Microbial DNA was extracted from each of the 24 samples using a FastDNA Spin Kit for soil (MP Biomedicals, Santa Ana, CA, United States), according to the manufacturer's protocols (Sun et al., 2022 (link)). The DNA quality and quantity were determined using a NanoDrop 2000 spectrophotometer (Bio-Rad Laboratories Inc., USA.). The V4–V5 region of the bacteria 16S ribosomal RNA gene and the ITS gene was amplified by PCR (95°C for 5 min, followed by 25 cycles at 95°C for 30 s, 30 s at C, and 72°C for 45 s, and a final extension at 72°C for 10 min and 10°C until halted by user) using the primers 515F 5′-GTGCCAGCMGCCGCGG-3′, 907R 5′-CCGTCAATTCMTTTRAGTTT-3′, ITS1F 5′-CTTGGTCATTTAGAGGAAGTAA-3′, and ITS2R 5′-GCTGCGTTCTTCATCGATGC-3′, where the barcode is an eight-base sequence unique to each sample. The PCR reactions were performed in triplicate in a 20 μl of a mixture containing 4 μl of 5× FastPfu Buffer, 2 μl of 2.5 mM dNTPs, 0.8 μl of each primer (5 μM), 0.4 μl of FastPfu Polymerase, and 10 ng of template DNA. The amplicons were extracted from 2% agarose gels, purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, AP-GX-4) according to the manufacturer's instructions, and quantified using QuantiFluor™-ST (Promega, US).
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