Creation Of Resynthesized Brassica Napus And Its Epigenetic Analysis

Brassica napus is one of the important oil crops in China, however, due to the short history of origin and occurrence, the genetic background of B. napus is relatively narrow. Artificial resynthesis of B. napus, using the abundant germplasms of B. rapa and B. oleracea as parental materials, is an efficient way to broaden B. napus germplasm and create model materials for study of plant polyploidization. In this study, we chose four varieties of B. rapa (AA) and one B. oleracea (CC) as parents to create resynthesized B. napus, using conventional reciprocal hybridization and embryo rescue. Seven B. napus lines were created from hybridization with different combination of diploid progenitors. Through SSR analyis, two hybrid lines of ’Yangzhouqing’(AA) X’Yonglv 7’(CC) were confirmed containing both A and C genomes. Besides,38 chromosomes were observed in the two lines of hybrids, indicating these lines as resynthesized allopolyploids. By comparing agronomic characters in resynthesized B. napus (F1 generation) and diploid parents, we found the hybrids were much branched, and the shape of leaf and flower, as well as color of flower of Fl were in-between the diploid parents. The fertility of F1 (32.7%) was significantly reduced compared with B. rapa (93.1%) and B. oleracea (81%). Moreover, the number of effective branches, height, pod number, and grain weight (4.402 g) in F1 were in-between the diploid parents. And locus of first branch was shift down in F1 compared with diploid parents.In this study, DNA methylation status was also studied during plant polyploidization, using the newly resynthesized B. napus (F1 generation) and the parents as materials. Firstly, HPLC-assisted quantification was carried out to assess the DNA methylation level of hybrids and their parents. The DNA methylation rate of’Yangzhouqing’and’Yonglv 7’was 8.33% and 15.88%, respectively. And the methylation rate of two hybrid lines was 10.29% and 12.83%, which were in-between the diploid parents. Secondly, using methylation-sensitive polymorphism (MSAP) analyis, we found obvious variation of DNA methylation in resynthesized B. napus (F1 generation) compared with the parent lines (Yangzhouqing<F 1 generation<Yonglv 7), which is consistant to the HPLC results. Meanwhile, we identified that the methylation rate of F1 was 23.71%,6.6% and 10.16% of these methylation changes were inherited from A and C genome, respectively. These results suggested that a large number of rapid methylation changes occurred in newly synthesized allotetraploid B. napus. Sequence analysis of MSAP polymorphic fragments revealed that genes involed in multiple molecular functions were changed during polyploidization, including genes encoding Cysteine/Histidine-rich C1 domain family protein, RING/U-box superfamily protein, CCT motif family protein, GDSL-like Lipase/Acylhydrolase superfamily protein, F-box/RNI-like/FBD-like domains-containing protein, Vacuolar protein sorting 55 (VPS55) family protein, Serine protease inhibitor (SERPIN) family protein, TRAF-like superfamily protein, IAA-amino acid hydrolase, protein kinase APK2b and KCS8. Expressional analysis of these genes was conistant to the corresponding methylation changes.