Dna extraction, , PCR

Han, Meiyan, Karunarathna, Samantha C., Lu, Li, Zheng, Dege, Dai, Dongqin, Suwannarach, Nakarin, Kumla, Jaturong, Elgorban, Abdallah M., Zhang, Lijuan, Chukeatirote, Ekachai & Tibpromma, Saowaluck, 2025, Pseudodictyosporium yunnanensis sp. nov. (Dictyosporiaceae, Pleosporales) from dead twigs of Coffea arabica in China, Phytotaxa 711 (1), pp. 28-42 : 30

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from fresh fungal mycelia that had been cultured on PDA for 2–4 weeks using the Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux, China), following the manufacturer's instructions. The extracted DNA was stored at -20 °C for subsequent use. Polymerase chain reaction (PCR) was conducted targeting four loci: SSU, ITS, LSU, and tef 1-α genes, with primer pairs LR5/LR0R ( Vilgalys & Hester, 1990), ITS5/ITS4, NS1/ NS4 ( White et al. 1990), and EF1-983F/EF1-2218R (Rehner & Buckley 2005). The PCR reactions were prepared in a total volume of 25 µL, consisting of 12.5 µL of 2× Bench Top™ Taq Master Mix, 8.5 µL of ddH 2 O, 2 µL of each forward and reverse primer, and 2 µL of the DNA template. The same PCR conditions for the LSU, ITS, SSU, and tef 1-α genes involved an initial denaturation at 94 °C for 3 minutes, followed by 35 cycles of 94 °C for 45 seconds, 55 °C for 50 seconds, and 72 °C for 60 seconds, with a final extension at 72 °C for 10 minutes. The PCR products were purified and sequenced by Sangon Biotechnology Co., Ltd. (Shanghai, China). All new sequences generated in this study were submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and are detailed in Table 1.