Читать книгу Handbook of Enology: Volume 1 - Pascal Ribéreau-Gayon - Страница 36

PCR‐RFLP associated with the MET2 gene can be used to demonstrate the existence of hybrids between the species S. cerevisiae and S. uvarum. This method has been used to prove the existence (Masneuf et al., 1998) of one such natural hybrid (strain S6U) among commercial dry yeasts sold by Lallemand Inc. (Montreal, Canada). Ciolfi (1992, 1994) isolated this yeast in an Italian winery, and it was selected for certain enological properties, in particular its aptitude to ferment at low temperatures, its low production of acetic acid, and its ability to preserve must acidity. The MET2 gene restriction profiles of this strain by EcoR1 and Pst1, composed of three bands, are identical (Figure 1.25). In addition to the amplified fragment, two bands characteristic of S. cerevisiae with EcoR1 and two bands characteristic of the species S. bayanus with Pst1 are obtained. The bands are not artifacts due to an impurity in the strain, because the amplification of the MET2 gene carried out on subclones (obtained from the multiplication of unique cells isolated by a micromanipulator) produces identical results. Hansen from the Carlsberg laboratory (Denmark) sequenced two of the MET2 gene alleles from this strain. The sequence of one of the alleles is identical to that of the S. cerevisiae MET2 gene, with the exception of one nucleotide. The sequence of the other allele is 98.5% similar to that of S. uvarum. The presence of this allele is thus probably due to an interspecific cross.

Subsequently, more recent research (Naumov et al., 2000b) has shown that the S6U strain is, in fact, a tetraploid hybrid. Indeed, the percentage germination of spores from 24 tetrads, isolated using a micromanipulator, was very high (94%), whereas it would have been very low for a “normal” diploid interspecific hybrid. The monospore clones in this first generation (D1) were all capable of sporulating. However, none of the ascospores of the second‐generation tetrads was viable. The hybrid nature of the monospore clones produced by D1 was confirmed by the presence of the S. cerevisiae and S. uvarum MET2 gene, identified by PCR/RFLP. Finally, measuring the DNA content per cell using flux cytometry estimation confirmed that the descendants of S6U were interspecific diploids and that S6U itself was an allotetraploid.

FIGURE 1.25 Electrophoresis in agarose gel (1.8%) of (a) EcoR1 and (b) Pst1 digestions of the amplified fragments of the MET2 gene of the hybrid strain. Bands 1, 2, 3, subclones of the hybrid strain; band 4, hybrid strain; band 5, S. cerevisiae control; band 6, S. uvarum control; M, molecular weight marker.

The molecular characterization of wine yeasts isolated on grapes and in must undergoing spontaneous fermentation revealed the existence of many natural hybrids between S. cerevisiae and S. uvarum (Le Jeune et al., 2007). This is also true between S. cerevisiae and Saccharomyces kudriavzevii (Sipiczki, 2008; Arroyo‐López et al., 2009; Erny et al., 2012). Interspecific hybridization leads to new gene combinations in a given cell and may confer a selective advantage with respect to parental strains. Interspecific hybrids possess interesting technological properties for winemaking. For example, S. uvarum and S. kudriavzevii are better adapted to low‐temperature growth, and S. cerevisiae presents high tolerance to ethanol. Natural hybrids between these species are more adapted to growth in a large range of temperatures and at high ethanol concentrations, thanks to the genetic heritage of one or both parents, thus yielding properties of interest (Da Silva et al., 2015). This mechanism for acquiring superior phenotypic characteristics in hybrid descendants during cross‐breeding of parental strains is well described in plants. This is called a heterosis effect.