Handbook of Enology: Volume 1. Pascal Ribéreau-Gayon
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 commercial yeast strains analyzed, 25 can be differentiated by theirPCR profile associated with δ sequences. Lavallée et al. (1994) also observed excellent discriminating power with this method while analyzing industrially produced commercial strains from Lallemand Inc. (Montreal, Canada). In addition, this method enables the identification of 25–50 strains per day; it is the quickest of the different strain identification techniques currently available. When used for native yeast strain identification in a given viticultural region, however, it seems to be less discriminating than karyotype analysis. PCR profiles of wild yeasts isolated in a given location often appear similar. They have several constant bands and only a small number of variable discriminating bands. Certain strains have the same PCR amplification profile while having different karyotypes. In a given location, the polymorphism witnessed by PCR associated with δ sequences is lower than that of the karyotypes. This method is therefore complementary to other methods for characterizing winemaking strains. PCR enables a rapid primary sorting of a native yeast population. Karyotype analysis refines this discrimination.
FIGURE 1.31 Electrophoresis in agarose gel (at 1.8%) of amplified fragments obtained from various commercial yeast strains. Band 1, F10; band 2, BO213; band 3, VL3c; band 4, UP30Y5; band 5, 522 D; band 6, EG8; band