Studies On Genomic DNA Methylation Pattern During Heterosis In Brassica Napus

The mechanisms underlying heterosis remain largely unknown regardless the fact that heterosis has been proven a powerful tool for agricultural development. After the word "heterosis" was first brought forward by Shull in 1914, many scientists poured a lot of effort in this area and two classical genetic explanations—dominance hypothesis and super overdominance hypothesis were held out. With the rapid development of molecular biology and epigeneitcs, scientists began to explore this area at molecular level from the angle of gene expression and regulation and DNA methylation.A Brassica napus hybrid and its parental lines W2-513 and W2-680 which were from the DH lines were studied for DNA methylation pattern. DNA methylation is known to play an important role in regulating gene expression. In this study, we explored the relationship between DNA methylation and heterosis. The genomic DNA methylation pattern of the hybrid Brassica napus and its parental lines was assessed by using the methylation-sensitive amplification polymorphism (MSAP), which is modified from the amplified fragment length polymorphism (AFLP) method using the differential sensitivity of a pair of isoschizomers (Hpall and Mspl) to cytosine methylation. The result showed that there existed some cytosine methylation patterns differing between the hybrid and its parents. Four classes of patterns were identified: class A, B, C and D. In class A, a band detected in the hybrid was also found in both of its parents. In class B, the fragment detected in the hybrid was detected in either of its parents. The patterns in class C and class D represented an increase or decrease in the level of methylation in the hybrid compared to the parents respectively. After sequencing, we got 38 fragments with different DNA methylation patterns, and 18 of these sequences were found to be homologous to some putative genes.Southern blotting is usually used in DNA methylation research. In this study, we employed Southern blotting to compare the DNA methylation level of 5SrDNA and a centromere repetitive sequence (805bp) between the hybrid and its parents. The result indicated that there were no markedly different methylation level between the hybrid and its parents. Bisulfite Sequencing PCR is another commonly used technique to detect specific DNA methylation state. After the bisulfite treatment to the 5SrDNA followed by sequencing, we found that there existed 3 CG sites and 1 CAG sites whose methylation levels were all over 50%. Though the methylation level differed somewhat in some of the specific cytosines within the sequences surveyed from the hybrid to its parents, the overall trends of the change in cytosine methylation level retained similar.DNA methylation patterns are established and maintained by DNA methyltransferases, which catalyze the transfer of the methyl group from S-adenosylmethionine (SAM) to C-5 position in the pyramiding ring of cytosine. In this process, DNA methyltransferase is indispensable. RT-PCR result revealed that the expression level of two putative methyltransferases (homologous to Arabidopsis Drml and Drm2) appeared the same.Taking together, we concluded that compared to its parents, the hybrid got changed and adjusted methylation pattern, to which the heterosis may be attributable. Though repetitive sequences have a close connection with the gene expression, but the differences of methylation level of 5SrDNA and a centromere repetitive sequence (805bp) between the hybrid and its parents are not drastic. Methyltransferases are of importance in the process of DNA methylation, but the expression level of the two putative methyltransferase genes looked the same.Heterosis in Brassica napus is an extremenly complex phenomenon, which involves the change of DNA methylation pattern in some specific regions of the genome. However, the molecular mechanisms underlying the regulation of methylation pattern await more investigation.