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
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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.
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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.
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