DNA extraction and sequencing
Soil and fecal DNA were extracted from the collected samples using the MoBio Powersoil and Qiagen Powerfecal isolation kits according to the manufacture instructions respectively (MoBio Laboratories, Carlsbad, CA, USA). The phyllosphere genomic samples were extracted using Mobio Powersoil isolation kits after ultrasonic leave washing. Leaf samples were aseptically placed in the polypropylene tubes containing 0.1 mol/L potassium phosphate buffer (pH 7.0) as the washing buffer. After the ultrasonic clean bath and the membrane filtration, the microorganisms were dislodged for the nucleic acid extraction (Zhang et al., 2008). Subsequently, a quality and quantification assessment of the purified DNA was conducted by the Nana-Drop ND-1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). The final DNA samples obtained from three biotopes were diluted and stored at –80 °C for further analysis.
For the Illumina Miseq High-throughput sequencing, PCR amplification of
16S rRNA were performed with universal bacterial primers 338F and 806R in the region V3-V4 (Mori et al., 2014), universal fungal primers ITS1F and ITS2R (Bokulich & Mills, 2013) and universal archaeal primers 524F-10-ext and arch958R in the region V4-V5 respectively (Pires et al., 2012). The amplicon procceded to be sequenced on an Illumina Miseq PE250 platform.