GREAT AT SMALL THINGS

DNA-sequence based identification

Different analyses are performed based on preliminary morphological assessment and in agreement with customers’ requirements. Preferably living pure cultures are to be submitted for identification together with all relevant information on the substrate of isolation.

The report will provide the name of the genus or species, the analyses on which the identification is based, and a brief statement of the substrates and/or habitats were the identified fungus is typically encountered, if this information is available from the scientific literature.

Ribosomal DNA sequencing is a standard method in fungal taxonomy and identification. It generates results at genus and potentially species level. The method is based on the detection of sequence differences in the internal transcribed spacer (ITS) regions 1 and 2 as well as in the D1/D2 regions of the large subunit (LSU) rRNA. An unknown isolate can be identified by comparing sequences with those of strains with known taxonomic identity that are documented in public databases (e.g. NCBI). However, results of similarity searches may be difficult to interpret due to the unconfirmed identity of certain publicly available sequences. For the reliable identification of an unknown fungal isolate, comparison with sequences of taxonomic reference or type strains of validly published species is performed. As far as available, dedicated reference databases (e.g. NCBI Reference Sequence (RefSeq) collection, YeastIP for ascomycetous yeasts) will be used, but often our specialised staff needs to find suitable reference sequences in the taxonomic literature.

The standard sequencing targets ITS and D1/D2 LSU might show limited resolution to differentiate members of closely related taxa. A higher genetic variation is usually provided by housekeeping genes, which thus enable a more accurate classification and identification. Different housekeeping genes are used in different fungal groups. This implies that the genus or family identity needs to be determined before a choice of the best suited housekeeping gene(s) can be made. For filamentous fungi, the choice of the most appropriate sequencing target is made based on morphological assessments. In yeasts, this assessment relies on standard sequence analysis.

Arbuscular mycorrhizal fungi are multinucleate and require a cloning step before sequencing. A single spore or a spores cluster is collected and submitted to nested PCR, cloning and sequencing of a region of 1500 bp covering part of the SSU, ITS and D1/D2 regions of the LSU ribosomal DNA, as described by Krüger et al. (2010).

Contact: Cony Decock (filamentous fungi), Heide-Marie Daniel (yeasts), Sylvie Cranenbrouck (AMF)

Literature:

Krüger M, Stockinger H, Krüger C, Schüssler A 2009 DNA-based species level detection of Glomeromycota: one PCR primer set for all arbuscular mycorrhizal fungi. New Phytol. 183(1): 212-23.

Schoch CL, Seifert KA, Huhndorf S, et al. 2012 Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. PNAS 109: 6241-6246.

Stielow JB, Lévesque CA, Seifert KA, et al. 2015 One fungus, which genes? Development and assessment of universal primers for potential secondary fungal barcodes. Persoonia 35: 242-263.

The NCBI handbook [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2002 Oct. Chapter 18, The Reference Sequence (RefSeq) Project. Available from http://www.ncbi.nlm.nih.gov/books/NBK21091/

Weiss S, Samson F, Navarro D, Casaregola S. 2013 YeastIP: a database for identification and phylogeny of ascomycetous yeasts. FEMS Yeast Res. 13:117-125; http://genome.jouy.inra.fr/yeastip/