Molecular analyses
Soil DNA was extracted using MoBio PowerSoil kits ® following
manufacturer’s instructions. Prokaryotic and fungal DNA were amplified
targeting the 16S and ITS rDNA regions, respectively. The primers used
for the amplification were the 515f-806R couple (targeting the
hypervariable V4 region) for 16S and the ITS1f-ITS2 couple for ITS
(targeting the ITS1 subregion) (White et al. , 1990; Gardes &
Bruns, 1993; Gilbert et al. , 2014). PCR amplifications were
conducted with 0.2 μM primers, 0.2mM dNTPs, 0.5 units of hi-fidelity Taq
polymerase (New England Biolabs), and 1 μL of DNA extract in 25 μL
reactions. Thermocycling was done as follows: initial denaturation at
95°C for 1 minute, 30 cycles of denaturation (95°C 30s) - annealing
(16S: 50°C / ITS: 52°C for 30s) – elongation (72°C 1 min), and a final,
5 min extension at 72°C. Blanks were included to ensure absence of DNA
amplification when no soil was added in DNA extraction kits. Positive
amplicons were purified and sequenced using the Illumina MiSeq
technology (Genome Québec Innovation Center).
Sequences were demultiplexed and paired-ends were merged in QIIME
(Caporaso et al. , 2010), after which we proceeded to a quality
filtering (quality score > 25) and a removal of sequences
with homopolymers longer than 10 bases. Chimera removal and operational
taxonomical unit (OTU) clustering was performed in the open source
software VSEARCH (Rognes et al. , 2016), at a 97% similarity
threshold. Taxonomy was assigned in QIIME using the curated databases
Silva and UNITE (Pruesse et al. , 2007; Abarenkov et al. ,
2010). We removed OTUs occurring in only one sample and occurrences with
5 reads or less, considering these as potential artefacts. We rarefied
our OTU tables (16S and ITS) to 8000 reads per sample for fungi and 1100
reads for prokaryotes to correct for imbalanced sequencing depth per
sample, due to the stochastic nature of sequencing. Sequence data were
deposited in SRA under project # 7716646.