2.3 DNA extraction and epiGBS library preparation
We isolated genomic DNA (gDNA) from four S. cataractaepopulations (Sc1, Sc2, Sc3, Sc4) with each exposed to three treatments (control, Cd-treated, Cu-treated) hereafter referred to as group (n = 12 groups) with each group replicated in triplicate (n = 36 samples in total). We followed the cetyltrimethylammonium bromide (CTAB) DNA extraction protocol for recalcitrant plant tissues (https://www.protocols.io/view/high-quality-dna-extraction-protocol-from-recalcit-i8jchun) with small modifications (detailed protocol available in the Supplementary Methods - SMs). We checked the quality of the gDNA with the NanoDrop (Nanodrop™ 8000 Spectrophotometer; Thermo Scientific), and quantified its concentration using the Qubit 3.0 Fluorometric dsDNA BR assay kit (Q32851; Life Technologies). We obtained high quality gDNA for all samples except for one replicate of Sc3 in control conditions, which we did not include in the sequencing library.
We prepared the epiGBS libraries following 71. We digested 400 ng of gDNA from each sample with the restriction enzymePstI . Then, we ligated methylated, non-phosphorylated barcoded adapters to both ends of the digested fragments. We concentrated the library using the NucleoSpin™ Gel and PCR Clean-up Kit (12303368, Macherey-Nagel™) and performed a fragment size selection with 0.8x SPRI beads (A63880, Agencourt AMPure XP Beckman coulter). We performed nick translation, bisulfite converted the DNA using the EZ Lightning methylation kit (Zymo Research), and amplified the library with the KAPA HIFI Uracil+ Hotstart Ready Mix (Roche) under the following PCR conditions: initial denaturation step at 95ºC for 3 min; 20 cycles of 98ºC for 10s, 65ºC for 15s, and 72ºC for 15s; final extension of 72ºC for 5 min. Finally, we quantified the library using the Qubit dsDNA assay kit, pooled all samples using equimolar concentrations and assessed its quality by analyzing 1 µL on a High Sensitivity DNA chip on an Agilent 2100 Bioanalyzer. The library was sequenced at Novogene (HK) Company Limited in Hong Kong on the Illumina HiSeq X-Ten System (PE-150 bp).