Optimizing Fungal ITS Primer Pairs

Optimal annealing temperatures for the selected fungal ITS primer pairs were explored in vitro. Three ascomycete and four basidiomycete fungal species collected on Yakushima Island, Kagoshima Prefecture, Japan, were subjected to the in vitro examination (A1–3 and B1–4: Table 2). DNA was extracted from fruiting body tissues of the fungal specimens using the CTAB method, as described elsewhere [32] . PCR was conducted using the buffer system of Ampdirect Plus (Shimadzu) with BIOTAQ HS DNA Polymerase (Bioline) under a temperature profile of 95°C for 10 min, followed by 35 cycles at 94°C for 20 s, 47°C, 50°C, 53°C or 56°C for 30 s, and 72°C for 20 s (40 s for the entire ITS region), followed by 72°C for 7 min. The concentration of MgCl₂, dNTPs, PCR primers and the template DNA in the reaction buffer were 1.5 mM, 200 µM, 0.5 µM and 1 ng/µl, respectively. Amplification of the DNA fragments was confirmed using the Flash Gel System for DNA (Lonza).
To evaluate the coverage of the selected fungal ITS primer pairs in vitro, an additional PCR assay was conducted under the optimal annealing temperature tested above using seven ascomycete and seven basidiomycete specimens (Table 2). All necessary permits of the sample collection were issued by Kyushu Regional Forest Office, Ministry of Agriculture, Forestry and Fisheries, Japan.