Fine Mapping And Cloning Of Purple Stigma Gene In Rice(Oryza Sativa L.)

Different colors, such as purple, red and white, which occured in rice different tissues, are the result of anthocyanidin accumulation.The anthocyanidin is the material base of different kinds of colors. In this study, we found that indica Xieqingzao(XQZ) has the phenotypes of purple stigma, red apiculus and purple leaf sheath. The dominance phenotype of F1 heterozygous plants and genetic analysis of F2 population derived from a cross between XQZ and the japonica cultivar Kitaake. When the apiculus and purple colors for each F2 individual were compared, only three types of segregants were classified: + +(apiculus and stigma both colored), +-(colorless stigma and red apiculus), and--(apiculus and stigma both colorless), and segregation of + + : +- :-- fitted a 9:4:3 ratio, indicating that the purple stigma phenotype was controlled by two complementary dominant genes, one of which was responsible for the red apiculus phenotypes. One dominant gene was mapped within a 120 kb interval flanked by two Indel markers Z8 and Z20 on chromesome 1 using 1300 F2 individuals with clorless stigma and red apiculus phenotype, and the other dominant gene was mapped within a 106 kb interval flanked by two SSR markers RM19556 and RM19561, using 500 F2 individuals with clorless stigma and colorless apiculus phenotype. Based on the Nipponbare sequences, the 120 kb interval on chromosome 1 contained 15 predicted open reading frames(ORF). Sequencing of these ORFs showed ORF5 had variations between XQZ and Kitaake gene sequence. The ORF5 gene encoded a key enzymes inanthocyanin synthesis, that is dihyrdroflavonol-4-eductase(DFR), named Ps-a. The target 106 kb interval on chromosome 6 contained the gene which was responsible for clored apiculus phenotype in rice,named Ps-b.In order to determine the functions of Ps-a and Ps-b and their roles in control of the formation of purple stigma. We cloned Ps-a and Ps-b genes from XQZ, and transformed them independently into japonica variety kitaake through a grobacterium and mature culture transformation system. Neither Ps-a nor Ps-b alone could recover purple stigma phenotype, even the Ps-b gene could recover the red apiculus phenotype. Further, we have transformed the Ps-a gene into one Ps-b-transgenic line with Kitaake background, and now are waiting for phenotype investigation.