Studies On The Mechanism Of Formation Of Seedcoat Colour In Brassica Napus L.

Brassica napus L.(2n=38, AACC) is Brassica of Cruclferae. B. napus is an amphidiploid species (AC genome, n=19) derived from interspecific of two diploid species, B. rapa (A genome, n=10) and B. oleracea (C genome, n=9). In the world, B. napus is not only one of the most important of oil crops, but is one of the most important oil crops worldwide. It is also an important source to edible oil and the vegetable protein feed. Besides, Brassica and the model plant Arabidopsis thaliana are belong to the same family, and have some similarities level at the genome sequence. The genes have high degree of sequence identity and remarkably conserved genome structure. Moreover, with the whole genome being sequenced of B. rapa and being sequencing of B. oleracea, we could analyze the genome synteny among them and get the differences and similarities in the structures of Brasica genome and genome structure. It also would provide a theoretical basis for the analysis of genetic evolution and practical implications for improvement of Brassica crops.Studies have demonstrated that in the same genetic background, yellow rapeseed seed has both thinner seed coat and higher seed oil and protein, and lower meal lignin content, cellulose and polyphenol than black seed varieties. In addition, other toxic materials or antinutritives, such as glucosinolates, phytic acid, tannin and sinapine, is lower than the black seed and easy to be digested by the animals and hindering its usage as feed. Therefore, development of yellow seeded lines and cultivars is one of the major breeding objectives in rapeseed, and the inheritance of seed coat colour has been investigated for many years in B. napus by several researchers around the world. However, there are no stable lines of yellow-seeded oilseed germplasm, and the major genes for the yellow-seeded were come from the B. rapa, B. oleracea, B. carinata, B. juncea and their mutation progeny in B. napus. Besides, the inheritance of seed coat color in B. napus is quite complicated, and affected by environmental factors greatly. Moreover, the molecular mechanism of yellow seed trait formation of Brassica is remaining poorly understood.In the process of seed coat development, we analyzed laws of the variations in accumulation of flavonol content and phenolic compounds, and identified differential expression of the transcription factors and main structural genes. It is useful to explain the laws of pigments accumulation and expression patterns or regulation mechanism of gene in B. napus. In addition, based on the constructed linkage groups, a stable major QTL was detected by composite interval mapping using Windows QTL Cartographer2.5, and affecting the seed coat colour of B. napus in different different environments. Moreover, according to the results of comparative genomic and bioinformatics analysis of the major QTL region with Arabidopsis chromosome sequences and related DNA sequences in chromosome A09of B. rapa, and analysis of gene expression response in the peak period of seed coat pigments synthesis in B. napus, some stable differential expression of genes were found and acted as the candidate gene. Then we constructed the overexpression vector and RNAi expression vector of engineering strains and transform by the transformation protocol technique. Some conclusions are as follows:1Accumulation of Proantnocyanins and polyphenols showing the significant difference in seed coat of the yellow-and black-seeded B. napusThe paraffin sections of the different development stages of seed in yellow-(GH06) and black-seeded (ZY821) of B. napus were stained with toluidine blue O. The results showed that the pigments mainly accumulated in the layer of palisade and pigment of seed coat. These compounds began to accumulate in the hilum of seed during the early development stages. The pigments will gradually increase and show the significant difference in the layer of palisade and pigment of seed coat during the seed mature. Moreover, proantnocyanins and polyphenols content is higher in the black-seeded than in the yellow-seeded of B. napus.2The function of enzymes and gene for the synthesis of epicatechin and their derivates may be strongly attenuated in B. napusIn this research, thirty-five phenolic compounds were detected in the flavonoid extracts from ZY821and GH06seed coat according to the retention time analysis of LC-UV-MS, including that13and one compounds could be detected only in ZY821and GH06, respectively. Four types of flavonols derivates were identified, including epicatechin, quercetin, isorhametin, and kaemferol derivates. But in the whole development stages, epicatechin derivates cannot be detected in seed coat of yellow seed coat parent, and the expression level of related genes (BnUGT2and BnTT12) for epicatechin synthesis was also significantly lower in yellow-seeded than in black-seeded. The result showed that the function of enzymes and gene for the synthesis of epicatechin and their derivates may be more strongly attenuated in yellow-seeded than in black-seeded in B. napus.3In-PAs accumulation may be involved in the difference in seed coat colour of B. napusSimilar to Arabidopsis, in-PAs cannot accumulate in the B. napus in early developmental stages of seed coat. Whereas, in-PA began to speedily accumulate and not showed the significant difference between the yellow-and black-seeded in B. napus during the middle-to-late stages. With the accumulation of in-PAs in cell walls, the seed coat colour gradually forming differences in B. napus. 4Correlation analysis among some traits of seed coatCorrelation analysis were carried out among the seed coat colour, seed hull content and seed coat pigments of RILs populations in B. napus L. The correlation studies showed seed coat colour had significant negative correlation with seed hull content and seed coat pigments in the different environments, including the anthocyanidin content, flavonoid content, total phenol content and melanin content. The results showed that it is possible that we can improve some quality traits according to select the yellow-seed traits. Moreover, the syntheses of seed coat colour and seed coat pigments are probably controlled by the same genes or have the same pathway partially.5Differential genes expression of flavonoid biosynthesis pathway may be the key factor for formation seed coat colourUsing the method of qRT-PCR, we analyzed and identified differential expression of the main structural genes and transcription factors of biosynthesis pathway of flavonoid. The results indicated that the expression of nine structural genes(BnPAL, BnC4H, BnTT4, BnTT6, BnTT3, BnTT18, BnTT12, BnTT10and BnUGT2) and three transcription factors (BnTTG1,BnTTG2and BnTT8) showed significant differences between the yellow-and black-seeded. BnPAL, BnC4H and BnTT12encode enzymes specifically involved in the steps of the pathway leading to the biosythesis of precursor of polyphenols and flavonoids compounds and proantnocyanins, and may be the key genes for formation seed coat colour. The BnTT4expression levels reached on the tops at the28DAP, and involved in the expression of downstream genes (BnTT5, BnTT6and BnTT7). Likewise, downstream genes of BnTT3and BnTT18were also regulated by upstream gene. This result indicated that the BnTT4is the first key gene in the flavonoid pathway in B. napus. Moreover, downstream genes were regulated by the upstream genes in the flavonoid biosynthetic pathway. In addition, BnTTGl can also be directly regulated by BnTTG2, and the co-expression of BnTT2, BnTT8and BnTTG1regulates flavonoid biosynthesis pathway in B. napus. Therefore, the function of and the expression patterns of transcription factors in B. napus were also similar with Arabidopsis thaliana.6Linkage map constructionThe recombinant inbred lines (RILs) population was used for the mapping population and derived from a cross between black-seeded male parent cultivar ZY821and yellow-seeded female parent line GH06was established by selfing for seven successive generations with single seed propagating from F2. A high-density genetic linkage map was constructed using JoinMap4.0.1089loci (456for SSR,97for RAPD,451for SRAP and75for IBP) were mapped on19linkage groups, covering2775cM of B. napus genome and the average distance between two adjacent markers was2.54cM. The numbers of markers were varied from6to184on each linkage group. The length of linkage group varied from47.22cM to243.46cM and the average genetic distance was between0.83cM/marker (A09)-7.87cM/marker (C02).7Comparative analysis of genetic mapping with published genetic mapping of B. napusIn this research, comparative mapping and analysis of linkage groups between the RILs and been published in B. napus, and the results showed that the nineteen chromosomes of linkage map showed the consistence of others.237markers of them were found and indicated that the linkage mapping could be universally used in the researches of B. napus. Meanwhile,196markers were integrated to the linkage maps and designed according to the genome sequences of B. rapa and B. oleracea.8Comparative the linkage map of B. napus with the genome of B. rapa and Arabidopsis thalianaThe sequences of all SSRs and IBP makers located on the linkage map were compared with the genome of B. rapa and Arabidopsis thaliana, respectively.237sequences had homologous with the B. rapa and located on the126Scaffold of genome. Extensive macrocolinearity and rearrangement have been found between the A subgenomes of B. napus (An) and B. rapa (Ar). The syntenic between An and Ar subgenomes showed that there were highly homologous in B. napus and B. rapa. Whereas, one marker of436sequences were not found the homologous in Arabidopsis thaliana genome, and some sequences of them were consistent with gene sequences of Arabidopsis thaliana. The result provided strong evidence to support the hypothesis that B.napus, B. rapa and the model plant Arabidopsis originated from the common ancestor.9QTL analysis seed coat colourThis study was to identify QTL which controlled seed coat colour components in RILs (SWU-2) using the composite interval mapping (CIM) method.18of QTLs were detected on7different linkage groups. One major QTLs explaining41.38%-64.17%of phenotypic variation were found and located in the linkage groups A09in different environments. In addition, the minor QTL were also detected and located in the groups A07, C03and C08in different environments, accounting for5.69%-11.04%of phenotypic variation, respectively. Three QTLs with relative smaller effects were also found in the linkage groups A01, A04and C05, respectively. These results have indicated that the seed coat colour was controlled by major and many minor-effect genes, with a pattern of quantitative trait inheritance, and the expression of the QTL was affected by environmental factors.10Analysis of gene expression in the peak period of seed coat pigments synthesis in B. napus In this study, gene expression profiles were analyzed in the peak period of seed coat pigments synthesis (42DAP) using to two parents (ZY821and GH06) and two near-isogenic lines of black-and yellow-seeded in B. napus. There are27genes expressed much higher in black-seeded than in yellow-seeded. These genes are the regulatory factors and transporter protein of gene in the biosynthesis of seed coat pigments, and might be candidates for the seed coat colour gene.11Selection of candidate gene for seed coat colour in the major QTL regionComparative genomic and bioinformatics analysis of the major QTL region with related DNA sequences in chromosome A09of B. rapa supplied by Korea, and covering about440kb interval in chromosome A09of B. rapa. The sequence was submitted to the NCBI website (http://www.ncbi.nlm.nih.gov/) and BRAD database (http://www.brassicadb.org/brad/) for BLAST search. The sequences of major QTL region were found high homologues in the B. rapa and B. oleracea. We extracted the interval sequences and made gene forecast according to the Arabidopsis thaliana genome. The sequence was also submitted to the NCBI website for BLASTP and completed the gene annotation.105genes were found in this region and selected40genes acting as the candidate gene for seed coat colour, according to the results of gene expression in the peak period of seed coat pigments synthesis and differential genes expression of flavonoid biosynthesis pathway. Most of these genes are the regulation factors for pigments biosynthesis and structure gene for coding transporters.12Molecular cloning and functional identification of candidate geneIn this research, we cloned11candidate genes fragments of B. napus. Through transformation of Agrobacterium tumefaciens LBA4404strain, we have received the overexpression vector and RNAi expression vector of engineering strains, respectively. With the transformation protocol technique, we transform the yellow-seed and black-seeded lines with the overexpression vector and RNAi expression vector of candidate genes, respectively. Nowly, we have got some Basta-resistantce plants and transgenic positive plants by multiple PCR identification. The results will provide the foundation for further research of genetic mechanism of yellow-seeded gene, and will be helpful to create the germplasm resources of yellow-seeded in B. napus.