| Rapeseed is one of the major oil crops in the world, and it is an important edible oil,protein feed and industrial energy crops. Improving oil content and quality of rape seeds is an important research topic. Numerous studies showed that the oil content of yellow-seeded is generally higher than the brown-seeded and black-seeded in the same genetic background.Yellow-seeds have some advantages, such as thin coat, clear and transparent oil and high protein content. However, there is no natural yellow-seeded material in Brassica napus. So far,breeders have studied the yellow-seeded formation mechanism and genetic characteristics in Brassica juncea and Brassica rapa, it is desired to cultivate yellow-seeded Brassica napus with stable inheritance through hybridization, gene transfer and so on.In our research, a yellow mustard local variety spread in northern Shaan xi, Wuqi yellow mustard(Brassica juncea L.), has been focused. Due to the special geographical location, the oil content of this yellow mustard is high, the inheritance is stable and the seeds perform pure yellow seed, therefore it is considered as the ideal material for studying yellow-seeded gene.In this study, a BC6S1 derived from Wuqi yellow mustard and Wugong mustard has been constructed, which were used for phenotype, genetic analysis, fine mapping of the yellow seeded gene, candidate gene analysis construction of over-expression vector and silencing vector of candidate gene. The results were as follows:1. Phenotypic characterization and genetic analysis: A BC6S1 segregated population consisting of 1216 individuals derived from a yellow-seeded land-race ’Wuqi mustard’ and a brown-seeded land-race ’Wugong mustard’ was developed for gene mapping. The phenotype underlying seed coat color of BC6S1 individuals were determined according to the phenotypes of the BC6S2 and BC6S3 seeds. There were 293 homozygous yellow-seeded individuals, 307 homozygous brown-seeded and 616 heterozygous brown seeded in the 1216 BC6S1 populations. The chi-square test showed that the segregation of 3 genotypes was consistent with the expected ratio of 1: 2: 1(χ2 = 0.22, p≤0.05). This result was further proof of yellow seed of northern Shaanxi yellow mustard controlling by a single gene, and the brown seed is dominant over the yellow seed.2. Fine mapping of the yellow-seeded gene: previous studies showed that the yellow-seeded gene of yellow mustared was located on the A09 chromosome. In this study,we enlarged the plant number of mapping populationto1216 individuals, 63 pairs of primers were designed. As a result, 10 IP and SCAR markers were successfully developed, IP4 and Y1 were located on either side of the yellow-seeded gene at a distance of 0.1 and 0.3cM,respectively. IP1, IP2, IP3 markers cosegregated with the target gene. BLAST analysis indicated that the sequences of newly developed markers showed good collinerity with those of the A09 chromosome, and that the target gene might exist between 27.079-27.616M(about 0.54Mb)3. Analysis of yellow seed candidate gene: 95 genes in the 0.54 Mb region have been selected for structure and function analysis. The results showed that only Bra036828 is related to flavonoid biosynthetic pathway, and it was highly homologous with F3 H gene from Arabidopsis, thus the Bra036828(designated as BjF3H-b1 in Brassica juncea) was considered as the candidate gene preliminary.4. Construction of over expression and silencing vector: the target gene was amplified for two times, pCAMBIA1301 expression vector and PCR products were connected by fast ligase, and the ligation products were transformed into E.coli DH5α, the recombinant plasmid containing the target gene was screened, and an overexpression vector BjF3H-b1-1301 and RNAi expression vector silence2-1301 of the candidate gene BjF3H-b1 were successfully constructed. PCR and digestion methods were used to validate the recombinants, and the verified plasmids were transferred into Agrobacterium tumefaciens GV3101 to construct an expression vector. |
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