H33YU2J01__TOMA_521__rbcL_F3
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 host-plant chloroplast rbcL sequences based on a tag-encoded massively-parallel pyrosequencing. For each root sample, a 0.5-kb rbcL gene fragment of plants was amplified using the forward primer rbcL_F3 (5'-AAY TCC CAA CCA TTY ATG CG-3') 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_R4 (5'-CAT ATG CCA AAC RTG AAT ACC-3') fused with the 454 Adaptor B (5'-CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG-3'). PCR was conducted using a temperature profile of 95??C for 10 min, followed by 40 cycles at 94??C for 20 s, 56??C for 30 s, 72??C for 90 s and a final extension at 72??C for 7 min with a buffer system of Ampdirect Plus (Shimadzu Corp., Kyoto, Japan) and BIOTAQ HS DNA Polymerase (Bioline, London, UK). 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.