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Saccharomyces species assignment by long range ribotyping

R Messner1, H Prillinger

  • 1Institute of Applied Microbiology, University of Agriculture, Wien, Austria.

Antonie Van Leeuwenhoek
|January 1, 1995
PubMed
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Ribotyping, a ribosomal DNA restriction fragment analysis, reliably differentiates Saccharomyces species. This reproducible method aids in accurate yeast identification and phylogenetic analysis, even detecting misidentified genera.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Yeast Taxonomy

Background:

  • Accurate identification of Saccharomyces species is crucial for various applications, including brewing, baking, and research.
  • Existing molecular methods for yeast differentiation vary in reproducibility and resolution.
  • Ribosomal DNA (rDNA) contains conserved and variable regions suitable for phylogenetic and taxonomic studies.

Purpose of the Study:

  • To develop and validate a ribotyping method for differentiating Saccharomyces species.
  • To assess the reproducibility and phylogenetic utility of the developed ribotyping technique.
  • To compare ribotyping with other molecular methods for yeast identification.

Main Methods:

  • Amplification of the full-length chromosomal ribosomal repeat in two parts: 18S rDNA (including ITS) and 25S rDNA.

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  • Restriction fragment analysis of amplified products using nine enzymes.
  • Monitoring of fragments longer than 200 bp for pattern generation.
  • Phylogenetic analysis using the PHYLIP program package based on fragment presence/absence.

    • Main Results:

    • Ribotyping successfully differentiated type strains of 10 genotypically distinct Saccharomyces species.
    • Generated characteristic restriction fragment patterns unique to each species.
    • Demonstrated absolute reproducibility, contrasting with methods like RAPD-PCR.
    • Phylogenetic analysis based on ribotyping data aligned with ribosomal RNA sequence analysis.
    • Identified misidentified genera, highlighting the method's ability to recognize distant relationships.

    Conclusions:

    • Long-range ribotyping is a reproducible and reliable method for Saccharomyces species differentiation and identification.
    • The phylogenetic resolution of ribotyping is comparable to DNA sequence analysis of the same locus.
    • Ribotyping is suitable for database creation and can detect distant evolutionary relationships, aiding in genus-level misidentification detection.