Rhizosphere Microbiome DNA Extraction and Amplification

The total microbial DNA of rhizosphere soil samples was extracted with PowerSoil® DNA Isolation Kit (MOBIO, United States). The rhizomes and leaves were chopped and ground with liquid nitrogen, and the total DNA was extracted by PowerSoil® DNA Isolation Kit. The DNA concentration and purity were determined by Nanodrop ultraviolet spectrophotometer (Thermo Scientific, United States). The quality of DNA extract was detected by 1.2% agarose gel electrophoresis, and then the DNA extraction solution was stored at −80°C for later use. The V5-V7 region of fungal 18S rRNA was amplified by using universal primers SSU0817F (5’-TTAGCATGGAATAATRRAATAGGA-3′) and 1196R (5’-TCTGGACCTGGTGAGTTTCC-3′; Jiang et al., 2017 (link)). The V3-V4 region of 16S rRNA was PCR amplified by using universal primers 338\u00B0F (5’-ACTCCTACGGGAGGCAGCA-3′) and 806 R (5’-GGACTACHVGGGTWTCTAAT-3′; Li et al., 2020 (link)). Pfu high-fidelity DNA polymerase from TransGen Biotech was used for PCR amplification with the number of amplification cycles strictly controlled to make cycle number as small as possible and the amplification conditions for the same batch of samples consistent. The PCR products were purified and recovered with the AxyPrep DNA Gel extraction kit. Subsequently, each sample was quantified using BioTek Flx800 microplate reader with Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, P7589).