Map-Based Cloning and Functional Analysis of Dark Brown Fiber Gene Lc1 in Upland Cotton

Functional Genomics

oral

yan, Qian ; Xiao, Yuehua; Liu, Housheng ; Ding, Hui; Li, Xin; Yao, Dan

yan, Qian

Xiao, Yuehua

Brown cotton fiber is the most widely used in the modern naturally-colored cotton industry. It was reported that brown pigments in cotton fiber belong to proanthocyanidins (PAs) and PA derives. Dark brown fiber gene (Lc1), mapped on Chr. 7, is a semi-dominant gene conferring dark brown color to fiber in upland cotton. To further understand the regulatory mechanism of brown pigmentation, we aimed to clone the Lc1 gene and to clarify its role in regulating PA biosynthesis in cotton fiber. R2R3 MYB protein TT2 plays a key role in controlling PA biosynthesis in Arabidopsis seed coat. To reveal the possible relatedness of cotton TT2 homologs to the regulation of PA biosynthesis in fibers, we identified 5 cotton TT2 homologous genes in D genome (GrTT2-1~5), and cloned 10 corresponding genes from G. hirsutum (GrTT2-1A, 1D~5A and 5D). Among these genes, only GhTT2-3A showed a significant variation in expression levels between dark brown (Lc1Lc1) and white fibers, suggesting that GhTT2-3A was related to the PA biosynthesis in dark brown fibers. Furthermore, GhTT2-3A was constructed downstream to a constitutive promoter (CaMv35S) and transformed into cotton. The expression levels of PA structural genes and PA content in GhTT2-3A overexpressing calli were significantly increased. This indicated that GhTT2-3A could activate the whole PA pathway and promote PA biosynthesis and accumulation in cotton. To determine the positional relationship between GhTT2-3A and Lc1, we perform fine-mapping in GhTT2-3A region using a RIL population, derived from a cross of a dark brown-fiber line T586 (Lc1Lc1) and a white-fiber cultivar Yumian No.1, and a secondary F2 population of 1698 individuals. Based on a BAC containing GhTT2-3A and comparative cloning of corresponding sequences in T586 and Yumian No.1, 9 polymorphic SSR/SNP makers were identified and further genetically linked to the Lc1 gene. Lc1 was finally mapped in a region between markers TT2-4a and TT2-3a3y. According to the sequenced D genome, this region harbored only two coding genes, GrTT2-3 and GrTT2-5. In conclusion, the data on expression analyses, transgenic cotton and genetic mapping collectively demonstrated that cotton TT2 homologous gene GhTT2-3A controlled PA biosynthesis in brown cotton fibers, and may be the dark brown fiber gene Lc1.