HESHY4K01__AACGGCTT__ITS
DNA extraction from individual soil sample (0.10 g dry weight per extraction) by a cetyl trimethyl ammonium bromide (CTAB) method.
First PCR: PCR-amplified with the fungus-specific high-coverage primer ITS1-F_KYO2 (5'-TAG AGG AAG TAA AAG TCG TAA-3'; Toju et al. 2012) and the universal primer LR3 (5'-CCG TGT TTC AAG ACG GG-3'; Vilgalys and Gonzalez 1990). The first PCR was performed with the buffer system of Ampdirect Plus (Shimadzu) and BIOTAQ HS DNA Polymerase (Bioline). The PCR condition for the first step was as follows: 95 degrees C for 10 m, followed by 30 cycles of 94 degrees C for 30s, 53 degrees C for 30 s and 72 degrees C for 90 s, and a final extension at 72 degrees C for 7 m. Second PCR: Targeting a 0.5-kb fragment of ITS2 and partial LSU regions with the forward primer ITS3_KYO2 (Toju et al. 2012) fused with the 454 Adaptor A sequence and a multiplex identifier (MID) tag sequence (Hamady et al. 2008) to identify source soil samples (5'-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG [Adaptor A] ?xxxxxxxx [MID] -GAT GAA GAA CGY AGY RAA-3' [ITS3_KYO2]), and the reverse primer LR_KYO1b fused with the 454 Adaptor B sequence (5'-CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG [Adaptor B] -MGC WGC ATT CCC AAA CWA-3' [LR_KYO1b]). The second PCR was conducted with the buffer system of Taq DNA Polymerase with Standard Taq Buffer (New England BioLabs) under a temperature profile of 95 degrees C for 2 m, followed by 40 cycles of 94 degrees C for 30 s, 50 degrees C for 30 s and 72 degrees C for 60 s, and final extension step at 72 degrees C for 7 m.