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. Five phylogenetic markers were targeted for amplification: ITS, SSU, LSU, TEF 1, and RPB2. 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; PNS1/NS41 ( Hibbett 1996) for SSU; EF1-983F/EF1-2218R for TEF 1 ( Carbone & Kohn 1999; Rehner & Buckley 2005); fRPB2-5F/fRPB2-7cR ( Carbone & Kohn 1999; Rehner & Buckley 2005) for RPB2, 11 μL sterile ddH 2 O, and 15 μL 2× Flash PCR Master Mix (containing Taq DNA polymerase, dNTPs, Mg²⁺, and optimized buffer; CoWin Biosciences, Nanjing, China). Thermal cycling parameters included: initial denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C (15 s), annealing at 55°C (25 s), and extension at 72°C (25 s); followed by final extension at 72°C for 5 min. Successful amplification was confirmed through electrophoretic separation on 1% agarose gels. Sanger sequencing was conducted by Tsingke Biological Technology ( Beijing, China). The newly generated sequences were deposited in GenBank, and their accession numbers are listed in Table 1.