ITS amplicon sequencing of rhizosphere microbiome in maizesoybean/peanut intercropping field
DNA was extracted from 200 mg of soil using the FastPure Soil DNA Isolation Kit (Majorbio BioPharm Technology Co., Ltd., Shanghai, China). The extraction process began with the addition of 750L Lysis Buffer A and 90L Lysis Buffer B to the soil sample, followed by homogenization in a grinding tube. The sample was centrifuged at 14,000rpm for 5 minutes, and the supernatant was transferred to a new 1.5 mL microfuge tube for purification. Purification involved the addition of Purification Buffers I and II, followed by the application of proprietary magnetic beads to ensure high-quality DNA recovery. DNA was eluted in 100 L Elution Buffer and incubated at 56C for 5 minutes. The purified DNA was quantified using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA) to assess concentration and purity. For 16S rRNA gene sequencing, the V3-V4 region was amplified using primers 338F (5-ACTCCTACGGGAGGCAGCAG-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3). For ITS sequencing, the ITS1 region was amplified using primers ITS1F (5-CTTGGTCATTTAGAGGAAGTAA-3) and ITS2R (5-GCTGCGTTCTTCATCGATGC-3). PCR reactions were performed out in a 20 L mixture containing template DNA, primers, dNTPs, FastPfu DNA Polymerase, and PCR buffer. Each amplification included 35 cycles for ITS and 27 cycles for 16S, with standard initial denaturation, annealing, and elongation steps. Amplicons were purified and sequenced on an Illumina MiSeq PE300 platform (Majorbio BioPharm Technology Co., Ltd. Shanghai, China).