RNA isolation from biological soil crusts: methodological aspects

Rippin, Martin; Komsic-Buchmann, Karin; Becker, Burkhard

doi:10.1127/algol_stud/2016/0256

Cited by 6 publications

(7 citation statements)

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“…The biocrust was removed from a dark brown soil rich in humic substance, covered with mosses. DNA was extracted following a CTAB protocol using RNase A (Rippin, Komsic‐Buchmann, & Becker, ) and further purified using the “DNA Clean & Concentrator” (Zymo Research, Irvine, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

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New barcoded primers for efficient retrieval of cercozoan sequences in high‐throughput environmental diversity surveys, with emphasis on worldwide biological soil crusts

Fiore-Donno

Rixen²,

Rippin

et al. 2017

Molecular Ecology Resources

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We describe the performance of a new metabarcoding approach to investigate the environmental diversity of a prominent group of widespread unicellular organisms, the Cercozoa. Cercozoa is an immensely large group of protists, and although it may dominate in soil and aquatic ecosystems, its environmental diversity remains undersampled. We designed PCR primers targeting the hypervariable region V4 of the small subunit ribosomal RNA (SSU or 18S) gene, which is the recommended barcode marker for Cercozoa. The length of the amplified fragment (c. 350 bp) is suitable for Illumina MiSeq, the most cost-effective platform for molecular environmental surveys. We provide barcoded primers, an economical alternative to multiple libraries for multiplex sequencing of over a hundred samples. In silico, our primers matched 68% of the cercozoan sequences of the reference database and performed better than previously proposed new-generation sequencing primers. In mountain grassland soils and in biological soil crusts from a variety of climatic regions, we were able to detect cercozoan sequences encompassing nearly the whole range of the phylum. We obtained 901 operational taxonomic units (OTUs) at 97% similarity threshold from 26 samples, with c. 50,000 sequences per site, and only 8% of noncercozoan sequences. We could report a further increase in the diversity of Cercozoa, as only 43% of the OTUs were 97%-100% similar to any known sequence. Our study thus provides an advanced tool for cercozoan metabarcoding and to investigate their diversity and distribution in the environment.

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Section: Methodsmentioning

confidence: 99%

“…The biocrust was removed from a dark brown soil rich in humic substance, covered with mosses. DNA was extracted following a CTAB protocol using RNase A (Rippin, Komsic-Buchmann, & Becker, 2016) . Biocrusts were only present on litter-free soil at exposed or disturbed sites.…”

Section: Provenance Of the Soil And Biocrust Samples And Dna Extracmentioning

confidence: 99%

New barcoded primers for efficient retrieval of cercozoan sequences in high‐throughput environmental diversity surveys, with emphasis on worldwide biological soil crusts

Fiore-Donno

Rixen²,

Rippin

et al. 2017

Molecular Ecology Resources

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“…Total RNA was extracted using the CTAB protocol as described by Rippin et al (2016). Genomic DNA was removed by incubating the solution with DNase I Thermo Scientific (Waltham, MA) and subsequently purifying it using the RNeasy MinElute Cleanup kit (Qiagen, Hilden, Germany) according to manufacturer's instructions.…”

Section: Metatranscriptomicsmentioning

confidence: 99%

Metatranscriptomic and metabolite profiling reveals vertical heterogeneity within a Zygnema green algal mat from Svalbard (High Arctic)

Rippin

Pichrtová

Arc

et al. 2019

Environmental Microbiology

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Summary Within streptophyte green algae Zygnematophyceae are the sister group to the land plants that inherited several traits conferring stress protection. Zygnema sp., a mat‐forming alga thriving in extreme habitats, was collected from a field site in Svalbard, where the bottom layers are protected by the top layers. The two layers were investigated by a metatranscriptomic approach and GC–MS‐based metabolite profiling. In the top layer, 6569 genes were significantly upregulated and 149 were downregulated. Upregulated genes coded for components of the photosynthetic apparatus, chlorophyll synthesis, early light‐inducible proteins, cell wall and carbohydrate metabolism, including starch‐degrading enzymes. An increase in maltose in the top layer and degraded starch grains at the ultrastructural levels corroborated these findings. Genes involved in amino acid, redox metabolism and DNA repair were upregulated. A total of 29 differentially accumulated metabolites (out of 173 identified ones) confirmed higher metabolic turnover in the top layer. For several of these metabolites, differential accumulation matched the transcriptional changes of enzymes involved in associated pathways. In summary, the findings support the hypothesis that in a Zygnema mat the top layer shields the bottom layers from abiotic stress factors such as excessive irradiation.

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“…All harvested filters were instantly quenched in liquid nitrogen and homogenized in a chilled mortar. Subsequently, the powder was processed with the Spectrum Kit (Sigma–Aldrich, St. Louis, MO, United States) according to Rippin et al (2016). The quality of the resulting RNA samples was assessed on a BioAnalyzer instrument (Agilent Technologies, Santa Clara, CA, United States) and subsequently used to prepare libraries.…”

Section: Methodsmentioning

confidence: 99%

Cold Acclimation Improves the Desiccation Stress Resilience of Polar Strains of Klebsormidium (Streptophyta)

et al. 2019

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Biological soil crusts (BSCs) are complex communities of autotrophic, heterotrophic, and saprotrophic (micro)organisms. In the polar regions, these biocrust communities have essential ecological functions such as primary production, nitrogen fixation, and ecosystem engineering while coping with extreme environmental conditions (temperature, desiccation, and irradiation). The microalga Klebsormidium is commonly found in BSCs all across the globe. The ecophysiological resilience of various Klebsormidium species to desiccation and other stresses has been studied intensively. Here we present the results of transcriptomic analyses of two different Klebsormidium species, K. dissectum and K. flaccidum , isolated from Antarctic and Arctic BSCs. We performed desiccation stress experiments at two different temperatures mimicking fluctuations associated with global change. Cultures grown on agar plates were desiccated on membrane filters at 10% relative air humidity until the photosynthetic activity as reflected in the effective quantum yield of photosystem II [Y(II)] ceased. For both species, the response to dehydration was much faster at the higher temperature. At the transcriptome level both species responded more strongly to the desiccation stress at the higher temperature suggesting that adaptation to cold conditions enhanced the resilience of both algae to desiccation stress. Interestingly, the two different species responded differently to the applied desiccation stress with respect to the number as well as function of genes showing differential gene expression. The portion of differentially expressed genes shared between both taxa was surprisingly low indicating that both Klebsormidium species adapted independently to the harsh conditions of Antarctica and the Arctic, respectively. Overall, our results indicate that environmental acclimation has a great impact on gene expression and the response to desiccation stress in Klebsormidium .

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RNA isolation from biological soil crusts: methodological aspects

Cited by 6 publications

References 0 publications

New barcoded primers for efficient retrieval of cercozoan sequences in high‐throughput environmental diversity surveys, with emphasis on worldwide biological soil crusts

New barcoded primers for efficient retrieval of cercozoan sequences in high‐throughput environmental diversity surveys, with emphasis on worldwide biological soil crusts

Metatranscriptomic and metabolite profiling reveals vertical heterogeneity within a Zygnema green algal mat from Svalbard (High Arctic)

Cold Acclimation Improves the Desiccation Stress Resilience of Polar Strains of Klebsormidium (Streptophyta)

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