Mapping And Cloning Of Yellow-petalled Gene BolC.cpc-1 In Brassica Oleracea L.

Cabbage(Brassica oleracea L. var. capitata) and Chinese kale(Brassica oleracea L. var. alboglabra) are two varieties of Brassica oleracea of cruciferae. Cabbage usually has yellow petals, while Chinese kale has two types, white and yellow. In order to reveal the molecular mechanism of petal color formation in B. oleracea, cabbge inbred line YL-1(yellow petal) and Chinese kale inbred line 11-192(white petal) were used in this study for inheritance analysis and fine mapping of petal color gene. Candidate gene was screened and cloned according to the genome annotations among the mapping region. The results are as follows:1. Genetic analysis of petal colorCabbge inbred line YL-1(yellow) was crossed with Chinese kale inbred line 11-192(white) to construct the F2 and BC population. Petal color traits were investigated during flowering time. In the 344 individuals of F2 population, 271 had white petals and 73 had yellow petals. This segregation conformed to a Mendelian ratio of 3:1 by the chi-square test. The segregation ratio(253 white: 238 yellow) in the BC1P1 conformed to the ratio of 1:1. These results suggested that in this study the inheritance of petal color followed a monogenic pattern, white petal being dominant over yellow petal.2. Fine mapping of petal color geneBSA method was used to screen 1093 pairs of InDel primers(including 95 pairs designed based on whole genome re-sequencing data of the parents). InDel marker M4131, linked to petal color trait, was located at chromosome C03 of cabbage. More primers were designed and larger population(containing 1251 recessive individuals) was constrcted for fine mapping of the gene. A genetic map and a physical map comprising 11 InDels were constructed. InDel markers M4085 and M4089 were closest to the gene, flanking it with genetic distances of 0.88 cM and 0.16 cM respectively, the interval of these two markers is about 207 kb. Moreover, the genetic map developed herein suggested that Scaffold000063 was a misanchored scaffold in Bolbase or BRAD cabbge database.3. Candidate gene analysisThe target region contained 14 genes according to annotations of B.oleracea and Arabidopsis thaliana. In which, Bol029878 encoding carotenoid oxygenase, is the most possible candidate gene, named BolC.cpc-1 in B.oleracea.Primers were designed for BolC.cpc-1 gene cloning. Sequence assembly showed that BolC.cpc-1 in Chinese kale with 1791 bp was the same as Bo3g158650, the reference gene in TO1000 genome sequence, while in YL-1, BolC.cpc-1 had three InDels(1bp insertion, 7bp deletion, 1bp deletion at position 308 bp, 771 bp, 1094 bp, respectively) and 34 SNPs. We inferred that these insertion/deletions and SNPs resulted in the function disruption of BolC.cpc-1 in YL-1. An internal marker D1 in the gene was developed according to the 7bp deletion, and was verified to be cosegregated with petal color. The candidate gene was verified through the determination of carotenoids in petals of two parents.