Genetic Analysis And Candidate Gene Mining Of Cluster Mutant In Cotton

Cotton（Gossypium spp.）is an important economic crop in the world.China is a huge country in cotton production,consumption and textile.Mutants are excellent germplasm resources for gene cloning and functional research.We found a stable dwarf cluster mutant（Cluster mutant:cl）in transgenic progenies.The phenotype of cl is compact plant type,low plant height,shrinking of all tissues and organs,yellow green leaves,growth of lateral branches close to the main stem,low height of the first fruit branch and low seedling rate of seeds.In this study,genetic analysis and fine mapping of candidate genes were carried out by using the newly discovered dwarf cluster mutants.The research results have important theoretical and practical significance for cotton dwarf cluster molecular mechanism,dwarf breeding,ideal plant type cotton breeding.1.Genetic analysis of cluster mutants.A dwarf cluster mutant was found in transgenic progenies of upland cotton in our previous studies,and could be inherited stably.We used transgenic receptor material G.hirsutum acc.W0 and the cluster mutant to construct segregation population for genetic analysis.The results showed that the F₁ plants were wild type,which indicated that the cluster character was controlled by recessive gene.We obtained two sets of F₂ populations in 2017 and 2018 by strict selfing of F₁ individuals,and verified their phenotypes with F_2:3 population.The results showed that there were 154 F₂ plants in the first set（W0×cluster mutant）,including 126 normal plants and 28 cluster plants.Combined with the investigation of F_2:3 plants,42 of the normal plant types were not isolated,and 84 of them had normal and cluster phenotype segregation,showing the segregation ratio of homozygous and heterozygous genotypes of 1:2.There were 817 F₂ plants in the second set（W0×cluster mutant）,among which 636 plants had normal plant type similar to W0 and 181plants had compact plant type similar to dwarf cluster mutant.Furthermore,the cluster mutants in F₂ population were selfed and planted into F_2:3 lines,which showed all cluster mutants in F₂ without segregation in F_2:3 lines.Combined withχ² analysis,the results showed that the cluster mutant was a recessive qualitative trait controlled by a single gene.2.Mapping analysis of cluster gene in upland cotton based on BSA-seq.Combined with the phenotype verification of F_2:3 population,50 homozygous plants with normal phenotype（similar to W0 phenotype of transgenic receptor parent）and mutant phenotype（similar to cl phenotype of dwarf cluster mutant）were selected respectively from F₂segregation population.Then,we extracted the DNAs from leaves and constructed two bulk pools.By bulk-sequencing analysis,we preliminarily mapped the target gene of cluster mutant to 42.41Mb-52.23 Mb region on Chr.D13,including a physical distance of 9.82 Mb.We used 14 pairs of polymorphic In Del loci in the initial mapping interval to carry out population detection.According to the statistics of the number of exchange individuals among all cluster phenotype individuals in the（W0×Cluster mutant）F₂ segregation population,the target gene of cluster mutant was further located between S9385 and S9848,corresponding to 44137954 to 45068039 on Chr.D13,with a physical distance of 930 Kb,containing 17 genes.Among these 17 genes,only four genes（GH＿D13G1383,GH＿D13G1388,GH＿D13G1390 and GH＿D13G1391）were expressed in all tissues and organs of TM-1.The q RT-PCR analysis further confirmed GH＿D13G1391 was differentially expressed between the two parents of W0 and cluster mutant.By designing SNP markers for GH＿D13G1391 with parental differences,we found that the marker corresponding to mutant was co-segregated with all clustered phenotype individuals in F₂ segregation population,and preliminarily speculated that the gene was a candidate gene for clustered mutants.3.Transcriptome analysis of W0 and cluster mutants.In this study,we extracted the RNA from the stem tip of W0 and cluster mutant,and sequenced the transcriptome.We obtained 778 differentially expressed genes between the two parents by sequencing,among them,283 were up-regulated and 495 were down regulated.Go enrichment analysis showed that the differentially expressed genes were mainly enriched in auxin biosynthesis and metabolism,protein transport,sugar metabolism,DNA template transcription,termination and glycine biosynthesis and metabolism.The q RT-PCR analysis of W0 and cluster mutant showed that there were significant differences in the expression of auxin synthesis,transportation and signal transduction related genes in the mutant.It was speculated that the genes related to cluster mutation mainly mediated auxin pathway to affect cotton growth and development.Based on the fine mapping results of mutant genes,the expression of 17 genes in the 930 Kb candidate region was further analyzed.The results showed that only GH＿D13G1391 gene was differentially expressed between W0 and cluster mutants,which further confirmed that GH＿D13G1391 was a candidate gene for the cluster mutant.4.Preliminary functional verification of GH＿D13G1391.GH＿D13G1391 gene encodes RNA helicase,has only one pair of genes in Gossypium hirsutum genome.There were SNP differences between W0 and cluster mutant in the CDS sequence and promoter region of GH＿D13G1391,and the difference in promoter region caused the change of cis regulatory elements,which may be the reason for the differential expression of the gene.In addition,we found that silencing of GH＿D13G1391 gene in W0 led to similar phenotype with the cluster mutants 35 days after cotyledon injection.Based on the above results,we speculate that GH＿D13G1391 gene is responsible for the dwarf cluster phenotype,further indepth verification and mechanism analysis are being carried out.