Dissecting The Molecular Mechanism Of Yellow-and Black-seeded Brassica Napus

Yellow-seeded Brassica napus has several admirable advantages over its black-seeded phenotype, including thinner seed coat, higher oil and protein content, lower lignin and fiber content, and purer oil quality. Development of yellow-seeded cultivars is becoming an optimum pathway to improve rapeseed quality and quantity. The objective of this study was to conduct a detailed comparison of yellow-seeded progenies from B. napus-Sinapis alba hybrids and black seeds of B. napus. With the assistant of NIRS, FTIR and NMR analysis, we compared the quality differences between yellow-and black-seeded materials. LC-ESI-MSn analysis of flavonoid extracted from yellow-and black-seeded B. napus revealed the different accumulation and content of these chemicals. Comparative proteomics of seed filling between yellow-and black-seeded materials was taken out using two-dimentional electrophoresis (2-DE). Oil body proteins extracted from yellow and black seed B. napus, S. alba were quantitatively compared using iTRAQ and Q-TOF MS/MS. DGE analysis was also applied to discriminate transcriptome difference between yellow-and black-seeded B. napus, and the differentially expressed genes related to flavonoid biosynthesized pathway were verified using qRT-PCR. The research would aid in dissecting the genes related to the formation of yellow-seeded B. napus. The main results were shown as followings:Quality analysis of yellow-and black-seeded B. napus NIRS analysis revealed that yellow seed lines used in this research possess higher oil and protein content, low glucosinolate and erucic acid than black-seeded Yangyou6. The content of NDF, ADF and ADL in yellow seeds was lower than Yangyou6. ATR-FTIR analysis showed many differences in lipid feature, protein feature were observed between yellow-and black-seeded B. napus, and the FTIR spectra was also different among yellow seed lines. Variation of carbohydrate features in yellow and black seed materials was not obvious. By comparing the NMR spectra of yellow and black seed materials, we found the difference between S. alba and Yangyou6were most obvious at peaks representing for lignin. The resonance peaks standing for lignin and fiber were stronger in Yangyou6than S. alba and yellow seed lines, which are consistent with the perspective that yellow seed material possess lower fiber and lignin than black B. napus.HPLC analysis of flavonoids in yellow-and black-seeded B. napus We applied LC-ESI-MSn to identify phenylpropanoid and flavonoid in rapeseeds. The most abundant phenolic compounds (sinapine and sinapic acid) and1,2-disinapoylglucose,16different flavonoids were identified and quantified, including (-)-epicatechin and its five monocharged oligomers, quercetin, kaempferol, isorhamnetin-dihexoside, kaempferol-sinapoyl-trihexoside, isorhamnetin-sinapoyl-trihexoside, isorhamnetin-hexoside-sulfate, and isorhamnetin-3-O-glucoside. Most of the flavonoids accumulated with seed development, whereas some rapidly decreased during maturation. The content of these flavonoids was lower in the yellow-seeded materials than in the black seeds. In addition, variations of insoluble procyanidin oligomers and soluble phenolic acids were observed among both rapeseed varieties.Proteomics analysis of seed filling between yellow-and black-seeded B. napus Comparative proteomics of seed filling between yellow and black seed were taken out to find proteins related to yellow seed character. By using two-dimensional electrophoresis (2-DE) and MALDI-TOF-MS analysis, eight out of the27discriminate proteins were identified by MASCOT comparison, including protein kinase, enolase, triosephosphate isomerase and dioxygenase. PCR primers contrived for the putative genes were applied for further identification of progenies and both parent, indicating H3-5might be the novel protein of intergeneric hybrid, H5-2derived from S.alba. Applying these specific primers, this study demonstrates that the new yellow seed germplasm is different from the existing yellow seed materials.Comparison of oil body proteins in yellow-and black-seeded B. napus Oil body from Yangyou6,S. alba, yellow seed line were purified for protein extraction. The oil body protein were digested and labeled with iTRAQ reagents, and peptides were analyzed and quantified by Q-TOF MS/MS mass spectrometer. Finally, we successfully identified3091unique peptides representing for1511proteins, including fifteen oleosin proteins, five steroleosin and three caleosin. Besides the main OB proteins, beta-glucosidase and alpha-glucosidase, myrosinase, myrosinase-binding protein, myrosinase-associated protein, embryo specific protein (ATS), heat shock protein (HSP) and peroxiredoxin were also identified. Obvious contamination of storage proteins, including cruciferin and napin were found in protein storage vesicles. Besides, mitochondrial import inner membrane translocase, mitochondrial import receptor, secretory carrier membrane protein, mitochondrial ATP synthase and ATP binding protein were also included as contamination from mitochondria. Among all the identified proteins,270proteins displayed significant differences (p<0.05) between W82and S. alba, of which92proteins were up-regulated and178proteins were down-regulated in W82.167proteins were up-regulated and86proteins were down-regulated in S. alba compared with Yangyou6.27proteins were up-regulated and37proteins were down-regulated in W82compared with Yangyou6. Clustering analysis of differentially expressed proteins between each comparison (Bn vs W82, Sa vs W82, Bn vs Sa) was carried out,85identified proteins were found to have commonly changed in each comparison, of which only three oleosins were included (oleosin S1-2, oleosin S4-1and oleosin S4-3).Digital gene expression (DGE) analysis on seed filling of yellow-and black-seeded B. napus We compared gene expression difference of yellow seed line (W82) and Yangyou6using DGE analysis. By GO annotation and pathway analysis of differentially expressed genes (DEG), we selected DEGs that might be related to yellow seed character. Obvious gene expression difference was found in the flavonoid biosynthesis pathway, fatty acid biosynthesis, fatty acid metabolism. Transcriptome changes of genes participating in the flavonoid pathway were discovered by qRT-PCR analysis. Consistent with flavonoid changes identified by HPLC analysis, the expression for most genes in the flavonoid biosynthetic pathway was also downregulated.Genetic analysis of the new yellow-seeded B. napus In this research, the yellow seed line was hybridized with Yangyou6to obtain Fi and F2, the seed coat color of this two generation was analyzed. F1generation was also backcrossed with the yellow seed parent and Yangyou6, respectively. By calculation of the seed coat color segregation, we deduced that the yellow seed character was controlled by two pairs of independent genes. The yellow seed phenotype was presented only when both pairs of genes were recessive.