Soil microbial DNA sequencing
DNA was extracted from soil samples using the MOBIO
PowerSoil® DNA Isolation Kit (MOBIO Laboratories,
Carlsbad, CA, USA). DNA quality, concentration and purification were
checked on 1% agarose gel electrophoresis and NanoDrop One UV-Vis
Spectrophotometer (Thermo Scientific, Wilmington, USA). The V4
hypervariable region of the 16S rRNA for bacteria and second internal
transcribed spacer (ITS2) region of the rRNA operon for fungi were
amplified using primer combinations 515F/806R and ITS3F/ITS4R
respectively. Polymerase chain reaction (PCR) were conducted using
BioRad S1000 (Bio-Rad Laboratory, CA), with a 50 μl mixture containing
25 μl of 2
×
Premix Taq, 10 mM of each primer, 60 ng DNA and nuclease-free water. The
PCR amplification had an initial denaturation step at 94 °C for 5 min,
followed by 30 cycles of denaturation at 94 °C (30 s), annealing at 52
°C (30 s), extension at 72 °C (30 s), and a final extension at 72 °C (10
min). PCR products were extracted and purified using
E.Z.N.A.® Gel Extraction Kit (Omega, USA). Amplicon
libraries were prepared using NEBNext® Ultra™ DNA
Library Prep Kit for Illumina® (New England Biolabs,
USA) followed the manufacturer’s protocol, and PE250 sequencing was
performed on NovaSeq 6000 Sequencing System (Illumina, San Diego, USA).
Operational taxonomic units (OTUs) were categorized at 97% sequence
similarity using the UPARSE package, and taxonomic assignment for
bacteria and fungi was performed using Silva and Unite database
respectively. Singleton sequences, chloroplast or mitochondria (16S
amplicon) sequences and sequences that could not assign to the kingdom
level were removed from the final OTU table. Each sample was rarefied to
the same number of reads as the smallest sample (52190 and 29940 reads
for bacteria and fungi respectively) for further analyses. Alpha
diversity for soil bacteria and fungi was measured using the exponential
Shannon-Weiner index based on the rarefied OTU tables. Fungal OTUs were
assigned to arbuscular mycorrhizal fungi (AMF) and putative plant
pathogens using FUNGuild (Nguyen et al. 2016) and further checked
based on literature (Roncero et al. 2003; Wu et al. 2008;
Mendes et al. 2013).