Читать книгу Handbook of Enology: Volume 1 - Pascal Ribéreau-Gayon - Страница 38

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 strain are compared. The absence of any extra bands, with respect to the yeast starter strain restriction profile, demonstrates that the yeast starter has been properly implanted, with an accuracy of 90%. In fact, in the case of a binary mixture, the minority strain must represent around 10% of the total population to be detected (Hallet et al., 1989).