to differentiate between S. cerevisiae and S. uvarum (formerly S. bayanus), when working on strain types of these two species. This gene, which codes for the synthesis of homoserine acetyltransferase, has different sequences in the two species. Part of the gene is initially amplified by using two complementary oligonucleotides of the sequences bordering the fragment to be amplified. The fragment obtained (about 600 bp) is the same size for typical strains of the S. cerevisiae and S. uvarum species. Different restriction endonucleases, which recognize certain specific DNA sequences, then digest the amplified fragment. Figure 1.23 gives an example of the mode of action of the EcoR1 restriction endonuclease. This enzyme recognizes the base sequence GAATTC and cuts at the location indicated by the arrows. Electrophoresis is used to separate the obtained fragments. As a result, restriction fragment length polymorphism (RFLP) can be assessed. The restriction profiles obtained differ between S. cerevisiae and S. uvarum.
FIGURE 1.22 Mode of action of DNA polymerase.
FIGURE 1.23 Recognition site and cutting mode of an EcoR1 restriction endonuclease.
FIGURE 1.24 Identification principles for the S. cerevisiae and S. uvarum species by the MET2 gene PCR‐RFLP technique, after cutting the amplified fragment with EcoR1 and Pst1 restriction enzymes.
This PCR‐RFLP technique associated with the MET2 gene has been developed and adapted for rapid analysis. The whole cells are simply heated in water to 95°C for 10 minutes before amplification. Only two restriction enzymes are used: EcoR1 and Pst1 (Figure 1.24; Masneuf et al., 1996a,b).
By applying this relatively simple and quick technique to different wine yeast strains studied by Naumov et al. (1993), we obtained perfect concordance between the MET2 gene PCR‐RFLP and hybridization tests in order to delimit S. cerevisiae and S. uvarum species.
We extended this type of analysis by PCR‐RFLP of the MET2 gene to different yeast strains selected from the market and often used in winemaking. Depending on their ability to ferment galactose or not, they are still sometimes called S. cerevisiae or S. bayanus by wine professionals around the world at the time this handbook is being written (Table 1.6). For all of these strains, we have obtained the same characteristic restriction profiles of the species S. cerevisiae.
TABLE 1.6 Characterization by PCR‐RFLP of the MET2 Gene of Various Commercial Strains of the S. cerevisiae Species Used in Winemaking (Masneuf, 1996)
| Strains | Commercial brand | Origin | Enological designation | Species |
|---|---|---|---|---|
| VL1 | Zymaflore VL1 | FŒB | Saccharomyces cerevisiae | Saccharomyces cerevisiae |
| VL3c | Zymaflore VL3 | FŒB | Saccharomyces cerevisiae | Saccharomyces cerevisiae |
| WET 136 | Siha levactif 3 | Dormstadt | Saccharomyces cerevisiae | Saccharomyces cerevisiae |
| 71B | Actiflore primeur | INRA Narbonne | Saccharomyces cerevisiae | Saccharomyces cerevisiae |
| F10 | Zymaflore F10 | FŒB | Saccharomyces bayanus | Saccharomyces cerevisiae |
| R2 | Vitlevure KD | NA | Saccharomyces bayanus | Saccharomyces cerevisiae |
| BO213 | Actiflore bayanus | Institut Pasteur | Saccharomyces bayanus | Saccharomyces cerevisiae |
| CH158 | Siha levactif 4 | NA | Saccharomyces bayanus | Saccharomyces cerevisiae |
| QA23 | Lalvin QA23 | UTM | Saccharomyces bayanus | Saccharomyces cerevisiae |
| IOC182007 | IOC 182007 | IŒC | Saccharomyces bayanus | Saccharomyces cerevisiae |
| DV10 | Vitlevure DV10 | CIVC | Saccharomyces bayanus | Saccharomyces cerevisiae |
| O16 | Lalvin O16 | UB | Saccharomyces bayanus | Saccharomyces cerevisiae |
| Epernay2 | Uvaferm CEG | NA | Saccharomyces bayanus |
Saccharomyces cerevisiae
|
