DNA extraction, PCR amplification and DNA sequencing

Genomic DNA was extracted from a 20 mg piece of dried tissue by a modified CTAB method (Lee et al. 1988). Loci examined during this study include the complete ITS region (ITS1–5.8S–ITS2) of the nuclear ribosomal RNA gene (hereafter ITS), the first ca. 900 bp of the nuclear 28S rRNA gene (nrLSU) and the mitochondrial small subunit rRNA gene (mtSSU).

Primers used for amplification were: ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) for ITS; LR0R and LR5 for nrLSU (Vilgalys and Hester 1990); and MS1 and MS2 for mtSSU (White et al. 1990). The amplification reaction mixture contained 2.5 µl Econo buffer, 0.5 µl dNTPs, 1.25 µl each primer, 0.125 µl Econo Taq, 14.375 µl of deionised water and 5 µl of template DNA. Thermal profile of PCR for ITS was initial denaturation at 94 °C for 1 min.; then 35 cycles of denaturation at 94 °C for 1 min, annealing at 53 °C for 1 min and extension at 72 °C for 1 min; and final extension at 72 °C for 8 min. For nrLSU: 94 °C for 2 min; then 40 cycles of 94 °C for 1 min, 52 °C for 1 min and 72 °C for 1:30 min; and 72 °C for 5 min. For mtSSU: 95 °C for 10 min; then 30 cycles of 95 °C for 30 sec, 52 °C for 30 sec and 72 °C for 40 sec; and 72 °C for 7 min.

PCR products were run on 1% agarose gel, stained with ethidium bromide and bands were visualised under a UV transilluminator. Amplified PCR products of the ITS region were sent for purification and bidirectional sequencing to Macrogen (Republic of Korea). PCR products of 28S and 16S were purified using QIAquick PCR purification kit (Qiagen, Stanford, California) as per manufacturer’s guidelines and sequencing reactions were performed using the Big Dye Terminator v3.1 Cycle Kit (Life Technologies, Carlsbad, California). Sequencing was carried out using the same primers as those used for PCR.