Chop-PCR for DNA Methylation Analysis

Chop-PCR was performed as previously described by [62 (link)]. Briefly, genomic DNA was extracted with the CTAB method from wheat roots after 48 h of Pb, Cd and Zn treatments. Then the DNA was digested with AciI (R0551S New England Biolabs, Ipswich, MA, USA) and hpaII (R0171S New England Biolabs, Ipswich, MA, USA) for CG DNA methylation, and with AluI (New England Biolabs, Ipswich, MA, USA USA) and haeIII (R0108S New England Biolabs, Ipswich, MA, USA) for CHH/CHG methylation. Equal amount of digested and undigested DNA were used as template for qPCR, and normalized to undigested DNA. Basal DNA methylation represents the levels of DNA methylation of each variety in control conditions. To compare the basal gene DNA methylation levels in Pirsabak 2004 and Fakhar-e-sarhad, the DNA methylation levels of Pirsabak 2004 were set to 1 for each gene and then the DNA methylation levels in Fakhar-e-sarhad were normalized to Pirsabak 2004. For total DNA methylation levels, the McrBC enzyme (M0272S New England Biolabs, Ipswich, MA, USA USA) was used that specially digested methylated DNA, therefore, bands represent the non-DNA methylation levels. Chop-qPCR primers are listed in the Table S1.