Evaluation of ribosomal regions sequencing as tool for wine yeast identification

MIRAS, SILVANA; MASSERA, ARIEL; MERCADO, LAURA; STURM, MARÍA ELENA; COMBINA, MARIANA

Carlos Paz

Congreso; 44th Annual Meeting of Argentine Society for Biochemistry and Molecular Biology Research; 2008

SAIB

Yeast identification is important in wine industry to resolve microbiological problems and improve fermentation management. Different molecular techniques have been proposed to identify yeast species. The aim of this work was to evaluate if direct sequencing of two ribosomal regions allow a rapid and accuracy identification of yeasts related to wine industry. Fifty yeasts were evaluated; reference and native strains were included. ITS1 and ITS4 primers were used to amplify a region of the rRNA gene 5,8S and ITS1-ITS2; and NL1 and NL4 primers to amplify the domain D1/D2 of the 26S rRNA gene. Sequencing was done by automatic sequencer. Sequences were aligned and compared in Genebank database using the BLAST tool. 5.8S-ITS RFLP was done to confirm the results. 5.8S-ITS sequencing allowed identify 84% of strains, 9 strains remained misidentified. 5.8S-ITS RFPL helped to resolve species assignation, but some strains remained doubtful. 26S sequencing allowed 90% of correct identification. Combining both techniques, complete identification was reach. Results showed that 5.8S-ITS RFPL and 26S sequencing could be suggested to unequivocal identification of yeast species closely related or not. Moreover, yeast database of RFLP is available and facilitate the species assignation; and the most commonly utilized D1/D2 region is also very accessible and it can be determined in one experiment.