Rapid Genomic DNA Extraction and PCR Amplification

Genomic DNA of each strain was extracted from fresh mycelium growing on PDA after 5 days of growth following the rapid “thermolysis” method described in Zhang et al. (2010) (link). For the amplification of ITS, RPB2, and TEF1-α gene fragments, ITS4 and ITS5 for ITS (White et al. 1990 (link)), EF1-728F (Carbone and Kohn 1999 (link)) and TEF1LLErev (Jaklitsch et al. 2005 (link)) for TEF1, and RPB2-5F and RPB2-7R for rpb2 (Liu et al. 1999 (link)) were used. Each PCR reaction consisted of 12.5 μl T5 Super PCR Mix (containing Taq polymerase, dNTP, and Mg²⁺, Beijing TsingKe Biotech Co. Ltd., Beijing), 1.0 μl of forward primer (10 μM), 1.0 μl of reverse primer (10 μM), 0.5 μl DMSO, 3 μl DNA template and 7 μl double sterilized water. PCR reactions were in Eppendorf Mastercycler, following the protocols described by Sun et al. (2016) (link). PCR products were purified with the PCR product purification kit (TIANGEN Biotech, Beijing, China), and sequencing was carried out in both directions on an ABI 3730 XL DNA sequencer (Applied Biosystems, Foster City, California) with primers used during PCR amplification.