FEMS Microbiology Letters 193 (2000) 255^259 www.fems-microbiology.org PCR di¡erentiation of Saccharomyces cerevisiae from Saccharomyces bayanus/Saccharomyces pastorianus using speci¢c primers Sabatë Josepa a , Josë M. Guillamon b , Josë Cano a a; * Unitat de Microbiologia, Facultat de Medicina i Cie©ncies de la Salut, Institut d'Estudis Avanc° ats, Universitat Rovira i Virgili, 43201-Reus Tarragona, Spain b Departament de Bioquimica i Biotecnologia, Facultat d'Enologia, Universitat Rovira i Virgili, Tarragona, Spain Received 26 June 2000; received in revised form 26 September 2000; accepted 30 September 2000 Abstract The aim of the present study was to design species-specific primers capable of distinguishing between Saccharomyces cerevisiae, Saccharomyces bayanus/Saccharomyces pastorianus. The 5P-specific primers were designed from the ITS-1 region (between positions 150 and 182 from the 3P-SSU end) and the 3P-specific primers were located in the LSU gene (positions 560^590 from the 5P-end of this gene). These primers were tested with different collections and wild strains of these species and the results showed that the primers were capable of distinguishing between S. cerevisiae strains and S. bayanus/S. pastorianus. Not enough sequence differences were found between S. bayanus and S. pastorianus to design specific primers for these species using this region. This method offers an effective tool for a quick differentiation of the Saccharomyces strains of the most common species involved in industrial processes. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Speci¢c primer; Saccharomyces cerevisiae ; Saccharomyces bayanus ; Saccharomyces pastorianus 1. Introduction The taxonomy of the genus Saccharomyces has undergone signi¢cant changes in the last two decades. However, recent progress in molecular biology has led to the development of new techniques which have made signi¢cant advances in the taxonomy of this group. DNA relatedness and fertility experiments have established that Saccharomyces sensu stricto complex [1] comprises four sibling species: Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces pastorianus and Saccharomyces paradoxus [2,3]. These species include the most important strains in yeast-based industries, such as brewing and winemaking. Traditionally, their identi¢cation and characterisation have been based on morphological traits and, especially, on physiological properties [4,5]. However, these characteristics tend to be in£uenced by cultural conditions and can give ambiguous results [6]. By contrast, molecular bi- ology techniques are an alternative to the traditional methods of identi¢cation and characterisation of yeasts and an important tool in solving industrial problems. These techniques include: DNA^DNA reassociation [2,3], chromosomal DNA pro¢les [6,7] mitochondrial DNA RFLPs [8], rDNA restriction analysis of the internal transcribed spacers and 5.8S rDNA [9], rDNA sequencing [10]. However, some of these methods are sophisticated and they cannot be easily employed in industrial laboratories. For this reason, we have developed a rapid and reliable method to di¡erentiate species within the Saccharomyces sensu stricto group. This method is based on the design of species-speci¢c primers to distinguish S. cerevisiae from the S. bayanus and S. pastorianus species. 2. Materials and methods 2.1. Yeasts strains * Corresponding author. E-mail : jfcl@astor.urv.es In this study we used collection strains of S. cerevisiae, S. bayanus and S. pastorianus and 15 wild strains isolated from di¡erent wine fermentations, previously identi¢ed as S. cerevisiae [11] (Table 1). 0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 4 5 8 - 4 256 S. Josepa et al. / FEMS Microbiology Letters 193 (2000) 255^259 Table 1 Collection strains of Saccharomyces used in the study Species Origin Other designations S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. CECT 1942T CECT 1941T CECT 1969 CECT 1991 CECT 1940NT CECT 1970 CECT 11037 FMR Ia FMR IIa FMR IIIa FMR IVa FMR Va FMR VIa FMR VIIa FMR VIIIa FMR IXa FMR Xa FMR XIa FMR XIIa FMR XIIIa FMR XIVa FMR XVa ATCC 18824, CBS 380 ATCC 76514, DSMZ 70411 ATCC 12752, CBS 1503 ATCC 76529, cerevisiae bayanus bayanus bayanus pastorianus pastorianus pastorianus cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae cerevisiae CBS 1171 2.3. PCR conditions CBS 395 CBS 1538 CBS 1513 Each strain was analysed with both pairs of primers (SB1/SB2 and SC1/SC2). The thermal cycling parameters were an initial denaturation at 94³C for 5 min and 30 s for subsequent cycles, primer annealing at 50³C for 1 min and primer extension at 72³C for 1 min. There were 30 cycles followed by a ¢nal extension at 72³C for 7 min. The ampli¢ed products were electrophoresed on 1.4% Multipurpose agarose gel (Boehringer) and stained with ethidium bromide. A 100-bp DNA ladder marker (Gibco BRL) was used as the size standard. 3. Results and discussion ATCC, American Type Culture Collection, Rockville, MD, USA. CBS, Centraalbureau voor Schimmelcultures, Delft, The Netherlands. CECT, Spanish Type Culture Collection, University of Valencia, Valencia, Spain. DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany. FMR, Faculty of Medicine Reus, Tarragona, Spain. T : Type strain. NT : Neotype strain. a Isolated from spontaneous fermentation in Porrera (Spain) in 1995. 2.2. Design of primers The di¡erent S. cerevisiae, S. bayanus and S. pastorianus rDNA sequences used were obtained from the EMBL (Table 2) and aligned with the Clustal W, version 1.5 of multiple sequence alignment computer program [12]. From these alignments, the S. cerevisiae (SC1 and SC2)- and S. bayanus/S. pastorianus (SB1 and SB2)-speci¢c primers were designed (Fig. 1). The respective sequence of these primers are: SC1: SC2: SB1: SB2: The expected PCR results and the thermal conditions were checked with the amplify version 1.2 program. 5P-AACGGTGAGAGATTTCTGTGC-3P 5P-AGCTGGCAGTATTCCCACAG-3P 5P-GCTATTCCAAACAGTGAGACT-3P 5P-CAGTTGGCAGTATTCCCACTA-3P The small (18S) and large (25S) rRNA sequences from the species of the Saccharomyces sensu stricto display high identity. Kurtzman and Robnett [10] reported that only 12 nucleotide sites were found to be variable when 900 nucleotide sequences from these species were compared. Furthermore, the 900-base sequences from S. pastorianus and S. bayanus were identical. The alignment of the rRNA sequences of S. cerevisiae, S. bayanus and S. pastorianus has us allowed to design species-speci¢c primers for S. cerevisiae and S. bayanus/S. pastorianus. We were not able to ¢nd enough nucleotide variation to design speci¢c primers for these latter species. Despite the postulated hybrid origin of S. pastorianus from the cross between S. cerevisiae and S. bayanus, the di¤culty in di¡erentiating this species from S. bayanus on the basis of other molecular markers [8] and phenotypic characters [13] has been widely reported. ITS regions are characterised by a lesser degree of evolutionary conservation than 18S and 25S rRNA genes and they exhibit far greater interspeci¢c di¡erences [14]. As expected, the multiple sequence alignments showed a higher level of nucleotide variation in this region than 18S gene (SSU) and the 5P-primers were designed based on the sequences ranging from 152 to 172 (SB1) and 161 to 181 Table 2 Sequence data of the strains used in the primers design Saccharomyces species Abbreviation in Fig. 1 Isolate number DNA fragment Size (bp) GenBank accession number S. S. S. S. S. S. S-cere-1 S-cere-2 S-cere-3 S-past-1 S-baya-1 S-baya-2 IFO10217 CBS 1171 clone py1ra3 IFO1167 IFO1127 IFO1127 18Sp*, ITS-1, 5.8S, ITS-2, 28Sp 28Sp 18Sp, ITS-1, 5.8S, ITS-2, 28S 18Sp, ITS-1, 5.8S, ITS-2, 28Sp 18Sp, ITS-1, 5.8S, ITS-2, 28Sp 28Sp 840 319 3911 827 791 132 D89886 L20827 J01355 D89889 D89887 D83573 cerevisiae cerevisiae cerevisiae pastorianus bayanus bayanus p*: Partial sequence. S. Josepa et al. / FEMS Microbiology Letters 193 (2000) 255^259 257 Fig. 1. Diagrammatic representation of position of the rDNA primers used for PCR ampli¢cation. (SC1) of the ITS-1 spacer. However, ITS-2 alignment did not reveal enough di¡erences to design the 3P-primers based on these sequences. These primers were selected from positions 562^582 (SC2) and 564^585 (SB2) of the 25S gene (LSU). The use of SB1/SB2 primers produced an ampli¢cation product of approximately 1170 bp in the S. bayanus and S. pastorianus reference strains but the S. cerevisiae type strain (CECT 1942) was never ampli¢ed (Fig. 2). On the other hand, the PCR process with the SC1/SC2 primers yielded an ampli¢cation product of the same size in the type strain of S. cerevisiae only. Non-speci¢c ampli¢cation occasionally appears in S. bayanus or S. pastorianus strains with the S. cerevisiae-speci¢c primers. This nonspeci¢c ampli¢cation, when it occurred, showed a constant pattern of ¢ve bands ranging from 360 to 1290 bp, and would not be confused with the speci¢c product obtained for S. cerevisiae. This unexpected ampli¢cation disappeared when the DNA concentration was adjusted to 1^ 10 ng in the PCR mixture. Recently, Sabatë et al. [11] studied the dynamics of the natural Saccharomyces strains present in wine fermentation. All the strains isolated in that work were identi¢ed as S. cerevisiae on the basis of the mtDNA restriction patterns [8]. In order to check the e¤ciency of the species-speci¢c primers, 15 of these strains were tested. All of the isolates yielded an ampli¢cation product of 1170 bp with SC1/SC2 primers and no amplicon was detected with SB1/SB2. The utility of this technique for the identi¢cation of natural Saccharomyces isolates, was therefore demonstrated. The availability of speci¢c primers is a useful tool for rapid identi¢cation of species of the genus Saccharomyces that could not be di¡erentiated on the basis of phenotypic characteristics [15]. Moreover, our method allowed processing of the ampli¢cation reaction without DNA extraction. An aliquot of a growth plate colony was directly added to the PCR mixture and the same results were obtained in 3 h. However, it should be taken into account, as mentioned above, that the lack of DNA quanti¢cation sometimes yielded unspeci¢c ampli¢cations. Therefore the cellular concentration used for the process should be tested. Unambiguous identi¢cation of natural and commercial yeast has always been a concern for the industry. We should keep in mind that, generally, industry cannot arrange sophisticated techniques for routine work. We have provided an easy and quick technique to employ in these laboratories. Acknowledgements This work was supported by Grant PM95-0160 from CICYT (Ministerio de Educaciön y Ciencia) and the Fundaciö Cie©ncia i Salut, Reus, Spain. 258 S. Josepa et al. / FEMS Microbiology Letters 193 (2000) 255^259 Fig. 2. PCR ampli¢cation of the studied strains. A: SC1/SC2 ampli¢cations. B: SB1/SB2 ampli¢cations. The selected molecular-size marker (100-bp DNA ladder) is indicated on the left and right. References [1] Van der Walt, J.P. (1970) Saccharomyces (Meyen) emend. Reess. 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