IRE1TFY01__C2-12__ITS3
Randomly selected five terminal roots (ca. 2 cm each) from each sample were used for DNA extraction. All soil was carefully removed from each root sample by immersion in 70 percent ethanol containing 1-mm-diameter zirconium balls followed by shaking for 2 min at a rate of 15 times per sec using a TissueLyser II (Qiagen, Venlo, The Netherlands) (Toju et al. 2013, Ecol Evol). Washed roots were frozen at -25 degC and then pulverized by shaking at a rate of 20 times per sec for 3 min with 4-mm-diameter zirconium balls using a TissueLyser II. Total DNA was extracted from each root sample using the cetyltrimethylammonium bromide (CTAB) method described elsewhere (Sato and Murakami 2008, Mol Phyl Evol).
We amplified the entire ITS region and the partial ribosomal large subunit region using the fungus-specific high-coverage primer ITS1F_KYO2 and the universal primer LR3 (see Toju et al. 2012, PLOSONE and citations therein). PCR was conducted with a temperature profile of 95 degC for 10 min, followed by 20 cycles at 94 degC for 20 s, 50 degC for 30 s, 72 degC for 120 s, and a final extension at 72 degC for 7 min using an Ampdirect Plus buffer system and Nova Taq. We subjected the PCR product from each root sample to a second PCR step that targeted the ITS2 region. The second PCR was conducted using the universal primer ITS3_KYO2 (Toju et al. 2012, PLOSONE) fused with 454 Adaptor A and sample-specific molecular ID; the reverse universal primer was LR_KYO1b (Toju et al. 2013, PLOSONE) fused with 454 Adaptor B. A buffer system of Taq DNA Polymerase together with Standard Taq Buffer (New England BioLabs, Ipswich, MA, USA) was used with a temperature profile of 95 degC for 1 min, followed by 40 cycles at 94 degC for 20 s, 50 degC for 30 s, 72 degC for 60 s, and a final extension at 72 degC for 7 min.