Fine Mapping And Cloning Of The Dominant Glandless Gene Gl₂^e In Cotton （Gossypium Hirsutum L.）

As one of the world’s most important economic crops, cotton has been cultivated for more than7000years, and it is not only an excellent source of natural fiber, but also an important source of nutrients.Pigment gland is unique to cotton. The toxic gossypol stored in pigment glands plays an important rolefor cotton in resistance to certain pests and diseases, but also limits the use of cottonseed nutrition, sothe low gossypol cotton breeding is an important direction of cotton breeding. Dominant glandless geneGle2can effectively suppress the formation of pigment glands, and the glandless character, comparedwith that controlled by the recessive genes, can avoid outcrossing that result in segregation of character.Gle2gene has a very broad application prospects in glandless cotton breeding, and it has importantsignificance to study the Gle2gene.China Cotton Research Institute12(CCRI12) and Dominant glandless line Dgl-CCRI12as well asLiao mian7(L7) and Dgl-L7are near-isogenic lines (NILs) which differ only in the pigment gland trait.In this study, the two pair NILs were used as parents to construct two F2populations for fine mapping ofGle2, which contains1051and2254individuals respectively. The gland character of individuals wasinvestigated and the two F2populations both contain gland, intermediate and glandless plants.Chi-square analys is showed that the segregation ratio of each F2population was1:2:1(χ2=0.831，4.705；χ2<χ20.05=5.991) which is consistent with observations previously reported.According to the previous work in our laboratory, Gle2was located in a0.9cM region on an800kbscaffold. Based on the results and genome sequence (http://cgp.genomics.org.cn), we developed newSSR markers and InDel markers, and a total of16molecular markers were used for fine mapping.Finally we narrowed down the candidate region to a15kb region between InDel markers CS2and CS4and obtained an InDel marker CS3co-segregated with Gle2gene. The mapping results of twopopulations were consistent, proving the accuracy each other.There was only one predicted gene without intron in the candidate region, which contained a1428bpORF and encoded475amino acids. Structure prediction revealed that this protein consisted oftwo domains: bHLH-MYC_N region and bHLH region, which belonged to the bHLH-MYCtranscription factor family. According to the gene sequence analysis in the two pair parents, the gene inCCRI12and L7was identical, as well as in Dgl-CCRI12and Dgl-L7. However, there were three SNPsbetween the genes from gland and glandless parents, one of which leaded to changing of an amino acidlocated in the bHLH-MYC_N region.Real-time PCR analysis revealed that the candidate gene was down-regulated in Dgl-CCRI12andDgl-L7compared with CCRI12and L7respectively. Furthermore, according to the transcriptomes data,the gene was also differential expression between gland and recessive glandless materials, suggestingthat the gene may be involved in the gland organogenesis.For the further functional analys is, the genes and antisense genes from gland and glandless parentswere connected with the pBI121vector respectively and placed under the control of35S promoter to construct expression vector. The recombinants were obtained successfully, which lay a good foundationfor the subsequent functional verification and the application of the gene in cotton breeding.