*4.2. Genotyping-by-Sequencing*

For each of the 192 genotypes, DNA was extracted from 0.1 g finely ground tissue following the protocols of NucleoSpin® Plant II Kit (Macherey-Nagel, Bethlehem, PA, USA), and was eluted in a 1.5 mL Eppendorf tube with Elution Buffer. NanoDrop 8000 (Thermo Fisher Scientific, Waltham, MT, USA) was used to measure the quality of the DNA by comparing the 260 and 280 nm absorptions. DNA samples were further quantified through the Quant-iTTM PicoGreen®dsDNA assay kit (Invitrogen, Carisbad, CA, USA) and diluted to 60 ng/µL with 1× TE buffer prior to sequencing analysis.

A genetic diversity-focused GBS (gd-GBS) protocol by Peterson et al. [3] was used for the preparation of multiplexed GBS libraries. In brief, for each library, 200 ng purified genomic DNA was first digested with the restriction enzyme combination *Pst*I and *Msp*I (New England Biolabs, Whitby, ON, Canada). Ligation of customized adapters onto the 50 and 30 ends of the restriction fragments by T4 ligase was subsequently carried out. Then, the ligation fragments were purified by an AMPure XP kit (Beckman Coulter, Brea, CA, USA). Following the purification, Illumina TruSeq HT multiplexing primers were added through PCR amplification. The amplicon fragments were further quantified, concentrated, and pooled to form 4 subgroups of 12 samples each. The samples in the subgroups were pre-selected using a Pippin Prep instrument (Sage Science, Beverly, MA, USA) for an insert size range of 250–450 bp, before pooling the samples into a library. Each pooled library was diluted to 6 pM, and denatured with 5% of sequencing-ready Illumina PhiX Library Control (Illumina, San Diego, CA, USA) that can serve for calibration. Sequencing was completed using an Illumina MiSeq Instrument with paired-ends of 250 bp in length. MiSeq runs generated 384 FASTQ sequence files from 192 genotypes of 12 lines (one forward and one reverse for each of 192 genotypes). All the raw pair-end sequencing data in FASTQ format were deposited into the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) with accession number SRP115373 as part of the larger sequencing effort to enhance crested wheatgrass breeding [41]. The sequencing information for all 192 assayed samples is described in the online Supplementary Material, Section A.
