Soil DNA Extraction and 16S rRNA Amplification

The total DNA of each sample was isolated from 0.25 g of soils using the PowerSoil DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA, United States). Prior to amplification, the DNA concentration was standardized to be the same. The 16S rRNA gene’s V3-V4 region was chosen to create the community library using the forward primers 338\u00B0F (Huse et al., 2008 (link)) and reverse primer 806 R (Li et al., 2019 (link)), using a six-base barcode that is particular to each sample. Supplementary Table S2 offers more information on the PCR conditions. PCR products from all samples were combined and identified by 2% agarose gel electrophoresis, and the PCR products were recovered by gel cutting using the AxyPrep DNA Gel Recovery Kit (AXYGEN). PCR products were quantitatively detected by QuantiFluor™-ST Blue fluorescence quantitative system (Promega), and the MiSeq sequencing library was constructed using TruSeqTM DNA Sample Prep Kit, which was sequenced based on PE300 strategy. The PCR products were purified, pooled in equimolar amounts, and paired-end sequenced (2 × 300) using an Illumina MiSeq platform at Shanghai Meiji Biological Medicine Technology Co Ltd., Shanghai, China.