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The rapid assignment of ruminal fungi to presumptive genera using ITS1 and ITS2 RNA secondary structures to produce group-specific fingerprints

Matthew J. Nicholson^2,3,

¹ F2G Ltd, Lankro Way, Eccles, Manchester M30 0BH, UK
² Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion, SY23 3EB, UK
³ School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK
⁴ CSIRO Livestock Industries, Queensland Bioscience Precinct, Carmody Road, St Lucia, Brisbane, Australia

Correspondence
Michael K. Theodorou
mike.theodorou{at}bbsrc.ac.uk

Identification of microbial community members in complex environmental samples is time consuming and repetitive. Here, ribosomal sequences and hidden Markov models are used in a novel approach to rapidly assign fungi to their presumptive genera. The ITS1 and ITS2 fragments from a range of axenic, anaerobic gut fungal cultures, including several type strains, were isolated and the RNA secondary structures predicted for these sequences were used to generate a fingerprinting program. The methodology was then tested and the algorithms improved using a collection of environmentally derived sequences, providing a rapid indicator of the fungal diversity and numbers of novel sequence groups within the environmental sample from which they were derived. While the methodology was developed to assist in investigations involving the rumen ecosystem, it has potential generic application in studying diversity and population dynamics in other microbial ecosystems.

A representative alignment of 12 ITS1 sequences plus flanking regions from anaerobic gut fungi showing Variable Regions I–IV is available as Supplementary Fig. S1. An alignment of the five Caecomyces and nine Cyllamyces sequences for ITS1 Variable Regions I–IV, together with the ITS1 Variable Regions II–IV sequences from the eleven of the forty-eight environmental samples that group together as Novel Group 1, is available as Supplementary Fig. S2 with the online version of this paper at http://mic.sgmjournals.org.

Present address: AgResearch Grasslands, Private Bag 11008, Palmerston North, New Zealand.

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