Fine Mapping And Predicting The Candicate Gene Of Orange Head In Chinese Cabbage

Because of orange head Chinese cabbage contains more carotenoids than the white head Chinese cabbage, many consumers and researchers have been focus on it. Orange head Chinese cabbage is the qualitative trait which is controlled by a single recessive gene. In this study, orange head Chinese cabbage and white head Chinese cabbage were selected as the test materials. Based on the previous work, we located the target gene which controlled orange head Chinese cabbage traits for fine mapping. The sequence characteristics of target genes and the reason of two both materials performed the different characters were analyzed. The gene expression pattern of orange head Chinese cabbage and the enzymes in carotenoid biosynthesis were analyzed using quantitative real-time PCR （qRT-PCR） technology. The molecular markers were developed in the promoter region and verified in different varieties Chinese cabbage. The main results are as follows:1. The white head Chinese cabbage Chiifu and the orange head Chinese cabbage 07A163 were used to build a larger F2 segregation population and identified 2200 homozygous recessive individuals as the fine mapping population. Two SSR markers, syau26 and syau28 positioned the target gene on A09 chromosome, and the physical distance was about19.9 kb. A total of six candidate genes were inside this region and Bra031539 gene was the predicted gene which is encoding a carotenoid isomerase enzyme. This enzyme is the specific isomerase in carotenoid biosynthesis.2. The full-length of Bra031539 gene was 3402 bp in white head Chinese cabbage Chiifu, thirteen coding sequences （CDS） encodes 589 amino acid sequences. TargetP analysis showed that Bra031539 protein was likely the chloroplast transport peptides. The protein sequence pattern analysis by Prosite showed that Bra031539 protein contains twelve casein kinase Ⅱphosphorylation sites, eight protein kinase C phosphorylation sites, one cAMP-and cGMP-dependent protein kinase phosphory-lation site, thirteen N-myristoylation sites, two tyrosine kinase phosphorylation sites, three N-glycosylation sites and aleucine zipper pattern. SMART analysis showed that Bra031539 proteins exist four domains:Pfam:DAO domain, FAD_binding₂ domain, NAD_binding₈ domain and Amino_oxidase domain.3. Sequence analysis showed that, comparing to Chiifu, Bra031539 in 07A163 have 88 bp deletion and 88 SNPs in the promoter region, six bases were deleted in CDS1 which resulted two amino acids translation were not normal, the mutation of 43 SNPs results in 15 amino acids were changed, the shift mutation was happened in the CDS 13 and leads to protein translation premature termination, the bases deletion occurred in the 3’UTR.4. Bra031539 was cloned and sequenced in three different colored head Chinese cabbage found that compared with white and yellow materials,88 bp and 7 bp were lost in six orange head Chinese cabbage and 30 SNPs were found in six orange materials. Coding region sequence analysis found that 6 bp were deleted in CDS 1 and 12 SNPs were found in six orange head Chinese cabbages and 15 SNPs were found in three materials containing 07A163, Ju65 and Ju62. The frameshift mutations occurred at CDS 13 terminal and resulted in premature termination of protein translation. The deletion was in the 3’UTR.5. According to the sequence difference of Bra031539 promoter region in different color head materials, an Indel marker-BrProl was developed and the tag can be used for marker-assisted selection in orange head trait.6. Analyzing the expression differences of Bra031539 in different color head materials found that the expression of Bra031539 gene of three color materials in vernalization stage, seedling leaves, roots, stems were relatively lower and the difference is not obvious. According to the effect of developmental stages, expression organ and optical signal conditioning, the expression of Bra031539 gene has a relatively high amount of expression of the leaves and petals and the difference is remarkable. Flowering period is the highest relative expression in Bra031539 gene, the expression level of the cabbage leaves in heading stage has reduce gradually from the outside to inside. The lowest expression level is inner leaves in 07A163.7. According to analysis the gene transcription of carotenoid biosynthesis in Chiifu and 07A163, the expression of 12 related genes between two materials were not significant difference in vernalization and seedling stage by the light and developmental stage and organ effects. However, PSY gene expressing significantly in two materials at seedling stage.8. The relative expression of carotenoid biosynthesis enzymes in both Chinese cabbage cabbage period were significantly different. According to the early response, the substrates of catalysing enzyme IPI, GGPS, PSY, PDS, and ZDS were expressed increasingly compared with the white material. The subsequent enzyme catalyzed substrates, such as LCYB, LCYE, CHXB, ZEP, VDE, CCD and NCED were expressed lower in orange material.