fungal ITS2 wheat leaves: pair 8 conventional extraction replicate b
Ten leaves were pooled into one sample from each field. DNA extraction was performed on macerated leaf tissue with three extraction replicates per field. The fungal ITS2 region was amplified using the forward primer fITS7 (Ihrmark et al. 2012, FEMS Microbiology Ecology, DOI: 10.1111/j.1574-6941.2012.01437.x) and the reverse primer ITS4 (White et al. 1990, in PCR Protocols: A Guide to Methods and Applications) tagged with an 8 bp barcode. Each sample was run in 3 x 50 ľl PCR reactions with 0.8 ng/ľl template, 200 ľM of each nucleotide, 2.75 mM MgCl2, forward primer at 500 nM, tagged primer at 300 nM and 0.02 U/ľl HotStar Taq polymerase (Qiagen, Germany) in PCR buffer. PCR conditions were 5 min at 94°C, 30-35 cycles of [30 s at 94°C, 30 s at 57°C, 30s at 72°C] and 7 min at 72°C. The number of cycles was adapted for each sample. Three PCR replicates from each extraction replicate were pooled and purified using AMPure (BeckmanCoulter, CA, USA) according to the manufacturers instructions. DNA concentration of the band corresponding to PCR product was measured using capillary electrophoresis on a MultiNA (Shimadzu, Japan) and the samples were pooled in equimolar amounts. The sample pool was sent to Eurofins MWG Gmbh (Ebersberg, Germany) for adaptor ligation and sequencing on 1/8th of a plate on a GS Junior 454 sequencer (Roche, Switzerland). Before sequencing the amplicon pool was purified by gel extraction.