Dna

DNA Extraction, PCR Amplification, and Sequencing

For molecular characterization, genomic DNA was isolated from fungal mycelia using the Trelief™ Plant Genomic DNA Kit (TSINGKE Biotech, Shanghai, China) following the manufacturer's instructions, with subsequent storage at -20°C for preservation. Four phylogenetic markers were used for amplification: ITS, LSU, RPB2, and SSU. The PCR amplification was performed in 30 μL reaction volumes containing: 2 μL genomic DNA template, 1 μL each of forward and reverse primers (ITS9mun/ITS4_KYO1 ( Egger 1995, Toju et al. 2012) for ITS; LR0R/LR5 ( Vilgalys & Hester 1990, Cubeta et al. 1991) for LSU; fRPB2-5F/fRPB2-7cR ( Carbone & Kohn 1999; Rehner & Buckley 2005) for RPB2; and PNS1/NS41 ( Hibbett 1996) for SSU, 11 μL double‐distilled water (ddH2O), and 15 μL 2× Flash PCR Master Mix (containing Taq DNA polymerase, dNTPs, Mg²⁺, and optimized buffer; CoWin Biosciences, Nanjing, China). The PCR program consisted of the following steps: initial denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C (20 s), annealing at 55°C (30 s), and extension at 72°C (25 s); followed by a final extension at 72°C for 5 min. The PCR product was examined by 1% agarose gel electrophoresis to confirm the successful amplification, and then sent to Tsingke Biological Technology, Beijing, China, for Sanger sequencing. The newly generated sequences were deposited in GenBank, and their accession numbers are listed in TABLE 1.