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Characterizationof natural hybridsofSaccharomyces cerevisiae andSaccharomycesbayanus var. uvarumChristine Le Jeune1, Marc Lollier1, Catherine Demuyter1, Claude Erny1, Jean-Luc Legras2, Michel Aigle3 &Isabelle Masneuf-Pomarede4
1Laboratoire Vigne Biotechnologie et Environnement, Universite de Haute-Alsace, Herrlisheim, Colmar, France; 2Institut national de la Recherche
Agronomique (INRA) UMR SVQV Œnologie, Herrlisheim, Colmar, France; 3UMR 5122, Universite de Lyon 1, Villeurbanne, France; and 4ENITA Bordeaux,
Gradignan, France
Correspondence: Le Jeune Christine,
Laboratoire Vigne Biotechnologie et
Environnement, Universite de Haute-Alsace,
32 rue de Herrlisheim, BP 568, 68 008 Colmar
Cedex, France. Tel.: 1 00 33 3 89 30 31 36;
fax: 1 00 33 3 89 30 31 36;
e-mail: [email protected]
Received 13 April 2006; revised 7 December
2006; accepted 7 December 2006.
First published online 15 February 2007.
DOI:10.1111/j.1567-1364.2007.00207.x
Editor: Cletus Kurtzman
Keywords
Saccharomyces cerevisiae ; Saccharomyces
bayanus var. uvarum ; hybrid cells.
Abstract
Nine yeast strains were isolated from spontaneous fermentations in the Alsace area
of France, during the 1997, 1998 and 1999 grape harvests. Strains were character-
ized by pulsed-field gel electrophoresis, PCR–restriction fragment length poly-
morphism (RFLP) of the MET2 gene, d-PCR, and microsatellite patterns.
Karyotypes and MET2 fragments of the nine strains corresponded to mixed
chromosomal bands and restriction patterns for both Saccharomyces cerevisiae
and Saccharomyces bayanus var. uvarum. They also responded positively to
amplification with microsatellite primers specific to both species and were
demonstrated to be diploid. However, meiosis led to absolute nonviability of their
spores on complete medium. All the results demonstrated that the nine yeast
strains isolated were S. cerevisiae� S. bayanus var. uvarum diploid hybrids.
Moreover, microsatellite DNA analysis identified strains isolated in the same cellar
as potential parents belonging to S. bayanus var. uvarum and S. cerevisiae.
Introduction
The Saccharomyces clade is composed of seven species:
S. bayanus, S. cariocanus, S. cerevisiae, S. kudriavzevii,
S. mikatae, S. paradoxus, and S. pastorianus (Kurtzman,
2003). Among these species, S. cerevisiae and S. bayanus are
known for their role in alcoholic fermentation. Saccharo-
myces bayanus has the following specific properties: cryoto-
lerance (Kishimoto et al., 1993; Massoutier et al., 1998;
Giudici et al., 1999; Rainieri et al., 1999); a typical fermenta-
tion profile in grape must that is clearly different from that
of S. cerevisiae; production of smaller amounts of acetic acid
and ethanol, but higher amounts of glycerol and succinic
acid; synthesis, but not degradation, of malic acid (Kishi-
moto et al., 1993; Bertolini et al., 1996); and significant
production of volatile fermentative compounds, such as
phenylethanol and its acetate (Masneuf et al., 1998). These
factors result in considerable organoleptic variations, de-
pending on the species present.
Saccharomyces bayanus strains were divided into two sub-
groups using molecular variability analysis (Nguyen & Gail-
lardin, 1997; Nguyen et al., 2000). The first contains strains
with homogeneous phenotypic and genotypic characteristics
(Rainieri et al., 1999) similar to those of the former S. uvarum
(type strain CBS 395). Strains in this subgroup exhibit an
electrophoretic karyotype characterized by the presence of two
bands of size 225 and 365 kb (Giudici et al., 1999) and a
fermentation profile resulting in characteristic amounts of
glycerol, succinic acid, and acetic acid (Castellari et al., 1994).
These strains are frequently found in the winemaking process.
The second group contains many more heterogeneous strains
and the CBS 380 type strain. Strain CBS 380 behaves
physiologically as an intermediate between the first subgroup
and S. cerevisiae (Rainieri et al., 1999). As hybrids between the
two S. bayanus subgroups are semisterile, Naumov has
suggested considering these two subgroups as varieties named
S. bayanus var. bayanus and S. bayanus var. uvarum (Naumov,
2000). Nguyen et al. (2000) demonstrated that CBS 380, the
type strain of S. bayanus var. bayanus, was, in fact, a natural
interspecific hybrid of S. cerevisiae and S. bayanus var. uvarum
with a composite genome. Recent studies have investigated the
DNA introgression between S. cerevisiae and S. bayanus
(Naumova et al., 2005a, b).
Saccharomyces bayanus is not the first hybrid species to be
described. Saccharomyces pastorianus (syn. S. carlsbergensis)
was shown to be a partial hybrid produced by a mating event
FEMS Yeast Res 7 (2007) 540–549c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
between baker’s yeast, S. cerevisiae, and a yeast belonging to
the S. bayanus complex (Borsting et al., 1997; Sipiczki, 2002;
Fernandez-Espinar et al., 2003). Mitochondrial DNA was
inherited only from the S. bayanus parent (Guillamon et al.,
1994; Piskur et al., 1998; Pulvirenti et al., 2000).
Natural hybrids have been isolated in diverse fermenta-
tion processes. Two native hybrids of S. cerevisiae and
S. bayanus var. uvarum have already been described: S6U,
isolated from wine, and CID1, isolated from cider (Masneuf
et al., 1998). Amplified fragment length polymorphism
analysis demonstrated that the cider yeast CID1 (de Barros
Lopes et al., 2002; Naumova et al., 2005a) was a triple hybrid
between S. cerevisiae, S. kudriavzevii, and S. bayanus var.
uvarum. The two lager hybrids, BRYC32 and NCYC 1324 (de
Barros Lopes et al., 2002), also contained nuclear DNA from
three separate species (Groth et al., 1999), two of which have
been identified as S. cerevisiae and S. pastorianus. De Barros
Lopes et al. (2002) also confirmed the results obtained by
Naumov et al. (2000), showing that S6U is an allotetraploid
hybrid. Sipiczki reviewed existing data on various hybrids
(Sipiczki, 2002). More recent work, based on molecular
genetic studies, showed that baker’s yeast and blackcurrant
isolates contained S. cerevisiae/S. bayanus var. uvarum
hybrids (Naumova et al., 2005b).
Novel combinations of genetic material and stable viable
hybrids were obtained in laboratory experiments (Marinoni
et al., 1999). In general, the less closely related the species,
the lower the frequency of zygotes. Hybrids self-propagate
through mitosis for many generations, indicating that two
genomes can coexist in the same cell and that yeasts can
combine their genetic material in nature. Marinoni et al.
(1999) emphasized that, as horizontal transfer of genetic
material occurs in nature, modern yeast species may contain
DNA of polyphyletic origin, and sequencing studies of a
single locus may therefore be misleading.
Numerous authors have shown that allodiploid hybrids
do not sporulate or produce nonfertile spores (Banno &
Kaneko, 1989; Hawthorne & Philippsen, 1994; Giudici et al.,
1998; Rainieri et al., 1998). In contrast, allotetraploid
hybrids sporulate and produce highly viable spores (Nau-
mov et al., 2000; Greig et al., 2002; Sebastiani et al., 2002).
Antunovics et al. (2005) proposed that viable spores were
produced in an allotetraploid genome, as each chromosome
had a matching partner for pairing. Therefore, characteriz-
ing the sporulation and germination capacities of hybrids is
an excellent way of determining their ploidy.
In previous work, we characterized yeast populations
present on grapes, in crush and tank, with the aim of
determining the origin of the yeasts responsible for sponta-
neous alcoholic fermentation in an Alsace winery (Demuy-
ter et al., 2004). Molecular biology techniques differentiated
the two species S. cerevisiae and S. bayanus var. uvarum.
Indeed, these two species have very different karyotypes
(Naumov et al., 1993; Vaughan-Martini et al., 1993; Kishi-
moto et al., 1994; Rainieri et al., 1999), and different
restriction patterns of the MET2 alleles (Hansen & Kiel-
land-Brandt, 1994; Masneuf et al., 1998); also, Ty1 transpo-
sons are specific to the S. cerevisiae genome but not present
in S. bayanus var. uvarum (Neuveglise et al., 2002).
We used both karyotypes and d-amplification of the
conserved sequences flanking Ty1 transposons to character-
ize the strains and, thus, determine the homogeneity of the
groups formed by the PCR technique. Three different types
of strain were identified. The first group had d-PCR
amplifications and karyotypes characteristic of S. cerevisiae,
and the second group did not show any d-amplification but
had specific S. bayanus var. uvarum karyotypes. Finally, the
third group had d-PCR amplifications and karyotypes with
an abnormal number of chromosomes (Table 1).
First, all third-group strains were characterized to deter-
mine their origin. Their karyotypes (Vezinhet et al., 1990),
PCR – restriction fragment length polymorphism (RFLP) of
the MET2 gene and d-amplification (Ness et al., 1993;
Hansen & Kielland-Brandt, 1994) and ploidy (Naumov
et al., 2000) were investigated, as well as their ability to
sporulate and germinate. This demonstrated their hybrid
nature and diversity.
Second, a possible relationship between S. cerevisiae and
S. bayanus var. uvarum strains present at the same time in
the same cellar was investigated by microsatellite analysis, as
previously described (Field & Wills, 1998; Gallego et al.,
1998; Gendrel et al., 2000; Young et al., 2000; Legras et al.,
2005) and recently adapted to S. bayanus var. uvarum
(Masneuf-Pomarede et al., 2007). This approach provides
elements for understanding the mechanisms underlying the
natural formation of these hybrids.
Materials and methods
Origin and isolation of yeast strains
The indigenous strains were isolated from a vineyard
belonging to the Rolly–Gassman estate, near Colmar, in
three consecutive years (1997–1999). Each isolate was con-
sidered to be a separate strain until it had been genetically
characterized.
Indigenous strains were isolated during spontaneous
alcoholic fermentation of sweet white wines, as previously
described (Demuyter et al., 2004). The strains used for
analysis and their origins are listed in Table 1. The S. bayanus
var. uvarum strain CBS 7001 was used as a reference for
microsatellite analysis (Masneuf-Pomarede et al., 2007), and
S. cerevisiae VKM-502 (Masneuf et al., 2002) was used as a
reference for the PCR/RFLP of the MET2 gene. The S6U2a
hybrid diploid strain and the S288Ca S. cerevisiae haploid
FEMS Yeast Res 7 (2007) 540–549 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
541Saccharomyces cerevisiae and S. bayanus var. uvarum hybrids
strain (Naumov et al., 2000) were used as references for flow
cytometry analysis.
DNA extraction
Total DNA was extracted from yeast colonies as described by
Demuyter et al. (2004). The extracted DNA was used for d,
MET2 and microsatellite amplification.
Microsatellite amplification and analysis
For the amplification of specific S. cerevisiae sequences, 11
microsatellite loci were chosen and amplified with the
corresponding primers: YKL172w, SCYOR267c, SCAAT1,
SCAAT5, YPL009c, C3, C4, C6, C8, C9, and C11 (Legras
et al., 2005). Primers corresponding to loci 1–4 were used
for amplification of specific S. bayanus var. uvarum se-
quences (Masneuf-Pomarede et al., 2007). Loci 1 and 3 are
not located in ORFs, whereas loci 2 and 4 are located in
ORFs homologous to two S. cerevisiae ORFs coding, respec-
tively, for an RNA polymerase1 enhancer-binding protein
(YBR049c), and for a putative protein of unknown function
(YKR045c). DNA microsatellite amplification and analysis
was carried out as described by the authors.
d, d0 and MET2 amplification and analysis
Primers d1 (50-CAAAATTCACCTATA/TTCTCA-30) and d2
(50-GTGGATTTTTATTCCAACA-30), based on the S. cere-
visiae-specific d sequences flanking the Ty1 retrotransposon,
were used to amplify the yeast genomic DNA (Ness et al.,
1993). Alternatively, primers d12 (50-TCAACAATG-
GAATCCCAAC-30) and d2 were used for more discrimina-
tive d0 amplification (Legras & Karst, 2003). Primers used
for amplification of the MET2 gene were 50-CGGCTCTA-
GACGAAAACGCTCCAAGAGCTGG-30 and 50-CGGCT-
CTAGAGACCACGATATGCACCAGGCAG-30 (Hansen &
Kielland-Brandt, 1994). A Perkin Elmer Gene Amp PCR
2400 was used for PCR amplification, under the following
conditions: 4 min at 97 1C for the first cycle, then 30 s at
94 1C, 1 min at 45 1C, and 2 min at 72 1C for the next 30
cycles, and 10 min at 72 1C for the last cycle, except for
MET2 amplification, when the annealing temperature was
50 1C.
A 50-mL reaction mixture was prepared with 1 U of
recombinant Taq polymerase (Invitrogen), 5 mL of Taq
polymerase 10� buffer, 200 mM each dNTP, 1mM each
primer, and 5mL of extracted DNA.
The MET2 PCR products were precipitated, and aliquots
were digested with EcoRI or PstI (Masneuf et al., 1998).
d-PCR products and MET2 restriction fragments were
analysed by electrophoresis in 1.5% agarose gel, according
to the standard procedure.
Chromosomal DNA preparation and pulsed-fieldgel electrophoresis (PFGE)
Karyotypes were obtained using the procedure described by
Demuyter et al. (2004).
Micromanipulation
Sporulation was induced by incubating cells on acetate
medium (1% CH3COONa, 0.5% KCl, 2% agar) for 2 days.
Following preliminary digestion of the ascus walls with
cytohelicase (Sigma), adjusted to 2 mg mL�1, ascospores were
isolated using a Singer MSM Manual micromanipulator.
Flow cytometry analysis
The basic procedure has been described by Paulovich &
Hartwell (1995). Yeast cells were grown in 10 mL of liquid
minimal medium under vigorous agitation. Early station-
ary-phase cells (c. 5� 108 cells mL�1) were harvested by
centrifugation, washed, and fixed in 70% ethanol at 4 1C
for 12 h. Fixed cells were washed once with 50 mM sodium
citrate (pH 7.5), and resuspended at 8� 108 cells mL�1 in
50 mM sodium citrate containing 25mL of RNAse
(10 mg mL�1). After incubation at 37 1C for 2 h, cells were
treated with 50 mL of proteinase K (20 mg mL�1) at 50 1C for
Table 1. List of yeasts strains
Strain no. Group Sources Year
First group
RP2-16� Press 1997
RP1-1� Press 1998
RP1-7� Press 1999
RC2-17 Tank 1999
RP2-2 Press 1999
RP1-17 Press 1999
RP1-5 Press 1999
Second group
RC4-5 Tank 1997
RC1-14 Tank 1998
RC2-20 Tank 1998
RP1-16 Press 1998
RP1-21 Press 1999
RC1-19 Tank 1999
RP1-8 Press 1999
Third group
RC4-87 Tank 1997
RC2-19 Tank 1998
RP1-4 Press 1999
RP2-5 Press 1999
RP2-6 Press 1999
RP2-17 Press 1999
RC1-1 Tank 1999
RC1-11 Tank 1999
RC2-12 Tank 1999
�Strain belonging to the d-PCR C7 family.
FEMS Yeast Res 7 (2007) 540–549c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
542 C. Le Jeune et al.
1 h, and 1 mL of sodium citrate (50 mM) containing
16 mg mL�1 propidium iodide was added to the preparation.
Samples were incubated in the dark at 4 1C for 12–48 h, and
analysed using a flow cytometer (Becton–Dickinson FAC-
scan analyser). Two independent samples of 10 000 cells
were analysed for each strain.
Results
Hybrid strain characterization
In a previous ecological study, we identified three different
groups of yeast strains in the same winery. The first group of
strains had d-PCR patterns and karyotypes typical of
S. cerevisiae. The second group had a karyotype specific for
S. bayanus var. uvarum but no d-amplification (Demuyter
et al., 2004). The last group of strains, labelled ‘non-
S. cerevisiae’ in our previous study, had positive d-PCR
amplification patterns (Fig. 1). The karyotypes of these
strains (Fig. 2, lane A) were much more complex than those
of the S. cerevisiae and S. bayanus var. uvarum strains already
identified in this winery (Fig. 2, lanes B and C). Nine of the
isolates fell into in this category. These isolates were
hypothesized to be natural hybrids of the two species present
in the winery.
PFGE of the nine isolates revealed a higher number of
bands than normally observed for S. cerevisiae (Fig. 2, lanes
1–9). The very high number of chromosomes comprising
the karyotypes of these isolates made profile analysis diffi-
cult. Nevertheless, it was possible to differentiate two
groups: strains RC2-19, RP1-4, RP2-17, and RC1-1 (lanes
2, 3, 6 and 7), and strains RC4-87 and RP2-6 (lanes 1 and 5).
The three other strains (lanes 4, 8, and 9) had unique
karyotypes.
Figure 3 shows the d-PCR amplification profiles for these
hybrids. Strains RC2-19, RP1-4, RP2-5, RP2-17, RC1-1,
RC1-11 and RC2-12 had the same amplification profile,
although RC1-1 seemed to be different, due to a variation in
the matrix DNA concentration. Lanes 8 and 9 (RC4-87 and
RP2-6, respectively) had two other different profiles.
PCR-RFLP analysis of the MET2 gene (Masneuf et al.,
1998) confirmed the hybrid nature of these nine isolates.
Restriction with EcoR1 (Fig. 4) resulted in profiles combin-
ing the typical patterns of S. cerevisiae and S. bayanus var.
Fig. 1. d-PCR amplification patterns obtained for the strains of Sacchar-
omyces cerevisiae (lane 1 and lanes 3–6) and for the strains of
Saccharomyces bayanus var. uvarum (lane 2) RP1-7. M: 1-kb plus DNA
ladder (Life Technology).
Chromosomespecific toS. cerevisiaekaryotypes
MMM A B C1 2 3 4 5 6 7 8 9
Fig. 2. Chromosomal patterns of the nine hybrid strains (RC4-87, RC2-
19, RP1-4, RP2-5, RP2-6, RP2-17, RC1-1, RC1-11, RC2-12, lanes 1–9
respectively) and of a control Saccharomyces cerevisiae YNN295,
denoted M. Karyotypes obtained for a strain of Saccharomyces cerevisiae
RP1-5 (lane B), for a strain of Saccharomyces bayanus var. uvarum RP1-8
(lane C), and for a hybrid strain RP1-4 (lane A).
M M M91 2 3 4 5 6 7 8
Fig. 3. d-PCR amplification of the RC2-19, RP1-4, RP2-5, RP2-17, RC1-
1, RC1-11, RC2-12, RC4-87 and RP2-6 hybrid strains, lanes 1–9
respectively. M: 1-kb plus DNA ladder (Life Technology).
FEMS Yeast Res 7 (2007) 540–549 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
543Saccharomyces cerevisiae and S. bayanus var. uvarum hybrids
uvarum. Restriction with Pst1 confirmed these results (data
not shown).
The capacity of these hybrids to sporulate was also tested.
Out of 100–150 cells of each strain, only 5.6–12.5% sporu-
lated, giving an average sporulation rate of c. 7%. Analysis of
10 complete tetrads of each of the nine hybrids showed
absolute nonviability of spores on complete medium.
Once the hybrid nature of these nine strains was con-
firmed, we investigated their genomic contents. The ploidy
of the nine isolates was determined by flow cytometry. This
technique measures the fluorescence fixed by one cell. The
ploidy of the cells tested was determined by comparison of
the DNA content per hybrid cell with those of haploid and
diploid reference cells. On comparison with the S6U2a
hybrid diploid strain (Naumov et al., 2000) and the S288Ca
S. cerevisiae haploid strain (Naumov et al., 2000), with
arbitrary values of 81 and 46, respectively, the nine hybrids
had values ranging from 75 to 85, consistent with a diploid
status (e.g. hybrid RC2-12).
Thus, our results, i.e. karyotypes, PCR-RFLP analysis of
the MET2 gene, and nonviability of spores, clearly indicated
the hybrid nature of these nine isolates. In our case, the nine
hybrids were diploid. Moreover, three different groups were
distinguished by PCR amplification, with a major group of
seven hybrids (RC2-19, RP1-4, RP2-5, RP2-17, RC1-1, RC1-
11, and RC2-12), as confirmed by d0-PCR (Legras & Karst,
2003) (data not shown). This major group was subdivided
into four subgroups by the more discriminating PFGE
(Table 2).
Hybrid strain relationships
The second part of this study concerned the origin of the
hybrids and their relationships with the S. cerevisiae and
S. bayanus var. uvarum strains isolated in the same geogra-
phical area. The d-PCR profile of the seven hybrids in the
larger group (Fig. 3) was identical to that of the C7
S. cerevisiae family, also isolated from this cellar (Demuyter
et al., 2004). The two other hybrids (RC4-87, RP2-6) did not
have the same d-PCR profile, and were thus eliminated from
that group. These two strains had two different amplifica-
tion profiles, totally unlike those of the S. cerevisiae strains
isolated in the cellar. These d-PCR results raised questions
about the relationships between the hybrids and the
S. bayanus var. uvarum and S. cerevisiae strains present in
the same cellar.
In order to answer this question, microsatellite loci
specific to S. cerevisiae and S. bayanus var. uvarum were
amplified for the hybrid strains and potential S. cerevisiae
and S. bayanus var. uvarum parents. For that purpose, we
selected 11 pairs of primers extracted from the S. cerevisiae
genome (Legras et al., 2005) and four pairs of primers
specific to S. bayanus var. uvarum (Masneuf-Pomarede
et al., 2007). Sequencing of the amplified loci was necessary
to confirm the length of the microsatellite DNA amplified
and the exact number of repetitions (n) for each motif.
Using the specific S. cerevisiae primers, we observed allele
variability among hybrids for four loci (SCYOR267c,
SCAAT1, C11, C3) tested, whereas the other seven loci were
invariant. Considering the 11 loci, the hybrids clustered into
three groups, g1 (RC1-1, RC1-11, RC2-12, RC2-19, RP1-4,
RP2-17), g2 (RP2-5), and g3 (RP2-6, RC4-87). Hybrid
RP2-5 had some common alleles with each of the other two
groups (Table 3).
Microsatellite primers specific to S. cerevisiae were also
used on six S. cerevisiae strains, three from the C7 family,
and three from other distinct d-PCR families (Table 3). We
observed two different alleles for several loci, showing that
these indigenous S. cerevisiae strains were partly hetero-
zygous. Two of the three S. cerevisiae strains in the d-PCR C7
family (RP2-16, RP1-7) were strictly identical, and were
692bp
404bp
242bp
M S c S u 1 2 3 4 5 6 7 8 9
Fig. 4. RFLP analysis of PCR-amplified MET2 gene fragments. Amplified
MET2 gene fragments were digested with EcoRI. Sc, control Sacchar-
omyces cerevisiae VKM502; Su, control Saccharomyces bayanus var.
uvarum CBS7001. Lanes 1–9: RC4-87, RC2-19, RP1-4, RP2-5, RP2-6,
RP2-17, RC1-1, RC1-11, RC2-12.
Table 2. Variability of the hybrid strain population
Hybrid
strains
Characterization methods
Resulting
groupd-PCR
S. cerevisiae
microsatellite
S. bayanus
var. uvarum
microsatellite PFGE
RC2-19 g1 g1 g1 g1 G1
RP1-4 g1 g1 g1 g1
RP2-17 g1 g1 g1 g1
RC1-1 g1 g1 g1 g1
RC2-12 g1 g1 g1 g2 G2
RC1-11 g1 g1 g2 g3 G3
RP2-5 g1 g2 g3 g4 G4
RC4-87 g2 g3 g4 g5 G5
RP2-6 g3 g3 g5 g5 G6
g1 to g5, groups of strains formed by each analysis used in the study.
G1 to G6, final groups of strains resulting from the synthesis of the four
methods.
FEMS Yeast Res 7 (2007) 540–549c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
544 C. Le Jeune et al.
considered to be two isolates of the same ‘RP2-16’ strain.
The third strain (RP1-1) only differed from the previous two
by homozygosity for the C9 locus (Table 3). The three
S. cerevisiae strains that were not from the C7 d-PCR family
(RC2-17, RP2-2, RP1-17) had very different, unique pat-
terns. The relationship between the hybrids and the
S. cerevisiae strains was investigated by comparing micro-
satellite patterns. All the alleles in the hybrids were present in
S. cerevisiae strains RP2-16 and RP1-7. However, the RP1-1
S. cerevisiae strain did not have allele 90 at locus C9. Hence,
a single S. cerevisiae strain was involved in forming the three
groups of hybrids via different recombination events, as
indicated by the different allele distribution. It is noteworthy
that only strains from the C7 family were involved in hybrid
formation, as it was not prevalent in the cellar, representing
0.5, 6.3 and 8.3% of the strains isolated from press and tank,
in 1997, 1998 and 1999, respectively.
In parallel, four pairs of specific S. bayanus var. uvarum
primers were tested on the hybrids and S. bayanus var.
uvarum strains isolated in the same cellar. The loci tested
showed different degrees of allele variability among the
hybrids, with two to four allele forms. Analysis of the four
microsatellite loci identified one major group, including
strains RC2-19, RP1-4, RP2-17, RC1-1, and RC2-12, and
four distinct hybrids (Table 4).
To find the potential parents of these hybrids, seven
strains of S. bayanus var. uvarum from the winery were
analysed by the same technique (Masneuf-Pomarede et al.,
2007). Five different patterns were obtained for these seven
strains (Table 4). We did not observe any allele polymorph-
ism, indicating that these seven strains were probably
homozygotes. The RC1-14 S. bayanus var. uvarum strain
Table 3. Length (bp) of amplified microsatellite loci for hybrid and Saccharomyces cerevisiae strains
Microsatellite loci
YKL172 SCYOR267c SCAAT1 C11 SCAAT5 C3 C4 C6 YPL009c C8 C9 Group
Hybrid strains
RC1-1 139 318 216 215 162 99 250 106 302 139 90 g1
RC1-11 139 318 216 215 162 99 250 106 302 139 90 g1
RC2-12 139 318 216 215 162 99 250 106 302 139 90 g1
RC2-19 139 318 216 215 162 99 250 106 302 139 90 g1
RP1-4 139 318 216 215 162 99 250 106 302 139 90 g1
RP2-17 139 318 216 215 162 99 250 106 302 139 90 g1
RP2-5 139 318 216 185 162 120 250 106 302 139 90 g2
RP2-6 139 351 264 185 162 120 250 106 302 139 90 g3
RC4-87 139 351 264 185 162 120 250 106 302 139 90 g3
S. cerevisiae
RP2-16� 139 318 216 185 162 99 244 106 302 139 90
351 264 215 120 250 93
RP1-1� 139 318 216 185 162 99 2442 106 302 139 93
351 264 215 120 50
RP1-7� 139 318 216 185 162 99 244 106 302 139 90
351 264 215 120 250 93
RP2-2 124 330 204 213 165 120 250 104 296 136 93
RP1-17 124 360 228 207 165 120 253 114 287 151 93
RC2-17 124 330 234 185 159 123 259 106 296 139 90
255 207 148
�Strain belonging to the d-PCR C7 family.
Table 4. Repeat numbers of Saccharomyces bayanus var. uvarum
microsatellite loci for hybrid and Saccharomyces bayanus var. uvarum
strains isolated in the same cellar
Strains
Microsatellite locus
(GT)n:
Locus 1
(TA)n:
Locus 2
(ATT)n:
Locus 3
(CTG)n:
Locus 4
Hybrid strains
RC2-19 12 8 10 13
RP1-4 12 8 10 13
RP2-17 12 8 10 13
RC1-1 12 8 10 13
RC2-12 12 8 10 13
RP2-5 13 13 10 13
RC1-11 12 8 10 11
RC4-87 13 8 15 9
RP2-6 13 8 15 7
S. bayanus var. uvarum
RC1-14 12 8 10 13
RP1-16 13 8 15 9
RP1-21 13 8 15 9
RC1-19 13 8 15 9
RC4-5 13 11 11 13
RP1-8 13 11 11 9
RC2-20 13 8 11 9
FEMS Yeast Res 7 (2007) 540–549 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
545Saccharomyces cerevisiae and S. bayanus var. uvarum hybrids
had the same pattern as the large group of hybrid strains.
The three S. bayanus var. uvarum strains, RP1-16, RP1-21,
and RC1-19, had a microsatellite pattern identical to that of
the RC4-87 hybrid strain. The RP1-16 and RP1-21 strains
also had the same karyotype, and were considered to be
isolates from the same ‘RP1-21’ strain, whereas the RC1-19
strain was different (results not shown). The potential
S. bayanus var. uvarum parents of the three last groups
of hybrids were not found in the cellar.
In terms of population diversity, four hybrids (RC2-19,
RP1-4, RP2-17, RC1-1) were identical, irrespective of the
method of analysis (Table 2), and were considered to be
different isolates of the same strain. A fifth strain, RC2-12,
only differed from the previous strains by the PFGE profile.
The remaining four strains could be distinguished from each
other by at least one of the tests used. Altogether, six
different hybrid strains were characterized among the nine
isolates (Table 2).
Regarding their potential parents, the RP2-16 strain of
S. cerevisiae was identified as possibly being involved in the
formation of all six hybrid strains, via different recombina-
tion events (Table 5). Five different S. bayanus var. uvarum
microsatellite patterns were observed among the hybrids,
probably indicating the involvement of five different strains
in hybrid formation. Three S. bayanus var. uvarum strains
(RC1-14, RP1-21, RC1-19) potentially involved in the for-
mation of two of the hybrid groups (g1 and g4, Table 2) were
identified in the cellar, whereas parental strains for the last
three groups of hybrids have yet to be isolated.
Discussion
According to the results obtained by PFGE, PCR-RFLP of
the MET2 gene, and microsatellite DNA analysis, the nine
isolates collected in 1997, 1998 and 1999 corresponded to six
S. cerevisiae/S. bayanus var. uvarum hybrid strains. These six
hybrids were characterized from a total of 143 yeast isolates
analysed by PFGE (Demuyter et al., 2004).
Cytometry flow analysis of the nine hybrid isolates
showed that they were diploids rather than polyploids, like
the other natural hybrids previously characterized (Masneuf
et al., 1998; Groth et al., 1999; Naumov et al., 2000; De
Barros Lopes et al., 2002). This was confirmed by the
nongermination of the very few spores produced by these
hybrid strains. Indeed, allotetraploid hybrids have been
shown to produce viable spores, whereas laboratory-gener-
ated diploid hybrids are unable to do so (Naumov, 1996;
Giudici et al., 1998; Naumov et al., 2000). Greig et al. (2002)
and Sebastiani et al. (2002) hypothesized that this infertility
was due to the extent of nonhomologous sequences in the
two species that prevent the normal chromosome pairings
required for viable spore production. The absence of viable
sporulation and, thus, of genetic rearrangements theoreti-
cally gives these strains greater stability, which may be an
advantage from the standpoint of industrial production.
However, the genetic instability of artificial diploid hybrids
and a gradual loss of parental DNA during successive mitosis
had been described (Antunovics et al., 2005). RC2-12 may
represent an example of this type of evolution, as only the
karyotype differs from those of G1 group hybrids (Table 2).
These natural hybrids isolated during this study will provide
valuable material for studying possible genome instability
through successive generations.
To investigate the phylogeny of the nine hybrids, we used
DNA microsatellite amplification, described as the most
representative method for analysing phylogenetic relation-
ships between individuals (Legras et al., 2005; Masneuf-
Pomarede et al., 2007), as these sequences follow an allele
distribution.
The initial results, obtained using specific S. cerevisiae
primers, showed that all nine hybrids originated from the
same C7 S. cerevisiae strain, but via at least three allele
segregation events. It should be emphasized that the RP2-16
and RP1-7 isolates of this strain were collected in 1997 and
1999, respectively, indicating that it is a resident strain in
this cellar. It is also noteworthy that the C7 family was not
dominant in the cellar, as it always represented less than 10%
of the isolates in a given year. It would be very interesting to
understand why this family was the only one involved in
hybrid formation, whereas a large number of other strains
were also present. This will require a study of sporulation
frequency and analysis of the homo/heterothallism of strains
in this family.
In the second stage, four specific pairs of S. bayanus var.
uvarum microsatellite primers were used to determine the
precise relationship between the hybrids and the strains of
this species present in the cellar. Only the RC1-14 strain,
isolated in 1998, had the same microsatellite profile as the
G1 and G2 hybrid groups. No potential parents for the three
remaining hybrids were identified among the S. bayanus var.
uvarum strains isolated in the cellar, or among strains
Table 5. Potential parents of the hybrid strains
Hybrid
groups Hybrid strains
Potential S. cerevisiae and S. bayanus
var. uvarum parents
S. cerevisiae
S. bayanus
var. Uvarum
G1 RC2-19/RP1-4/
RP2-17/RC1-1
‘RP2-16’ a RC1-14
G2 RC2-12 ‘RP2-16’ a RC1-14
G3 RC1-11 ‘RP2-16’ a Sbu1?
G4 RP2-5 ‘RP2-16’ b Sbu2?
G5 RC4-87 ‘RP2-16’ c ‘RP1-21’/RC1-19
G6 RP2-6 ‘RP2-16’ c Sbu3?
a, b and c, type of allele segregation.
Sbu1, 2 and 3: S. bayanus var. uvarum allele sources.
FEMS Yeast Res 7 (2007) 540–549c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. No claim to original French government works
546 C. Le Jeune et al.
isolated in other vineyard regions (Masneuf-Pomarede et al.,
2007). This did not mean that the S. bayanus var. uvarum
parents were not present in the cellar, but simply that the
number of isolates tested was not sufficient.
To further complete the relationship between parental
and hybrid strains, sequenced-based techniques, such as the
multilocus sequence typing approach (Ayoub et al., 2006),
should be a complementary method. In the present study,
three isolates of S. bayanus var. uvarum had the same
microsatellite amplification profile as the RC4-87 hybrid,
but two distinct PFGE profiles. To elucidate the parental
origin of this hybrid S. bayanus var. uvarum strain, coding or
intergenic regions should be sequenced and analysed
further.
Once the nature and characterization of these hybrids has
been clearly established, the mechanism leading to their
formation will still require clarification.
The production of these six natural hybrids requires
sexual reproduction of the yeast. Conditions in the medium
must be suitable for the production of ascospores by the two
species, leading to interspecific crossing. The conditions
required for sexual reproduction and germination of the
spores are still unknown. The sporulation mechanism is
usually expected in a starved medium, but Mortimer et al.
(1994) showed that it could also take place without starva-
tion. Pulvirenti et al. (2002) also suggested that ascus walls
could be hydrolysed during passage through the digestive
tracts of various animals, resulting in the release of free
spores into the environment. Among the nine hybrids, four
were isolated in the press and five in tanks, but none directly
on the grapes. These data indicate that hybridization prob-
ably takes place in the cellar.
Hybridization was easily detected, as two different species
were implicated. It could also occur between other Sacchar-
omyces species (such as S. kudriavzevii), but these hybrids
could not be identified under our experimental conditions
or even at an intraspecies level. New genomes, produced by
genetic mixing, constitute a possible source of biodiversity
within the genus Saccharomyces in a given ecosystem,
leading to the emergence of more vigorous, competitive
strains. Hybrids are useful from a technological standpoint,
as they combine the specific properties of each of the
parents. The G1 group of hybrids contained different
isolates of the same strain. The fact that these isolates were
collected not only in 1998 but also in 1999 indicated that
this strain was able to colonize the cellar and, therefore, that
it probably had a competitive advantage over S. cerevisiae
and S. bayanus var. uvarum strains. The physiological and
fermentation characteristics of artificial S. cerevisiae/
S. bayanus var. uvarum hybrids (H9, S6u2a) were previously
studied by Serra et al. (2005). The same experiments should
be repeated on the six natural diploid hybrids to evaluate
their technological properties and long-term stability. In
parallel, our collection should be subjected to a systematic
screening for hybrids. It would be interesting to see whether
the S. cerevisiae part of the hybrid genome always originates
from the ‘RP2-16’ strain and, if this is the case, to investigate
the reasons for this phenomenon. The widespread existence
of allodiploids in a given ecosystem may be significant from
an evolutionary point of view. It is now well established that
the genome in the Saccharomyces branch of yeasts was
doubled (Byrne & Wolfe, 2006). The origin of this duplica-
tion is still obscure, but it may be due to autodiploidization
or allodiploidization. The latter, resulting in the combina-
tion of two already slightly different sets of alleles, opens the
way for the subsequent loss of specific orthologous genes.
This demonstration that this type of situation is not rare in
nature supports this hypothesis.
Acknowledgements
In part, this study was the subject of an abstract: ‘Le Jeune C,
Masneuf I, Demuyter C, Lollier M, Aigle M. Characteriza-
tion of nine hybrid strains between Saccharomyces cerevisiae
and S. uvarum. ISSY, 26–29 August 2003, Budapest, Hun-
gary.’
The results are not under consideration for publication
elsewhere.
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