GBS-based genomic characterization of a wheat–Thinopyrum 3J(3D) chromosome substitution line (GLA3)
Young leaf samples were collected from hydroponically grown seedlings of the GLA3 introgression line for genomic DNA extraction. Samples were immediately processed or stored at –20 °C until use. Wheat (Triticum aestivum genotype Mv9kr1) and the wheat–Thinopyrum 3J(3D) chromosome substitution line GLA3 were grown hydroponically under standard greenhouse conditions. Root tip meristems were used for cytogenetic analyses, and young leaves were collected for genomic DNA extraction at the seedling stage. Genomic DNA was extracted from young leaves of the GLA3 introgression line using the BioSprint DNA kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA concentration and quality were assessed prior to library construction. GBS libraries were prepared using a modified double-digest RAD-seq (ddRAD-seq) protocol based on Yang et al. (2023). Genomic DNA was digested with restriction enzymes MspI and SphI. Adapter and index designs followed Poland et al. (2012). Size selection of 350–390 bp fragments was performed using a BluePippin system (1.5% gel cassette; Sage Science, Beverly, MA, USA). Equimolar sub-libraries were pooled prior to sequencing.