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M I N I R E V I E W

Interspecies hybridization and recombination in Saccharomyces

wine yeasts

Matthias Sipiczki

Department of Genetics and Applied Microbiology, University of Debrecen, Debrecen, Hungary

Correspondence:Matthias Sipiczki,

Department of Genetics and Applied

Microbiology, University of Debrecen, PO Box

56, 4010 Debrecen, Hungary.

Tel.: 136 52 316666; fax: 136 533690;

e-mail: [email protected]

Received 10 November 2007; revised 10

January 2008; accepted 23 January 2008.First published online 18 March 2008.

DOI:10.1111/j.1567-1364.2008.00369.x

Editor: Graham Fleet

Keywords

Saccharomyces cerevisiae;Saccharomyces

uvarum;Saccharomyces kudriavzevii; alloploid

hybrid; recombination; wine.

Abstract

The ascomycetous yeasts traditionally referred to as the Saccharomyces sensu stricto

complex are a group of closely related species that are isolated by a postzygotic

barrier. They can easily hybridize; and their allodiploid hybrids propagate by

mitotic divisions as efficiently as the parental strains, but can barely divide by

meiosis, and thus rarely produce viable spores (sterile interspecies hybrids). The

postzygotic isolation is not effective in allotetraploids that are able to carry out

meiosis and produce viable spores (fertile interspecies hybrids). By application ofmolecular identification methods, double (Saccharomyces cerevisiae Sacchar-

omyces uvarum and S. cerevisiae Saccharomyces kudriavzevii) and triple

(S. cerevisiae S. uvarum S. kudriavzevii) hybrids were recently identified in

yeast populations of fermenting grape must and cider in geographically distinct

regions. The genetic analysis of these isolates and laboratory-bred hybrids revealed

great variability of hybrid genome structures and demonstrated that the alloploid

genome of the zygote can undergo drastic changes during mitotic and meiotic

divisions of the hybrid cells. This genome-stabilization process involves loss of

chromosomes and genes and recombination between the partner genomes. This

article briefly reviews the results of the analysis of interspecies hybrids, proposes a

model for the mechanism of genome stabilization and highlights the potential of

interspecies hybridization in winemaking.

Introduction

The early stages of the alcoholic fermentation of grape must

are characterized by the simultaneous growth of a broad

spectrum of yeast species. As the alcohol concentration

increases, the yeast population gradually becomes domi-

nated by strains ofSaccharomyces(Fleet & Heard, 1993). The

principal species of alcoholic fermentation in grape wine is

S. cerevisiae, but the closely related Saccharomyces uvarum

(Saccharomyces bayanus var. uvarum) can also participate.

Both yeasts belong toSaccharomyces sensu stricto, a complex

of seven related species (for a review see Rainieri et al.,

2003). A recent taxonomic revision reduced the genus

Saccharomycesto these species (Kurtzman, 2003). The other

five members of the group (Saccharomyces cariocanus,

Saccharomyces kudriavzevii, Saccharomyces mikatae, Sac-

charomyces paradoxus and Saccharomyces pastorianus) are

not likely to play important roles in wine fermentation on

their own. Nevertheless, S. paradoxus has been found on

grapes in a north-western region of Croatia (Redzepovic

et al ., 2002), and genetic elements originating from

S. kudriavzeviiwere detected in certainSaccharomyceswine

strains (Groth et al., 1999; Gonzalez et al., 2006; Heinrich,

2006; Lopandicet al., 2007).

Both S. cerevisiae and S. uvarum are able to grow on

substrates characterized by high sugar and ethanol content,

low pH, high sulphur dioxide concentrations and remains

of fungicides, demonstrating that their genomes are well

adapted to the oenological conditions. From oenological

point of view, these species differ in a number of properties.

Saccharomyces uvarumis more cryotolerant, produces smal-

ler amounts of acetic acid, low amounts of amyl alcohols,

but higher amounts of glycerol, succinic acid, malic acid,

isobutyl alcohol, isoamyl alcohol and numerous secondary

compounds (for a review see Sipiczki, 2002). Wines pro-

duced byS. uvarumstrains have a higher aromatic intensity

than those produced by S. cerevisiae (e.g. Henschke et al.,

2000; Coloretti et al., 2006). Saccharomyces uvarum is less

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Detection of natural interspecies hybrids

The occurrence of interspecies hybrids in natural fermenta-

tions of must is difficult to assess because hybrid strains can

only be detected by molecular methods, which have not

been applied to taxonomical identification of wine yeasts

until recently. Certain commercial wine strains are alsointerspecies hybrids (Bradburyet al., 2006). These will also

be considered in this review as natural hybrids because of

their selection from yeast populations of natural fermenta-

tions.

Hybrids have been identified using various molecular

methods (for a review see Sipiczki, 2002), including PCR-

restriction fragment length polymorphism analysis of nucle-

ar and mitochondrial genes, ribotyping, d-PCR, micro-

satellite analysis, hybridization with subtelomeric and

transposable repetitive elements, electrophoretic karyotyp-

ing, random amplification of polymorphic DNA (RAPD),

amplified fragment length polymorphism (AFLP) finger-

printing, macroarray karyotyping and their combinations

(Table 1). Most of these techniques rely on testing of a few

loci in the chromosomes or in the mitochondrial genome,

which can be misleading by suggesting that something has

an alloploid genome, although we only know that it has

extra copies of certain genes. Application of multilocus

markers, such as AFLP (Azumi & Goto-Yamamoto, 2001; de

Barros Lopeset al., 2002; Lopandic et al., 2007) or RAPD

(Fernandez-Espinaret al., 2003), is more effective because it

maps the whole genome. However, these methods do not

discriminate between more conserved and more variable

regions and may not detect fine (few-nucleotide) differences

at conserved loci that are used for hybrid analysis in othermethods. Microarray karyotyping (array karyotyping; array-

CGH) maps the genome best (e.g. Bondet al., 2004); hence

it has great potentials in the analysis of genome structures of

natural isolates.

Table 1 lists natural interspecies hybrids isolated from

wine or cider fermentation. Most strains are supposed to be

double hybrids of S. cerevisiae with either S. uvarum or S.

kudriavzevii. A hybrid is usually identified as a heterozygous

strain that possesses alleles of one or a few genes character-

istic of two species. However, more detailed analyses can

then reveal elements from additional species. The case of the

cider isolate CID1 is a good example of this process. It was

originally described as a hybrid containing two versions of

the nuclear MET2 gene: an S. cerevisiae-like allele and an

S. bayanus-like allele (Masneuf et al., 1998). However, a

different line of research revealed that the CID1 mtDNA was

not from these species but from a yeast similar to Sacchar-

omycessp. IFO 1802 (Groth et al., 1999).Saccharomycessp.

IFO 1802, isolated in Japan, later became the type strain ofS.

kudriavzevii (Naumov et al., 2000a). Then, using AFLP

analysis, de Barros Lopez et al. (2002) detected amplified

fragments in the CID1 nuclear genome that were neither

fromS. uvarumnor fromS. cerevisiae. Finally, the sequence

analysis of the nuclear genes ACT1 CAT8, CYR1, GSY1,

MET6and OPY1revealed that the non-cerevisiaeand non-

uvarum nuclear sequences must also have derived from S.

kudriavzevii(Naumova et al., 2005; Gonzalez et al., 2006).

Thus, CID1 is a triple (or perhaps a quadruple) hybrid.

Genome structure in natural hybrids

Molecular analyses of natural hybrids revealed an extensive

variation in the genome organization. The hybrid genomes,

which consist of complete sets of chromosomes from the

partners, can be allodiploid or allotetraploid. Other hybrids

have only portions of the partner genomes in the form of

extra (supernumerary) chromosomes (alloaneuploids) or

translocations (interspecies recombinants).

Allotetraploids

As suggested by Johnston et al. (2000) and proved by

Naumovet al. (2000b), S6U (Table 1) is most probably an

allotetraploid hybrid because it contains genes ofS. cerevi-

siaeand S. uvarum and produces viable F1 spores. The F1

clones formed by the F1 spores also sporulate but the spores

are dead, which indicates that S6U segregates into allodi-

ploids during meiosis. The nonviability of the F2 spores

further indicates that the F1 spores are heterozygous at the

mating type locus MAT. MAT heterozygosity (a/a) sup-

presses conjugation, and consequently prevents the restora-

tion of the parental ploidy by autofertilization, a process

operating in haploid spores (genome renewal, Mortimer

et al., 1994). The alloploidy of S6U is corroborated by thefinding that it contains both S. cerevisiae and S. uvarum

alleles of five nuclear genes. Somewhat contradictory to

these results is the finding that S6U is not heterozygous at

the region ITS1-5.8S-ITS2: it has the S. uvarum-type allele

only (Gonzalezet al., 2006).

Allodiploids

The hybrids RC1-1, RC1-11, RC2-12, RC2-19, RC4-87, RP1-

4, RP2-5, RP2-6, and RP2-17 (Table 1) isolated in an Alsace

winery had diploid or near-diploid amounts of DNA,

S. cerevisiae-typed sequences,MET2alleles and microsatel-

lites both from S. cerevisiae and from S. uvarum, highnumbers of chromosomes and produced dead F1 spores

(Le Jeuneet al., 2007). These results suggestedS. cerevisiae

S. uvarumallodiploid genomes.

Alloaneuploids and interspecies recombinants

CECT 1885 (Table 1) appears to be an S. cerevisiae

S. uvarum alloaneuploid because it does not have both

parental alleles of the six nuclear genes tested. For three



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genes it does not have S. cerevisiae alleles (Gonzalez et al.,

2006). Three S. cerevisiae S. kudriavzevii hybrids out of

the eight hybrids described by Gonzalezet al. (2006) are also

alloaneuploid-like because they do not have S. cerevisiae-

type ITS1-5.8S-ITS2 regions. The industrial strains Lalvin

W46, Assmannshausen, DSM Fermicru VB1, Anchor Vin7

and the Austrian natural isolates HA1835 and HA1844 areS. cerevisiae S. kudriavzeviihybrids of aneuploid genome

size. All haveS. kudriavzevii-type ITS1-5.8S-ITS2 and prob-

ably complete or nearly complete S. cerevisiae genomes

(Bradbury et al., 2006; Lopandic et al., 2007). In Lalvin

W46 the S. cerevisiae genome is incomplete because this

strain does not haveS. cerevisiae-type ITS15.8SITS2. The

lack of thisS. cerevisiaesequence might be due to the loss of

the relevant chromosomal region or to concerted evolution

(gene conversion) characteristic of the multicopy regions

coding for rRNAs (Eickbush & Eickbush, 2007). The un-

equal participation of the partners indicates that the hybrid

genomes can change with time. The difference found

between the genome of the commercial strain UVAFERM

CEG and the genome of its progenitor isolate AWRI 1116

(Heinrich, 2006) is an example of such changes. Both strains

are S. cerevisiae S. kudriavzeviiunequal hybrids in which

theS. kudriavzeviipart was estimated to amount to c. 10%

of the genome. However, AWRI 1116 has somewhat more

S. kudriavzevii sequences than its derivative UVAFERM

CEG. This difference can be interpreted as indicating that

the AWRI 1116 genome is still unstable and can change.

Triple hybrids

The S. cerevisiae S. uvarum S. kudriavzevii triple

hybrids CID1 and CBS 2834 (Table 1) tested for the presence

of the parental alleles of nuclear genes also appear to be

alloaneuploids (Masneufet al., 1998; Naumovaet al., 2005;

Gonzalez et al., 2006). In the CID1 genome, no S. kudriav-

zevii-typeMET2allele has been detected and the S. cerevi-

siae-type and the S. kudriavzevii-type ITS15.8SITS2

sequences are also missing. However, it contains the ACT1

alleles of all three species and was suggested to have the

ACT1-carrying chromosomes of all parents. Its near-triploid

amount of DNA also indicates that it might have sets of

chromosomes from each parental species (Naumovaet al.,

2005). The other triple hybrid, CBS 2834, does not have S.

cerevisiae-type ITS15.8SITS2 and GSY1sequences, and italso lacksS. kudriavzevii-type ITS15.8SITS2.

Mitochondrial genome

In contrast to the heterozygosity in the nuclear genomes, the

mitochondrial genomes seem to be pure (homoplasmic) in

all hybrids tested so far. Among the hybrids analysed by

Gonzalezet al. (2006), theS. cerevisiae S. uvarumstrains

had either S. cerevisiae-like orS. uvarum-like COX2alleles,

whereas all hybrids withS. kudriavzevii had S. kudriavzevii-

likeCOX2sequences. TheCOX2sequence of CID1 clustered

only loosely with those of the type strain ofS. kudriavzevii

and the other S. kudriavzevii hybrids, which indicates that

the mtDNA of CID1 might originate from a hybridization

event different from that of the nuclear genomes. The

Austrian S. cerevisiae S. kudriavzevii isolate HA1841(Table 1) also had a COX2gene similar to that of the type

strain of S. kudriavzevii (Lopandic et al., 2007). Similar

uniparental inheritance of mitochondrial genomes has been

found in the alloploid lager yeasts BRYC 32 and NCYC (de

Barros Lopes et al., 2002) and between laboratory strains

(Marinoniet al., 1999).

The geographical paradox

The presence of S. kudriavzevii mosaics in certain hybrid

genomes is difficult to interpret becauseS. kudriavzevii has

not been found in a wine-related environment and has not

been detected outside of Japan. Four out of the five known

S. kudriavzeviistrains were isolated from decaying leaves or

from soil in Japan (www.nbrc.nite.go.jp). These facts suggest

that the hybridization event(s) could possibly have occurred

in natural environment and in localities from which the

S. kudriavzevii isolates originate. The hybrids must have

then lost large parts of the S. kudriavzevii genome and

spread over the wine-growing regions of Europe and per-

haps other continents. In this context, it would be interest-

ing to test sake yeasts for the presence of S. kudriavzevii

sequences in their genomes. The vineyard and sake yeasts

have been estimated to separate c. 11 900 years ago (Fay &

Benavides, 2005).

Hybridization of species in laboratory:artificial hybridization

Both S. cerevisiae and S. uvarum can be hybridized under

laboratory conditions with other species ofSaccharomyces

sensu stricto (Greig et al., 2002). Hybrid construction

between wine strains ofS. uvarum and various S. cerevisiae

strains was reported by numerous authors (e.g. Cummings

& Fogel, 1978; Naumov, 1987; Banno & Kaneko, 1989; Jolly

et al., 1993; Zambonelliet al., 1993, 1997; Kishimoto, 1994;

Giudiciet al., 1998; Rainieriet al., 1998; Caridi et al., 2002;

Masneufet al., 2002; Satoet al., 2002; Sebastianiet al., 2002;

Nakazawa & Iwano, 2004; Torriani et al., 2004; Antunovics

et al., 2005b). Hybrid wine yeasts have also been pro-

duced between S. cerevisiae wine strains and strains of

S. kudriavzevii and S. paradoxus (http://www.awri.com.au/

information_services/publications/).

Under laboratory conditions, interspecies hybrids can be

obtained by conjugating (mating) spores, spores with hap-

loid cells, by making use of rare mating occurring between



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diploid vegetative cells or by fusing protoplasts. Mating of

spores with spores or vegetative cells can be carried out by

micromanipulation (e.g. Cummings & Fogel, 1978; Banno

& Kaneko, 1989; Giudici et al., 1998; Rainieri et al., 1998;

Caridi et al., 2002; Masneuf et al., 2002; Sebastiani et al.,

2002; Colorettiet al., 2006): single spores or single haploid

cells of the strains to be hybridized are placed close to oneanother on agar surface and the conjugating pairs are

identified by microscopic examination. The alternative

possibility is mass mating of partner populations carrying

complementary (complementing) genetic markers (e.g.

Hawthorne & Philippsen, 1994; Satoet al., 2002; Nakazawa

& Iwano, 2004; Antunovicset al., 2005b). In this case, the

hybrids are identified as colonies produced under culturing

conditions restrictive for both parents (e.g. minimal med-

ium if the partners are auxotrophic). Rare mating (also

called illegal mating) means conjugation between cells

heterozygous for the mating-type alleles at theMAT locus.

Such cells are normally unable to conjugate but can turn to

mating-competent owing to rare interchromosomal mitotic

gene conversion that abolishes mating-type heterozygosity

(Gunge & Nakatoni, 1972). de Barros Lopes et al. (2002)

proved that rare mating is also possible between species of

Saccharomyces sensu stricto. Protoplast fusion may be useful

for hybridization of nonmating strains and heterothallic

strains of identical mating type (Nakazawa & Iwano, 2004).

Genome structure of laboratory-bredS. cerevisiae S. uvarum hybrids

The artificial hybrids are viable, propagate by vegetative

division as efficiently as the parental strains, and show

combinations of the phenotypic traits of the parental strains,

but there is a considerable variability in their genome

structure. Basically, three categories can be distinguished.

Sterile hybrids

These hybrids either do not sporulate or sporulate poorly,

and the spores produced are mostly dead (a property usually

referred to as sterility). It is hypothesized that these hybrids

are allodiploids containing single copies of the partner

chromosome sets. The production of dead spores is ascrib-

able to the differences between the chromosomes of the

partners, which prevents their pairing in meiosis I (e.g.

Hawthorne & Philippsen, 1994). The sterility is not abso-

lute; viable spores were found in numerous cases, although

with much lower frequencies (usually o 1%) than in the

intraspecies hybrids (e.g. Hawthorne & Philippsen, 1994;

Marinoni et al., 1999; Greig et al., 2002; Sebastiani et al.,

2002). Most hybrids described in the literature belong to this

category (e.g. Banno & Kaneko, 1989; Zambonelli et al.,

1993, 1997; Hawthorne & Philippsen, 1994; Kishimoto,

1994; Giudici et al., 1998; Rainieri et al., 1998; Marinoni

et al., 1999; Sebastianiet al., 2002; Colorettiet al., 2006). The

sterile hybrids usually contain all chromosomes of the

parents but can undergo genomic changes. For example,

Giudici et al. (1998) and Coloretti et al. (2006) described

sterile hybrids with electrophoretic profiles containing chro-

mosomal bands missing in the karyotypes of the parents.

F1-sterile hybrids

The hybrid produces viable spores, but the spore clones

(referred to as F1 generation) do not produce viable spores

(F1 sterility). It is proposed that viable spores can be

produced if each chromosome has a matching partner in

meiosis I. This requires the presence of at least two copies

from both partner chromosome sets; hence these hybrids

must be at least allotetraploid (Cummings & Fogel, 1978;

Antunovics et al., 2005b). Meiosis of allotetraploid cells

produces spores of allodiploid chromosomal sets. If homo-

thallic, these allodiploids are unable to conjugate (hetero-

zygosity at theMATlocus) and develop allodiploid F1 clonesincapable of producing viable spores. Numerous laboratories

reported on hybrids producing viable spores (Cummings &

Fogel, 1978; Marinoni et al., 1999; Greig et al., 2002;

Sebastiani et al., 2002), but the F1 generation was usually

not tested for fertility. Sterile F1 spores were reported by

Cummings & Fogel (1978) and Sebastiani et al. (2002).

Fertile hybrids

The hybrid and its filial generations produce viable spores

(fertile filial generations). In the few instances in which

genetic analysis was performed, tetraploid marker segrega-

tion was observed (Cummings & Fogel, 1978; Banno &Kaneko, 1989; Sebastiani et al., 2002; Antunovics et al.,

2005b). How can an allotetraploid strain retain its genome

over multiple meiotic divisions? If a diploid spore is hetero-

zygous at the MAT locus, as suggested above, it and its

vegetative progeny cells should not be able to conjugate.

However, Sebastiani et al. (2002) managed to cross about

30% of the F1 alloploid spores of a presumably allotetra-

ploid homothallic hybrid with each other, which indicates

that allodiploid spores are not necessarily heterozygous

for the MAT alleles at birth. If an allodiploid spore is

homozygous for one of the MAT alleles, it can conjugate

with another allodiploid spore homozygous for the

opposite MAT allele and restore the parental allodiploid

hybrid genome. However, this possibility has to be verified

experimentally.

Antunovicset al. (2005b) performed a genetic analysis of

a fertile hybrid and monitored the changes of its genome

over four filial generations of viable spores. From the

segregation of three genetic and eight molecular markers,

they concluded that its nuclear genome was allotetraploid.

Although all filial generations were also fertile, the genome

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underwent a gradual reduction over the four successive

meiotic divisions. The S. uvarum part became gradually

smaller through recurrent losses of complete chromosomes

and genetic markers. OtherS. uvarumchromosomes under-

went rearrangements in interactions with S. cerevisiaechro-

mosomes, demonstrating that genetic recombination can

take place between nonhomologous genomes. The gradualelimination and alteration of large parts of the S. uvarum

genome was associated with a progressive increase of

sporulation efficiency and karyotype homogeneity in spores,

suggesting a causal relationship between the reduction of the

S. uvarum components and the stabilization of the hybrid

genome.

Masneuf-Pomaredeet al. (2007) also observed the loss of

an S. uvarum chromosomal segment from a laboratory-

made hybrid. Preferential elimination ofS. uvarumchromo-

somes was also detected in triploid (S. cerevisiae S.

uvarum) S. cerevisiaesporulation (Sebastianiet al., 2002).

Coloretti et al. (2006) and Sato et al. (2002) described

hybrids that contained chromosomes not present in the

parents, which also indicates that interspecies recombina-

tion can take place in alloploids. In addition to meiotic

changes, Satoet al. (2002) also detected mitotic instability in

one of their hybrids during prolonged vegetative propaga-

tion. It is pertinent to mention here that hybrids formed

between species ofSaccharomyces sensu strictoand species of

the more distantly related Saccharomyces sensu lato also

tended to kick out most of the chromosomes from one of

the parents (Marinoniet al., 1999).

All artificial hybrids analysed were homoplasmic for the

mitochondrial genomes. They inherited their mtDNA either

from theS. uvarumor from theS. cerevisiaeparent but neverfrom both (Masneufet al., 2002; Antunovicset al., 2005b).

Oenological aspects

Interspecies hybridization of Saccharomyces wine strains

appears to have important biotechnological potentials in

winemaking. Numerous industrial strains (starters) devel-

oped from natural isolates have proved to be interspecies

hybrids and similar hybrids have also been detected in

natural wine fermentation in various wine-growing regions

(Table 1). Apparently, favourable combinations of positive

properties, including better adaptation, can arise from the

mixing of two or more genomes.

Each of the two major wine yeast species,S. cerevisiaeand

S. uvarum, has characteristic contribution to the composi-

tion of the wine, and distinct technological abilities that

make it better suited than the other species for fermentation

under particular conditions (see Introduction). Their hy-

brids, either natural or laboratory-made, possess these

properties in new combinations that can be superior to

those of the parents. Hybrids that ferment at both low and

high temperatures and produce minor fermentative com-

pounds in intermediate quantities have been constructed,

and have intermediate ability to interact with phenolic

compounds, with respect to the individual species (Naumo-

va et al., 1993; Zambonelli et al., 1993, 1997; Kishimoto,

1994; Caridiet al., 2002; Colorettiet al., 2006). Caridiet al.

(2002) observed low production of acetic acid and highproduction of glycerol in a hybrid, two traits characteristic

of the cryotolerantS. uvarum parent. Masneufet al. (2002)

reported on hybrids that had an enhanced ability to liberate

sulphur varietal aromas in Sauvignon blanc wines. Two of

these hybrids were tested for growth at various temperatures

and were found to have optimal growth at 30 1C, like the

parentalS. uvarumstrain (Serraet al., 2005), suggesting that

the temperature sensitivity of S. uvarum was dominant.

However, other hybrids made in different laboratories

showed temperature tolerance similar to that of the

S. cerevisiae parent (Rainieri et al., 1998; Antunovicset al.,

2005b; Coloretti et al., 2006). Coloretti et al. (2006) con-

structed flocculent hybrids for sparkling wine production.

An advantageous feature of the allodiploid interspecies

hybrids in industrial application is the absence of viable

sporulation and thus of genetic rearrangements, which give

these strains greater stability.

In order to exploit genetic resources from more species,

the Australian Wine Research Institute hybridized S. cerevi-

siaewith additional members ofSaccharomyces sensu stricto

(http://www.awri.com.au/information_services/publications/

). TheS. cerevisiae S. kudriavzeviihybrid AWRI 1503 has

retained the fermentation vigour of the S. cerevisiaeparent

and is well suited for building aroma and palate complexity.

It shows high alcohol tolerance, low volatile acidity, moder-ate foaming and excellent sedimentation properties after

alcoholic fermentation. The S. cerevisiae S. paradoxus

hybrid AWR 1501 is better at building flavour complexity.

Genome stabilization and phylogeneticaspects

Mating barriers between species can be either prezygotic or

postzygotic. In the Saccharomyces sensu stricto group of

yeasts, the barriers are postzygotic: different species from

this group can mate, but their hybrid offspring are almost

completely sterile, producing o 1% viable spores (gametes)

(Greiget al., 2002). Hybrid sterility is thought to be mainly

due to the inability of the chromosomes of the partner

genomes to pair in the prophase of meiosis I, which prevents

normal meiotic division (Hawthorne & Philippsen, 1994;

Sebastianiet al., 2002; Antunovicset al., 2005b).

This postzygotic barrier does not seem to be effective in

allotetraploids. Greig et al. (2002) produced allotetraploid

hybrids ofSaccharomyces sensu strictospecies in all possible

combinations and found 7599% spore viability. The



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allotetraploid S. cerevisiae S. uvarum hybrid analysed by

Antunovics et al. (2005b) produced four generations of

viable spores. The high spore viability was explained as

being due to the presence of a matching homologous

partner for each chromosome, which makes meiosis possi-

ble (Greiget al., 2002; Antunovicset al., 2005b).

As shown above, the partner genomes are not isolated inthe hybrid cell and can interact. This interaction is mani-

fested in recombination accompanied by extensive reduc-

tion of the genome size through the loss of large parts of one

or both partner genomes. Owing to these changes, the

hybrid genome becomes more stable. Fig. 1 summarizes the

postzygotic genetic events, whose existence can be deduced

from the analysis of the hybrids and which may lead to

genome stabilization. The genome of the founding alloploid

zygote can be diploid or tetraploid depending on whether it

arises from conjugation between haploids or between di-

ploids. Allotetraploids may also arise from allodiploids

by endomitosis (Sebastiani et al., 2002). The allodiploid is

sterile but not necessarily stable; it can undergo mitotic

recombination and can lose chromosomes during vegetative

propagation. It stabilizes with a chimerical genome consist-

ing of DNA from both partners. The size of the stabilized

genome and the proportion of the partners are variable. The

allotetraploid hybrid is fertile. However, its allodiploid

spores can be sterile, most probably because of their inability

to fertilize each other. As shown forS. cerevisiae laboratory

strain, diploids heterozygous for the mating types do not

conjugate. But a diploid spore is not necessarily heterozy-

gous at birth, and thus conjugation between allodiploid

spores may take place. This self-fertilization can restore

tetraploidy. The DNA content and marker composition ofthe triple hybrids CID1 indicates that allotriploids can also

be formed, most probably if one of the partners is haploid

and the other is diploid at conjugation. Consistent with this,

Sebastiani et al. (2002) managed to cross an allodiploid F1

derivative of an S. cerevisiae S. uvarum hybrid with S.

cerevisiae spores and obtained hybrids showing triploid

segregation. Multiple hybrids may arise from rare-mating

events of cells of a double hybrid with cells (or spores) of a

third species or a different hybrid.

Can a stabilized hybrid be considered a distinct taxo-

nomic entity? If species are defined on the basis of sequence

differences between certain conserved genes (e.g. LSU d1/

D2, ITS, 18S, actin, etc), such a hybrid is not a distinct

species because it does not have new alleles sufficiently

divergent from the corresponding sequences of the parental

species. Nevertheless, it might become the founder of a

population that gradually evolves into a new species. How-

ever, when considering this possibility one has to bear in

Sp 1

(n)

Sp 2

(n)

Sp 1

(2n)

Sp 2

(2n)

Allodiploid Allotetraploid

Low spore

viability

High spore

viability

Allodiploid Alloaneuploid

Loss of

chromosomes

Allotetraploid

Recombinant

haploid

Hybridization

Stable

genome

Genome

stabilization

(Infertility)

Meiosis Mitosis

Mating Rare mating

Meiosis

Recombination

(+ loss of

chromosomes)

Mitosis

Loss of

chromosomes

Rare

endomitosis

Mitotic

recombination

Mitotic

recombination

Fig. 1. Postzygotic genetic events leading to genome stabilization in interspecies hybrids ofSaccharomyces sensu strictowine yeasts. Sp 1, species 1;

Sp 2, species 2.




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mind that the Saccharomyces sensu stricto species are sup-

posed to have diverged fromS. cerevisiaebetween 5 and 20

million years ago (Kurtzman & Piskur, 2006) and are

regarded as still being in the early stages of species differ-

entiation (Delneri et al., 2003). Nevertheless, interspecies

hybridization may play an important role in the evolution of

the existing species. The extensive reduction of one partnergenome in laboratory-made hybrids and the predominance

of the S. cerevisiae genome in certain natural hybrids

indicate that interspecies hybridization mediates horizontal

transfer of genetic material among Saccharomyces sensu

strictospecies. Due to such transfer events, certain strains of

these species contain DNA of polyphyletic origin, which

increases intraspecies biodiversity and thus enhances the

genetic flexibility of the species and promotes its adaptive

change. The presence of S. kudriavzevii sequences in wine

isolates of various geographical origin raises the possibility

that genome fractions may leave the genome of a species and

gain ground in related genomes all over the world by

horizontal transfer.

If hybridization promotes adaptation, why do Sacchar-

omycesinterspecies hybrids occur rarely in nature? It may be

the consequence of their sterility, i.e. the very low frequency

of viable ascospores. Ascospores act not only as gametes but

also as resting forms that can withstand harsh environmen-

tal changes lethal to vegetative cells. Allodiploid and alloa-

neuploid hybrids sporulate poorly and their spores have

very low viability, which reduces their prospects for survival

under unfavourable environmental conditions (e.g. between

two vintages). It is possible that many sterile interspecies

hybrids die after fermentation and are newly formed during

the next vintage season. A recent report (Le Jeune et al.,2007) onS. cerevisiae S. uvarumhybrids, whose putative

parents were found in the same winery, seems to corroborate

this possibility.

Conclusion

Advances in molecular genetic methods have provided new

tools for studying yeasts associated with wine and revealed

great variability in the genome structures of interspecies

hybrids of the postzygotically isolated species of Sacchar-

omyces sensu stricto. The hybrid genome can be allodiploid,

allotetraploid, alloaneuploid or an interspecies recombinant

composed of mosaics of genomes of two or more species. In

allodiploids the postzygotic barrier acts efficiently; it makes

the hybrids almost completely sterile. However, it seems less

effective in allotetraploids, which usually produce viable

spores. Alloaneuploids and interspecies recombinants arise

from postzygotic loss of chromosomes and recombination

between the partner genomes. The alloaneuploids and

recombinants seem to retain a complete or almost complete

genome of one of the hybridizing partners, which can be

interpreted as demonstrating that interspecies hybridization

is also a mode of horizontal gene transfer. Because the

hybrids usually possess oenological properties of the par-

ental strains in new combinations, interspecies hybridiza-

tion has great potential in genetic improvement of wine

yeasts without the application of methods of recombinant

DNA. However, to make it a powerful technique of directedand controllable modification of the yeast genome, we shall

have to go much deeper into the postzygotic events of

genome stabilization.

Acknowledgements

This work was supported by grants NKTH KPI (NKFP-4/

017/2005) and RET-06/2004 (GENOMNANOTECH).

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