title: Soil Fungal Community under Eucalyptus plantation
design: The soil DNA was extracted using a PowerSoil® DNA Isolation Kit (Mobio, CA, USA), according to the manufacturer’s instructions (except in the lysis step, which was modified to be performed using FastPrep® equipment (Bio 101, CA, USA) with a frequency of 5.5 for 40 seconds).  The fungal communities were studied using pyrosequencing. The extracted DNA was submitted to PCR amplification targeting a fragment of the large sub-unit of the rRNA gene (28S rRNA), using MID adapted primers (barcodes with 10 nucleotides added to each primer) LR0R (5′-ACCCGCTGAACTTAAGC-3′) (Cubeta et al., 1991) and LR3 (5′-CCGTGTTTCAAGACGGG-3′) (Vilgalys and Hester, 1990). The primers amplify the region 26 to 651 of the 28S rRNA (in relation to Saccharomyces cerevisiae). PCR reactions of 20 μl containing Buffer 1X, MgCl2 3.37 mM, dNTP 5 nM each, BSA 0.1 mg ml-1 and GoTaq 2.5u (Promega) were performed in triplicate per sample, with the following program 94 ˚C for 3 min followed by 30 cycles of 94 ˚C for 1 min, 51 ˚C for 40 s and 72 ˚C for 1 min and a final extension step of 72 ˚C for 10 min. After amplification the triplicate PCR reactions of each sample were combined, analysed and extracted in 1.6% agarose gels, and subsequently purified in two steps using the QIAquick Gel Extraction and QIAquick PCR product purification kits (QIagen). Equimolar amounts of the PCR products from the different samples were combined and submitted to pyrosequencing on a Genome Sequencer Titanium system (454 Life Sciences, USA) at Utah State University facility, and were sequenced after a ligation step to insert adaptors to the amplicons.