Methods by which strains of the same species can be differentiated have been shown to be very important for yeast strain characterization. In winemaking, several studies have analyzed the diverse microflora of grapes and musts and several interesting methods have been developed (Figure 2).
PCR amplification using intron splicing site (consensus sequences that flank introns) displays polymorphism mainly at the within-species level (de Barros Lopes de et al. 1996); PCR amplification of delta elements (repeat sequences that flank the TY1 retrotransposons) has been widely used to characterize wine yeast strains of Saccharomyces cerevisiae (Ness et al. 1993). Another PCR-based approach, known as random amplified polymorphic DNA (RAPD), consists of the amplification of random segments of DNA with a single and short (from 5- to 15-mer) primer of an arbitrary nucleotide sequence. The level of differentiation, either interspecific or intraspecific, depends highly on the primer used. However, one of the most important problems with this technique is the low stable (nonrepetitive) patterns obtained.
The amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in commercial strains and winery isolates (de Barros Lopes de et al. 1999). It was proposed that AFLP is a very useful method for discriminating yeasts at both species and subspecies levels, and also to characterize hybrids (de Barros Lopes de et al. 2002). Although initially more labor intensive than other PCR techniques, the reproducibility of the results is the advantage over the previous PCR method described.
The analysis of mitochondrial DNA restriction fragment length polymorphisms (mtDNA-RFLP) has long been used as a method for characterizing wine yeast strains (Vezinhet et al. 1990). However, due to the complexity associated with the procedure required to isolate a sufficient amount of mtDNA, its use for routine analysis was limited until quite recently. The last decade has seen improvements in the process (Querol et al. 1992b), removing the need for specialized equipment and reducing the complexity and time scale, while retaining the discriminatory power and reproducibility (Figure 3). This simplified technique has been used successfully to characterize yeast strains of species belonging to genera Brettanomyces, Candida, Debaryomyces, Kluyveromyces, Saccharomyces, and Zygosaccharomyces (see Table 2).
Chromosomal DNA profile analysis (electrophoresis karyotyping). The analysis of chromosomal DNA polymorphisms due to chromosome rearrangements has proved to be useful for the differentiation of species (and also strains) belonging to several genera, e.g., Candida, Kluyveromyces, Saccharomyces, and Zygosaccharomyces. In the case of wine yeast, it has been used for yeast characterization and was applied to analyse the dynamics of the yeast populations (Querol and Ramon 1996) and in the characterization of the industrial wine yeast (Fernandez-Espinar et al. 2001).
In the following section we will discuss the resolution of some of these techniques and their industrial application for the study of yeast population dynamics during natural and inoculated wine fermentations and for the characterization of S. cerevisiae strains of industrial interest.
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