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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 7, L‐1597; band 8, WET 136; M, molecular weight marker; T, negative control.

Saccharomyces uvarum strains cannot be distinguished by this technique because their genome contains only a few Ty elements.

Lastly, because of its convenience and rapidity, PCR associated with δ sequences facilitates verification of the implantation of yeast starters used in winemaking. The analyses are conducted on the entire biomass derived from lees, placed beforehand in a liquid medium in a laboratory culture. The amplification profiles obtained are compared with inoculated yeast strain profiles. They are identical with a successful implantation, and different if the inoculation fails. Figure 1.32 gives examples of successful (yeasts B and C) and unsuccessful (yeasts A, D, and E) implantations. Contaminating strains have a different amplification profile than the yeast starter.

The detection threshold of a contaminating strain was studied in the laboratory by analyzing a mixture of two strains in variable proportions. In the example given in Figure 1.33, the contaminating strain is easily detected at 1%. In winery fermentations, however, several native strains can coexist with the inoculated strain in small proportions. When must undergoing fermentation or lees is analyzed by PCR, the yeast implantation rate is at least 90% when the amplification profiles of the lees and the yeast starter are identical.