Detection of DNA methylation changes during seed germination in rapeseed (Brassica napus)

DNA methylation is known to play a crucial role in regulating plant development and or- gan or tissue differentiation. In this study, we as- sessed the extent and pattern of cytosine methylation during rapeseed (Brassica napus L.) seed germina- tion, and compared the methylation level of various tissues in seedling, using the techniques of methyla- tion-sensitive amplified polymorphism (MSAP) and HPLC separation and quantification of nucleosides. In all, 484 bands, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified by 12 pairs of selective primers in DNA ob- tained from dry seeds. A total of 76 sites were found to be differentially digested by the isoschizomers, indicating that approximately 15.7% of 5′-CCGG-3′ sites in the genome were cytosine methylated. Four classes of patterns were observed in a comparative assay of cytosine methylation in the dry and germi- nating seeds; a small number of hypermethylation events occurred at 5′-CCGG-3′ sites in germinating seeds compared with dry seeds, while many more hypomethylation events were detected after seed germination. Differences in DNA methylation level in various tissues were also detected; radicel was less methylated than hypocotyl and cotyledon. These observations were further confirmed by HPLC analy- sis. In addition, sequencing of eleven differentially methylated fragments and the subsequent blast search revealed that cytosine methylated 5′-CCGG- 3′ sequences were equally distributed between cod- ing and non-coding regions. These results clearly demonstrate the power of MSAP technique for large-scale DNA methylation detection in rapeseed genome, and the complexity of DNA methylationchange during seed germination. DNA Hypomethyla- tion going with seed germination appears to be a necessary step toward transcriptional activation in gene expression, and may well contribute to the de- velopmental gene regulation.