Ekhaga_Sunflower_Soil_nosZII
DNA was extracted from soil and root samples using the FastDNA™ SPIN Kit for Soil (MP Biomedicals, NY, USA) following the manufacturer’s instructions with 200-300 mg of soil and roots, respectively. In addition, DNA was extracted from 200-300 mg each of the barley and sunflower seeds. The DNA concentration was determined using a Qubit® fluorometer and the Qubit® dsDNA BR assay kit (Life Technologies Corporation, USA).  Sequences of nosZ Clade II were PCR amplified from the DNA extracts in a two-step procedure as recommended by (Berry et al., 2011).  For each sample, three replicate reactions using 10 ng template DNA, 2X Dream Taq Green PCR master Mix (Thermo Fisher Scientific, USA), 25 mM MgCl2, 10 % BSA and 0.8 μM of the nosZ Clade II  primers by (Jones et al., 2013) with 20 cycles of amplification. In the second PCR, 2 μL of the pooled PCR products from each sample in the first reaction were amplified in five replicates of a 15-cycle PCR using the forward primer preceded by a 10 bp-long barcode, a sequencing key and a forward sequencing adapter; the reverse primer being only preceded by the sequencing key and the reverse sequencing adapter. The PCR products from the second PCR were agarose gel verified, pooled and purified using the QIAquick PCR Purification Kit (Qiagen; France). Pyrosequencing was performed by Microsynth (Balgach, Switzerland) on a 454 FLX Genome Sequencer (Roche Applied Science, Penzberg, Germany).