Studies On The Molecular Mechanism Of Yellow-seeded Formation In Brassica Juncea

Rapeseed is one of the major oilseed crops over the world. High quality is the focus of rapeseed breeding in many countries, including decreasing the contents of erucic acid, glucosinolates, fiber and linolenic acid, and increasing the contents of oleic acid and protein. Yellow seeds have such advantages as thinner seed coat, higher oil content, clearer oil, higher protein and lower fiber contents compared with black seeds of the same genetic background. However, most of current commercial rapeseed cultivars are not yellow-seeded but black-seeded. It will therefore have a bright future to develop yellow-seeded rapeseed varieties. Unfortunately, there is no report on indigenous yellow-seeded germplasm in Brassica napus which is widely grown in the world at present. Although some yellow-seeded Brassica napus cultivars have been developed through wide cross or induced mutation, their yellow-seeded character is genetically unstable and their seed coats are not evenly yellow. Furthermore, the mechanism of formation of yellow seeds in Brassica remains unknown. Hence, it is difficult to develop superior yellow-seeded Brassica napus cultivars nowadays.The seed coat color is a qualitative character in Brassica juncea. The yellow seed is recessive to the black seed and is shown to be controlled by one or two loci. In this study, the inheritance of seed coat color was investigated; the molecular markers tightly linked to the gene for seed coat color were found; the chemical composition of seed coat was analyzed; the genes for pathway of flavonoid biosynthesis were cloned; and expression of these genes was analyzed through reverse transcription-polymerase chain reaction (RT-PCR) in Brassica juncea. The results obtained are summarized as follows:The embryos are yellow whether the yellow-seeded or black-seeded lines. The seed coat of the yellow seed is transparent, but that of black seeds is black. The apparent color of yellow seed is determined by the embryo color. The seed coat color is controlled by 2 duplicate loci, with plants having a dominant allele produce black seeds. Using an inbred line of Sichuan Yellow (SY) as the recurrent parent and Purple-leaf Mustard (PM) as the donor parent, one BC₅F₂ segregating population was developed and used for mapping the gene controlling seed coat color. The two markers, designated as SZ1-₃₃₁ and SCAR57-₃₈₃, were found to be linked to the allele for brown seed coat. The recombination frequency between either of these markers and locus for seed coat color was 2.35%. These markers were linked to the marker RA2-A11 previously published, located at the same side of the locus and 2.5cM away from the target gene.Contents of polyphenol, anthocyanidins, melanin, cellulose and lignin in the seed coat were spectrophotometrically determined. The PM seed coat has 3.26 times as anthocyanidins, 3.73 times melanin, 3.26 times lignin, 1.16 times polyphenol and 0. 99 time cellulose as the SY seed coat. On basis of the shift direction and the shift quantity of the spectral bands I & II, it could be deduced that ring A of the flavanol-phenolic molecules might contain o-dihydroxyI in the polyphenol of black seed-coats. It is proposed that the structural differences of the polyphenol might be the major factor resulting in the color difference between the yellow and the black seed coats. Staining of the developing seed coat by vanillin, which specifically bind to flavanols, showed that flavan-3,4-diols (leucoanthocyanidins) and flavan-4-ols are present in the black seed coat while they are absent in the yellow seed coat. Staining of mature seed coats by p-dimethylaminocinnamaldehyde, which specifically bind to proanthocyanidins, showed that the black seed coat stained blue while the yellow seed coat remained unchanged, indicating that there is no proanthocyanidins in the yellow seed coat. It is proposed that proanthocyanidins is a major component which brings about color difference between the yellow and the black seed-coats in Brassica juncea.Homology-based cloning strategy was used to clone the genes for pathway of flavonoid biosynthesis in Brassica juncea. The primers were designed according to the sequences of homologous genes of Arabidopsis thaliana. The 26 gene copies were sequenced and analyzed by BLAST and were shown to be homologous to 21 known Arabidopsis thaliana functional genes. The genes CHS and PAP have each 3 copies, while the gene CHI has 2 copies. Only one copy was obtained for the remaining 18 genes. Search in the database of GenBank revealed that these genes except the gene for GST were cloned for the first time in Brassica juncea. No data were found in GenBank about the genes F3H, LAC, TT12, TT1, TT8, TT16, TTG1, TTG2, AHA10, BAN, PAP and FLS in Brassica species.On the basis of the cloned genes for flavonoid biosynthesis in Brassica juncea, the primers were re-designed and used for RT-PCR analysis of the seeds and seed coats of SY, PM, NILA and NILB 15 and 25 days after pollination (DAP). The expression of PAP and AHA10 were not detected in all the seeds and seed coats studied. The expression of structural genes PAL, C4H, CHI, F3H, F3'H, LAC, TT12, GST, FLS and regulatory genes TT1, TT2, TT8, TT16, TTG1, TTG2 was detected in the seed and seed coat, but no drastic difference was observed among the 4 lines examined. The black-seeded lines have higher CHS expression in the seed coats 15 and 25 DAP compared with the yellow-seeded line. The expression of the genes DFR, BAN and ANS was detected in the seed coats of the black-seeded lines while their expression was not detected in the seed coat of the yellow-seeded line. However, the expression of the genes DFR,BAN and ANS were detected in seeds of the all lines studied.It has been proposed from these results that the seeds produce transparent testa and appear yellow because of no expression of DFR,ANS and BAN in the seed coat of Brassica juncea.