The Fate Of Key Genes Related To Flavonoids Biosynthetic Way In Resynthesized Brassica Napus

Polyploidization plays extremely important roles in plant evolution and speciation. In recent years, it’s very popular in researching the evolution and expression of polyploidy genome. Synthetic allopolyploid Brassica had been model plants for studying genetic and epigenetic changes of polyploidy due to the exact origin of their parents. Additionally, yellow seed breeding was one of the most essential components for high oil content breeding. However, the genetic module of yellow seed was very complicated. It was influenced not only by the environment but also the genetic background. In this study,6different generations of artificially synthesized B. napus (AACC,2n=38) within yellow/black seed and their parents, B. rapa (1151, JB2, DB; AA,2n=20) and B. oleracea (T1, T2, T3,03K04; CC,2n=18), were used as materials. Then, the gene structure and expression changes of several key genes, which related to flavonoid. synthesis and underwent polyploidy evolution, in polyploid B. napus were investigated by SCAR and Real-time PCR. Our results were contributed to reveal the forming of yellow seed B. napus and the evolution of polyploid Brassica, and they were listed as followed:1. The bias of sequence elimination.47pairs of primers were designed according to15key genes of flavonoid synthesis in A. thaliana and B. napus. They were used for PCR amplification among different generations in all crosses. The results showed that loss bands in all the offspring, whose maternal plants were B. rapa (1151×T2,1151×03K04, JB2x T3), major came from A genome. However, it’s inconsistent for the offspring, whose maternal plants were B. oleracea. In T1×DB, they had a same results with above; in03K04xDB, they had equal frequency of loss banks between A and C genome; in T1×JB2, the loss banks major came from C genome.2. Sequence elimination tended to stability. The statistics of those genes’amplifications in each stain of1151xT2(S1-S6and S8) told us that, F1had higher frequency of losing A, C genomic banks than other generations. Then, the losing frequency was depressed and stable subsequently. Meanwhile, there were some new banks appeared in each strain and they occupied12.1%of all the amplified banks.All the gene changes were regularity in each cross. For instance, TT2had no loss banks, which came from C genome, in all crosses. And,TT12had more loss banks in A genome among all crosses. There were four crosses having more TT8loss banks in A genome, even though they were equal between A and C genome in T1×JB2and1151x03K04. In TT16, the loss banks were rare in1151×03K04, but had more C genome in other crosses. There were more new banks came from1151x T2(S1-S6and S8), and less in1151x03K04and T1×JB2. Simultaneously, there were no new banks of TT16in JB2×T3, T1×DB and03K04xDB.4. In all generations of the6crosses,103new banks, which came from TT6, TT10, TT12and TT18, were sequenced.200-300bp InDels were found in introns of TT18. The insertion was identified as DNA transposon (TcMar-Stowaway) using BLAST. By contrast, there were few bases changes in other3genes.5. For three yellow or black seed strains of S8and their parents in1151xT2, seed coats were attained after10days,20days and30days from pollination separately. Then, we studied the expression of7key genes in flavonoid synthesis (TT3, TT6, TT8, TT10, TT12, TT18and TT19) by Real-Time PCR. By doing this, we found that the expressions of TT6, TT8, TT18were temporal up-regulated consistently in strains with black seeds and parental T2(B. oleracea, yellow seed). Besides, strains with yellow seeds and their parental1151(B. rapa, yellow seed) had the same temporal-regulated expression in TT6, TT8and TT12. They all obtained the expressive peak after20days from pollination. Additionally, the expression of TT10and TT19were also temporal-regulated consistently in both parents and all their offspring, respectively. However, the expressive peak was obtained after30days from pollination in TT10, and after20days in TT19.In comparison with yellow seed strains, the expressions of TT3, TT6, TT8, TT12, TT18and TT19in black seed strains were up-regulated4-30times.