Genomic DNA extraction, PCR amplification, and high-throughput amplicon sequencing

Total DNA was extracted from soil samples using the PowerSoil DNA Isolation Kit (MoBio, USA), according to the manufacturer’s protocol. The concentration and purity of the obtained DNA samples were then determined using a UV-1200 UV spectrophotometer (Shanghai Mapada, China) and agarose gel electrophoresis. The V3–V4 region of the 16S rRNA gene was amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) for bacterial community analysis⁵⁴. PCR amplification was carried out in a total volume of 25 μL containing 5 μL 5 × reaction buffer, 5 μL 5 × GC buffer, 2 μL dNTPs (2.5 mmol), 1 μL 338F primer (10 μmol), 1 μL 806R primer (10 μmol), 2 μL DNA template, 8.75 μL ddH2O, and 0.25 μL Q5 High-Fidelity DNA Polymerase (NEB, USA)⁵⁵. The PCR protocol consisted of an initial denaturation step at 98 °C for 2 min, followed by 28 cycles of denaturation at 98 °C for 15 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s; a final extension at 72 °C was performed for 5 min and reaction mixtures were held at 10 °C until further analysis⁵⁶. Three replicates were used per sample, and these were pooled to minimize PCR bias. PCR products were detected via electrophoresis on 2.0% agarose gels, and fragments of about 450 bp were purified with the AxyPrep DNA Gel Extraction Kit (Axygen, USA). PCR amplicons were quantified using the PicoGreen dsDNA Assay Kit (Invitrogen, USA). Finally, paired-end 2 × 300 bp sequencing of bacterial amplicons was carried out on the MiSeq platform (Illumina, USA) at Personal Biotechnology Co., Ltd (Shanghai, China). The detailed experimental procedure and pipeline chart are shown in Supplementary Figures ^S3 and ^S4, respectively.