2.4.1 DNA extraction, PCR amplification and sequencing
Soil biological samples representing different treatments were frozen at
-80 °C for further DNA analysis. DNA was directly extracted using Power
Soil kit 152 (MoBio Laboratories, Carlsbad, CA, USA) following the
manufacturer’s instructions for specific amplification and high
throughput sequencing.
The V3-V4 region of bacterial 16S rRNA gene was amplified by a
polymerase chain reaction (PCR) using the primer 341F
(5’-CCTAYGGGRBGCASCAG-3’) and 806R (5’-GGACTACNNGGGTATCTAAT-3’). PCR
reactions were carried out in a 25 µl mixture with three replicates per
DNA sample, containing 5 μl of Q5 reaction buffer (5×), 5 μl of Q5
High-Fidelity GC buffer (5×), 0.25 μl of Q5 High-Fidelity DNA Polymerase
(5 U/μl), 2 μl (2.5 mM) of dNTPs, 1 μl (10 uM) of each Forward and
Reverse primers, 2 μl of DNA Template, and 8.75 μl of dd
H2O. The fungal ITS rRNA genes were amplified with
ITS1F(5’-CTTGGTCATTTAGAGGAAGTAA-3’)/ITS2R(5’-GCTGCGTTCTTCATCGATGC-3’)
primers. PCR reactions were carried out in a 25 µl mixture with three
replicates per DNA sample, containing 2 μL of 10× Buffer, 2 μL of 2.5 mM
dNTPs, 0.8μL of each Primer (5 μM), 0.2 μL of rTaq Polymerase, and 10 ng
Template DNA. PCR amplification was performed under the following
cycling conditions: initial denaturation at 98 °C for 2 min, followed by
25 cycles consisting of denaturation at 98 °C for 15 s, annealing at 55
°C for 30 s, and extension at 72 °C for 30 s, with a final extension of
5 min. All PCR amplifications were performed in triplicate and then
combined. PCR amplicons were then pooled in equimolar concentrations on
a 1% agarose gel, and purified PCR products were recovered using a Gel
Extraction Kit (Omega Bio-tek, Norcross, GA, USA). High-throughput
sequencing of the PCR products was performed on an Illumina Miseq
platform (Miseq PE250)(Zhang et al ., 2020).