Mapping Of A Novel Anthocyanin Locus (Anm) And Screening Of Candidate Genes In Brassica Rapa L.

Anthocyanins are flavonoid pigments that are responsible for the purple leaves of Chinesecabbage(Brassica rapa)，which is derived from the interspecific hybridization between Chinese cabbageand red leaf mustard (Brassica juncea).Besides the color attributes, interest in anthocyanins hasintensified because of their possible health benefits.The aim of our work was to analyse and tentatively identify anthocyanin species in Chinesecabbage, map and screen anthocyanin-related candidate genes. The genes will be useful in plantbreeding and regulation of anthocyanin synthesis understanding in Chinese cabbage. The results were asfollows:1. We crossed an inbred line of the purple Chinese cabbage ‘B90335-5’, which is pigmented withanthocyanin, with a Chinese cabbage inbred line,‘H165A’, which lacks anthocyanin pigmentation. F2plants derived from a cross between ‘B90335-5’ and ‘H165A’ was examined and qualitative traitsphenotype of color was separated into two groups (purple or no-purple), The presence and absence ofanthocyanin pigmentation was segregated116:172in the F2population, indicating that can notbe readily explained by Mendelian geneticprinciples.2. Using HPLC-MS method, we tentatively identified and analysed anthocyanin species in purpleChinese cabbage and other4Brassica vegetables. The total anthocyanin contents were604.03μg·g-1FWand20anthocyanins were separated and identified in purple Chinese cabbage，including17cyanidin，2delphinidin and1petunidin. In4vegetables except red cabbage，there were three common majoranthocyanins：two acylated cyanidin3-sophoroside-5-glucosides and one acylated cyanidin3，5-diglucosides. Two acylated cyanidin3-sophoroside-5-glucosides and one petundin-3，5-diglucosidewere identified for the first time in purple Chinese cabbage.3. We screened the two bulked DNA samples (purple or green) of the F2plants with SSR and Indelprimers. Nine Indel markers on chromosome A02were found to be linked to the anthocyanin-relatedgene. They were BrID10199，BrID11739，BrID101125，BrID111333，BrID10207，BrID11737，BrID11893，BrID101151，BrID11131. These fragments revealed polymorphism were cloned andsequenced. On the basis of those sequences, we conducted that the anthocyanin-related gene wasderived from the red leaf mustard and located on chromosome A02and named Anm.4. Based on the B.rapa and Arabidopsis reference genome information and blast homologyanthocyanin-related gene between B.rapa and Arabidopsis, the anthocyanin-related gene was located onA02between scaffold063and scaffold007. We analysed the Anm candidate gene in the mapped regionand seven potential candidate genes were identified. They were Bra028793，Bra023441，Bra023594，Bra023602，Bra022700，Bra039763and Bra007957.5. Purple and no-purple Chinese cabbage were tested for identifying candidate genes responsible forthe biosynthesis of anthocyanin by A combination of RNA-seq and analysis of gene differentialexpression based on the next generation sequencing technology. Six genes on A02were selected as potential candidates involved in anthocyanin biosynthesis.They were Bra023441， Bra020688，Bra023594，Bra039763，Bra007957and Bra023602.6. Thirteen anthocyanin-related genes on A02were evaluated for gene expression by quantitative realtime polymerase chain reaction (Q-RTPCR). The expression levels of seven potential candidate genes inpurple Chinese cabbage were greater than in no-purple Chinese cabbage. These potential candidategenes were Bra023441，Bra020688，Bra007957，Bra023602，Bra0202700，Bra023594and Bra032010.7. Based on the results summarized above，we selected four as candidate genes involved inanthocyanin biosynthesis on chromosome A02in Chinese cabbage. They were Bra023441，Bra023594，Bra007957and Bra023602.