DNA-metabarcoding of soil fungi
The full ITS region was amplified by PCR, using the universal fungal primers ITS1f-Kyo2 (forward): TAGAGGAAGGAAGTAAAAGTCGTAA and LR3-I (reverse): TGGTCCGTGTTTCAAGAC (Mafune et al. 2020). The Phusion Plus DNA Polymerase (Thermo Fisher Scientific) was used to amplify 10 ng of template DNA following the polymerase standard program: initial denaturation at 98C for 30 seconds, 30 amplification cycles at 98C for 10 seconds, 56C for 10 seconds and 72C for 30 seconds, final extension at 72C for 5 minutes. PCR products were visualized by electrophoresis on a 1% agarose gel. PCR products were purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega) according to the manufacturer's instructions.