Restriction profiles of 26S rDNA as a molecular approach for wine yeasts identification

the complex microbial ecosystem existing in grape, must and wine comprises a wide diversity of yeast species. the knowledge of composition and dynamics of yeast biota occurring along vinification process would provide a better control of wine quality. the sequence of d1/d2 domain of 26s ribosomal dna (rdna), reflects ascomycetous yeast phylogenetic relationships and enables their separation at the species level. a region of the 26s rdna, with around 1100 bp comprising domain d1/d2, was amplified by pcr and then digested with restriction endonucleases (apai, hinfi, msei, haeiiiand cfoi) in order to differentiate yeast species frequently isolated from grape surfaces, wine and cellar equipments. a total of 78 yeast strains (including 36 type strains) belonging to 53 species were used to generate the restriction profiles. numerical analysis of the profiles generated by the five restriction enzymes enabled to group the strains in 47 different clusters and 42 of them clearly corresponded to different yeast species. the remaining groups comprise closely related species. the enzymes msei, haeiiiand cfoirevealed a high discrimination power and the restriction profiles generated were sufficient to clearly identify the 42 species mentioned above. despite one of the clusters included different yeast genera, with different wine characteristics, the common wine spoilage yeasts zygosaccharomyces bailii and z. lentus could be separatedto one distinctive cluster through the use of apai restriction profiles. since the analysis of restriction profiles of amplified 26s rdna showed to be a valuable method to identify oenological yeast species, a database comprising the majority of wine yeast biota was created to be applied both at research and industrial environment.