Patterns of cytosine methylation in an elite rice hybrid and its parental lines detected by a methylation-sensitive amplification polymorphism technique
Xiong, L. Z., C. G. Xu, M. A. Saghai Maroof, Q. Zhang.
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1999. Mol. Gen. Genet. 261: 439-446.
DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we assessed the extent and patterns of cytosine methylation in the rice genome using a method of methylation-sensitive amplified polymorphism (MSAP) that was modified from the amplified fragment length polymorphism (AFLP) technique making use of the different sensitivity of a pair of isochizomers to cytosine methylation. The tissues assayed included seedlings and flag leaves of an elite rice hybrid, 'Shanyou 63', and the parents 'Zhenshan 97' and 'Minghui 63'. Overall, 1076 fragments, each representing a recognition site cleaved by either or both of the isochizomers, were amplified with 16 pairs of selective primers. A total of 195 sites were found to be cytosine-methylated in one or both parents, and the two parents had approximately the same degree of methylation (16.3%), as revealed by the differential digestion of the isochizomers. Four classes of patterns were identified in the comparative assay of cytosine methylation in the parents and hybrid; increased methylation was detected in the hybrid compared to the parents in some of the recognition sites while decreased methylation in the hybrid was detected in other sites. A small proportion of the sites was found to be differentially methylated between seedlings and flag leaves, DNA from young seedlings was methylated to a greater extent than from flag leaves. Almost all of the methylation patterns detected by MSAP could be confirmed by Southern analysis with the amplified fragments recovered as probes. The results clearly demonstrated that the MSAP technique is highly efficient for large scale detection of cytosine methylation in the rice genome. It is also believed that this technique can be adapted for detecting cytosine methylation in other plant species.
DNA methylation, hybrid rice, tissue specificity, isochizomers, PCR amplification