ELSEVIER
FEMS Microbiology Letters 138 (I 996) 239-244
Development of a polymerase chain reaction/restriction
fragment
length polymorphism method for Saccharomyces cerevisiae and
Saccharomyces bayanus identification in enology
Isabelle Masneuf a3*, Michel Aigle ‘, Denis Dubourdieu
a
d Fuculte’ d’CFnologie de Bordeaux. 351, Cours de la Libe’ration, 33400 Talence, France
’ IBGC, I, me Camille Saint Sai;ns. 33077 Bordeaux, France
Received 23 February 1996; revised I9 March 1996; accepted I9 March 1996
Abstract
S. bayanus and S. paradoxus,
first identified by
Several yeast strains of the species Saccharomyces
cerecisiae,
hybridization
experiments
and measurements
of DNA/DNA homology, were characterized
using polymerase chain
reaction/restriction
fragment length polymorphism (PCR/RFLP)
analysis of the MET2 gene. There was no exception to the
agreement between this method and classical genetic analyses for any of the strains examined, so PCR/RFLP of the MET2
gene is a reliable and fast technique for delimiting S. cerwisiae and S. bayanus. Enological strains classified as S. bayanus,
S. checalieri, and S. capensis gave S. cerevisiae restriction patterns, whereas most S. uc~arum strains belong to S. hayanus.
Enologists should no longer use the name of S. bayanus for S. cerer,isiae Gal strains, and should consider S. bayanus as a
distinct species.
Keywords: Molecular taxonomy; Saccharom.vces cerel,isiae; Saccharomyces
bayanus; PCR/RFLP;
charomyces
1. Introduction
Electrophoretic
cerecisiae
strains, are at present used to
between four species of the genus Saccharomyces
sensu strict0 [6,7]: S. cereoisiae,
S.
bayanus, S. paradoxus
and S. pastorianus. Until
now, these four species have been differentiated by
hybridization
experiments
and measurements
of
DNA/DNA
homology [8-l I].
Wine yeasts belong to a few species and physiological races. Enologists usually designate them as
varieties of S. cerecisiae,
i.e. S. cerecisiae
var.
cerecisiae,
var. bayanus, var. ucarum, var. chevalieri, var. cupensis, etc., differentiated by ferrnentation tests of various sugars, although Yarrow [12]
considered all these yeasts to be synonyms of S.
cerecisiae. However, this designation is inconsistent
discriminate
For the last 10 years, genetic characterization
of
wine yeast strains has progressed by applying different molecular
biology techniques:
mitochondrial
DNA restriction
fragment
length polymorphism
(RFLP) analysis [l,3], separation of yeast chromosomes by pulsed-field electrophoresis
13,181. DNA
fingerprinting
[2,5] and polymerase chain reaction
@CR) [4,51.
These methods, first applied to differentiate Sac-
* Corresponding
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with a more recent classification [ 131. which distinguished 10 species in the genus S~~~c~hcrrom~c~rs,
including
S. cerel~isiur and S. ba~urtlrs. Thus. S.
baymms is used by enologists to designate a physiological race of S. crrrl~isiue. whereas taxonomists
now consider it a separate species.
Hybridization
experiments [l4] show that most
wine yeast strains which ferment melibiose (Mel ’ )
belong to S. buyznus, although some Mel’ strains
must be included in S. cerer,isiur. Moreover. according to Naumov and his colleagues. S. buyrllrs displayed a species-specific chromosomal pattern which
could be easily distinguished from that of S. wrc~~isiae [ 141.
In a recent study of the phylogenetic origin of S.
carlsbergensis. the use of PCR/RFLP
of the MET2
gene was proposed for differentiating
S. crrer,isiar
from S. buyanus [I s].
In the present study. we intended to verify the
concordance
between
hybridization
tests and
PCR/RFLP
analysis of MET2 gene for delimiting
S. cem~isiue and S. bqanu.s species. in order to help
characterize a large number of wine yeast strains.
2. Materials and methods
2. I. Yeust struins
Yeast strains from different culture collections are
listed in Table I (CBS: Centraalbureau voor Schimmelcultures, Delft, The Netherlands: VKM: National
Collection
of Microorganisms.
Moscow. Russia:
MUCL: Mycotheque de I’Universite
de Louvain:
DBVPG: culture collection at Dipartimento di Biologia Vegetale, Universit’a di Perugia, Italy). Yeast
strains from CLIB collection (Collection de Levures
d’IntCr&t Biotechnologique)
VKM Y-502. VKM Y1146,581,SCU
ll,SCU74.SCU
13.L 19.L99.L
490, DBVPG 1642. DBVPG 1643. DBVPG 1689. L
579. L 1425 are monosporic cultures of S. cvrec~isiue
and S. buyanu.s [14]. Industrial or wild yeasts come
from the collection of the Faculte d’(Enologie de
Bordeaux.
2.2. Primers
Synthetic oligonucleotide
primers for MET2 am
plification were defined by Hansen and Kielland-
Table
I
Reference htrains of Soc,c,h~i,v,,lf?t,~.\ apeciea (‘I.: type strain)
Speck
s.
c‘err!~i.Ykf,
Strain designation
CBS 5287 = VKM
MUCL
Y-502
2783 I
.s. ho~wrut
VKM
s. /‘c”‘/tlow.c
DBVPC
6466
DBVPG
6307
DBVPG
6389
Y-I 146
DBVPG
6490
DBVPG
649 I
DBVPG
6493
DBVPG
6494
s. //CdiC~U.S
CBS 459 r
s. drrlu/irri
CBS 500 T
s. uipw7.\ic
MUCL
27830
Brdndt [ 151. The sequences of primer I and 2 are
T-CGAAAACGCTCCAAGAGCTGG
and S-GACCACGATATGCACCAGGCAG
respectively.
The amplification
reaction was carried out on
entire yeast cells, after cultivating
them on solid
YPG medium (20 g/l L-glucose, IO g/I bactopeptone. IO g/l yeast extract, 20 g/l agar) for 72 h until
stationary phase [ 161. After two successive washes,
cells were maintained at 95°C for IO min.
Amplification
reactions were performed with a
Perkin Elmer DNA thermal Thermocycler 480, under
the following conditions: a 100 ~1 reaction mixture
was prepared with 2 units of APPLIGENE (Strasbourg, France) Taq DNA polymerase, IO ~1 APPLIGENE IO X C buffer (MgCI? I .S mM), 100 pmol of
each primer. 200 PM of each dNTP and about
2-4 X lOh cells. The reaction was cycled 30 times
using the following program: 95°C for 30 s to denature the DNA, 60°C for 30 s for the annealing cycles
and 72°C for 2 min for the extension reaction.
PCR products were precipitated and aliquot samples were restriction digested. Amplified fragments
and restriction fragments were analyzed by electrophoresis on a I .8% agarose gel in 1 X TBE buffer
(0.09 M Tris-borate, 0.002 M EDTA), visualized
after ethidium bromide staining by short UV (524
nm) and photographed
with Polaroid 665 film.
Boehringer Mannheim DNA molecular mass marker
VIII was used.
I. Masneufer al./ FEMS Microbiology Letters 138 (1996) 239-244
2.4.
CHEF
(contour
clamped
homogeneous
electric
Fig.
PstI
VKM
(lane
lb respectively corresponding
to EcoRI and
cleavage. The reference strain S. cereuisiae
Y-502 (lane 8) and S. bayanus VKM Y-l 146
9) and seven wine Mel+ yeasts named S.
cerecisiae
var. uuarum (lanes l-7) were analyzed.
Among these strains, five were previously identified
as S. bayanus (lanes l-5) and two as S. cerevisiae
(lanes 6 and 7) by hybridization
tests [14]. For all
four restriction enzymes used, the restriction profiles
of lanes l-5 were identical to that of lane 9 (reference strain S. bayanus), and those of lanes 6 and 7
to that of lane 8 (reference strain S. cerecisiae).
Additionally, we verified that EcoRI cleaves the S.
cerevisiae MET2 fragment (two bands of 219 bp and
369 bp) and does not cleave the S. bayanus MET2
fragment. The behavior of PstI is strictly contrary,
since PstI cleaves the S. bayanus MET2 fragment
but not that of S. cereuisiae. A Mae111 restriction
pattern of two bands (246 bp and 334 bp) is obtained
for S. cerevisiae strains identical to that of reference
strain S. cerevisiae and a Mae111 cleavage pattern of
three bands (390 bp, 130 bp and 60 bp) for S.
bayanus strains identical to that of reference strain S.
bayanus (data not shown). Finally, NcoI cleaves the
MET2 fragment of S. cerevisiae and reference strain
(two bands 466 bp and 114 bp) but does not cleave
the MET2 fragment of S. buyanus and the reference
strain (data not shown). Complete comparison between the results obtained by hybridization
experi-
field) gel electrophoresis
Chromosomal DNA was prepared in agarose plugs
[ 171 and separated on a 0.8% agarose gel in 1 X TBE,
at 165 V, temperature
lo”C, using the following
program [ 181:
Switch
Switch
12.5 h
16.5 h
40 to 90 s
80to 120s
The gel was stained with ethidium
photographed with Polaroid 665 film.
bromide
241
and
3. Results
Hansen and Kielland-Brandt
[ 153 have studied the
following
strains by PCR/RFLP:
S. cerevisiae
S288C, S. bayanus CBS 380 and S. uuarum CBS
395. We have extended the PCR/RFLP
analysis of
the MET2 gene to 12 Mel+ strains (ex. ucarum)
which had already been identified as S. cerecisiae
and S. bayanus by hybridization
experiments
by
Naumov et al. [14].
The enzyme PstI was associated with EcoRI,
NcoI and MaeIII, because while there is a PstI site
in the MET2 sequence of S. bayanus, there is none
in the MET2 sequence of S. cereuisiae.
Some results obtained are shown in Fig. la and
tG92pb
+
t
404 pb
242 pb
Fig. 1. MET.2 DNA from Mel+ Saccharomyces wine yeast strains, identified by Naumov et al. [14]. Digestion by EcoRl (a) and PsrI (b).
Lane I: S. bayanus SCU 11; lane 2: S. bayanus SCU 13; lane 3: S. bayanus SCU 73; lane 4: S. bayanus L 19; lane 5: S. ba.yanus L490;
lane 6: S. cerecisiae L 579; lane 7: S. cerevisiae L 1425; lane 8: S. ceredsiae VKM Y-502; lane 9: S. bayanus VKM Y -1146; M:
molecular mass marker.
ments [14] and PCR/RFLP
of the MET2 gene for
the strains analyzed are listed in Table 2. The concordance between these two methods for S. cerec,isiae and S. bayanu.s species delimitation do not
have any exceptions for the 14 strains analyzed.
PCR/RFLP
of the MET2 gene has been applied
to strains identified as S. paradoxus by DNA/DNA
hybridization
experiments [ 191 and electrophoretic
karyotyping [20]. In the absence of data on the S.
paradoxus MET2 sequence, the same restriction enzymes were tested for these analyses. Results obtained for four S. paradoxus strains are shown in
Fig. 2a and Fig. 2b, corresponding to the EcoRI and
Mae111 cleavage, respectively.
For each of these
figures, the restriction patterns of the reference strains
S. bayanus (lane 5) and S. cerecisiae (lane 6) are
shown. Although of the same size as those of S.
cerecisiae and S. bayanus. the S. paradoxus amplified MET2 fragment differs in that it is not cleaved
by EcoRI or PstI (data not shown). The restriction
by Mae111 gives a two band pattern (334 bp and
about 220 bp) (Fig. 2b). Lastly, the amplified MET2
gene is not cleaved by NcnI (results not shown). The
same results were obtained for all the S. paradoxus
strains studied (Table I ).
By the same methods, we analyzed 17 industrial
dry yeast strains, designated as S. cerelisiae, four
fermenting
galactose (Gal+) (VLI; VL3c; 7 I B;
WET 136) and I3 not fermenting galactose (Gall )
(VL3a; FIO: KD; B0213; CH 158; N 96; Vin 13:
QA23; IOC; SBI; 016: CIVC 8130: DVIO). The
usual enological designations
for those two cate1
2
3
4
5
6
Table 2
Specica delimitation of S. c~rrr~~i.sitrr/S. hqurw
by hybridizing
and PCR/RFLP
of the MET2 gene tests (R: reference strain)
Strain
Hybridization
CLIB 2 19: VKM Y-502 R
GLIB 2 18: VKM Y- 1 146 R
CLIBIOI: SCU I I
CLIB 103: SCU 73
GLIB 102: SCU I?
CLIB 108: L I9
CLIB I IO: L 490
GLIB 94: L 579
CLIB 95: L 1425
S.
S.
s.
s.
s.
s.
s.
S.
S.
cvrwisiar
ha~unus
htr~trnu.\
htr~trr1u.s
hqww.s
htr!unu.s
hqcmus
test
PCR-RFLP
S.
S.
S.
s.
s.
s.
s.
test
crrerisiar
hrryatw
tqwul.\
hawlilu.~
hu,wrnu.s
htr~clnu.s
htrwnus
crrec~isitre
S. crrwisirw
crrrf,isiae
S. cem~isitrr
The smx results were obtained for the S. hcrwnus strains 5x1,
CLIB 109: L 99. CLIB 113: DBVP 1642, CLIB 114: DBVPG
1633 and CLIB I 15: DBVPG 1689.
gories are S. cerelisiae
var. cerecisiae (Gal’) and
S. cerecisiae var. bayanus (Gal-). Results for five
strains are given in Fig. 3a,b. All the strains, Gal+ or
Gal-, have an EcoRI and PstI restriction pattern of
the amplified MET2 gene typical of S. cerecisiae.
We also analyzed many wild wine yeast strains.
isolated from spontaneous
fermentations:
8 Gal ’
strains, 45 Galstrains, 2.5 Mel’ strains, 2 S.
cerecisiae
var. capensis, Gall
and Mall
strains
(which do not ferment maltose), 3 S. cerecisiae var.
cheralieri Gal + and Mal
strains and one S. italicus
strain (Table 1). Restriction digests of the MET2
PCR/amplified
DNA produced by EcoRI, Mae111
and PstI are typical of S. cererisiae. On the other
hand. most Mel+ strains. named S. cerecisiae var.
w’arum (22 strains/25),
gave a restriction digest
M
-692
pb
4-404
pb
242 pb
Fig. 2. MET2 DNA restriction analysis of S. pwtiduxm
strains with the endonucleases
Ec‘coRI(a) and Mae111 (b). Lanes l-4:
6466, 6489, 6490, 6493; lane 5: S. huwnus VKM Y-l 146. lane 6: S. crrwisiue
VKM Y-502; M: molecular mass marker,
S. pcwudo.rus
I. Masneuj’et al. / FEMS Microbiology
Letters 138 (1996) 239-244
243
(b)
(4
Fig. 3. MER DNA restriction analysis of industrial wine yeast strains with restriction endonucleases
EcoRI (a) and PHI (b). Lane 1: FlO;
lane 2: KD: lane 3: B0213; lane 4: DVIO; lane 5: QA23; lane 6: S. buyanus VKM Y-l 146; lane 7: S. cerevisiae VKM Y-502; M:
molecular mass marker.
typical of S. buyanus species; three strains, however,
must be included in the species S. cerecisiae (data
not shown).
In previous works, many authors have shown that
electrophoretic karyotyping analyses can be used to
differentiate species belonging to the genus Succharomyces sensu strict0 [20], especially S. cerecisiae
and S. bayanus species [ 14,21,22]. Karyotype analyses of wine yeast strains studied in the present work
corroborate results obtained by PCR/RFLP
of the
MET2 gene. Chromosomal
DNA patterns of S.
buyanus strains previously identified by PCR/RFLP
of the MET2 gene displayed band patterns similar to
1
2
3
4
5
6
7
8
9
10
Fig. 4. Karyotypes of S. cerevisiae and S. buyanus wild wine
yeast strains. Lanes 1, 3, 4, 5, 7, 8, 9: S. cereL,isae; lanes 2, 6, IO:
S. bapznus.
one another
cereuisiue
but different from band patterns
strains (Fig. 4).
of S.
4. Discussion and conclusion
Hybridization
experiments and DNA/DNA
hybridization,
which are difficult to implement
and
time consuming, are hardly relevant to fast identification of wine yeast species. On the other hand,
PCR/RFLP
of the MET2 gene technique is useful
and fast. Direct amplification on entire cells and the
use of PstI enzyme have enabled us to improve this
technique
for delimiting
S. cerevisiue
and S.
buyunus. The findings obtained are perfectly consistent with the results of hybridization
experiments.
They are also in concordance
with differentiation
based on karyotype analysis, but this method takes
much longer and its results are tedious to interpret
because of the intraspecific polymorphism
of wine
yeast karyotypes [3,18].
The first results show that S. paradoxus species
can be identified by PCR/RFLP
of the MET2 gene.
Nevertheless, the MET2 gene sequence of S. purudoxus species has to be established
in order to
improve the restriction enzyme choice for the RFLP
analyses.
From a taxonomic point of view, this method
gives some new information for identifying enological yeast species. After analyzing a large number of
industrial and wild strains, we show that enological
strains classified as S. cereuisiue var. buyunus, S.
cereuisiae var. cupensis, S. cerevisiue var. chevulieri are in fact S. cerevisiue that have probably lost
I. M~istwrfrt
244
<I/. / FEMS Miuohrolo~~
certain characteristics: the ability to ferment galactose in the case of S. cerecisiue var. buyunus and S.
cerersisiae var. cqwnsis, maltose in the case of S.
var.
cerei,isiue
var. cupensis and S. cerecisiae
cher’alieri. However, the majority of wine Mel’
yeast strains (ex. S. cerevisiae var. ucarum) are S.
ba.wznu.s, in accordance with Naumov’s and Kielland-Brandt’s findings and the Mel ’ phenotype can
no longer be used to differentiate S. cereuisiae from
S. bayanus. To avoid confusion in enological terminology, the name S. bqunus
should no longer be
used to refer to S. cerecisiae Galstrains, but
should be applied only to strains belonging to the
distinct species S. bagnus.
Letter.\ 138 (IYY6I
239-244
~oIII\T’~.$ sensu stricto complex according to mitochondrial
DNA patterns. Int. J. Syst. Bacterial. 44, 708-713.
[8] Vaughan-Martini,
A. and Kurtzman,
C.P. (1985)
Deoxyri-
bonucleic acid relatedness among species of the genus .Sr/c,-
I I.
chtrrorttvces sensu stricto. Int. I. Syst. Bacterial. 35. 508-5
[9] Vaughan-Martini,
A. and Martini,
A. (1987)
delimited species of Saccharom~crs
Three newly
sensu stricto. Antonic
Van Leeuwenhoek 53, 77-84.
[IO] Naumov,
C.I.
(1987)
Genetic
basis for classification
and
identification of the ascomycetous yeasts. Symposium on the
Expanding Realm of Yeast-like Fungi, Elsevier, Amsterdam.
pp. 469-475.
[I
I] Kaneko, Y. and Banno. I. (199 I) Reexamination
rornvces havunuc strains by DNA-DNA
of Strc~chtr-
hybridization
and
electrophoretic karyotyping. I.F.O. Res. Commun. 15, 30-41.
[ I21
Yarrow.
( 1984)
D.
Genus 22.
Rij,
Strcchnrom,wrs
N.J.W.
(Ed.),
The
Meyen
ex
Yeasts.
A
Reess. In
Kreger-van
Taxonomic
Study. 3rd edn., Elsevier, Amsterdam, pp. 379-
395.
Acknowledgements
[I31 Barnett,
J.A..
Payne,
R.W.
Yeasts: Characteristics
We wish to thank R.K. Mortimer (Berkeley),
LALLEMAND Inc. (Canada), H.V. Nguyen (GLIB,
Paris) and G.I. Naumov (Moscow) for strains and
J.C. Gandar (IBGC, CNRS, Bordeaux) for the synthesis of oligonucleotide
primers. This research was
in part supported by SARCO (Bordeaux, France).
and Yarrow.
D.
and Identification.
(1990)
The
2nd edn.. Cam-
bridge University Press, Cambridge.
[I41 Naumov, G.I.,
Naumova.
netic and karyotypic
haycmu\
E. and Caillardin,
identification
C. (1993)
Ge-
of wine Srtc~churon~wes
isolated in France and Italy. Syst. Appl. Microbial.
16. 274-279.
[I51 Hansen,
J. and Kielland-Brandt.
c~urkhergensis
M.C. (1994)
Strccharomywr
contains two functional ME72
to homologues from S. ceret,isioe
alleles similar
and 5. monctcerwis. Gene
140, 33-40.
[t61
References
Masneuf. 1. and Dubourdieu, D. (1994) Comparaison de deux
techniques d’identification
des souches de levures de vinifi-
cation has&es sur le polymorphisme
[]I
de I’ADN
gtnomique:
Dubourdieu, D.. Sokol. A.. Zucca, J.. Thalouarn, A.. Dattec.
reaction de polymerisation en chaine (PCR)
A. and Aigle. M. (1987) Identification de souches de levures
caryotypes (Clectrophortse en champ pulse). J. Int. Sci. VI-
isolees de vins par l’analyse de leurs ADN
mitochondrial.
Conn. Vigne Vin 21, 267-278.
gnes Vin 28, l53-
Identification
par la caracterisation
rapid method for preparing yeast chromosomes for pulsed
de souchea de levures anologiques
de leur
ADN
a I’aide
de sondes
field electrophoresis. Nucleic Acids Res. 15, 6749.
[I81 Frezier,
moleculaires specifiques. Am. J. Enol. Vitic. 40, 309-315.
mal DNA patterns and mitochondrial DNA polymorphism a\
for
ron*lws
identification
of
cenological
cerec~isiur. Appl. Microbial.
strains of
Biotechnol.
V.
and Dubourdieu,
strain Saccharomwes
[31 Vezinhet, F., Blondin, B. and Hallet, J.N. (1990) Chromosotool
Swchtr-
[a
L. (1993) Identification of yeast strains using the polymerase
Cardinalli,
Korhola,
romyces cererisiae
3, 413-426.
G. (1993)
[71 Guillamon,
(I 994)
Antonie van Leeuwenhoek 43, 145-156.
J.M.,
Barrio,
E.,
Huetta,
T.
and Querol,
parudoxu.s
cmh.
sensu
homologies. Syst.
A. (1994)
Electrophoretic
kary-
Int. J. Syst. Bacterial. 44, 791-797.
[211 Naumov, G.I., Naumova, E., Lantto, R.A., Louis, E.J. and
printing J. Basic Microbial.
Electrophoretic karyotyping as a taxonomic tool in the genus
Saccharomyces
12, 179-182
G. and Martini,
cation and differentiation of yeasts by DNA and PCR finger-
Saccharomyces.
A. (1989)
genus Saccharomyes.
A. and Cardinali,
of yeast
otypes of authentic strains of the sensu strict0 group of the
chain reaction. J. Sci. Agric. 62, 89-94.
A., Martini.
Ecology
during spontaneous fermen-
strict0 complex based upon nDNA/nDNA
Appl. Microbial.
El Lieckfeld, E., Meyer, W. and Boer, T. (1993) Rapid identifi-
D. (1992)
nav.. a newly separated species of the Sacchuromyces
32, 568-
141 Ness, F.. LavalICe, F.. Dubourdieu, D.. Aigle, M. and Dulau.
cererisiae
tation in a Bordeaux winery. Am. J. Enol. Vitic. 43, 375-380.
[I91 Vaughan-Martini,
571.
[61 Vaughan-Martini,
160.
t171 Bellis, M., Pages, M. and Roizes, G. (1987) A simple and
El Degre, R., Thomas, D.Y., Mailhot, K.. Morin, A. and Dubord.
C. (1989)
et analyae des
M.
(1992)
Genetic
homology
between
Sacch~~-
and its sibling species .S. paradoxus
and
S. hayanus: electrophoretic karyotypes. Yeast 8, 599-612.
WI
Kishimoto, M. and Goto, S. (1995) Growth temperatures and
electrophoretic karyotyping as tools for practical discrimina-
A.
Rapid characterisation of four species of the Saccho-
tion of Srrcchorom\ces
hqwnus
vi~ine. J. Gen. Appl. Microbial.
and Saccharomycrs
41. 239-247.
cere-