Carotenoid identification and molecular analysis of carotenoid isomerase-encoding BrCRTISO, the candidate gene for inner leaf orange coloration in Chinese cabbage

摘要

In Chinese cabbage (Brassica rapa L. ssp. pekinensis), the locus associated with orange inner leaves has been mapped. The mechanism responsible for carotenoid pigment synthesis in orange inner leaves of Chinese cabbage is still unclear, however, and limited molecular research has been conducted on the candidate gene. In this study, high-performance liquid chromatographic analysis indicated that orange leaf coloration in Chinese cabbage is due to accumulation of prolycopene and other carotenoid pigments. Mapping of simple sequence repeat and insertion/deletion markers from 269 individuals of an F-2 population pinpointed the Bror locus within a 1.0-cM interval, with the physical distance of 65.555 kb, on B. rapa chromosome A09. Using B. rapa genome annotation information, the candidate gene BrCRTISO (Bra031539) was predicted to reside within the mapped Bror locus. Coding and promoter regions were sequenced in white-and orange-type homozygous lines. A 6-bp deletion and 53 single nucleotide polymorphisms (SNPs), corresponding to two glutamic acid deletions and 12 amino acid mutations, were identified in the coding sequence of the orange-type cultivar. The orange-type promoter was approximately 141 bp shorter than that of the white type, and many SNPs were located in the promoter region. Two functional markers were designed and found to cosegregate with the Bror locus in F-2 individuals. Expression analysis demonstrated that BrCRTISO transcription levels were higher in white-type Chinese cabbage than in the orange-type cultivar. These results offer insights into the molecular mechanism of carotenoid biosynthesis and lay the foundation for molecular breeding of orange-type Chinese cabbage.