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.
1.9 Identification of Wine Yeast Strains
1.9.1 General Principles
The principal yeast species involved in grape must fermentation, particularly S. cerevisiae and S. uvarum, comprise a very large number of strains with extremely varied technological properties. The yeast strains involved during winemaking influence fermentation speed, the nature and quantity of secondary products formed during alcoholic fermentation, and the aroma characteristics of the wine. The ability to differentiate between the different strains of S. cerevisiae is required for the following fields: the ecological study of wild yeasts responsible for the spontaneous fermentation of grape must, the selection of strains presenting the best enological qualities, production and marketing controls, the verification of the implantation of selected yeasts used as yeast starter, and the constitution and maintenance of wild or selected yeast collections.
The initial research on infraspecific differentiation within S. cerevisiae attempted to distinguish strains by electrophoretic analysis of their exocellular (Bouix et al., 1981) or intracellular (Van Vuuren and Van Der Meer, 1987) proteins or glycoproteins. Other teams proposed identifying the strains by the analysis of long‐chain fatty acids using gas chromatography (Tredoux et al., 1987; Augustyn et al., 1991; Bendova et al., 1991; Rozes et al., 1992). Although these different techniques differentiate between certain strains, they are irrefutably less discriminating than genetic differentiation methods. They also present the major drawback of depending on the physiological state of the strains and on cultural conditions, which must always be identical.
In the late 1980s, owing to the development of genetics, certain techniques from molecular biology were successfully applied to characterize wine yeast strains. They are based on the clonal polymorphism of the mitochondrial and genomic DNA of S. cerevisiae and S. uvarum. These genetic methods are independent of the physiological state of the yeast, unlike the previous techniques based on the analysis of metabolism by‐products.
1.9.2 Mitochondrial DNA Analysis
The mtDNA of S. cerevisiae has two remarkable properties: it is extremely polymorphic, depending on the strain; and stable (it mutates very little) during vegetative reproduction. Restriction endonucleases (such as EcoR5) cut this DNA at specific sites. This process generates fragments of variable size that are few in number and can be separated by electrophoresis on agarose gel.
Aigle et al. (1984) first applied this technique to brewer's yeasts. Since 1987, it has been used for the characterization of enological strains of S. cerevisiae (Dubourdieu et al., 1987; Hallet et al., 1988).
The extraction of mtDNA comprises several stages. The protoplasts obtained by enzymatic digestion of the cell walls are lysed in a hypotonic buffer. The mtDNA is then separated from the chromosomal DNA by ultracentrifugation in a cesium chloride gradient, in the presence of bisbenzimide, which acts as a fluorescent intercalating stain. This agent amplifies the difference in density between chromosomal DNA and mtDNA. The mtDNA has a high number of adenine and thymine base pairs, for which bisbenzimide has a strong affinity. Finally, the mtDNA is purified by a phenol–chloroform extraction and an ethanol precipitation.
Defontaine et al. (1991) and Querol et al. (1992) simplified this protocol by separating the mitochondria from the other cell constituents before extracting the DNA. In this manner, they avoided the ultracentrifugation step. The coarse cellular debris is eliminated from the yeast lysate by centrifuging at 1,000 g. The supernatant is then recentrifuged at 15,000 g to obtain the mitochondria. The mitochondria are then lysed in a suitable buffer to liberate the DNA.
Unlike the industrial brewer's yeast strains analyzed by Aigle et al. (1984), which have the same mtDNA restriction profile, implying that they are of common origin, the winemaking yeast strains have a large degree of mtDNA diversity. This method easily differentiates between most of the selected yeasts used in winemaking as well as wild strains of S. cerevisiae found in spontaneous fermentations (Figure 1.26). This method may also help differentiate S. uvarum strains (Naumova et al., 2010).
FIGURE 1.26 Restriction profile by EcoR5 of mtDNA of different strains of S. cerevisiae. Band 1, FIO; band 2, BO213; band 3, VLI; M, marker; band 4, 522; band 5, Sita 3; band 6, VL3c.
This technique is very discriminating and not too expensive, but it is long and requires several complex manipulations. It is useful for the subtle characterization of a small number of strains. Inoculation effectiveness can also be verified by this method. In the laboratory, the lees, sampled during or toward the end of alcoholic fermentation, are cultured in a liquid medium. The mtDNA restriction profiles of this total biomass and of the yeast starter
