Dna

DNA Extraction, PCR Amplification, and Sequencing

Fungal genomic DNA was extracted from mycelia using the Trelief™ Plant Genomic DNA Kit ( Beijing Qingke Biotech) in accordance with the manufacturer’s protocol. Based on a comprehensive review of the pertinent literature, suitable DNA barcodes and corresponding primer sequences were selected, and PCR reactions were carried out. Four DNA barcodes were utilized, and the primers employed included ITS9mun and ITS4_KYo1 ( Toju et al. 2012) for the internal transcribed spacer (ITS), LR0R ( Vilgalys & Hester 1990) and LR5 ( Cubeta et al. 1991) for the large subunit ribosomal RNA (LSU), fRPB2-7cR and fRPB2-5F ( Voglmayr et al. 2016) for the second largest subunit of RNA polymerase II ( rpb2), and EF1-728F and EF1-986R ( Rehner et al. 2005) for translation elongation factor 1-alpha ( tef1-α). The final PCR reaction system was set at 30 μL, consisting of 15 μL PCR Master Mix (CoWin Biosciences, Taizhou, China), 11 μL ddH 2 O, 1 μL each of forward and reverse primers (10 μM), and 2 μL of DNA template. The thermal cycling conditions for ITS, LSU, tef1-α, and rpb2 were set as follows: an initial denaturation occurred at 94 °C for 5 minutes, followed by 35 cycles. For ITS, LSU, and tef1-α, each cycle consisted of denaturation at 94 °C for 45 seconds, annealing at 56 °C for 50 seconds, and extension at 72 °C for 1 minute. For rpb2, each cycle included denaturation at 95 °C for 60 seconds, annealing at 56 °C for 120 seconds, and extension at 72 °C for 90 seconds. A final extension at 72 °C for 10 minutes was performed to ensure complete amplification. The PCR products were analysed using 1% agarose gel electrophoresis, followed by Sanger sequencing conducted by Sangon Biotech Co., Ltd. After obtaining the raw sequencing data, the SeqMan v.7.1.0 software was employed to evaluate the chromatogram files, check sequencing quality, trim the unstable terminal sequences, and compile the bidirectional sequencing results.