2.4 DNA extraction, amplification, sequencing and data processing
Soil total DNA was extracted from 0.3 g of homogenized soil samples using a DNA extraction kit (MoBio Laboratories Inc., CA, USA) following the manufacturer’s instructions. The quantity and quality of the extracted DNA were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, MA, USA), and were further checked with 1% (w/v) agarose gel. A primer set of ITS5-1737F (5’-GGAAGTAAAAGTCGTAACAAGG-3’) and ITS2-2043R (5’-GCTGCGTTCTTCATCGATGC-3’) with adaptors and barcodes was used to amplify fungal ITS hypervariable region (White, Bruns, Lee, & Taylor, 1990). Polymerase chain reaction (PCR) followed the thermal-cycling conditions: initialization for 5 min at 94 °C, 30 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 52 °C, and extension for 30 s at 72 °C, followed by a final elongation for 10 min at 72 °C. PCR products were mixed in equal density ratios according to the Gene Tools Analysis Software (Version4.03.05.0, SynGene), and were purified using an E.Z.N.A® Gel Extraction Kit (Omega Bio-Tek, Norcross, Georgia, USA). The resultant PCR products were sequenced on the IlluminaHiSeq2500 (250-bp paired-end reads) platform at Magigene Biotechnology Co., Ltd. (Guangzhou, China).
The paired-end raw reads were proceeded using Quantitative Insights Into Microbial Ecology (QIIME) pipeline (Caporaso et al., 2010) for quality filtering, trimming, and chimera checking. Low quality sequences (ambiguous bases, nucleotide mismatches, an average quality score< 20, length < 100 bp, total expected errors > 0.1) were filtered. After discarding chimeras and singletons, operational taxonomic units (OTUs) were identified using UPARSE with a similarity threshold of 97% (Edgar, 2013). Finally, representative sequences of fungal OTU were selected for taxonomic assignment by using BLAST against the UNITE database (http://unite.ut.ee/index.php) (Abarenkov et al., 2010), using the Ribosomal Database Project (RDP) classifier (Caporaso et al., 2010). Fungal functional guilds were tentatively assigned using the ‘FUNGuild’ algorithm (Nguyen et al., 2016). All raw sequences were deposited in the NCBI Sequence Read Archive (SRA) database under accession number SRR (SUB9976505).