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.
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References
Gonzalgo, M. L., Jones, P. A., Mutagenic and epigenetic effects of DNA methylation, Mutat. Res., 1997, 386(2): 107–118.
Holliday, R., The inheritance of epigenetic defects, Science, 1987, 238(4824): 163–170.
Finnegan, E. J., Brettell, R. I. S., Dennis, E. S., The role of DNA methylaiton in the regulation of plant gene expression, in DNA Methylation: Molecular Biology and Biological Significance (eds. Jost, J. P., Saluz, H. P.), Basel: Birkhauser, 1993, 218–261.
Ronemus, M. J., Galbiati, M., Tchnor, C. et al., Demethylation-induced developmental pleiotropy in Arabidopsis, Science, 1996, 273(5275): 654–657.
Jacobsen, S. E., Sakai, H., Finnegan, E. J. et al., Ectopic hypermethylation of flower-specific genes in Arabidopsis, Curr. Biol., 2000, 10(4): 179–186.
Sherman, J. D., Talbert, L. E., Vernalization-induced changes of the DNA methylation pattern in winter wheat, Genome, 2002, 45(2): 253–260.
Bastow, R., Mylne, J. S., Lister, C. et al., Vernalization requires epigenetic silencing of FLC by histone methylation, Nature, 2004, 427(6970): 164–167.
Messeguer, R., Ganal, M. W., Steffens, J. C. et al., Characterization of the level, target sites and inheritance of cytosine methylation in tomato nuclear DNA, Plant Mol. Boil., 1991, 16(5): 753–770.
Lund, G., Messing, J., Viotti, A., Endosperm specific demethylation and activation of specific alleles of alpha-tubulin genes of Zea may, Mol. Gen. Genet., 1995, 246(6): 716–722.
Dhar, M. S., Pethe, V. V., Gupta, V. S. et al., Predominance and tissue specificity of adenine methylation in rice, Theor. Appl. Genet., 1990, 80(3): 402–408.
Labra, M., Grassi, F., Imazio, S. et al., Genetic and DNA-methylaiton changes induced by potassium dichromate in Brassica napus L., Chemosphere, 2004, 54(8): 1049–1058.
Goubely, C., Arnaud, P., Tatout, C. et al., S1 SINE retroposons are methylated at symmetrical and non-symmetrical positions in Brassica napus: Identification of a preferred target site for asymmetrical methylation, Plant Mol. Biol., 1999, 39(2): 243–255.
Lu, G. Y., Yang, G. S., Fu, T. D., Silver-stained AFLP-a novel assay for DNA fingerprinting in rapeseed, J. Huangzhong Agri. (in Chinese), 2001, 20(5): 413–415.
Chakrabarty, D., Yu, K. W., Paek, K. Y., Detection of DNA methylation changes during somatic embryogenesis of Siberian ginseng (Eleuterococcus senticosus), Plant Sci., 2003, 165(1): 61–68.
Portis, E., Acquadro, A., Comino, C. et al., Analysis of DNA methylation during germination of peper (Capsicum annuum L.) seeds using methylation-sensitive amplification polymorphism (MSAP), Plant Sci., 2004, 166(1): 169–178.
Sharpe, A. G., Parkin, I. A. G., Keith, D. J. et al., Frequent nonreciprocal translocations in the amphidiploid genome of oilseed rape (Brassica napus), Genome, 1995, 38(6): 1112–1121.
McClelland, M., Nelson, M., Raschke, E., Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases, Nucleic Acids Res., 1994, 22(17): 3640–3659.
Palmgren, G., Mattson, O., Okkels, F. T., Employment of hydrolytic enzymes in the study of the levels of DNA methylation, Biochem. Biophys. Acta, 1990, 1049(3): 293–297.
Reyna-Lopez, G. E., Simpson, J., Ruiz-Herrera, J., Differences in DNA methylation patterns are detectable during the dimorphic transition of fungi by amplification of restriction polymorphism, Mol. Gene Genet., 1997, 253(6): 703–710.
Xiong, L. Z., Xu, C. G., Saghai, M. A. et al., Patterns of cytosine methylation in an elite rice hybrid and its parental lines detected by methylation sensitive amplification polymorphism technique, Mol. Gen. Genet, 1999, 261(3): 439–446.
Cervara, M. T., Ruin-Garcia, L., Martinez-Zapater, J. M., Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers, Mol. Genet. Genomics, 2002, 268(4): 543–552.
Xu, M. L., Li, X. Q., Korban, S. S., AFLP-based detection of DNA methylation, Plant Mol. Boil. Rep., 2000, 18(4): 321–330.
Riddle, N. C., Richards, E. J., The control of natural variation in cytosine methylation in Arabidopsis. Genetics, 2002, 162(1): 355–363.
Siroky, J., Ruffini-Castiglione, M., Vyskot, B., DNA methylation pattern of Melandrium album chromosomes, Chromosome Res., 1998, 6(6): 441–446.
Grunau, C., Renault, E., Rosenthal, A. et al., MethyDB-a public database for DNA methylation data, Nucleic Acid Res., 2001, 29(1): 270–274.
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Lu, G., Wu, X., Chen, B. et al. Detection of DNA methylation changes during seed germination in rapeseed (Brassica napus). CHINESE SCI BULL 51, 182–190 (2006). https://doi.org/10.1007/s11434-005-1191-9
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DOI: https://doi.org/10.1007/s11434-005-1191-9