Fine Mapping Of Major QTLs For Lint Percentage And Fiber Strength And Functional Verification Of Important Candidate Genes In Gossypium Hirsutum

Cotton fiber development is a highly programmed process controlled by multiple genes.It is very important to identify the genes related to lint percent（LP）and fiber strength（FS）and analyze their regulatory mechanisms to accelerate the genetic improvement of cotton.In this study,G.hirsutum cv.Zhongmiansuo 127（CCRI127）,characterized by high quality,was selected as the shared male to cross with G.hirsutum cv.EZ60 and G.hirsutum cv.L177,both of which were featured with low quality.The resultant EZ60/CCRI127 and L177/CCRI127 F₂populations contained respectively 1141 and 1041individuals.F_2:3inbred lines were derived from self-pollination F₂plants.Then,this study used the NGS-BSA method to identify respectively LP-and FS-related QTLs in upland cotton,followed by fine mapping of candidate genes.The specific results are as follows:（1）RNA-seq analysis of cotton fiber samples from parentsIn this study,RNA seq analysis was performed on ovule/fibrous samples of EZ60 and CCRI127 at 0,5,10,15,20,and 25 days post anthesis（DPA）.A total of 2545 genes with differential expression（DEGs）between parents were detected.DEGs can be grouped into five clusters according to the expression dynamics model.GO terms enriched by DEGs were all associated with cell membrane and cell wall biosynthesis.Accordingly,DEGs were significantly enriched in KEGG pathways involved in carbon fixation,carbon metabolism,and carbon partition.DEGs,enriched in different GO terms,were correlated with each other and synergistically controlled the fiber development through KEGG pathway network.（2）Expansion of NGS-BSA and mapping of LP-related QTL in upland cottonIn this study,the F₂plants used for constructing BSA bulks were reaffirmed by the phenotype of corresponding F_2:3plant lines to ensure that the selected region of two DNA libraries were both consistent with the expected genotype.In addition,this study suggested that BSA analysis should be performed on two（or more）F₂half-sib populations,and the overlapped intervals of QTLs obtained by multiple BSA analyses were used as the final genomic candidate intervals.This strategy can be used to identify stable QTLs significantly associated with target traits at high resolution.In this study,the aforementioned scheme was applied to the EZ60/CCRI127 and L177/CCRI127 F₂populations,and a LP-related QTL was mapped to D09:37,098,560 bp-41,509,122 bp,which covered 4.41 Mb.This region can be narrowed to 30,976 bp via constructing a high-density linkage map,and synergistic genes were all conferred from the high-LP EZ60.（3）Identification of the LP-related candidate gene and variation analysis of this gene causing low LP in CCRI127.The gene Ghalg7,Gh PAP16,GhIQD14,and Gh GNS1 in the fine-mapped interval have all been functionally annotated.According to the characteristic expression profile of LP functional gene group,GhIQD14 was finally identified as a candidate gene.SNP-39,560,387（G→T）,a coding SNP（c SNP）was in GhIQD14^CCRI127,can cause a non-synonymous substitution of amino acid.The allele frequency of GhIQD14^EZ60in each parental and BSA library was significantly positively correlated with the corresponding LP value.Therefore,GhIQD14 may play an important role in regulating LP formation in upland cotton.（4）Identification of FS-related QTLs in upland cotton based on PCAMP strategyThe PCAMP technique perform BSA analysis via comparing multiple graded bulks（n≥3）in pairs,and the overlapping intervals of the QTLs obtained by multi-analysis were also used as candidate loci related to the target phenotype.In this study,the FS variation in F₂progeny of EZ60/CCRI127 was analyzed by PCAMP,and the QTL interval was narrowed from 8,141,986 bp of traditional BSA analysis to 2,579,003 bp.Moreover,a saturated genetic map was constructed in the PCAMP interval,and the FS-related QTL was further narrowed to 88,493 bp.The synergistic gene was conferred by the high-FS CCRI127.（5）Variation analysis of FS-related candidate genes between parents and those variation effects on the fiber developmentThe four genes in the candidate interval were NA（GH＿D09G1317）,GhCKX1,Ghgat B and Gh DNAJC7,which have all functionally annotated.According to the expression profile data of these four genes from RNA-seq analysis,GhCKX1 was finally identified as a candidate gene.There are two c SNPs in exon 1 of GhCKX1^EZ60,both of which cause non-synonymous substitution of amino acids.The frequency of GhCKX1^CCRI127allele increased successively in the order of L-bulk,M-bulk and H-bulk.（6）Functional verification of GhCKX1GhCKX1 gene in upland cotton has a high homologous gene AT2G41510 on chromosome2 in A.Thaliana.This study replaced the promoter of GhCKX1^CCRI127gene with a 35S constitutive one and genetically transformed wild-type A.thaliana.q PCR analysis showed that the GhCKX1^CCRI127expression driven by 35s promoter was significantly up-regulated in two transgenic lines with a high homeostasis level of its m RNA.For genetically modified（GM）Arabidopsis plants and control ones,the average thickness of stem TE wall at 1.0 cm from the root was 1.9633μm and 1.4675μm,at 6.0 cm from the root was 1.9126μm and 1.2778μm,at 11.0 cm from the root was 1.6933μm and 0.8533μmμm,respectively,dictating a statistical difference between GhCKX1-overexpression plants and wild type ones（P<0.0001）.The GhIQD14 and GhCKX1 genes reported for the first time in this study provided novel gene resources for LP and FS genetic improvement in upland cotton,and the In Del markers developed provided the basis for marker-assisted selection and molecular design breeding of yield and quality.