Map-based Cloning And Functional Analysis Of Li₃ Gene Controlling Fiber Development In Cotton

Cotton with cellulose-enriched mature fibers is the largest source of natural textile in the world.Through a map-based cloning strategy,we isolated an industrially important lint fiber development gene(Lis)that encodes a MYB-MIXTA like transcription factor(MML)on chromosome D12(GhMML4_D12).VIGS-silencing or decreasing the expression of the GhMML4_D12 gene in nZNSM plants resulted in a significant reduction in epidermal cell prominence and lint fiber production.The MIXTAs expanded in Malvaceae during their evolution and produced a Malvaceae-specific family that regulates epidermal cell differentiation,different from the gene family that regulates leaf hair trichome development.The research contents and results are as follows:1.For fine mapping li3,two segregated populations were constructed,(n2NSM ×XZ142FLM)F2 and(nZNSM × XZ142FLM)BC1.This produced a 3487 fuzzless-linted and 1175 fiberless segregation in(n2NSM × XZ142FLM)F2,and a 2343 fuzzless-linted and 2325 fiberless segregation in(n2NSM × XZ142FLM)BC1.Using 8869 pairs of simple sequence repeats(SSRs),we could primarily anchor li3 on Chr.D12.Based on the genome sequence of the D-genome progenitor(G.raimondii),544 SSRs/indel primers within the li3 gene were mined and their polymorphisms screened to fine-map this GhLINT gene.Finally,the li3 locus could be narrowed down to a 79.8-kb region spanning three annotated genes flanked by SSR markers,W1644/1654 and W1824/1834,with genetic distances of 0.071 cM and 0.284 cM,respectively.Three putative open reading frames(ORFs)encoding two MITXA genes,GhMML4 and GhMML3,a homoeologous GhMYB25-like gene responsible for fuzz development,and a NAC protein,were predicted by blast2go based on the D subgenome sequence of allotetraploid Upland cotton(G.hirsutum acc.TM-1).2.We isolated the full length genomic DNA of these three putative ORFs and found that there were no differences in the genome sequences of GhMML3 and NAC protein between the two mapping parents,nZNSM and XZ142FLM.However,one mutation occurred at the 500 bp site;from cytosine(C.)in n2NSM to adenine(A)in XZ142FLM,which resulted in the production of a TAA stop codon and the early termination of GhMML4_D12 translation or a causative mutation in li3 in XZ142FLM.We previously isolated the dominant fuzzless gene N1 using map-based methods and found that GhMML3_A12 regulates fuzz fiber development.Since GhMML4_D12 was specifically expressed during the initial fiber development stage,it is likely that GhMML4_D12 is a candidate gene for the mechanism underlying lint fiber development.This suggests that they may be all related to fiber development.3.Through the VIGS system,to determine the GhMML4 gene is the target gene li3.We therefore isolated the 3’-end 538bp fragment(410～948bp)of GhMML4_D12 from n2NSM and inserted it into the pTRV2 vector for transformation into n2NSM.The results showed that the expression of GhMML4 gene in ovules of-1DPA was significantly decreased after silencing.Much less lint fiber initiation was observed in 1 DPA ovules of VIGS plants,suggesting a role for this gene in the regulation of lint fiber production.At the boll opening stage,lint fiber production was significantly reduced in GhMML4_D12-downregulated plants.It was proved that GhMML4 is the key gene to control the development of fiber.4.The evolutionary tree includes 54 MIXTA proteins from A.thaliana,A.majus,U vinifera,T.cacao,G.raimondii and H.syriacus aligned using ClustalW in MEGA 5.05.Evolutionary analysis of the MIXTA gene family revealed that 10 cotton MML genes are distributed in four distinct clades(SBG9-1～SBG9-4).Two Malvaceae-specific clades were formed,SBG9-2,which includes MML3,MML7 and MML9,and SBG9-3,which includes MML4,MML8 and MML10,According to the function of MML3 and MML4 to regulate the development of fibroblasts,it is inferred that the two subgroups genes may be differentiated from Malvaceae in the process of evolution to regulate ovarian epithelial cell differentiation.In this study,a gene GhMML4,which regulates the initiation of fiber development,was successfully obtained by map-based eloning.And provide invaluable experience for map-based cloning of polyploid materials with complicated genome structure such as cotton.li3 gene is one of the key genes regulating fiber initiation,which is of great value in the future study of fiber initiation.