Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) reg
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© Springer-Verlag 1998
O R I G I N A L PA P E R
José Manuel Guillamón · Josepa Sabaté · Eladio Barrio · Josep Cano · Amparo Querol
Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region
Received: 2 July 1997 / Accepted: 7 December 1997
Abstract In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation. Key words Wine yeast identification · rRNA gene · RFLP-PCR · Anamorph state · Teleomorph state
Introduction The fermentation of grape juice in wine is a complex microbiological process in which yeasts play a central role.
J. M. Guillamón Unitat d’Enologia del Centre de Referència de Tecnologia d’Aliments, Departament de Bioquímica i Biotecnologia, Escola d’Enologia de Tarragona. Universitat Rovira i Virgili, Tarragona, Spain J. Sabaté · J. Cano Unitat de Microbiologia, Escola d’Enologia de Tarragona, Universitat Rovira i Virgili, Tarragona, Spain E. Barrio Departament de Genética, Universitat de València, Burjassot, Spain A. Querol (Y) Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (C.S.I.C.), P.O. Box 73, E-46100 Burjassot, Valencia, Spain Tel. +34-6-3900022; Fax +34-6-3636301 e-mail: [email protected]
Traditionally, identification and characterization of yeast species and strains have been based on morphological traits and especially on their physiological abilities (Barnett et al. 1990; Kreger-Van Rij 1984). These characteristics are strongly influenced by culture conditions and can give uncertain results (Yamamoto et al. 1991), and it is frequently necessary to conduct approximately 50–100 tests to identify most yeasts to the species level reliably (Lin and Fung 1987). In contrast, molecular biology techniques provide alternative and additional methods and are becoming an important tool in solving industrial problems. In recent years, the application of these techniques has generated a large number of studies on the classification, identification, and ecology of the species and strains of the genus Saccharomyces, the main yeast species in wine fermentation (Guillamón et al. 1994, 1996; Querol et al. 1992 b; Schütz and Gafner 1993, 1994). However, wine fermentation is not the action of a single species or single strain of yeast (Martini and VaughanMartini 1990; Fleet and Heard 1993). In grape musts and during the early phase of fermentation, apiculate
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