Pulsed field agarose

Verification of the karyotype was conducted using a pulsed-field gel electrophoresis system (DRII; Bio-Rad, Munich, Germany). Plugs containing intact chromosomes were produced from a Z. tritici cell suspension by centrifugation (3,500 × g, 10 min) and resuspension of the cell pellet in 1 ml double-distilled water (ddH₂O). Whole cells were embedded in 1 ml 2.2% Low-Range Ultra agarose (Bio-Rad, Munich, Germany)–0.5× Tris-borate-EDTA (TBE) buffer and incubated twice for 24 h each time at 55°C in 5 ml lysis buffer (1.5 mg/ml proteinase K, 1% SDS, 0.45 M EDTA, pH 8.0). Small chromosomes were separated in 1.2% pulsed-field agarose (Bio-Rad, Munich, Germany)–0.5× TBE buffer and were processed for 48 h at 14°C using an angle of 120°, 5 V/cm, and a switching time of 50 to 150 s. Midsize chromosomes were separated in 1% pulsed-field agarose–1× TBE buffer and were processed for 72 h at 14°C using an angle of 106°, 3 V/cm, and a switching time of 250 to 1,000 s. Large chromosomes were separated in 0.8% pulsed-field agarose–1× TRIS-acetate-EDTA (TAE) buffer and were processed for 92 h at 13°C using an angle of 106°, 2 V/cm, and a switching time of 1,000 to 2,000 s. Gels were stained for 30 min in a 0.5 µg/ml ethidium bromide solution followed by 10 min of destaining in H₂O.