HXVJWSB01__Yaku_0695__ITS3
All soil was carefully removed from the samples by placing them in 70% ethanol with 1-mm zirconium balls and then shaking the sample tubes 15 times per second for 2 min using a TissueLyser II (Qiagen, Venlo, The Netherlands). The washed root was frozen at _25??C and then pulverised by shaking with 4-mm zirconium balls 20 times per second for 3 min using a TissueLyser II. Plant and fungal DNA was extracted from each root sample by a cetyl trimethyl ammonium bromide (CTAB) method.
We sequenced fungal ITS sequences based on a tag-encoded massively-parallel pyrosequencing. For each root sample, the entire ITS region and partial ribosomal large subunit region was amplified using the fungus-specific high-coverage primer ITS1F_KYO2 and the universal primer LR3 (http://www.biology.duke.edu /fungi/mycolab/primers.htm). PCR was conducted using a temperature profile of 95??C for 10 min, followed by 20 cycles at 94??C for 20 s, 50??C for 30 s, 72??C for 120 s and a final extension at 72??C for 7 min with a buffer system of Ampdirect Plus and BIOTAQ HS DNA Polymerase (Shimadzu, Kyoto, Japan). The PCR product of each root sample was subjected to a second PCR step targeting the ITS2 region. The second PCR was conducted with a universal primer ITS3_KYO2 fused with the 454 Adaptor A and each sample-specific molecular ID, and the reverse universal primer LR_KYO1b (5'-MGC WGC ATT CCC AAA CWA-3') fused with the 454 Adaptor B. A buffer system of Taq DNA Polymerase with Standard Taq Buffer (New England BioLabs, Ipswich, MA, USA) was used under a temperature profile of 95??C for 1 min, followed by 40 cycles at 94??C for 20 s, 50??C for 30 s, 72??C for 60 s and a final extension at 72??C for 7 min. The ITS amplicons of the second PCR step were subjected to pyrosequencing. The first 576 and the second 624 samples were sequenced separately using a GS Junior sequencer (Roche). The rbcL amplicons of the first 480 root samples were pooled and purified using ExoSAP-IT (GE Healthcare) and QIAquick PCR Purification Kit (QIAGEN). The sequencing of the first 576 samples was conducted as instructed by the manufacturer. Likewise, the amplicons of the remaining 624 samples were pooled and purified, and then sequenced in the second run.