The importance of yeast identification is paramount, especially in the wine industry, where purity of the inoculated yeast culture dictates the efficacy of fermentation and the quality of the final wine. Molecular comparisons are increasingly being used as a method of yeast identification given that they are less laborious and have greater reliability. Initially, nuclear DNA was used as a method of determining relatedness between strains with members of a biological species generally exhibiting 70% or greater nDNA complementarity. Sequencing remains one of the more preferred methods as it is easy to compare strains and the choice of DNA allows one to resolve close or distant relationships between strains or biological species. The D1/D2 domain is a 600 nucleotide domain at the 5’ end of a large subunit of (26S) rDNA. A study by Kurtzmann and Robnett (1995) showed that most yeast species can be identified from sequence divergence of the D1/D2 domain. Studies of over 500 species of ascomycetous yeasts have shown that conspecific strains generally have fewer than 1% nucleotide substitutions in this domain, whereas biological species are separated by greater than this number of substitutions.
Many authors report the use of different protocols for this assay. In general, the steps remain as follows: the cells are grown on a suitable media and harvested, usually using centrifugation techniques. The cells are suspended in distilled water and maybe lyophilized and stored in a freezer. The cells are fractured using different methods (physically by centrifuging with glass beads, or by use of enzymes that cause lysis of cell walls) and the DNA is then precipitated (using isopropanol). O’Donnell in 1993 developed a method in which the divergent D1/D2 domain (nucleotides 63–642 for Saccharomyces cerevisiae) at the 50 end of the LSU rRNA gene was symmetrically amplified with primers NL-1 (50-GCATATCAATAAGCGGAGGAAAAG) and NL-4 (50-GGTCCGTGTTTCAAGACGG). PCR methods are then used to amplify the DNA and then strands of DNA are compared using sequencing kits that are commercially available. Softwares are then used to align the sequence data as well as to calculate phylogenetic relationships.
Ramos et al, 2006 developed a HMA (HeteroDuplex Mobility Assay) using the above described PCR amplified D1/D2 region of the 26S rDNA. This method accounts for potential heteroduplex formation between strains of the same species when the PCR products are mixed. This method also uses universal primers instead of combinations of specific ones and reduces manipulation errors. Many modifications to the method of sequencing the domain have been proposed in literature and thus D1/D2 sequencing method is being extensively used to identify yeasts with both ascomycetic and basidiomycetic affinities.
Application in Wine Microbiology:
The number of yeasts associated with the winemaking process –winery and vineyard- are numerous and include bothSaccharomyces and non- Saccharomyces strains. Such molecular techniques allow the winemaker to distinguish and clearly identify the strains that constitute the microflora of the winery. The effects of the microbial populations on the wine produced cannot be stressed enough. Correct understanding of the microbial populations can help understand various problems arising during fermentation and microbial spoilage as well. This is important for both native and inoculated fermentations. The correct Saccharomyces strain needs to be used for inoculated fermentations. Also, this technique is very important when yeast strains are genetically modified to improve their fermentation characteristics. From a taxonomic standpoint, the sequencing of this region helps understand the phylogenetic relationships between the various species and strains and this information can be used in the preparation of various mixed starter cultures.
The sequencing of this region has been used extensively by many researchers. Strains of most of the yeast species associated with the winemaking process have been identified and closely related strains which were considered the same before have been differentiated. These include Saccharomyces, Candida, Hanseniaspora, Kloeckera, Debaryomyces,Hansenula, Metschnikowia, Pichia and many more.
- Kurtzman CP & Robnett CJ (1991) Phylogenetic relationships among species of Saccharomyces, Schizosaccharomyces, Debaryomyces and Schwanniomyces determined from partial ribosomal RNA sequences. Yeast 7: 61–72.
- Cletus P. Kurtzman_ & Christie J. Robnett (1998). Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73: 331–371.
- Jesus P. Ramos1, Carlos A. Rosa2, Elaine M. M. Carvalho1, Orilio Leoncini1 and Patricia Valente. (2006). Heteroduplex mobility assay of the 26S rDNA D1/D2 region for differentiation of clinically relevant Candida species. Antonie van Leeuwenhoek. 89: 39 –44.
- O’Donnell K (1993) Fusarium and its near relatives. In: Reynolds DR & Taylor JW (Eds) The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics (pp 225–233). CAB International, Wallingford, UK.