Library construction and sequencing

ddRAD libraries were constructed according to the method described by Peterson et al.²⁸. Briefly, 500 ng of DNA template from each individual was double-digested using two restriction enzymes, EcoRI and NlaIII (New England Biolabs [NEB], Ipswich, MA, USA; 20 U/reaction) in one combined reaction for 30 min at 37 °C. Subsequently, each fragmented sample was purified using a QiagenMinElute Reaction Cleanup Kit (Qiagen, Valencia, CA, USA) and eluted in 20 μL elution buffer (EB). Fragments were then ligated to P1 adapters (including a unique 4–8-bp multiplex identifier [MID] used to distinguish each individual) that bound to EcoRI-created restriction sites and P2 adapters that bound to overhangs generated by NlaIII. In each reaction, 500 ng DNA, 1 μL P1 adapter (10 mM), 1 μL P2 adapter (10 mM), 1 μL T4 ligase (1,000 U/mL), 4 μL of 10 × T4 ligation buffer, and double-distilled water were combined into a total volume of 40 μL. The ligation was processed on a polymerase chain reaction (PCR) machine using the following conditions: 37 °C for 30 min, 65 °C for 10 min, followed by a decrease in temperature by 1.3 °C/min until the temperature reached 20 °C. Samples were pooled and size-selected (400–600 bp) from an agarose gel. Then DNA product was purified using a QiagenMinElute Gel Purication Kit and eluted in 10 μL EB. Paired-end (150 bp) sequencing of the ddRAD products from the 202 individuals was performed using an IlluminaHiSeqXten sequencing platform (Illumina, Inc., San Diego, CA, USA). Sequencing data for each individual were then extracted according to the specific MID.