Molecular methods for identification of beer-spoiling yeast species
As described above (see ‘Detection and identification'), methods for identification of yeasts were historically based on physiological characteristics, including sexual reproduction (where present), cell morphology, growth characteristics, and biochemical features. Some of these criteria are still utilized today for characterizing yeasts, although for identification purposes analysis of DNA homology is increasingly the most widely accepted method. Once a beer-spoiling yeast has been isolated, it can be identified to the genus and species level by analysing specific regions of DNA. These regions are typically selected based on specific criteria and should demonstrate a high degree of interspecific polymorphism and a low or non-existent intraspecific polymorphism. This enables the sequence to be used to accurately differentiate between species, but also provides a robust means of ensuring that all strains within a species yield identical results. There are several regions of the genome that meet these criteria, with the most widely used being those located within the ribosomal DNA (rDNA) sequence, coding for the ribosomal RNA. The rDNA contains tracts designated as 26S, 5S, 18S, 5.8S and 26S, which are highly conserved and arranged in tandem units (Fig. 11.7). These genes are separated by internal transcribed spacer (ITS) regions and non-transcribed spacer (NTS) regions, both of which are non-coding, highly variable, and match the criteria outlined above for classification purposes. Studies have shown that the ITS region is particularly useful for identification of yeasts to the genus and species level and this has recently been proposed for adoption as the primary fungal barcode marker (Schoch et al., 2012). In addition, a small region of the 26S subunit known as the D1/ D2 domain can be employed to accurately identify yeast species. Analysis of this region is important from a phylogenetic perspective as it has previously acted as one of the predominant means of confirming existing taxonomic groupings and in identifying novel yeast species (Kurtzman and Robnett, 1998; Weiss et al., 2013).