The absence of any extra bands, with respect to the yeast starter strain restriction profile, demonstrates that the yeast starter has been properly implanted, with an accuracy of 90%. In fact, in the case of a binary mixture, the minority strain must represent around 10% of the total population to be detected (Hallet et al., 1989).
1.9.3 Karyotype Analysis
Saccharomyces cerevisiae has 16 chromosomes with a size range between 250 and 2,500 kb. Its genomic DNA is very polymorphic; thus, it is possible to differentiate strains of the species according to the size distribution of their chromosomes. Pulsed‐field electrophoresis is used to separate S. cerevisiae chromosomes and to compare karyotypes of the strains. This technique uses two electric fields oriented differently (90–120°). The electrodes placed on the sides of the apparatus apply the fields alternately (Figure 1.27).
FIGURE 1.27 CHEF pulsed‐field electrophoresis device (contour clamped electrophoresis field).
The user can define the duration of the electric current that will be applied in each direction (pulse). With each change in the direction of the electric field, the DNA molecules reorient themselves; the smaller chromosomes reorient themselves more quickly than the larger ones (Figure 1.28).
Blondin and Vezhinet (1988), Petering et al. (1988), and Dubourdieu and Frezier (1990) applied this technique to identify wine yeast strains. Sample preparation is relatively easy. The yeasts are cultivated in a liquid medium, collected during the log phase, and then placed in suspension in a warm agarose solution that is poured into a partitioned mold to form small plugs.
Figure 1.29 gives an example of the identification of S. cerevisiae strains isolated from a grape must undergoing spontaneous fermentation. Vezhinet et al. (1990) have shown that karyotype analysis can distinguish between strains of S. cerevisiae as well or better than the use of mtDNA restriction profiles. Furthermore, karyotype analysis is much quicker and easier to use than mtDNA analysis. In the case of ecological studies of spontaneous fermentation microflora, pulsed‐field electrophoresis of chromosomes is extensively used today to characterize strains of S. cerevisiae (Frezier and Dubourdieu, 1992; Versavaud et al., 1993, 1995).
FIGURE 1.28 Mechanism of DNA molecule separation by pulsed‐field electrophoresis.
Very little research on the chromosomal polymorphism in other species of grape and wine yeasts is currently available. Naumov et al. (1993) suggested that S. uvarum and S. cerevisiae karyotypes can be easily distinguished. Other authors (Vaughan Martini and Martini, 1993; Masneuf, 1996) have confirmed their results. In fact, a specific chromosomal band systematically appears in S. uvarum. Furthermore, there are only two chromosomes whose sizes are less than 400 kb in S. uvarum but generally more in S. cerevisiae, in all of the strains that we have analyzed.
Non‐Saccharomyces species, in particular apiculate yeasts (H. uvarum and K. apiculata), are present on grapes and are sometimes found at the beginning of fermentations. These species have fewer polymorphic karyotypes and fewer bands than in Saccharomyces. Versavaud et al. (1993) differentiated between strains of apiculate yeast species and Candida famata by using restriction endonucleases at rare sites (Not1 and Sfi1). The endonucleases cut the chromosomes into a limited number of fragments, which were then separated by pulse‐field electrophoresis.
FIGURE 1.29 Example of electrophoretic (pulsed field) profile of S. cerevisiae strain karyotypes.
1.9.4 Genomic DNA Restriction Profile Analysis Associated with DNA Hybridization by Specific Probes (Fingerprinting)
The yeast genome contains DNA sequences that repeat from dozens to hundreds of times, such as the δ sequences or Y1 elements of the chromosome telomeres. The distribution, or more specifically, the number and location of these elements, has a certain intraspecific variability. This genetic fingerprint is used to identify strains (Pedersen, 1986; Degre et al., 1989).
The yeast strains are cultivated in a liquid medium and are sampled during the log phase, as in the preceding techniques. The entire DNA is isolated and digested by restriction endonucleases. The generated fragments are separated by electrophoresis on agarose gel and then transferred to a nylon membrane (Southern, 1975). Complementary radioactive probes (nucleotide sequences taken from δ and Y1 elements) are used to hybridize with fragments having homologous sequences. The result gives a hybridization profile containing several bands.
Genetic fingerprinting after hybridization is a more complicated and involved method than mtDNA or karyotype analysis. It is, however, without doubt the most discriminating strain identification method and may even discriminate too well. It has correctly indicated minor differences between very closely related strains. In fact, in the Bordeaux region (Frezier, 1992), S. cerevisiae clones isolated from spontaneous fermentations in different wineries have been encountered, which have the same karyotype and the same mtDNA restriction profile. Yet their hybridization profiles differ depending on sample origin. These strains, probably descendants of the same mother strain, have therefore undergone minor random modifications, maintained during vegetative reproduction.
1.9.5 PCR Associated with δSequences
This method consists of using PCR to amplify certain sequences of the yeast genome (Section 1.8.4), occurring between the repeated δ elements, whose separation distance does not exceed a certain value (1 kb). This method was developed (Ness et al., 1992; Masneuf and Dubourdieu, 1994; Legras and Karst, 2003) to characterize S. cerevisiae strains. The amplification is carried out directly on whole cells. They are simply heated to make the cell envelopes permeable. The resulting amplification fragments are separated according to their size by agarose gel electrophoresis and viewed using ultraviolet fluorescence (Figure 1.30).
FIGURE 1.30 Principle of identification of S. cerevisiae strains by PCR associated with δ elements.
This analysis can distinguish between most S. cerevisiae ADY strains used in winemaking (Figure 1.31). Out of the 26 selected
