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

Abstract

DNA methylation is known to play a crucial role in regulating plant development and organ or tissue differentiation. In this study, we assessed the extent and pattern of cytosine methylation during rapeseed (Brassica napus L.) seed germination, and compared the methylation level of various tissues in seedling, using the techniques of methylation-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 obtained 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 germinating 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 analysis. 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 coding 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 methylation change during seed germination. DNA Hypomethylation going with seed germination appears to be a necessary step toward transcriptional activation in gene expression, and may well contribute to the developmental gene regulation.