PCR-RFLP analysis of ITS region

A quick DNA extraction method was used for the PCR-RFLP analysis. Briefly, mushrooms were washed with MilliQ water, and took an approximately 100-mg portion was placed in a 1.5-mL Eppendorf tube. The mushrooms were homogenated using a BioMasher II in 400 μL PrepMan Ultra Sample Preparation Reagent, and incubated at 100 C for 10 min. Then, samples were centrifuged at 13,000 × g for 2 min. The supernatant was used as PCR template for PCR-RFLP. PCR amplification for the ITS region was performed using BIOTAQ HS DNA polymerase (Bioline, London, UK) and primers, ITS1 and ITS4. PCR reaction conditions were the same as those in Fungal sampling, DNA isolation, PCR, sequencing and dataset assembly section. For restriction endonuclease reaction, purified PCR product was digested using MslI, DdeI, or a combination of HaeIII and HincII (10U per 1 μg PCR product; FastDigest, ThermoFisher Scientific, MA, USA) for 5 min to 30 min at 37 °C, and the digested product was run on a 3% agarose gel in TAE buffer.