DNA extraction, PCR, and sequencing

Each root sample was washed in 80% ethanol by sonication for 5 min. DNA extraction was then performed with a cetyltrimethylammonium bromide (CTAB) method (Sato and Murakami, 2008). We amplified the internal transcribed spacer 1 (ITS1) region of root-associated fungi using the forward primer ITS1F-KYO1 (Toju et al., 2012) fused with 3–6-mer Ns for improved Illumina sequencing quality (Lundberg et al., 2013) and the forward Illumina sequencing primer (5′- TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG- [3–6-mer Ns]–[ITS1-KYO2]−3′) and the reverse primer ITS2-KYO2 (Toju et al., 2012) fused with 3–6-mer Ns and the reverse sequencing primer (5′- GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G [3–6-mer Ns]—[ITS2_KYO2]−3′). In the PCR, the buffer and DNA polymerase kit of KOD FX Neo (Toyobo) was used with a temperature profile of 94°C for 2 min, followed by 35 cycles at 98°C for 10 s, 50°C for 30 s, 68°C for 50 s, and a final extension at 68 °C for 5 min. The ramp rate through the thermal cycles was set to 1°C/s in order to prevent generation of chimeric sequences (Stevens et al., 2013). To add Illumina sequencing adaptors to respective samples, supplemental PCR was performed using the forward fusion primers consisting of the P5 Illumina adaptor, 8-mer indexes for sample identification (Hamady et al., 2008), and a partial sequence of the sequencing primer (5′- AAT GAT ACG GCG ACC ACC GAG ATC TAC AC—[8-mer index]—TCG TCG GCA GCG TC−3′) and the reverse fusion primers consisting of the P7 adaptor, 8-mer indexes, and a partial sequence of the sequencing primer (5′- CAA GCA GAA GAC GGC ATA CGA GAT—[8-mer index]—GTC TCG TGG GCT CGG−3′). KOD FX Neo was used with a temperature profile of 94°C for 2 min, followed by 8 cycles at 98°C for 10 s, 55°C for 30 s, 68°C for 50 s (ramp rate = 1°C/s), and a final extension at 68°C for 5 min. The PCR amplicons of the 247 root samples and a negative control sample were pooled with equal volume after a purification/equalization process with AMPureXP Kit (Beckman Coulter). The ratio of AMPure reagent to amplicons was set to 0.6 (v/v) in order to remove primer dimers (i.e., sequences shorter than 200 bp).

To discriminate Chamaecyparis and Pinus root samples, we performed, another set of PCR targeting plant chloroplast rbcL region using the rbcL_F3 and rbcL_R4 primers (Toju et al., 2013a) with the same DNA polymerase system, temperature profiles, and purification processes used in the fungal ITS analysis. The sequencing libraries of fungal ITS and plant rbcL regions were processed in an Illumina MiSeq sequencer (run center: KYOTO-HE) with the 2 × 250 cycle sequencing kit (20% PhiX spike-in).