title: Soil microbiome
design: Five bulk soil samples were collected in each plot from the upper 10 cm of mineral soil and pooled. DNA was extracted from 0.25 g of each soil subsample using the Powersoil DNA Isolation Kit (Qiagen) following the manufacturer's protocol and eluted into a final volume of 100 _l. Microbial communities were assessed using DNA metabarcoding to characterize the distribution and abundance of soil bacteria and fungi in the monoculture plantations. To amplify bacteria, we targeted the V4 hypervariable region of the 16S rRNA gene using the 515F-806R primer pair [43]. For the fungal analysis, we amplified the first internal transcribed spacer region (ITS1) of the rRNA operon, using the primers ITS1F [44] and ITS2 [45]. All primers included all necessary Illumina adapters with barcodes to distin-guish samples. The bacterial 806R primer included a 10 bp barcode sequences while the ITS1F and ITS2R primers each included an 8 bp index sequence, allowing us to multiplex multiple samples. We used 5Prime Hot MasterMix (QuantaBio) in PCR reactions with a final volume of 12.5 ul, with 25 cycles and an annealing temperature of 50_ C for both loci. PCR reactions were done in triplicate, pooled and products were cleaned and normalized using Sequelprep Normalization plates (Thermo Fisher). Libraries were sequenced on the Illumina MiSeq at the STRI Naos Molecular Laboratory using 2x250 v2 reagent cartridges.
identifiers.namespace: SUB15511657
identifiers.id: 72_ITS_Pq