sequencing of soil bacterial 16S rRNA gene
Total genomic DNA was extracted from soil samples using the PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA), following the manufacturers protocol . PCR amplification targeted the ITS2 region of fungal rRNA (primers: ITS3F/ITS4R) (Toju et al., 2012). Reactions were conducted with Phusion High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA) under the following conditions: initial denaturation at 95 C for 2 min, 30 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s, and extension at 72 C for 1 min, followed by a final extension at 72 C for 10 min. PCR products were purified using a GeneJET Gel Extraction Kit (Thermo Fisher Scientific) and sequenced on an Illumina MiSeq platform (San Diego, CA, USA) using paired-end 250 bp reads.