BioFun: Biodiversity in the dark: High-throughput sequence analyses of arctic fungal communities
Fungi are important players in most terrestrial ecosystems, acting as decomposers of organic material, parasites/pathogens, or as mutualists. Most arctic plants are highly dependent on mutualistic root symbiosis for survival in these dry, cold, and nutrient-poor environments (Gardes & Dahlberg 1996). In contrast to warmer ecosystems, in which herbaceous plants rely on arbuscular mycorrhiza, arctic herbs and shrubs associate predominately with ectomycorrhiza (Betula nana, Bistorta vivipara, Salix polaris and Dryas octopetala), ericoid mycorrhiza (Ericaceae) or root endophytes (other plant groups, including non-mycorrhizal species). Recent studies have confirmed that a high and largely unexplored fungal diversity exists in the arctic ecosystems (Bjorbækmo et al. 2010; Geml et al. 2012; Timling et al. 2012). These fungi are adapted to harsh environment and low concentration of soil nutrients that occur mostly in the organic form. Because of these adaptations, the arctic fungi may play a key role in releasing fixed soil carbon from retreating permafrost areas due to global warming and they offer great potential in pharmacology and enzyme production (Robinson 2001).
Since most fungi reside hidden in soil or plant tissues, DNA-based methods are required to detect and study these organisms in their natural habitats. The term ‘DNA barcoding’ is widely used for DNA-based identification of any organisms. Most of the research on fungi relies on the nuclear ribosomal internal transcribed spacer (ITS) region that was recently suggested as the main barcoding marker for fungi that allows to separate taxa at the species level (Schoch et al. 2012). In addition to ca. 80,000 described fungal species, 1.5-5 million species are estimated to exist in the world (Blackwell 2011). The presence of ca. 300,000 fungal sequences (sequenced by Sanger method) in databases and a look at the above figures suggest that only a tiny proportion of the fungal branch of life has been described or sequenced. All molecular studies of fungi suffer from the poor capacity of reliable identification due to the paucity of closely related reference sequences in public databases and numerous misidentifications (Nilsson et al. 2008, Abarenkov et al. 2010a). Many of the sequence accessions in public databases are artefacts of PCR and sequencing – either chimeras or containing multiple read errors (Tedersoo et al. 2011; Nilsson et al. 2012a). The need for more trustworthy reference sequence data for fungal DNA barcoding led us to the establishment of the UNITE database (Kõljalg et al. 2005). UNITE is a Baltic-Nordic initiative that has rapidly grown to include high quality sequences from all fungi and all genes in all parts of the world. Sequences submitted to UNITE directly and those downloaded from the International Nucleotide Sequence Databases (INSD) can be readily annotated for quality and environmental metadata via an attached PlutoF workbench (Abarenkov et al. 2010b). UNITE has been widely used for identification of fungi and, as of March 2013, it is being integrated into the major platforms for high-throughput sequencing such as QIIME.
The earliest molecular studies based on Sanger sequencing have provided a firm basis for populating sequence databases and inferring patterns in fungal ecology. Due to their high costs, traditional DNA-based approaches are poorly suited for intensive sampling of hyperdiverse microbial communities. During the last years, several high-throughput DNA sequencing techniques have evolved and become available to the scientific community. The 454 pyrosequencing technique has been successfully implemented in numerous studies by the project participants to analyze fungal communities, detect artefacts and propose standard approaches (e.g. Tedersoo et al. 2010; Nilsson et al. 2011; Carlsen et al. 2012; Kauserud et al. 2012; Lindahl et al. 2013). The Ion Torrent technology, introduced in 2011, resembles the 454 technology but has higher yields and lower running costs compared to 454 sequencing. The shorter read lengths has hitherto made Ion Torrent unsuitable for the analysis of the ITS region, but up to 400 bp sequences have recently been generated. Illumina sequencing is currently the most successful and widely adopted next-generation sequencing platform in metagenomics, but so far it has not been reported in successfully recovering patterns in fungal communities due to its limited read length. However, the recently available paired-end sequences on the MiSeq platform now enable coverage of 2 x 250 base read length and a yield of ~30 million reads, which render it potentially suitable for application in fungi. So-called “third generation sequencing platforms” such as PacBio RS (available since 2011) rely on real-time sequencing of a single DNA molecule. The reads readily span over 3000 bp, but sequence quality remains substandard for diversity analyses. However, by sequencing the template several times (circular consensus sequencing), the resulting reads may be of sufficient quality. Other upcoming techniques that probably will have a substantial impact on the field are based on registering the DNA (or RNA) as it migrates through nanopores placed in artificial membranes. The new sequencing techniques enable processing of hundreds of environmental samples in parallel with deep sequence coverage. However, these techniques have brought with them many new methodological challenges (Logares et al. 2012).
Abarenkov K, Nilsson RH, Larsson K-H, Alexander IJ, Eberhardt U, Erland S, Høiland K, Kjøller R, Larsson E, Pennanen T, Sen R, Taylor AFS, Tedersoo L, Ursing B, Vrålstad T, Liimatainen K, Peintner U, Kõljalg U. 2010a. The UNITE database for molecular identification of fungi – recent updates and future perspectives. New Phytol. 186: 281-285.
Abarenkov K, Tedersoo L, Nilsson RH, Vellak K, Saar I, Veldre V, Parmasto E, Prous M, Aan A, Ots M, Kurina O, Ostonen I, Jõgeva J, Halapuu S, Põldmaa K, Toots M, Truu J, Larsson K-H, Kõljalg U. 2010b. PlutoF – a web based workbench for ecological and taxonomic research with an online implementation for fungal ITS sequences. Evol. Bioinform. 6: 189-196.
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