Molecular Genotyping of Verticillium dahliae

High conidial suspensions (1 × 10⁸ conidia/mL) of V. dahliae strains P48, P50, and Vd991 were placed on a PDA medium and incubated at 25 °C, and the mycelium was collected after 7 days. The total genomic DNA was extracted for the identification of mating types, races, and D and ND types. Genomic DNA of each isolate was extracted using a FastPure Plant DNA Isolation Mini Kit (Vazyme, Nanjing, China) following the manufacturer’s instructions and was stored at −20 °C for polymerase chain reaction (PCR) assays.
For the genotype identification assays, D/ND, race1/2, and MAT1-1/MAT1-2 were determined by PCR with previously developed primers [35 (link),73 (link),74 (link)] (Table S2). All PCR assays in this study were performed in 20 μL reaction volumes using 2×Taq Mester Mix (Dye Plus) P112-AA (Vazyme, Nan Jing, China). PCR was performed under the following conditions: an initial 94 °C denaturation step for 10 min, followed by 30 cycles at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, and a final extension of 10 min at 72 °C. The PCR products were detected by 1% agarose (Sigma-Aldrich, St. Louis, MO, USA) gel dyed with GelStain (TransGen, Beijing, China) and electrophoresis for 20 min at 120 V in 1×TAE buffer. Then the image was obtained with the Bio-Rad’s ChemiDoc XRS system.