Mapping And Cloning Of The Orange Inner Leaf Gene Or In Brassica Rapa L.

Chinese cabbage originates and widely cultivated in China, which has become one of the mostimportant vegetable crops in the world. The inner leaf of Chinese cabbage is closed to white. Orangehead cabbage was obtained by the hybridization of Chinese cabbage and turnip through the use ofbiotechnologies. Chinese cabbage is rich in nutritional value.In this study, white head Chinese cabbage A21445and orange head one A21530were used as theexperimental materials, for genetic analysis and fine mapping. The high performance liquidchromatography (HPLC) was used for the the analysis of carotenoids components in different materialsleaves. Candidate gene was screened and cloned according to Chinese cabbage genome annotationinformation on targeted area. And the results are as follows:1. A F2population was constructed by homozygous breeding lines white inner leaf cabbageA21445and orange inner leaf cabbage A21530, which was observed during mature time. Thesegregation data from the F2generation (61orange individuals and208white individuals) fitted theexpected segregation for a Mendelian model based on the action of a single recessive allele (χ2c=0.77<χ20.05=3.84).2. BSA method, SSR and InDel markers were used for marker screening and fine mapping. Afterscreened1000pairs of InDel primers and200pairs of SSR primers,8InDel markers were found, thegene was positioned between scaffold000077and scaffold000082.9InDel primers and27SSR primerswere designed in this region. Moreover,11of them were closely linked with the orange gene. Therefore,a gnentic linkage map with19markers and or gene was builded. Mapping of SSR and InDel markers on269individuals of F2population，the or gene was located to a1.4cM interval with the physical distanceof98.904kb on chromosome A09of the B. rapa genome. There were23genes in this region, and thecandidate gene was named or.3. High-performance liquid chromatography (HPLC), coupled with photodiode array detection andcarotenoid standards were used to analyze caotenoid composition. A bit of violaxanthin and zeaxanthinwas detected in the inner leaf of white head Chinese cabbage.9caotenoid compositions were identifiedin the inner leaf of orange variety, such as violaxanthin, lutein, prolycopene (7Z,9Z,7’Z,9’Z-tetra-cis-lycopene), proneurosporene (7,9,9’-tri-cis-neurosporene), pro-ζ-carotene, ζ-carotene,9-cis-β-carotene, β-carotene and neurosporene. HPLC analysis indicated that orange inner leaf ofChinese cabbage is due to the accumulation of prolycopene.4. According to B. rapa genome annotation information and the results of carotenoids pigmentsanalysis, a candidate gene Bra031539, designated CRTISO, was predicted within the mapped or locus,encodes a carotenoid isomerase.5. According to the genome reference information of Bra031539, primers were designed for gDNAand cDNA cloning.200bp sequence of the3’ end was not obtained at the gDNA level. cDNA segmentamplification, cloning and sequencing assembly results showed that there was a deletion of6bp and53 SNPs in coding sequence of the orange type，corresponding to two glutamic acids (Glu) deletion and12amino acids mutations. An internal marker in the gene was developed and verified in F2population.Thefunctional marker was cosegregated with or loucs in F2individuals.