Soil and Root Microbial Community Analysis

Total DNA was extracted from the rhizosphere soil and root samples according to the instructions of the PowerSoil DNA extraction kit (Mobio Laboratories, Carlsbad, CA, USA). The concentration of the resulting DNA was measured and quantified with a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The specific primers 799F (5’-AACMGGATTAGATACCCKG-3´), 1193R (5´-ACGTCATCCCCACCTTCC-3´), and 1392R (5´-ACGGGCGGTGTGTRC-3´), targeted to the V5–V7 regions of 16S rDNA, were used for PCR. PCR was performed using phusion high-fidelity PCR master mix with GC buffer (NEB, Ipswich, MA, USA) and the reaction system and amplification program described [23 (link),24 (link),25 (link)]. The PCR products were detected by agarose gel electrophoresis, and the target DNA band was purified with the EasyPure Quick Gel Extraction Kit (Transgen Biotech, Beijing, China). The purified DNA was submitted to Majorbio Bio-pharm Technology Co., Ltd., (Shanghai, China) for paired-end library construction and sequencing using the Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA).