*4.7. DNA Methylation Patterns in Specific Regions of Chalcone Synthase Gene*

The methylation patterns of the chalcone synthase gene sequence were established in the control and ODN-treated plants. The DNA was incubated with restriction enzymes MspI and HpaII for at least 3 h (New England Biolabs, Ipswich, MA, USA); it differs in sensitivity to cytosine methylation. The genomic DNA digested by the restriction enzymes and undigested DNA were used as a templates for the real-time PCR reaction. The reaction was performed similar to the gene expression analysis. The primers for the reaction (Supplementary data Table S1) were designed for specific sites of methylation predicted by the NEB cutter V2.0. In the chalcone synthase gene, six CCGG islands were analyzed. The sites of analyzed -CCGG- motifs were indicated by their positions towards the ATG site (+1) as follows: 50UTR (site −232), non-coding region (+217) and coding region (+996, +1219, +1273, +1606).

The quantity of DNA measured by the real-time PCR was calculated in order to estimate the methylation of cytosines. The "CCGG" non-methylated DNA was calculated as the difference between undigested DNA and DNA incubated with HpaII. The single methylated cytosine "CCmGG" was estimated as the difference between samples digested by HpaII and digested by MspI. The level of the "CmCmGG" was equal to the DNA incubated with MspI. The values were presented as a percentage referring to the undigested DNA, set as 100%.
