title: GBS of GLA7: Triticum aestivum /'Agropyron glael' addition line
design: Aside from identifying potential chromosomal deletions and rearrangements, the objective was to precisely determine the homoeologous relationship of the added Thinopyrum chromosome with wheat. Genomic DNA was extracted from fresh young leaves of wheat donor genotypes MV9 and KAR, the parental Thinopyrum hybrid A. glael, and the GLA7 line in 96 well plates using BioSprint 96 DNA Plant Kit (Qiagen, Hilden, Germany) following the manufacturers protocol.  As the basis for double-digest restriction site-associated DNA (ddRAD) library protocol development, the protocol of Poland et al. (2012) was followed. First, 200 ng of DNA was cleaved with MspI (has a more common recognition site, CCGG) and SphI (rare-cutting enzyme, recognition sequence GCATG/C) restriction endonucleases in rCutSmart buffer (New England Biolabs, Ipswich, MA, USA) 3h at 37C. P1 and P2 barcoded adapters corresponding to the restriction site of the enzymes were ligated immediately afterwards with T4 ligase (Promega, Madison, WIS, USA) at 23C for 120 minutes. The enzymes were inactivated at 65C for 20 min. All samples with the same P2 and 12 different P1 adapters were pooled, purified by 1.5x AMPure XP Bead-Based Reagent (Beckman Coulter, Brea, CA, USA), and size selected with a dye-free Blue Pippin 1,5% gel cassette (BDF1510) (Sage Science, Beverly, MA, USA) for fragment sizes of 350390 bp. To add Illumina flowcell annealing sequences, multiplexing indices, and sequencing primer annealing regions to all fragments and to increase concentrations of sequencing libraries, PCR amplification with a Q5_ High-Fidelity DNA Polymerase enzyme (New England Biolabs, Ipswich, MA, USA) was performed. For large-scale combinatorial multiplexing of samples, we include a set of 8 uniquely indexed PCR2 primer sequences. Each of these primers will add a unique index sequence to all fragments in the PCR. Therefore, it is possible to uniquely label 96 individuals [12 (adapter P1 barcodes) x 8 (PCR2 indices)], to combine in a single lane of sequencing. PCR enrichment was performed in 10 cycles in 25 _L reactions containing 1.25-1.25 _L NEBNext Index/Universal Primer, 0.25 _L Q5 High-Fidelity DNA polymerase, 0.5 _L dNTP, 5 _L Q5 Reaction Buffer Pack, and 16.75 _L template. Each sublibrary was purified with 0.9x AMPure XP Bead-Based Reagent (Beckman Coulter, Brea, CA, USA) and quantified on Qubit (ThermoFisher Scientific, Waltham, MA, USA). All sublibraries were pooled equimolarly and sequenced on the NovaSeq 6000 platform (Illumina, San Diego, CA, USA) using the SP reagent kit v.1.5 in 2 _ 150 bp configuration at the Institute of Experimental Botany, Czech Academy of Sciences, Olomouc, Czechia.