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.