Nucleospin plant 2 molecular kit

Plant DNA was isolated by a NucleoSpin Plant II Molecular Kit (Macherey–Nagel GmbH & Co. KG, Germany) as described by Zemanová et al.^{34 (link)}. The global DNA methylation status of DNA was determined using 100 ng of isolated DNA and a MethylFlash Methylated DNA Quantification Kit (Fluorometric; Epigentek Group Inc., Farmingdale, USA) according to the manufacturer’s instructions. A spectrophotometer (Tecan Infinity M200, Tecan Deutschland GmbH) with excitation at 530 nm was used to measure the fluorescence at 590 nm using the Magellan program.

The fronds were weighed, frozen in liquid nitrogen and stored at − 80 °C prior to DNA methylation analysis. To isolate total DNA, the fronds (1 g fresh weight) were ground to a fine powder in liquid nitrogen by mortar and pestle. DNA was extracted from 100 mg of powdered tissue using a NucleoSpin Plant II molecular kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany), as instructed in the user manual. The global DNA methylation status of DNA was determined using 100 ng of isolated DNA and a MethylFlash Methylated DNA Quantification Kit (Fluorometric; Epigentek Group Inc., Farmingdale, NY, USA) according to the manufacturer’s instructions. A SpectraMax MiniMax 300 Imaging Cytometer (Molecular Devices LLC, San Jose, CA, USA) with excitation at 530 nm was used to measure the fluorescence at 590 nm.