIGS was carried out.This study revealed the genetic basis and molecular mechanism of fiber quality and yield,and provided a basis for the improvement of cotton fiber quality and yield.The main results of this study are as follows:1.355 upland cotton accessions were planted for many years.Five indexes related with fiber quality and two traits related with yield were collected:fiber length?FS?,fiber strength?FS?,fiber uniformity?FU?,fiber micronaire?FM?,fiber elongation?FE?,lint percentage?LP?and single boll weight?BW?.Statistical analysis showed that the character data of seven fiber quality and yield showed near-normal distribution,and there was significant phenotypic variation in these seven traits,and the results of correlation analysis showed that micronaire value was significantly negatively correlated with the other six traits.There was a positive correlation among the other six traits.Heritability analysis showed that the heritability of seven traits related to fiber quality and yield were generally high,ranging from 0.72 to 0.89,indicating that genotypes had a great influence on these traits.2.Using 355 simplified sequencing data of upland cotton and fiber length phenotype data in eight environments for GWAS analysis,14 SNP loci that significantly associated with fiber length were screened,which were distributed on 6 chromosomes?A03,D02,D03,D04,D09 and D11?.Among them,two significant SNP?D03chromosome 41720764 and D03chromosome 41721072?on chromosome D03 were most significantly associated with fiber length,which could explain 13.13-14.37%of the phenotypic variation.There are two haplotypes AA and TT in both SNPs.In 8 environments,the fiber length of materials carrying A haplotype was significantly shorter than that of materials carrying T haplotype.Linkage analysis of fiber length-related pedigree populations showed that two significant QTL?QFL-D03-1 and QFL-D08-1?were detected in both F2 and F2:3 populations.According to the physical location of the SNP,we found that both GWAS and linkage mapping detected an overlapping region on the D03 chromosome that could explain a relatively high phenotypic variation.The phenotypic variation interpretation rates were 13.75%and 19.28%,respectively,indicating there is a possible major effect site that controls fiber length traits,and a candidate gene Gh_D03G1338?F2KP?was selected based on the annotation information.3.From 3 DPA to 20DPA,there was a significant difference in the expression of GhF2KP in standard line TM-1 and long and short fiber materials,and it was found that the overexpression of this gene in Arabidopsis thaliana could significantly increase the hair density of leaf epidermis and the main root of Arabidopsis thaliana.The gene was silenced by virus-induced gene silencing?VIGS?technique.It was found that after inhibiting the expression of GhF2KP in different fiber materials,the fiber length became shorter,especially in long fiber materials.Combined with previous studies,it is speculated that GhF2KP positively regulates the elongation of fiber cells by participating in sucrose synthesis.4.Based on the genome-wide association analysis between 355 resequencing data of upland cotton and fiber quality and yield traits in 11 environments,the marker loci significantly associated with fiber quality and yield were obtained,and the number of markers above threshold 5 was 1825.The number of markers above threshold 6 was 361.Among them,10 SNP loci were significantly associated with multiple fiber traits at the same time,indicating that the inheritance of fiber quality was multiple.The SNP marker Ghir_A10₁14618736 located on chromosome A10 was significantly associated with fiber length and strength in multiple environments.LD block was drawn and combined with annotation,gene expression and other data,a closely linked gene GhTBL38,was found at the distance from the marker 78.7kb as a candidate gene for functional verification.5.GhTBL38 was predominantly expressed in cotton fiber elongation stage?5 DPA?,and the expression of GhTBL38 in long fiber materials was significantly higher than that in short fiber materials.The gene was overexpressed in Arabidopsis thaliana,and the leaf villi became denser.In the T1 generation plants after gene editing,lines with different degrees of deletion were observed,and one of the lines showed late flowering phenotype.The fiber processes of the ovules in 5DPA are collapsed.These evidences suggest that GhTBL38 participates in the regulation of upland cotton fiber length,and may play a positive role in cotton fiber elongation through acetylation modification.In this study,we find 155 fiber length and 434 fiber strength related loci by GWAS and simplified genome sequencing in natural and genetic populations.Combined with transcriptome and functional annotation,two fiber related candidate genes?GhF2PK and GhTBL38?were screened.Furthermore,through Arabidopsis transformation,cotton transient transformation,cotton gene knockout and other means to verify that GhF2PK and GhTBL38 genes have a positive regulatory effect on fiber length traits.This study provides a basis for analyzing the genetic mechanism of cotton fiber development,clarifying the molecular regulation mechanism of GhF2PK and GhTBL38,and providing theoretical support for high yield and high-quality cotton breeding.