G6YD1NQ01__AGGACTAG__rbcL
All soil was carefully removed from each root sample (ca. 2 cm in length) by placing them in 70% with 1-mm zirconium balls ethanol and then shaking the sample tubes 15 times per second for 2 min using TissueLyser II (QIAGEN). This procedure removed soil from the terminal root samples. The washed root was frozen at -25 degrees C and then pulverized by shaking it with 4-mm zirconium balls 20 times per second for 3 min using TissueLyser II. Plant and fungal DNA was extracted from each root sample by a cetyl trimethyl ammonium bromide (CTAB) method.
We sequenced host-plant chloroplast rbcL sequences based on a tag-encoded massively-parallel pyrosequencing. For each root sample, plant rbcL sequences were amplified using the primers rbcL_rvF (5'-CCA MAA ACR GAR ACT AAA GC-3') and rbcL_R1 (5'-CGR TCY CTC CAR CGC AT-3') with the buffer system of Ampdirect Plus (Shimadzu) and BIOTAQ HS DNA Polymerase (Bioline). PCR was conducted under a temperature profile of 95 degrees C for 10 min, followed by 30 cycles of 94 degrees C for 20 s, 50 degrees C for 30 s, and 72 degrees C for 30 s, and final extension at 72 degrees C for 7 min. The PCR product of each root sample was subjected to the second PCR amplification of 0.5-kb rbcL gene fragment using the rbcL_rvF primer fused with the 454 pyrosequencing Adaptor A (5'-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG-3') and the 8-mer molecular ID of each sample, and the reverse primer rbcL_R2 (5'-CCY AAT TTT GGT TTR ATR GTA C-3') fused with the 454 Adaptor B (5'-CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG-3'). 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 1 min, followed by 40 cycles of 94 degrees C for 20 s, 50 degrees C for 30 s, and 72 degrees C for 30 s, and final extension at 72 degrees C for 7 min. The rbcL amplicons of the second PCR step were subjected to pyrosequencing. The first 480 and the second 480 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 480 samples was conducted as instructed by the manufacturer. Likewise, the amplicons of the remaining 480 samples were pooled and purified, and then sequenced in the second run.