Dna extraction, , PCR

DNA extraction, PCR amplification, and sequencing

The TreliefTM Plant Genomic DNA Kit ( Beijing Qingke Biotech) was used to extract fungal genomic DNA from mycelium, following the steps outlined in its instruction manual. Polymerase Chain Reaction (PCR) was performed to amplify the ITS, LSU, and tef1-α regions using the primer pairs and conditions listed in Table 1. The final PCR reaction was prepared in a total volume of 30 µL, containing 15 µL of PCR Master Mix (CoWin Biosciences, Taizhou, China), 11 µL of double-distilled water (ddH₂O), 1 µL each of forward and reverse primers (10 µM), and 2 µL of DNA template. PCR products were visualized using 1% agarose gel electrophoresis and subsequently sent to Sangon Biotech Co., Ltd. ( Shanghai, China) for Sanger sequencing. The raw sequencing data were processed using SeqMan software version 7.1.0 to manually trim unstable regions at both ends and to assemble the bidirectional sequences.