Fungi (LSU) in soils from the South Shetland island, Antarctic Peninsula island, and Union Glacier

Study Extent

Soil samples were gathered during several expeditions to Antarctica, including Union Glacier, Lagotellerie island, King George, Deception, Snow, Dee, Livingstone, Greenwich, Robert, Nelson and Litchfield islands.

Sampling

Samples were collected in sterile 50 ml plastic tubes, which were sealed and shipped at -20°C to the Genetics Laboratory of the Faculty of Science at the Universidad de Chile. Once samples arrived at the laboratory, they were maintained at -80°C until processing.

Method steps

1. A PowerSoil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA, United States) was used for direct DNA extraction from soil samples. The manufacturers’ instructions were followed, with only one modification, the disruption step, was performed using a Mini Beadbeater-16 cell disrupter (BioSpec Bartlesville, United States) instead of vortex agitation, as no PCR-amplicons were obtained in reactions using samples obtained through vortex agitation.
2. The PCR reactions were performed using 1 μl of DNA sample (direct or 1/10 dilution), 5 units of Taq DNA polymerase, dNTP mix at 0.4 mM each, forward and reverse primers at 1 mM final each, PCR buffer and MgCl2 2 mM. Amplification was performed using a 2720 (Applied Biosystems) thermal cycler using the following protocol: initial denaturation at 94°C for 3 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 3 min, and extension at 72°C for 3 min; and a final extension step at 72°C for 10 min. The universal primers F63 (5′-GCA TAT CAA TAA GCG GAG GAA AAG-3′) and LR3 (5′-GGT CCG TGT TTC AAG ACG G-3′) were used; these primers have been described as specific for amplifying the fungal D1/D2 region of the large subunit ribosomal gene (LSU). In all PCR reactions, the same forward primer (F63) was used, but the reverse primer (LR3) was specific for each soil sample as a 454 adapter, a specific barcode and a linker sequence were added.
3. Sequencing was performed at OMICS Solutions (Santiago, Chile) using the Ion 314TM Chip Kit v2 (Thermo Fisher) and Ion Torrent personal genome machine (PGM), according to the manufacturer’s instructions (Rothberg et al., 2011). Three independent runs were performed: (i) samples from King George Island; (ii) samples from Deception, Snow, Dee, Livingstone, Greenwich, Robert, Nelson and Litchfield islands; and iii) samples from Lagotellerie and Union Glacier.

LSU marker gene for microbial soil Fungi

1. Fungi
   common name: Fungi rank: phylum

South Shetland Islands, Union Glacier (Antarctica)