2.3. Double‐digest RAD (ddRAD) sequencing

Double‐digest restriction site‐associated (ddRAD) libraries were prepared according to Peterson et al. (2012). DNA extracts were run on an agarose gel to check for DNA quality. Samples with low molecular weight smears were further purified using paramagnetic beads (SPRIselect; Beckman Coulter). Approximately 150 ng DNA from individual samples was digested with MluCI and MspI and purified using AMPure XP (Beckman Coulter). Custom‐barcoded adapters P1 and P2 (see Peterson et al. (2012) for sequences) were then ligated to ~50 ng of DNA. The P1 adapter includes a 5bp inline unique sequence for individual barcoding. Groups of 48 samples with unique barcodes were pooled (equal volumes of each sample), purified, and size‐selected using a BluePippin system (target insert size of 400–500 bp). Unique external indices were added to each pool by PCR amplification. PCR products were purified, fragment sizes were verified, qPCR was quantified, and PCR products were further pooled in equimolar quantities. Sequencing of DNA libraries was performed using the Illumina NovaSeq™ 6,000 Sequencing System with S4 flow cell type. Library construction and sequencing was performed at the Genomics Core Lab, Texas A&M University, Corpus Christi.