Genomic DNA Extraction and Molecular Markers for Aegilops villosum

Genomic DNA of D. villosum accession PI 491576, common wheat CS, CD-3, 22-12, and 24-20 was extracted from 2-week-old leaves by using the CTAB method [67 (link)]. V-genome-specific SCAR markers of DV1 were synthesized, as described by Zhang et al. [31 (link)]. The PLUG primers for the homologous group 3 and the EST primers specific to 3V#4 chromosomes were synthesized using the procedures described by Ishikawa et al. [43 (link)] and Li et al. [37 (link)], respectively. PCR was conducted using the T100^TM Thermal cycler (Bio-RAD Laboratories, Emeryville, CA, USA) in a 25 μL reaction mixture containing 2.5 μL of 10X buffer (50 mM KCl, 1.5 mM MgCl2, and 10 mM Tris-HCl; pH 8.3), 200 nmol of each dNTP, 100 ng of genomic DNA, 0.2 U of Taq polymerase (TianGen, Beijing, China), and 400 nmol of each primer. The cycling parameters were: 94 °C for 3 min for the initial denaturation, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C (dependent on different primer sets) for 1 min, extension at 72 °C for 2 min, and a final extension at 72 °C for 10 min. The amplified products were separated on 2% (w/v) agarose gels and visualized by EtBr staining.