Map-based Cloning Of PURPLE HULL 2(Pr2) And Fine Mapping Of PURPLE AWN 1(PA1) In Rice(Oryza Sativa L.)

Accumulation of pigment is an important marker trait in rice.It has significant values in rice classification and genetic breeding.Various studies revealed that the major component of purple pigment in rice was cyanidin-3-glucoside,one of three major types of anthocyanins.Anthocyanin synthesis is an extensively studied secondary metabolic pathway which can provide a classical model of regulatory system in plants.Although several genes participated in anthocyanin biosynthesis pathway have been cloned up to now,the regulatory mechanism of anthocyanin biosynthesis in rice is still not clear.To study the molecular mechanism of pigment distribution in rice,two backcross populations were developed and two genes,PURPLE HULL 2(Pr2)and PURPLE AWN 1(PA1),were fine-mapped and cloned following the strategy of map-based cloning.An introgression line with segregation of purple hull phenotype was obtained through backcross using Xiaozhanjiangmidao(with purple hull)as a donor parent and Nipponbare(colorless)as a recurrent parent.The segregation ratio of BC2F2 population indicated that the purple hull trait is controlled by a dominant gene,named PURPLE HULL 2(Pr2).Following the methods of map-based cloning,Pr2 was finally mapped to a 49.03-kb region between two Indica-Japonica polymorphic markers,Chr01MM3965 and Chr01MM3971,using BC2F2 population and BC2F3 families.In this region,there are 4 genes according to the prediction of RAP-DB.Sequence analysis showed that only one gene with 7 SNPs in the predicted exon regions was found.Further sequences alignment analysis and conserved domain search indicated that a variation from C to A in the second exon leads to a TAG stop codon that could chang the function of Pr2.To confirm the function of Pr2,the complementary and RNAi vectors about Pr2 were constructed.The expression analysis showed that the Pr2 had a high expression in the hulls at the stage of flowering and leafs.In order to explore the mechanism of light-induced expression of Pr2,the 1.5-kb promotor region of Pr2 was screened and a G-box motif that is a detailed studied light response element in plants was found.5 genes were screened out by the phylogenetic analysis among 26 GT-family genes and 3 GT-1 clade genes in Arabidopsis.Expression analysis showed that Os04g40930,Os04g51320 and Pr2 had a similar pattern.We conjecture that two genes may participate in the light-induced expression of Pr2.A near-isogenic line with purple awn phenotype was obtained through backcross using Damangdao(with purple awn)as a donor parent and Nipponbare(colorless)as a recurrent parent.The segregation ratio of BC5F3 population indicated that the purple awn trait is controlled by a dominant gene,currently named as PURPLE AWN 1(PA1).Genetic mapping by bulked segregation analysis and linkage analysis revealed that the PA1 locus was located in the region between two SSR markers,RM19552 and RM19583,on the short arm of chromosome 6.For physical mapping,5 polymorphic markers and 642 recessive plants of BC5F3 population were used and the PA1 locus was finally narrowed to a 50.17-kb interval between Indica-Japonica polymorphic marker,Chr06MM0667,and SSR marker,RM 19565.In this region,5 genes were predicted by RAP-DB,and one gene OsC1 associating with anthocyanin synthesis was found to have a 3-bp(GAG)insertion in the second exon from Purple Awn NIL compared with that of Nipponbare.Futher analysis of conserved domains showd that glutamic acid derived from 3-bp insertion located in the high conserved domain,MYB binding domain.Thus,we speculate that the 3-bp insertion in OsC1 is responsible for the purple awn characteristic in rice(Oryza sativa L.).