DNA extraction, PCR amplification, and illumina sequencing

We extracted total DNA from 500 mg of each sample (bulk soil, rhizosphere soil, root, stem, and leaf) using the PowerSoil DNA Isolation kit (Mo Bio Laboratories, Carlsbad, CA, United States) and Invisorb Spin Plant Mini Kit (Stratec Biomedical AG, Birkenfeld, Germany) following the manufacturer’s instructions. The quantity and quality of the DNA were assessed using the ratios of 260 nm/280 nm and 260 nm/230 nm. The DNA was later stored at −80°C until further processing.

The bacterial 16S rRNA gene V3–V4 region was amplified using a primer pair (Forward primer, 5′-ACTCCTACGGGAGGCAGCA-3′; reverse primer, 5′-GGACTACHVGGGTWTCTAAT-3′) combined with adapter sequences and barcode sequences. PCR amplification was performed in a total volume of 50 μl, which consisted 10 μl Buffer, 0.2 μl Q5 High-Fidelity DNA Polymerase, 10 μl High GC Enhancer, 1 μl dNTP, 10 μM of each primer, and 60 ng genome DNA. Below were the thermal cycling conditions: initial denaturation at 95°C for 5 min, followed by 15 cycles at 95°C for 1 min, 50°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 7 min. The PCR products obtained from the initial step of the PCR were purified using VAHTSTM DNA Clean Beads. The second round of PCR was then conducted in a 40 μl reaction, which consisted of 20 μl 2 × Phusion HF MM, 8 μl ddH₂O, 10 μM of each primer, and 10 μl PCR products obtained from the initial step. Thermal cycling conditions were as follows: an initial denaturation at 98°C for 30 s, followed by 10 cycles at 98°C for 10 s, 65°C for 30 s and 72°C for 30 s, with a final extension at 72°C for 5 min. Lastly, Quant-iT™ dsDNA HS Reagent was employed to quantify all PCR products and pooled together. Illumina Hiseq 2500 platform (2 × 250 paired ends) at Biomarker Technologies Corporation, Beijing, China, was employed to conduct a high-throughput sequencing analysis of bacterial rRNA genes on the sample. Finally, the raw data were submitted to the NCBI Sequence Read Archive (accession no. PRJNA848385).

FLASH was adopted to conduct merge paired-end reads of the DNA fragments, using a sample-specific barcode appropriated to each sample. The sequences were then clustered at the same operational taxonomic unit (OTU) using 97% similarity. Later, sequences were accordingly chosen for each OTU to conduct the annotation of the taxonomic information for each sequence by employing the Ribosomal Database Project (RDP) (Cole et al., 2009). We also removed sequences with low quality if they did not correspond to the primer and barcode or if they did not exceed 200 nucleotides consisting of a high average quality score (Q ≥ 20) or no ambiguous base pairs. Sequences were then clustered at 97% nucleotide similarity. Finally, SILVA database (SILVA Release 138, Bacterial) was leveraged for the taxonomic classification of the bacteria respective sequences (Quast et al., 2013).