Dna extraction, , PCR

Chen, Yanpeng, Liao, Yuechi, Shami, Ashwag, Alharbi, Nada K., Liu, Jian-Kui & Maharachchikumbura, Sajeewa S. N., 2025, Advancements in fungal and fungi-like diversity with a new species and updated records, Phytotaxa 701 (2), pp. 127-147 : 128

DNA extraction, PCR amplification, and sequencing

For unculturable specimens, fungal material, including mycelium, conidiophores, and conidia, was directly harvested from the substrates and transferred into 1.5 mL Eppendorf tubes. In contrast, sufficient mycelium was collected from culturable specimens. DNA extraction was performed using the Trelief™ Plant Genomic DNA Extraction Kit (TSINGKE Biotech, Shanghai, China), following the manufacturer’s instructions. If the initial DNA extraction failed, the process was repeated thrice. Two barcodes, viz., the nuclear ribosomal internal transcribed spacer (ITS) region and the partial sequence of the large-subunit rRNA gene (LSU), were amplified by polymerase chain reaction (PCR). The pairwise primers were ITS9mun and ITS4_KYO1 ( Toju et al. 2012) for ITS, LR0R ( Vilgalys & Hester 1990) and LR5 for LSU. The PCR reaction volume was 25 μL containing 2 μL of DNA template, 1 μL each of the forward and reverse primer (10 μM), 8.5 μL of double‐distilled water (ddH 2 O), and 12.5 μL of 2 × Flash PCR MasterMix (mixture of DNA Polymerase , dNTPs, Mg 2+ and optimized buffer; CoWin Biosciences, Taizhou, China). The PCR products were checked by agarose gel electrophoresis and then sent to Tsingke Biological Technology (Beijing, China) for Sanger sequencing. Based on the Sanger sequencing chromatograms, raw data were manually trimmed and assembled into consensus sequences using SeqMan Pro version 7.1.0 (DNASTAR, Inc. Madison, WI, USA).