root-associated Fungal Community Analysis
For Fungi, the amplicon libraries were constructed using the fungal primers ITS1F and ITS4 targeting the whole ITS region (White et al., 1990; Gardes & Bruns, 1993). Each primer pairs contained a 5 6 bp-long barcode sequence used to identify each sample in multiplexed pyrosequencing runs. Four independent PCRs were performed for each DNA sample under the following conditions: 2 L of template DNA (at 2ng/l), 0.5 M of each primer, 0.1 g l1 bovine serum albumin (BSA), 0.2 mM dNTPs and the supplied reaction buffer, with 2 U of High Fidelity Taq (Roche) to obtain a final volume of 25 l. Amplifications were run on a (Biometra, Gttingen, DE) following this programme: 95 C for 10 min, 35 cycles of 30 s at 95 C (denaturation), 52 C for 1 min (annealing) and 72 C for 40 s (extension), followed by 10 min at 72C. The DNA was amplified using a T3000 thermal cycler (Biometra, Gttingen, DE) and amplification success was checked on a 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.