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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: lipovy@tigris.unideb.hu
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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Published by Blackwell Publishing Ltd. All rights reserved
1003Interspecies hybridization inSaccharomyces
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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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