Identification Of QTLs (BnSCA09) And Candidate Genes For Yellow-seed Trait In Brassica Napus L.

Brassica napus L. is not only one of the most important oilseed crops in worldwide, but also is an important source of vegetable protein feed and the edible vegetable oil in our country. In the same genetic background, studies have confirmed that yellow-seeded lines have significantly thinner seed coat, with more transparent oil, higher protein content, lower hull proportion and lower fiber, lignin and polyphenol content than in black-seeded lines. Meanwhile, other toxic materials content, like that glucosinolates and tannin is lower than the black seeds. Therefore, development of yellow seeded cultivarsis acted as an important breeding objective with rapeseed. Howerer, the inheritance of seed coat color is very complicated, existing polygene, allotetraploidy and maternal effects, and involving the influence of lighting and temperature factors. Recently, the development of molecular marker technology accelerated the gene mapping for B. napus seed coat color and the selection of linked markers, and numerous loci associated with seed coat color have been mapped using different populations of B. napus, accounting for 40-60% phenotypic variation by single QTL.Although many molecular markers linked to these loci have also been identified, the major gene for seed coat color was still not successfully cloned in B. napus untill update.Therefore, in this research, a SNP high-density genetic linkage map of B.napus was constructed for QTL mapping, and then to select more candidate genes controlling seed coat colour in B. napus. In addition, using the homology-based cloning strategy, each of full-length ORF sequences was sequenced and analyzed at the levels of both nucleic acid and protein. Besides, the transcript profile of candidate genes was identified using the quantitative real-time PCR. Finally, we constructed the overexpression vector and RNAi expression vector of candidate genes, and transformed them into the black-seeded and yellow-seeded rapeseed with the transformation protocol technique. It will lay the foundation for further research of them. The main conclusions are as follows:1. Linkage map construction and QTL detection for seed coat color in B. napusUsing genome-wide single nucleotide polymorphism (SNP) markers assayed analyzed by the Brassica 60 K Infinium BeadChip Array, a high-density genetic linkage map was constructed in the recombinant inbred lines (RILs) population, which drived from a cross between the black-seeded male parent Zhongyou (ZY) 821 and yellow-seeded female parent GH06. The map consists of 34 linkage groups,9014 SNP markers and covers 1209.52 cM of B. napus genome with the average distance of 0.13 cM between two adjacent markers. The length of chromosome A09 was 76.30 cM including 384 SNP markers with the average distance of 0.19 cM between two adjacent markers. Here, using the CIM method, one stable major QTL associate with seed coat colour was detected on A09 in different environment, accounting for 41.38%-64.17% of the phenotypic variation. And then the likelihood interval was reduced by 200 kb in the flanking QTL region by screening the genotype of each RIL, a large population of F2 and BC3F2 progeny using additional genetic markers, which were syntenic to regions of the B. rapa genome and Chr.1 of Arabidopsis genome. According to the Arabidopsis genome, the extracted interval sequences were submitted to the NCBI website for BLASTP analysis and completed the gene annotation. Eventually,23 annotated genes were found in the major QTL region, using for the further study.2. Selection and identification of candidate genes for seed coat colourUsing the homology-based cloning strategy, each of full-length ORF sequences was abtained from the seed coats of yellow- and black-seeded B. napus with different genetic background and analyzed in the levels of both nucleic acid and protein.The results showed that SNPs in BnSC04, BnSC10, BnSC11 and BnSC20 were identified among the yellow- and black-seeded lines, and part of these SNPs resulted in mutations amino acids. Consistence with the SNP loci, the corresponding mutation amino acid sites were detected only in BnSC10 (52,116,123,142,184,188,198,206,217 and 222). These results indicated that the mutation of SNPs polymorphism may not be involved in the mutation of amino acid, and consistency difference in BnSC10 may involved in the difference formation of the seed coat colour between the yellow- and black-seeded B. napus. In addition, for unraveling the temporal and spatial specificity of candidate gene, the expression patterns of BnSC04, BnSC10, BnSC11 and BnSC20 were detected in the yellow- and black-seeded rapeseed of different development stages. The results showed that the similar expression pattern was found between BnSC04 and BnSC20, but the expression level showed differences among the yellow- and black-seeded rapeseed. Additionally, the expression levels of BnSC10 and BnSC11 in black-seeded rapeseed were significantly higher than in yellow-seeded. The expression levels of BnSC10 in black-seeded rapeseed was gradually increasing during the seed coat development, and peaking at 50 days after flowering, which was later than in yellow-seeded rapeseed. The transcript profile of BnSC11 peaked at early stages of seed development, but was lower or no expression in later stages. Moreover, the expression levels of these genes showed different patterns in different yellow- and black-seeded lines, indicating that the action sites or mechanism of these candidate genes may be different during the seed coat development in rapeseed. Recently, we had constructed the overexpression vector and RNAi expression vector of four candidate genes, and transformed into rapeseed using the transformation protocol technique and got the transgenetic plants. And the function identification of them will be further studied in the future work.