paddy soil AMF Community Analysis
For 18S AMF libraries we performed a nested PCR approach to overcome the difficulties of mixed starting material. The 18S primer sets AML1/AML2 (Lee et al. 2008) were used in the primary amplification followed by nested amplification with AMADF/AMDGR specific primers (Berruti et al., 2015; Sato et al., 2005) targeting the V3-V4 region of the SSU rRNA gene. This primer pair was chosen because of its suitable characteristics for the GS-FLX System, amplifyinga target sequence of approximately 450 bp including the SSU rDNA variable domain V3 and V4 from a broad spectrum of AMF taxa (Glomerales, Diversisporales and Archaeosporales, (Berruti et al. 2018). Each primer pairs contained a 5 6 bp-long barcode sequence used to identify each sample in multiplexed pyrosequencing runs. Four independent primary PCRs were performed for each sample under the following conditions: 20 l reaction volume containing 2 L template DNA (standardized at 2ng/l) and 18 l reaction mix, Final concentrations in the PCR were 0.2mM of each dNTPs, 0.5 M of each primer, 1x Phusion HF Buffer, and 0.02 U/l Phusion polymerase. The amplification program was as follows: 5 at 99 C, followed by 30 cycles of 10 s at 99 C, 30 s at 58 C, 30 s at 72 C and a final extension step at 72 C for 7 min. The DNA was amplified using a T3000 thermal cycler (Biometra, Gttingen, DE) and amplification success was checked on a agarose gel. The first PCR products were diluted at 1/50 and 1/100 in sterile water for nested PCR that were carried in duplicate with primers AMADF/AMDGR. The nested PCR was performed with 0.02 U/l Phusion polymerase, 2l PCR product, 1x Phusion HF Buffer, 0.5M of each primer (Barcode + primer), 0.2mM of each dNTPs, a total volume of 25 l. Cycling parameters were 30 s at 98c, 25 cycles of 10 s at 98C, 30 s at 61C, 30 s at 72C, followed by 10 min at 72C. The PCR product of the nested approach was run on a 1.5 % agarose gel. Each of the technical replicate was amplified in duplicate, pooled, checked on 1.5% agarose gel and concentration estimated spectrophotometrically as previously described. Libraries were sequenced using a Genome Sequencer (GS) FLX Titanium 454 System (Roche) at Beckman Coulter Genomics (Beverly, MA, USA). The two 454 pyrosequencing forward primer (Primer A-Key) and reverse primer (Primer B-Key) were added to the amplicons by random ligation, so that each amplicon library will generate reads from both the 5 or 3 ends.