Handbook of Enology: Volume 1. Pascal Ribéreau-Gayon
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.
1.9.6 PCR with Microsatellites
Microsatellites are tandem repeat units of short DNA sequences (1–10 nucleotides), i.e. in the same direction and dispersed throughout the eukaryote genome. The number of motif repetitions is extremely variable from one individual to another, making these sequences highly polymorphic in size. These regions are easily identified, thanks to knowledge of the full sequence of the S. cerevisiae genome. Approximately 275 sequences have been listed, mainly AT dinucleotides and AAT and AAC trinucleotides (Field and Wills, 1998; Hennequin et al., 2001; Perez et al., 2001). Furthermore, these sequences are allelic markers, transmitted to the offspring in a Mendelian fashion. Consequently, these are ideal genetic markers for identifying specific yeast strains, making it possible not only to distinguish between strains but also to arrange them in related groups. This technique has many applications in humans: paternity tests, forensic medicine, etc. In viticulture, this molecular identification method has already been applied to Vitis vinifera grape varieties (Bowers et al., 1999).
FIGURE 1.32 Electrophoresis in agarose gel (1.8%) of amplified fragments illustrating examples of yeast implantation tests (successful: yeasts B and C; unsuccessful: yeasts A, D, and E). Band 1, negative control; band 2, lees A; band 3, ADY A; band 4, lees B; band 5, ADY B; band 6, lees C; band 7, ADY C; band 8, lees D; band 9, ADY D; band 10, lees E; band 11, ADY E; M, molecular weight marker.
FIGURE 1.33 Determination of the detection threshold of a contaminating strain. T, negative control; band 1, strain A 70%, strain B 30%; band 2, strain A 80%, strain B 20%; band 3, strain A 90%, strain B 10%; band 4, strain A 99%, strain B 1%; M, molecular weight marker; band 5, strain A 99.9%, strain B 0.1%; band 6, strain A; band 7, strain B.
The technique consists in amplifying the region of the genome containing these microsatellites, and then analyzing the size of the amplified portion to a level of detail of one nucleotide by capillary electrophoresis. This size varies by a certain number of base pairs (approximately 8–40) from one strain to another, depending on the number of times the motif is repeated. A yeast strain may be heterozygous for a given locus, giving two different‐sized amplified DNA fragments. Using six microsatellites, Perez et al. (2001) were able to identify 44 different genotypes within a population of 51 strains of S. cerevisiae used in winemaking. Other authors (Gonzalez Techera et al., 2001; Hennequin et al., 2001, Klis et al., 2002) have shown that the strains of S. cerevisiae used in winemaking are weakly heterozygous for the loci studied. However, interstrain variability of the microsatellites is very high. The results are expressed in numerical values for the size of the microsatellite in base pairs or the number of repetitions of the motifs on each allele. These digital data are easy to interpret, unlike the karyotype images on agarose gel, which are not really comparable from one laboratory to another. Based on 41 microsatellites, Legras et al. (2005) selected six very discriminating and reproducible loci. They highlight the relationships between strains from different geographical zones or industrial environments.
Microsatellite analysis has also been used to identify the strains of S. uvarum (Masneuf‐Pomarède et al., 2007, 2016) and of S. kudriavzevii (Erny et al., 2012) used in winemaking. As the S. uvarum, S. kudriavzevii, and S. cerevisiae microsatellites have different amplification primers, this method provides an additional means of distinguishing between these species and their hybrids.
The development of new‐generation sequencing methods for yeast genomes has made sequences available for non‐Saccharomyces species. A typing method of yeast strains by microsatellite marker analysis is now offered for B. bruxellensis, T. delbrueckii, H. uvarum, and Starmerella bacillaris (Albertin et al., 2014a,b, 2016; Masneuf‐Pomarede et al., 2015, 2016). When applied to the study of a great number of yeast isolates, these methods help to better describe the genetic diversity and the population structure of winemaking yeasts. Factors influencing this structure as well as their life cycle and reproduction mode have also been described. From an applied point of view, this molecular typing method is a useful tool in winemaking yeast strain identification, ecological surveys, and quality control of industrial production batches.
1.9.7 Genome Sequencing
With the development of new‐generation sequencing methods, new approaches to yeast characterization have been suggested. The multilocus sequence typing (MLST) method is a standardized approach to full or partial sequence analysis of certain gene expressions in yeast. These genes are characterized by a slow accumulation of mutations, which help differentiate between individuals, as well as deduce phylogenetic relationships between strains. Applied to S. cerevisiae, the results obtained do not indicate a superior ability to discriminate among yeast strains when compared with analysis by repetitive‐element PCR or microsatellite marker polymorphism (Ayoub et al., 2006). However, studies have revealed the specific population structure of wine yeasts, confirming the domestication of these yeasts (Fay and Benavides, 2005). Other approaches consist in establishing sequences of regions located between randomly selected restriction sites in the genome (restriction site‐associated DNA sequencing or RAD‐seq). Many positions of variation of a base, or single nucleotide polymorphism (SNP), can thus be used for phylogenetic analyses. The RAD‐seq method has established the diversity and genetic structure of S. cerevisiae strains from a variety of ecological niches (Cromie et al., 2013; Hyma and Fay, 2013).
1.10 Ecology of Grape and Wine Yeasts
1.10.1 Succession of Grape and Wine Yeast Species
A large amount of research was focused on the description and ecology of wine yeasts. It concerned the distribution and succession of species found on the grape and then in wine during fermentation and conservation (Ribéreau‐Gayon et al., 1975; Lafon‐Lafourcade, 1983).
The ecological study of grape and wine yeast species represents a considerable amount of research. De Rossi began his