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).