Читать книгу Handbook of Enology: Volume 1 - Pascal Ribéreau-Gayon - Страница 40

The yeast genome contains DNA sequences that repeat from dozens to hundreds of times, such as the δ sequences or Y1 elements of the chromosome telomeres. The distribution, or more specifically, the number and location of these elements, has a certain intraspecific variability. This genetic fingerprint is used to identify strains (Pedersen, 1986; Degre et al., 1989).

The yeast strains are cultivated in a liquid medium and are sampled during the log phase, as in the preceding techniques. The entire DNA is isolated and digested by restriction endonucleases. The generated fragments are separated by electrophoresis on agarose gel and then transferred to a nylon membrane (Southern, 1975). Complementary radioactive probes (nucleotide sequences taken from δ and Y1 elements) are used to hybridize with fragments having homologous sequences. The result gives a hybridization profile containing several bands.

Genetic fingerprinting after hybridization is a more complicated and involved method than mtDNA or karyotype analysis. It is, however, without doubt the most discriminating strain identification method and may even discriminate too well. It has correctly indicated minor differences between very closely related strains. In fact, in the Bordeaux region (Frezier, 1992), S. cerevisiae clones isolated from spontaneous fermentations in different wineries have been encountered, which have the same karyotype and the same mtDNA restriction profile. Yet their hybridization profiles differ depending on sample origin. These strains, probably descendants of the same mother strain, have therefore undergone minor random modifications, maintained during vegetative reproduction.