10
Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum Christine Le Jeune 1 , Marc Lollier 1 , Catherine Demuyter 1 , Claude Erny 1 , Jean-Luc Legras 2 , Michel Aigle 3 & Isabelle Masneuf-Pomare ` de 4 1 Laboratoire Vigne Biotechnologie et Environnement, Universit ´ e de Haute-Alsace, Herrlisheim, Colmar, France; 2 Institut national de la Recherche Agronomique (INRA) UMR SVQV Œnologie, Herrlisheim, Colmar, France; 3 UMR 5122, Universit ´ e de Lyon 1, Villeurbanne, France; and 4 ENITA Bordeaux, Gradignan, France Correspondence: Le Jeune Christine, Laboratoire Vigne Biotechnologie et Environnement, Universit ´ e 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) 540–549 c 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. No claim to original French government works

Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

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Page 1: Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

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

Page 2: Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

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

Page 3: Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

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.

Page 4: Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

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

Page 5: Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

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.

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544 C. Le Jeune et al.

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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

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545Saccharomyces cerevisiae and S. bayanus var. uvarum hybrids

Page 7: Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

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.

Page 8: Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

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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