Mapping-Based Cloning And Functional Analysis Of Cotton Fiber Development Related Gene QFS10.1

Cotton is one of the most important natural fiber crops in the world.China is the largest country for the production of cotton.However,its fiber quality is still poorer than other countries,which results in lower international competitiveness.Both fiber yield and fiber quality are quantitative traits.Due to the narrow intraspecific genetic base of upland cotton,traditional breeding methods are inefficient for improving fiber quality,while transgenic breeding and molecular marker-assisted breeding can significantly improve breeding efficiency.Therefore,it is of great significance to identify QTLs or functional genes for improvement of fiber quality.In this study,we designed new primers for QTL region based on the reference sequence and resequence data.Then,these primers were used to enrich the density of the genetic map.New genetic map,combined with the phenotypic data of population,was used to narrow the QTL interval.Then,candidate gene was identified based on fine-mapping result,and expression profile data.Subsequently,over-expression,RNAi interference,E6,Fb L2A and CRISPR Cas9 vectors were constructed and transformed into cotton receptor material.The function of GhROPGEF5 was clarified by comparing fiber yield and quality between transgenic material and control.Besides,the interaction between GhROPGEF5 and ROPs was analyzed.The main results are as follows:1.Genetic map enrichment of QTL intervalA total of 125 pairs of primers were designed based on the genome reference sequence and the reseqencing results of the two parents.After polymorphism analysis of these primers,five primers show difference between two parents.Then,genotype of fine-mapping population was identified with five polymorphic primers.A new genetic map was constructed combined the results of this study with 43 markers located on the genetic map before.This map contains 48 loci,covering 32.53 c M with an average distance of 0.68c M between adjacent markers.2.Fine mapping of QTL controlling fiber yield and quality on A10QTL controlling fiber yield and quality was identified on A10 of upland cotton based on new genetic map build in this study and the phenotypic data of population.The size of QTL region is 0.7 c M,with two flanking markers SWU9011 and SWU8326 after identifying QTL controlling fiber yield and quality with F₂,F_2:3 and F_2:4population.In addition,the recombinant lines F_2:4 population was used for substitution mapping to further narrow the QTL interval.In final,the size of QTL region is 0.59 c M,with two flanking markers A10-98.22 and SWU8326.3.Candidate genes identification for fiber yield and qualityIn this study,QTL was mapped between A10-98.22 and SWU8326,corresponding to 98.22Mb-98.38Mp on A10 of reference genome.There are seven annotated genes in this region.For these genes,only A10G2035 and A10G2036 were expressed in fibers after analysis by expression profile sequencing and RT-PCR.Beside,it showed that only a frameshift deletion at the exon of A10G2036 was found in the QTL region.To sum up,it is suggested that A10G2036（GhROPGEF5）is the candidate gene for QTL controlling lint percentage and fiber quality on A10.There is a deletion of G base at the third exon of RIL179,which resulted in premature termination and loss of gene function.4.GhROPGEF5 function analysisIn order to verify the function of GhROPGEF5,over expression,RNAi interference,E6,Fb L2A and CRISPR cas9 knockout vectors were constructed and transformed into cotton by Agrobacterium-mediated method.Result showed that lint percentage was increased after over expression of GhROPGEF5.However,fiber quality is lower than control.There is no significant difference for lint percentage after decreasing the expression level of GhROPGEF5 by RNAi interference,while the fiber strength increased by 1.87-2.94c N/tex compared with the control.After improving the expression level of GhROPGEF5 at the early stage of fiber development by E6promoter,the lint percentage of mature fiber increased while the fiber strength decreased.Besides,lint percentage and fiber strength both decreased when the expression level of GhROPGEF5 was improved at secondary wall thickening stage by Fb L2A promoter.In addition,we identified servel ropgef5 mutation by CRISPR Cas9gene editing system.For these mutation lines,lint percentage reduced,while fiber strength and fiber length increased.5.Interaction between GhROPGEF5 and ROPs proteinIn upland cotton,there are 30 ROP genes.And 18 ROP genes were expressed during fiber development.Then,the interaction between GhROPGEF5 and 18 ROP proteins was analyzed by yeast two-hybrid and bimolecular fluorescence complementary techniques.Results showed that ROP9 and ROP10 had a strong interaction with GhROPGEF5.These results suggested that GhROPGEF5 might regulate fiber development by regulating the activity of ROP9 and ROP10.