Eucalyptus roots microbiome under different management conditions (Eucal 4)
The diversity of endophytic bacteria associated with samples of Eucalyptus roots was studied by pyrosequencing of a fragment of the 16S rRNA gene. The processed fragment roots were macerated in liquid nitrogen, and the powder resulting from the milling was used for DNA extraction using the PowerSoil® DNA Isolation Kit (Mobio, CA, USA) according to the manufacturer's instructions except for the initial amount of material that was adjusted to 0.5 g, and the lysis step was modified replacing the vortex by a FastPrep® equipment (Bio 101, CA, USA) at the rate of 5.5 per 40 seconds. The product from extraction was subjected to a further purification step with the ZR DNA purification kit Clean & Concentrator™ (Zymo Research Corporation, Irvine, CA) following the manufacturer's standards. DNA was subjected to PCR using the primers 1114f (5'-GGGTTGCGCTCGTTRC-3 ') and 801R (5'AACMGGATTAGATACCCKG-3'). The pair of primers was designed by James M. Kremer and has been designed especially for the study of endophytic bacteria, to avoid mitochondria and chloroplast amplification. The first primer covers 96.6% of the sequences of bacteria deposited in the Ribosomal Database Project (Cole et al., 2009), the second cover 91.8% of them. The primers were synthesized with MIDs sequences (10 nucleotides coding sequences) for identification of the samples and with the libl adapters for the viability of pyrosequencing. Each root sample (four per treatment) was subjected to PCR reactions in triplicate, in order to normalize possible variations of the individual reactions. The reactions were performed in 20 uL aliquots containing 1X reaction buffer, MgCl2 (3 Total mM), dNTPs (0.2 mM each), 1 U FastStart High Fidelity Taq (Roche), primers (0.2 mM each) and DNA (approximately 10ng). Amplification was performed using the following program: 94 ° C for 2 min followed by 30 cycles of 94 ° C for 30 s, 56 ° C for 40 to 72 ° C for 3 min followed by a final extension of 72 C for 7 min. Following amplification, the triplicate reactions were combined and analyzed by 1.6% agarose gel, extracted from the gel and purified in two steps, using the QIAquick Gel Extraction kit and QIAquick PCR product purification kit (Qiagen). Equimolar amounts of the purified reactions were then combined and sequenced using a Genome Sequencer Titanium system (454 Life Sciences, USA) at Utah State University (USA).