GBS of popcorn diversity panel
Leaf samples were collected by bulking tissue from five individual seedlings of each accession, collected from the youngest leaf of each individual approximately ten days after germination, placed on ice, and stored at -80C. DNA was extracted from each sample using a modified CTAB DNA extraction protocol and resuspended in water. DNA was quantified in 96-well plates using PicoGreen (Invitrogen, Carlsbad, CA), and DNA concentrations were normalized to 50ng/L by diluting with 10 mM Tris, then stored at 4C. Restriction digestion and ligation were performed in 96-well plates using ApeKI by adding 250 ng of DNA from each sample, 1.5L of 0.1 M rare barcoded adapter, and 0.5L of 10 M common barcoded adapter per well (Elshire et al. 2011). Libraries were pooled, size-selected and cleaned with Agencourt AMPure XP beads (Beckman Coulter Inc., Brea, CA), and amplified for 15 cycles using KAPA HiFi HotStart ReadyMix (KAPA BIOSYSTEMS). Following a second purification with Agencourt AMPure XP beads, libraries were quantified using PicoGreen. A 1 ng/L dilution of the library was made using 10 mM Tris, and 5 L was run on a 1% agarose gel to verify size selection and primer-dimer removal. Fragment size was estimated using a Bioanalyzer 2100 (Agilent, Santa Clara, CA) High Sensitivity DNA Analysis. Pooled libraries were diluted to 10 nmol using 10 mM Tris and sequenced with 100-bp, single-end reads on an Illumina NovaSeq 6000 (Illumina, San Diego, CA).