Root metagenome of Oryza sativa:ITS metabarcoding Bama site
Less than 24h after sampling, root samples (rice roots including rhizosphere) stored at 4C were processed. In order to separate the different root compartments, the tubes were vortexed vigorously one minute and then, roots were removed from the PBS solution using sterile forceps. Roots were then surface-sterilized with 70% alcohol (30s), 1% bleach (30s) and finally rinsed three times in sterile water. We considered these surface-sterilized roots as the root compartment, which comprises both endosphere microorganisms as well as persistent DNA from the rhizoplane. DNA extraction from root samples were performed on the same day as the process of compartment separation. 0.25g of root samples (crushed in liquid nitrogen beforehand) was extracted using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany), following manufacturers recommendations. DNA quality and quantity was verified using a NanoDrop ND-1000 spectrophotometer. PCR amplification, library and MiSeq Illumina sequencing were performed on a composite sample (3 plant samples were pooled to result in one sample per field), by Macrogen (Seoul, South Korea) using primers ITS1f: CTTGGTCATTTAGAGGAAGTAA and ITS2: GCTGCGTTCTTCATCGATGC to amplify the internal transcribed spacer 1 (ITS1) region.