Microorganisms (Bacteria 16S; Eukaryota 18S) from soil crusts in polar (Arctic and Antarctic) ecosystems

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Pesquisadores deveriam citar esta obra da seguinte maneira:

Rippin M, Lange S, Sausen N, Becker B (2019): Microorganisms (Bacteria 16S; Eukaryota 18S) from soil crusts in polar (Arctic and Antarctic) ecosystems. v1.1. SCAR - Microbial Antarctic Resource System. Dataset/Metadata. https://ipt.biodiversity.aq/resource?r=microorganisms_polar_biological_soil_crusts&v=1.1

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O editor e o detentor dos direitos deste trabalho é SCAR - Microbial Antarctic Resource System. This work is licensed under a Creative Commons Attribution (CC-BY 4.0) License.

GBIF Registration

Este recurso foi registrado no GBIF e atribuído ao seguinte GBIF UUID: a9a93a1e-4470-49c3-a98c-72f36c382090.  SCAR - Microbial Antarctic Resource System publica este recurso, e está registrado no GBIF como um publicador de dados aprovado por Scientific Committee on Antarctic Research.

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Metadata

Contatos

Cobertura Geográfica

Biological crusts were sampled in Antarctica at the Spanish Juan Carlos I Antarctic Base, and on Svalbard at Breinosa Gruve 7, Endalen, Ny-Alesund and Todalen

Cobertura Taxonômica

Bacteria were targeted by sequencing the 16S ssu rRNA gene (v2-v3 region)

Eukaryotes were targeted by sequencing the 18S ssu rRNA gene (v4 region)

Cobertura Temporal

Dados Sobre o Projeto

Nenhuma descrição disponível

O pessoal envolvido no projeto:

Métodos de Amostragem

The nucleic acid content of the samples was preserved using the LifeGuard Soil Preservation Solution (MO BIO Laboratories, Carlsbad, CA, USA) according to the manufacturer's instructions and stored at −80°C.

Descrição dos passos do método:

1. Total nucleic acids were extracted from the samples (all in triplicates, except NA that was only one sample) using the cetyltrimethylammonium bromide protocol according to Rippin, Komsic-Buchmann and Becker (2016) with several modifications: The LifeGuard Soil Preservation Solution was not removed prior to the extraction and the aqueous phase, retained after the second chloroform washing step, was further processed using the peqGOLD Plant RNA Kit (peqlab/VWR International, Erlangen, Germany) according to the manufacturer's instructions.
2. To obtain DNA, the extract was treated with RNase A (Thermo Fisher Scientific, Waltham, MA, USA) as suggested in the manual and purified using the illustra MicroSpin S-400 HR Columns (GE Healthcare, Little Chalfont, UK). RNA-based metabarcoding was only performed for the samples NÅ and JC as the sampling sites are most comparable in terms of climate (seaside, similar precipitation patterns etc.). RNA was purified by DNA removal using DNase I (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. cDNA synthesis was performed using the Revert Aid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA).
3. The extraction of DNA from all cultivated algae except K. crenulatum was carried out using the DNeasy Plant Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. The harvested filaments of K. crenulatum were processed using a modified version of the cetyltrimethylammonium bromide protocol described by Rippin, Komsic-Buchmann and Becker (2016). After the second chloroform washing step, the upper phase was collected. The DNA was precipitated with isopropanol at −20°C for at least 1 h. After a washing step with 75% ethanol, the DNA was eluted in RNase-free water.
4. Amplicons were generated for all triplicates (for NA three technical replicates were produced) using the Kapa HiFi HotStart DNA Polymerase (Roche, Basel, Switzerland). To analyze the eukaryotic diversity, we targeted the ribosomal small subunit (SSU) V4 region with the universal eukaryotic primers TAReuk454FWD1 (5′-CCAGCASCYGCGGTAATTCC-3′) and TAReukREV3 (5′-ACTTTCGTTCTTGATYRA-3′) designed by Stoeck et al. (Stoeck et al. 2010) (Amplicon size ≈ 380 bp). Similarly, the prokaryotic 16S rRNA gene V2-V3 region was amplified using the universal primers 104F (5′-GGCGVACGGGTGMGTAA-3′) and 515R (5′-TTACCGCGGCKGCTGGCAC-3′) taken from Lange et al. (Lange et al. 2015) (Amplicon size ≈ 400 bp). The eukaryotic amplicons of the BSC samples were amplified employing the following protocol: an initial denaturation step at 95°C for 3 min and 25 3-step cycles at 98°C for 20 s, at 49°C for 15 s and 72°C for 35 s were followed by a final elongation at 72°C for 1 min. The prokaryotic target was amplified in a similar way; however, the 25 cycles included 20 s at 98°C, 10 s at 65°C and 15 s at 72°C. Each template was amplified three times (NA was amplified nine times) with each primer pair and these triplicates were pooled in an equimolar manner to minimize PCR bias. For the MC, the templates, obtained from the cultivated algae, the bryophyte Physcomitrella patens and the liverwort Marchantia polymorpha (both supplied by O. Artz, AG Höcker, Botanical Institute, University of Cologne), were only targeted with eukaryotic primers and the PCR protocol consisted of 30 cycles of 20 s at 98°C, 15 s at 51.8°C and 35 s at 72°C. All PCR products were purified using the Agencourt® AMPure® XP system (Beckman Coulter, Brea, CA, USA) according to the manufacturer's protocol.

Citações bibliográficas

1. Rippin, M., Lange, S., Sausen, N., & Becker, B. (2018). Biodiversity of biological soil crusts from the Polar Regions revealed by metabarcoding. FEMS microbiology ecology, 94(4), fiy036. https://doi.org/10.1093/femsec/fiy036
2. Williams, L., Borchhardt, N., Colesie, C., Baum, C., Komsic-Buchmann, K., Rippin, M., ... & Büdel, B. (2017). Biological soil crusts of Arctic Svalbard and of Livingston Island, Antarctica. Polar Biology, 40(2), 399-411.

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