Читать книгу Handbook of Enology: Volume 1 - Pascal Ribéreau-Gayon - Страница 42

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.