Patterns of cytosine methylation in an elite rice hybrid and its parental lines, detected by a methylation-sensitive amplification polymorphism technique

Abstract

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 pattern of cytosine methylation in the rice genome, using the technique of methylation-sensitive amplified polymorphism (MSAP), which is a modification of the amplified fragment length polymorphism (AFLP) method that makes use of the differential sensitivity of a pair of isoschizomers to cytosine methylation. The tissues assayed included seedlings and flag leaves of an elite rice hybrid, Shanyou 63, and the parental lines Zhenshan 97 and Minghui 63. In all, 1076 fragments, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified using 16 pairs of selective primers. A total of 195 sites were found to be methylated at cytosines in one or both parents, and the two parents showed approximately the same overall degree of methylation (16.3%), as revealed by the incidence of differential digestion by the isoschizomers. Four classes of patterns were identified in a comparative assay of cytosine methylation in the parents and hybrid; increased methylation was detected in the hybrid compared to the parents at some of the recognition sites, while decreased methylation in the hybrid was detected at other sites. A small proportion of the sites was found to be differentially methylated in seedlings and flag leaves; DNA from young seedlings was methylated to a greater extent than that from flag leaves. Almost all of the methylation patterns detected by MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrate that the MSAP technique is highly efficient for large-scale detection of cytosine methylation in the rice genome. We believe that the technique can be adapted for use in other plant species.

DNA methylation polymorphism and stability in Chinese indica hybrid rice

“…Therefore, 14.78% (275.25/1842) of the 5′-CCGG sites in the genomes of the 20 rice parental lines were methylated. This proportion is slightly smaller than that reported by Xiong et al [19] (16.3%) and Ashikawa [20] (16.2%). However, these two previous studies only considered two rice accessions and any fragment absent from both accessions would not be counted.…”

Differential Methylation during Maize Leaf Growth Targets Developmentally Regulated Genes

“…More than 50% of the investigated CCGG sites were found to be stably methylated. Because the use of restriction enzymes does not allow us to distinguish some forms of methylation, MSAP results tend to yield an underestimation of genomic DNA methylation (Xiong et al, 1999). Indeed, recent wholegenome bisulfite sequencing of the maize genome in unfertilized ears revealed that of all Cs in CG and CHG contexts, 86% and 74%, respectively, are methylated (Gent et al, 2013).…”

“…These gradual methylation changes are most likely caused by unequal changes in the methylation state of the different cell types that make up the tissues of the harvested samples (Xiong et al, 1999;Peraza-Echeverria et al, 2001;Cervera et al, 2002). For our objectives, the most interesting loci were the CCGG sites that abruptly changed their methylation from one zone to the next.…”

Polyploid formation in cotton is not accompanied by rapid genomic changes

“…To explore the genomic composition of the synthetics relative to their parents, we used two types of AFLP analysis, i.e., standard (Vos et al 1995) and methylation-sensitive (Reyna-Lopez et al 1997;Xiong et al 1999). Standard AFLP analysis was carried out essentially according to the protocol of the AFLP ™ Plant Mapping kit of PE/Applied Biosystems (Foster City, Calif.), with slight modifications (M. Gitzendanner and the Soltis lab, personal communication).…”