2.3 High-throughput amplicon sequencing
Amplicon sequencing of the bacterial and fungal rhizosphere populations
for 20 soil samples was conducted with the V4 fragment of the 16S rRNA
gene and the ITS1 gene fragment, respectively, using the Illumina HiSeq
platform (Illumina, San Diego, CA, USA). The broadly conserved primers,
515F, 5′-GTGCCAGCMGCCGCGG-3′, and 806R, 5′-GGACTACHVGGGTWTCTAAT-3′, were
used for PCR to amplify the sequencing region of the 16S rRNA gene. PCR
primers for amplifying fungal ITS1-2 are: ITS1-1737F,
5′-GGAAGTAAAAGTCGTAACAAGG-3′; ITS2-2043R, 5′-GCTGCGTTCTTCATCGATG C-3′.
PCR reactions were carried out in 30 µL reactions with 15 µL of Phusion®
High-Fidelity PCR Master Mix (New England Biolabs, USA), 0.2 µM of
forward and reverse primers, and about 10 ng of template DNA. Thermal
cycling consisted of initial denaturation at 98 °C for 1 min, followed
by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for
30 s, elongation at 72 °C for 30 s, and finally 72 °C for 5 min. The
same PCR conditions were used for ITS, except that the second stage had
35 cycles. PCR products were subsequently subjected to electrophoresis
on a 2% agarose gel, stained with ethidium bromide, and the targeted
fragment size purified with an AxyPrepDNA gel extraction kit (Axygen,
China). Sequencing libraries were generated using Ion Plus Fragment
Library Kit 48 rxns (Thermo Scientific, USA), following the
manufacturer’s recommendations. The library quality was assessed on the
QuantiFluorTM-ST fluorometer (Promega) and Agilent 2100 Bioanalyzer
system (Agilent, USA). Lastly, the library was sequenced on an Ion S5TM
XL platform, and 400 bp/600 bp single-end reads were generated. All the
high-throughput sequencing was done by Novogene Technology Co, Ltd.
(Beijing, China).