Miseq sequencing of arbuscular my corrhiza in soil
Genomic DNA was extracted from 0.25 g fresh soils with a PowerSoil DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA, USA) following the manufacturers  instruction. An approximately 334 bp region of the 18S rDNA gene was amplified with two-step PCR. The first amplification with primers GeoA-2 (Schwarzott and Sch  ler 2001) and AML2 (Lee et al., 2008) was carried out in a final 25  L reaction solution including 2.5  L 10   buffer, 1.5 mM MgCl2, 200  M of each dNTP, 0.75  M of each primer, 0.75 U PrimeSTAR HS DNA Polymerase (Takara, Japan), and 1 L template DNA. The thermal cycling was followed by an initial denaturation at 94  C for 5 min, 30 cycles of denaturation at 94  C for 45 s, annealing at 56  C for 1 min, and extension at 72  C for 1.5 min, followed by a final extension at 72  C for 10 min. The products of the first amplification were diluted for 10 times with sterilized deionized water and 1.0  l diluted solution was used as the template for the nested PCR. Primers AMDGR (Lumini et al., 2010) and NS31 (Simon et al., 1992) were used for the nested PCR, and the forward primer was modified with a unique 12 nt barcode at the 5 end. Conditions for the nested PCR were similar to the first PCR, except for 58  C annealing temperature and extension for 1 min. PCR products were purified using an agarose gel DNA purification kit (TaKaRa, Japan) and quantified using Nanodrop 2000 (Thermoscientific, USA). Only PCR products with concentration > 10 ng/ L and OD 260 / OD 280 1.8 were used, others were discarded and re-amplified to ensure the Miseq sequencing quality. The finnal PCR products from all samples were mixed at equimolar concentrations and then subjected to Illumina Miseq platform at Environmental Genome Platform of Chengdu Institute of Biology, Chinese Academy of Sciences.