Genomic DNA Extraction and Library Preparation

Tissue samples were collected from young leaves, freeze-dried, and pulverized using GenoGrinder 2000 (Spex CertiPrep Inc. Metuchen, NJ, USA) at 650 strokes/min for 15 s. Genomic DNA was isolated using 96-well silica filter plates (Epoch Life Science #2020-001). DNA was quantified using PicoGreen (Thermo Fisher Scientific, Waltham, MA) on a Synergy™ HT plate reader (Bio-Tek Instruments, Winooski, VT, USA) and diluted to 50 ng/µl in EB buffer. Genomic libraries were constructed using the two-enzyme GBS approach^{12 (link)} using simultaneous restriction-ligation with HindIII-HF, MseI, and T4 DNA Ligase (New England Biolabs). Restriction-ligation reactions were pooled by 96-well plate, purified using AMPure XP beads, PCR-amplified using Phusion Master Mix (NEB #M0531), and purified again using AMPure XP beads. A DNA7500 chip in an Agilent 2100 Bioanalyzer was used to determine average library size and concentration, and to dilute each library to 10 nM before submission for sequencing on an Illumina Hiseq 2500 (SR100) at the UC Davis Genome Center (Davis, CA).