Genome Wide Association Study On Fiber Fineness And Candidate Genes Identify In Ramie

Fiber fineness is an important index for fiber quality of ramie(Boehmeria nivea(L.)Gaudich.),which directly related to the textile value of the fiber.Therefore,detected QTLs of fiber fineness was important for ramie breeding with higher fiber fineness.In this study,a total of 319 elite ramie germplasms were used to construct an associated group,and the whole genome SNPs were obtained using whole-genome resequencing technology.Based on the phenotypic data obtained from the field experiments of 2 locations with several years,and the genotyping data,the GWAS was conducted.Candidate genes were identified from those significantly associated locies,and further overexpression vector construction and transgenic research were conducted on candidate gene Marker09783.The main findings are as follows:1.The fiber fineness and fiber diameter of 319 ramie accessions were analysis at field experiment.The results showed that the 319 ramie accessions with a wide variation in fiber fineness and fiber diameter,with abundant genetic diversity.Based on the correlation analysis of different environments and the average value of broad sense heritability for fiber fineness and fiber diameter,indicate that the fiber finenesee and diameter was mainly affected by the genotype.2.After filtering,a total of 3,494,975(maf ? 0.05 and miss ? 0.2)high-quality SNPs was obtained,used for kinship and population structure analysis.After further filering,1,336,689 SNPs were obtained for GWAS analysis,with an average spacing of 246.80 bp,which was smaller than the ramie LD decay value of 1.27 Kb.The kinship values were calculated using SPAGeDi software indicate that most ramie germplasms with a genetic relationship between 0.3-0.4.Phylogenetic analysis showed that the 319 ramie can be divided into four groups,while not significantly regional distribution among those four groups.PCA analysis showed that the top four principal components(PC1,PC2,PC3,and PC4)only explained 13.60% of the phenotypic variation,and there were no obvious block-like clustered among germplasms.Population structure analysis shows that the 319 accessions no obvious population stratification.3.Based on the GLM+Q model,a total of 82 trait SNPs in environment 2017 Lb,361 trait SNPs in environment 2018 Lb,and 18 trait SNPs in environment 2018 La for fiber fineness were detected,respectively.A total of 227 trait SNPs in environment 2018 Lb and 28 trait SNPs in environment 2018 Lb were identified.Integrated the GWAS results and the previous QTLs mapping results of F1 populations(Zhongzhu1 × Hejiangzhuma),four promising QTLs for fiber fineness were detected(Chr.9: 16.16-16.50 Mb,Chr.9: 3.13-3.61 Mb,Chr.8: 13.90-14.17 Mb,Chr.8: 15.62-15.86 Mb).4.Combining the RNA-seq data of five developmental stages of stem bark in two different fiber fineness accessions(Chuanzhu2 and Quxianzhuma),the expression values(FPKM)of genes in the four QTLs region was analyze.Furthermore,quantitative PCR was perform for the promise candidate genes.Finally,six promising candidate genes were identified.Among the six genes,the Marker00009591 encoding a 1-aminocyclopropane-1-carboxylic acid oxidase,indicating that ethylene maybe plays an important role in the regulation of fiber fineness.5.Based on the paraffin sections of two different fiber fineness accessions in five developing stages,we found that ramie fiber cells were gradually dissolved from the ends of several fiber cells to form a long fiber cell,and the number of fiber cells gradually increases with the development time.Comparison analysis shows that the fiber cell diameter of Chanzhu 2(low fiber fineness accession)was higher than that of Quxianzhuma(high fiber fineness accession)in five development stages,which indicating that the ramie fiber fineness had already formed at the beginning of fiber development.6.A study on the regeneration system of leaf tissue in Chuanzhu 2 found that the optimal medium for callus induction was MS + 0.5 mg/L TDZ + 0.03 mg/L 2,4-D + 0.01 mg/L IAA,the optimal callus differentiation medium was MS + 0.3 mg/L TDZ + 0.03 mg/L 2,4-D + 0.03 mg/L IAA.Furthermore,the pBI121-Marker09783 overexpression vector was constructed for the candidate gene Marker09783,and 8 transgenic lines were obtained by Agrobacterium-mediated transformation of the leaf discs of Chuanzhu 2.7.qRT-PCR analysis found that the expression levels of Marker09783 gene in three transgenic lines were higher than the control,and further paraffin section showed that the fiber diameter of the transgenic lines was smaller than that of wild-type plants.