Microbiome (Archaea, Bacteria and Fungi) in soils from King George Island (Antarctica)

Study Extent

The 12 permanent quadrat plots (1.5 m × 1.0 m each) investigated in this study were established on the Fildes Peninsula and Ardley Island between 2013 and 2015. Each quadrat plot was fenced to minimize disturbance. The distance between quadrat plots ranges from approximately 1.6 to 8.2 km. Sampling occurred during China’s 33rd Antarctic expedition in January 2017.

Sampling

Soils were sampled from the A-horizon (10 cm) at an internal distance of approximately 3–5 m, and samples were collected in triplicate around each quadrat plot. Soil samples collected for each replicate were taken from five soil cores (5 cm diameter) and mixed thoroughly. A total of 36 soil samples were placed in sterile plastic bags, and soil DNA was extracted within 2 h in the laboratory of the Great Wall Station. The remaining soils were stored in the freezer until further soil physico-chemical property analyses were performed.

Method steps

1. The bacterial hypervariable V4 region of the 16S rRNA genes was amplified using primers 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) with a 7-nucleotide barcode and 907R (5’-CCGTCAATTCMTTTRAGTTT-3’). Amplifications of the bacterial 16S rRNA genes were performed, consisting of an initial denaturation at 98 °C for 30 s, followed by 25 cycles of denaturation at 98 °C for 15 s, annealing at 50 °C for 30 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 5 min.
2. The archaeal V5-6 region of 16S rRNA genes was amplified using primers 524F-10-extF (5’-TGYCAGCCGCCGCGGTAA-3’) with a 7-nucleotide barcode and Arch958-modR (5’-YCCGGCGTTGAVTCCAATT-3’). Amplifications of the archaeal 16S rRNA genes were performed, consisting of an initial denaturation at 95 °C for 30 s, followed by 25 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 5 min.
3. The fungal rDNA ITS1-5.8S-ITS2 region was amplified using primers ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) with a 7-nucleotide barcode and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’). Amplifications of the fungal ITS regions were performed, consisting of an initial denaturation at 95 °C for 30 s, followed by 25 cycles of denaturation at 95 °C for 15 s, annealing at 50 °C for 30 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 5 min. The PCR 25 μl reaction mixture contained 0.25 μl Q5 high-fidelity DNA polymerase (NEB), 5 μl reaction buffer, 5 μl high GC buffer, 0.5 μl of 10 mM dNTP, 1 μl template DNA, 1 μl of each primer (10 μM), and 11.25 μl ddH2O.
4. PCR products were purified using an AxyPreDNA Gel Extraction Kit (Axygen Biosciences, Corning, NY, USA) according to the manufacturer’s instructions. The purified PCR amplicons from each sample were then mixed after quantification using a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) in the Microplate reader (Bio Tek, FLx800). Sequencing was performed on the Illumina Miseq Platform.

Bacteria, profiled by targeting the 16S ssu rRNA gene

1. Bacteria
   common name: Bacteria rank: domain

Archaea, profiled by targeting the 16S ssu rRNA gene

1. Archaea
   common name: Archaea rank: domain

Fungi, profiled by targeting ITS gene

1. Fungi
   common name: Fungi rank: phylum

Fildes region on the King George island, Antarctica