Comparative Analysis On The Difference Of Seed Coat Color Regulating Mechanisms Between Brassica Rapa L. And Brassica Napus L.

Rapeseed is one of the most important oil crops in the world.Compared with black-seed rapeseed,yellow-seed rapeseed has many advantages,such as clear oil,high fat and protein content,low husk percentage and high feed value of cake.At preasent,there are natural yellow-seed resources in Brassica rapa,Brassica juncea and Brassica carinata,but there are no natural yellow-seed resources in Brassica napus,which is the most widely planted.The major seed coat color gene in B.napus is still unclear,which limits the application of yellow seed in this field.It is difficult to study the causes of seed color in B.napus by using yellow-seeded B.napus materials alone.B.rapa is an important yellow-seeded gene donor of Yellow-seeded B.napus.As one of diploid parents of B.napus,it is closely related to its genetic evolution.Compared with B.napus,the genetic regularity of seed coat color in B.rapa is relatively simple.It is also widely cultivated with abundant genetic resources.It was found that seed color formation was closely related to flavonoid metabolism pathway,and flavonoid pathway genes(TT genes)also played a certain role in the formation of seed color in Arabidopsis thaliana.In summary,this study was to compare the difference of seed coat color control mechanism between B.napus and B.rapa,including the comparative identification of family members of flavonoid pathway genes in A.thaliana,B.rapa,B.napus and Brassica olereace;Analysis of expression characteristics of important seed color related genes in different yellow-and black(brown)-seeded materials,comparative analysis of metabolites and transcripts in the seeds of B.rapa and B.napus at different developmental stages of seeds,in order to explore the causes of complex seed color formation mechanism in B.napus and lay the foundation for breeding yellow-seeded B.napus,the results are as follows:1.Identification of family members of flavonoid pathway genes in Cruciferous speciesFlavonoid biosynthetic pathway genes(TT genes)have been demonstrated to play an important role in seed color formation in Arabidopsis.Apart from some important TT genes which have been identified to play a major role in B.rapa,whether all TT genes in B.rapa and B.napus homologous to At TT genes in Arabidopsis also play an important role is still unknown.Therefore,homologous TT genes in Arabidopsis,Brassica crops and other sequenced species of Cruciferae were systematically identified,sorted and analyzed in this study.A total of 649 homologous genes were detected for 21 flavonoid pathway genes in 17 cruciferous plants.Most of the flavonoid pathway genes were detected in all species,but there were differences among the flavonoid pathway genes among different species,including chromosomal recombination and loss of some gene family members,which indicates that most flavonoid pathway genes continue to play an important role in plants,and also to evolve among different species.Phylogenetic analysis of the homologous genes of each flavonoid pathway gene in 17 species revealed that the flavonoid pathway genes in 17 cruciferous plants could also be divided into two groups which were closely related to the evolution of Brassica and Arabidopsis respectively,suggesting that although some TT genes have been confirmed to play an important role in Brassica so far,there are still some differences in flavonoid biosynthetic pathways between Brassica crops and Arabidopsis.Homologous comparison of flavonoid pathway genes in B.rapa,B.napus and B.olereace showed that the A and C subgenomes in Brassica napus were more similar to those in B.rapa and B.olereace,respectively,but the difference between the A and C subgenomes was greater.Compared with the two diploid parents,the positions of some important transcription factors and structural genes in chromosomes of B.napus has changed,which needs further analysis to verify the gene function variation.2.Comparative analysis on the expression patterns of seed coat color-related genes in B.rapa and B.napus with different genetic backgroundsA large number of studies have confirmed that flavonoid pathway genes play an important role in seed color formation.In addition,several important candidate genes were screened out from the mapping of seed coat color genes in B.napus in the early stage of our research group.In order to explore the differences of genes’ regulating mechanism of flavonoid pathway between B.rapa and B.napus,in this study,the expression of flavonoid pathway genes and seed color candidate genes in seeds during different development stages of B.napus and B.rapa were systematically analyzed using yellow-and black-seeded materials from different sources.Firstly,the overall expression difference of flavonoid pathway genes between B.rapa and B.napus was analyzed.It was found that the expression level of several structural genes and regulatory genes from flavonoid pathway in yellow-seeded materials of B.rapa was significantly lower than that of corresponding black-seeded materials,but in yellow-seeded materials of B.napus,although the expression level was significantly lower than that of black-seeded materials,there was still a certain amount of expression.The expression of major seed coat color genes in B.rapa showed significant expression differences between yellow and black seed materials of B.napus,on the contrary,it is not,which indicated that there are some differences between main seed coat color genes in B.rapa and that in B.napus.The analysis of the expression of different members of seed coat color related genes showed that the homologous genes from the subgenomes of An and Cn were expressed in a certain amount,and the expression patterns were similar.It was speculated that the introduction of flavonoid pathway genes in C genome would affect the expression of flavonoid pathway genes in A genome.3.Comparative analysis of metabolic components in seeds of B.rapa and B.napus with different genetic backgroundsFlavonoid metabolic pathway is one of the most important secondary metabolic pathways in plants,and it is the source of many pigment metabolites in plants.Proanthocyanidins,one of the final products of Flavonoid metabolic pathway,are the most important pigment metabolites affecting seed coat color.LC / MS and pigment content analysis were carried out to determine the main flavonoid metabolites in seeds of different development stages,and to analyze the differences of metabolites for seed coat color formation between B.rapa and B.napus.Among the varieties of B.napus,more substances were detected separately in one or two varieties,which indicated that the difference of flavonoid pathway among varieties of B.napus was greater than that among varieties of B.rapa.It was inferred that the differentiation of flavonoid pathway among varieties of B.napus was greater,while the flavonoid metabolic pathway in B.rapa was more stable among varieties.Based on the metabolites identified by predecessors,35 flavonoids or Sinapyl compounds were identified in our research,including 9 epicatechin monomers or polymers,8 quercetin and its derivatives,3 isorhamnetin and its derivatives,6 kaempferol and its derivatives,9 kinds of erucic acid and metabolites containing sinapyl.These flavonoids and some unknown metabolites were quantitatively analyzed and classified.epicatechins and proanthocyanidins with different polymeric states form by the condensation of epicatechin were the most significant compounds between B.rapa and B.napus.The dynamic changes of other flavonoid pathway intermediates in B.rapa and B.napus were different,possibly due to the fewer identified flavonoids,the different genetic backgrounds or the different seed coat color genes among different varieties.4.Comparative analysis of the differences of transcriptome in different developing seeds and major genes in B.rapa and B.napusIn order to explore the difference between the major genes of seed coat color in B.rapa and B.napus in the regulation of flavonoid pathway,the main gene of seed coat color in B.rapa were further refined by using a segregating population of B.rapa.The main genes of seed coat color in B.rapa and B.napus were further summarized and sorted out.The difference of gene transcription level between yellow and black seed materials of B.rapa and B.napus was compared,and the regulation mechanism of major genes of seed coat color on flavonoid pathway was preliminarily predicted.11 candidate genes related to seed coat color formation in B.rapa were screened out by fine mapping and transcriptome sequencing.The major genes of seed coat color in B.rapa and B.napus were summarized.The major genes for seed coat color in B.napus were mostly located at the end of A9 chromosome,while those in B.rapa were mostly located on the middle of A9 chromosome.The results of transcriptome sequencing in B.rapa and B.napus showed that the proportion of differentially expressed genes in the total number of genes in B.napus was much higher than that of differentially expressed genes in B.rapa,which indicates that the main genes of seed coat color in B.napus played more roles than in B.rapa.The most significant enriched and down-regulated metabolic pathways in B.rapa and B.napus were same,including phenylpropanoid pathway and flavonoids metabolic pathway.However,significantly enriched up-regulated metabolic pathways were different between the two species.By comparing the expression levels of major seed coat color genes and flavonoid pathway genes in two RNAi lines of candidate major seed coat color genes in B.napus and the NILs of B.rapa,the homologous of major seed coat color genes of B.napus showed little influence on the flavonoid pathway in B.rapa,which might control the flavonoid pathway by regulating important transcription factors in flavonoid pathway genes in B.napus.In conclusion,the results of TT gene expression pattern analysis,metabolomic analysis further proved that flavonoid metabolic pathway was closely related to the formation of seed color in B.rapa and B.napus.Proanthocyanidin was the important pigment affecting the difference of seed color between B.rapa and B.napus.Compared with the yellow-seeded materials in B.rapa,the flavonoid pathway of B.napus has not been completely inhibited after the silence of the main gene of seed color,which may be related to the genetic model of the main gene plus the minor genes for seed color trait in B.napus.In addition,the introduction of Cn subgenome doesn’t only change the physical location of some TT gene in the An subgenome,but also participates in the regulation of flavonoid metabolism pathway,which may be the reason why the regulations mechanism of seed color in B.napus is more complex than that in B.rapa.The homologous genes of new major genes in B.napus do not participate in the regulation of flavonoid pathway in B.rapa.but the homologous genes may directly or indirectly regulate the expression of known important TT genes to control the synthesis of seed coat pigments in B.napus.