MINIREVIEW
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 of
molecular identification methods, double (Saccharomyces cerevisiae Saccharomyces 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 dominated by strains of Saccharomyces (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 to Saccharomyces sensu stricto, a complex
of seven related species (for a review see Rainieri et al.,
2003). A recent taxonomic revision reduced the genus
Saccharomyces to these species (Kurtzman, 2003). The other
five members of the group (Saccharomyces cariocanus,
Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces paradoxus and Saccharomyces pastorianus) are
not likely to play important roles in wine fermentation on
their own. Nevertheless, S. paradoxus has been found on
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grapes in a north-western region of Croatia (Redzepovic
et al., 2002), and genetic elements originating from
S. kudriavzevii were detected in certain Saccharomyces wine
strains (Groth et al., 1999; Gonzalez et al., 2006; Heinrich,
2006; Lopandic et 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 uvarum is more cryotolerant, produces smaller 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 produced by S. uvarum strains 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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Interspecies hybridization in Saccharomyces
common; it occurs mainly in colder areas where fermentation takes place at low temperatures (e.g. Minarik & Laho,
1962; Naumov, 1987; Gainvors et al., 1994; Massoutier et al.,
1998; Sipiczki et al., 2001; Antunovics et al., 2003, 2005a;
Demuyter et al., 2004). Its presence in warmer regions is
much less frequent and may also be associated with fermentation at low temperature (e.g. Torriani et al., 1999; Fernandez-Espinar et al., 2003; Rementeria et al., 2003; Sipiczki
et al., 2004; Gonzalez et al., 2006). It occurs frequently in
sweet wines produced from partially dried and/or botrytized
grapes, such as wines in Alsace (France) (e.g. Demuyter et al.,
2004), Jurancon and Sauternes (France) (Masneuf-Pomarede
et al., 2007), Tokaj (Hungary and Slovakia) (e.g. Minarik
& Laho, 1962; Sipiczki et al., 2001; Antunovics et al.,
2003, 2005a), Val de Loire (France) (e.g. Masneuf et al.,
1996), Valpolicella (Italy) (Torriani et al., 1999; Dellaglio
et al., 2003), Yalta (Ukraine) (Naumov & Nikonenko,
1989), etc.
Because of the development of molecular methods in
strain characterization over the past decade, many Saccharomyces wine strains have turned out to have genomes
composed of genetic elements originating from two or more
species. These are usually referred to as interspecies or
interspecific hybrids. Interspecies hybrids can also be constructed ‘artificially’ in the laboratory. The genomes of
these laboratory-bred hybrids are usually unstable and
undergo a process of genome stabilization through gradual
loss of chromosomes and recombination between chromosomes of the partner genomes. This review is intended
to give a summary of the recent advances in the genetic
analysis of natural and laboratory-made interspecies hybrids, and propose a general model for the ‘postzygotic’
processes and events that lead to stable genomes. The reader
is referred to reviews by Guthrie & Fink (1991) and
Herskowitz et al. (1992) or to the web site http://
www.phys.ksu.edu/gene/a2.html for definitions of genetic
terms used in this article.
and S. uvarum (Nguyen et al., 2000; Pulvirenti et al., 2000).
Because neither modification is generally accepted, the
taxon names S. bayanus, S. uvarum, S. bayanus var. uvarum,
etc. are used inconsistently in the literature, and it is often
impossible to sort out which strain belongs to which
taxonomic name. Given that all putative S. bayanus wine
strains subjected to molecular analyses have proved to have
uvarum-type characteristics, the species name S. uvarum will
be used throughout this review.
Fertility--infertility
Saccharomyces sensu stricto natural strains are usually homothallic and live as single-celled diploids that can undergo
meiosis when starved, forming a tetrad of four haploid
spores (gametes). The ability to produce viable spores is
referred to as fertility. The spores act as gametes, as they can
fertilize each other by conjugation (mating) to produce
zygotes. There are two haploid mating types, a and a, and
each set of four spores consists of two of each type (for a
review see Herskowitz et al., 1992). The spores conjugate
upon germination with germinating spores of opposite
mating type, frequently from the same tetrad (a process also
called self-fertilization or autodiploidization). If unfertilized, the haploid spore propagates mitotically, producing
haploid cells that can also act as gametes to conjugate with
germinating spores or haploid cells of opposite mating type.
Diploids are heterozygous for the mating type alleles a and
a, and are unable to conjugate (for a review see Walton &
Yarranton, 1989). If the diploid does not produce viable
spores, it is considered infertile. Occasionally, unviable
spores are also called infertile, which is misleading. Heterothallic wine yeast strains are rare (e.g. Thornton, 1986;
Miklos et al., 1997) and incapable of sporulation and
autodiploidization on their own.
Interspecies/interspecific hybrid
Terminology
Saccharomyces bayanus --S. uvarum
Saccharomyces bayanus is a controversial species that contains quite diverse strains with different genetic and physiological traits (for a review see Sipiczki, 2002). Recently two
taxonomic modifications have been proposed to make clear
distinction between the wine strains that form a homogeneous group and the rest of S. bayanus. Naumov (2000)
suggested considering these groups as varieties named
S. bayanus var. uvarum and S. bayanus var. bayanus. In
contrast, other researchers proposed to reinstate S. uvarum
as a species name for the homogeneous group, including
the former type strains of Saccharomyces abuliensis
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By definition, the genetic hybrid is the result of the fusion of
two gametes differing in their genetic constitution (Rieger
et al., 1976). Hybridization of different species produces
an alloploid genome consisting of copies of the genomes
of the hybridizing species. The hybrid genomes of the
Saccharomyces wine yeasts can undergo extensive modifications including loss of chromosomes and various types
of recombination events, resulting in various alloaneuploids
or recombinants containing the genome of one of
the partners and mosaics from the genome of the other
partner. I will use the term hybrid in this review for all
strains in which genetic elements from more than one
species have been detected, regardless of the structure of
their genomes.
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998
Detection of natural interspecies hybrids
The occurrence of interspecies hybrids in natural fermentations 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 also
interspecies hybrids (Bradbury et al., 2006). These will also
be considered in this review as natural hybrids because of
their selection from yeast populations of natural fermentations.
Hybrids have been identified using various molecular
methods (for a review see Sipiczki, 2002), including PCRrestriction fragment length polymorphism analysis of nuclear and mitochondrial genes, ribotyping, d-PCR, microsatellite analysis, hybridization with subtelomeric and
transposable repetitive elements, electrophoretic karyotyping, random amplification of polymorphic DNA (RAPD),
amplified fragment length polymorphism (AFLP) fingerprinting, 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 Lopes et al., 2002; Lopandic et al., 2007) or RAPD
(Fernandez-Espinar et 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 other
methods. Microarray karyotyping (array karyotyping; arrayCGH) maps the genome best (e.g. Bond et 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 characteristic 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 Saccharomyces sp. IFO 1802 (Groth et al., 1999). Saccharomyces sp.
IFO 1802, isolated in Japan, later became the type strain of S.
kudriavzevii (Naumov et al., 2000a). Then, using AFLP
analysis, de Barros Lopez et al. (2002) detected amplified
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M. Sipiczki
fragments in the CID1 nuclear genome that were neither
from S. uvarum nor from S. cerevisiae. Finally, the sequence
analysis of the nuclear genes ACT1 CAT8, CYR1, GSY1,
MET6 and OPY1 revealed that the non-cerevisiae and nonuvarum 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
Naumov et al. (2000b), S6U (Table 1) is most probably an
allotetraploid hybrid because it contains genes of S. cerevisiae and 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 allodiploids 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) suppresses conjugation, and consequently prevents the restoration 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 the
finding 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 (Gonzalez et al., 2006).
Allodiploids
The hybrids RC1-1, RC1-11, RC2-12, RC2-19, RC4-87, RP14, RP2-5, RP2-6, and RP2-17 (Table 1) isolated in an Alsace
winery had diploid or near-diploid amounts of DNA,
S. cerevisiae-type d sequences, MET2 alleles and microsatellites both from S. cerevisiae and from S. uvarum, high
numbers of chromosomes and produced dead F1 spores
(Le Jeune et al., 2007). These results suggested S. cerevisiae
S. uvarum allodiploid 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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Double
Triple
Triple (or quadruple) S. cerevisiae, S. uvarum,
S. kudriavzevii, (unknown
species)
B10/39
CBS 2834
CID1 (CBS 8614)
Double
HA1856
Double
Double
HA1841
HA1869
Double
HA1835
S. cerevisiae, S. kudriavzevii
Lalvin W46, Assmannshausen, Double
DSM Fermicru VB1, Anchor
Vin7
HA1829
Double
S. cerevisiae
S. kudriavzevii
S. cerevisiae
S. kudriavzevii
S. cerevisiae
S. kudriavzevii
S. cerevisiae
S. kudriavzevii
S. cerevisiae
S. kudriavzevii
S. uvarum, S. cerevisiae
S. cerevisiae, S. uvarum,
S. kudriavzevii
S. cerevisiae, S. kudriavzevii
S. cerevisiae, S. kudriavzevii
S. cerevisiae, S. kudriavzevii
Double
Double
Double
Valladolid,
Spain
S. cerevisiae, S. uvarum
AWRI 1116
UVAFERM CEG
W27, W46, SPG 14-91, SPG
16-91, 126, 172, 319, 441
Alsace,
France
S. cerevisiae, S. uvarum
RC1-1, RC1-11, RC2-12, RC2- Double
19, RC4-87, RP1-4, RP2-5,
RP2-6, RP2-17
CECT 1885
Double
Karyotyping; Southern analysis of karyotypes;
nuclear genes ACT1, CAT8, CYR1, GSY1,
MET2, MET6 and OPY1; ITS1–5.8S–ITS2;
mitochondrial gene COX2; AFLP, flow
cytometry
Karyotyping; MET2; d-amplification;
microsatellite analysis; flow cytometry
Methods of analysis
Brittany, France
Pfaffstatten,
Austria
Perchtoldsdorf,
Austria
Perchtoldsdorf,
Austria
Eisenstadt,
Austria
Halbturn,
Austria
Tokaj, Hungary
Wadenswill, Switzerland
Karyotyping; nuclear genes CAT8, CYR1,
GSY1, MET6 and OPY1; ITS1–5.8S–ITS2;
mitochondrial gene COX2
Karyotyping; nuclear genes ACT1, CAT8,
CYR1, GSY1, MET2, MET6 and OPY1; ITS15.8S-ITS2; mitochondrial genes ATP8, ATP9,
COX2 and SSU; AFLP
AFLP; 26S rRNA gene; ITS1–5.8S–ITS2; flow
cytometry; growth at 40 1C
AFLP; 26S rRNA gene; ITS1–5.8S–ITS2; flow
cytometry; growth at 40 1C
CAT8, CYR1, GSY1, MET6 and OPY1;
mitochondrial gene COX2
AFLP; 26S rRNA gene; ITS1–5.8S–ITS2; flow
cytometry; growth at 40 1C
AFLP; 26S rRNA gene; ITS1–5.8S–ITS2; flow
cytometry; growth at 40 1C
Karyotyping; nuclear genes CAT8, CYR1,
GSY1, MET6 and OPY1; ITS1–5.8S–ITS2;
mitochondrial gene COX2
Epernay, France
AFLP
Epernay, France
AFLP
Wadenswill, Switzerland Karyotyping; nuclear genes CAT8, CYR1,
GSY1, MET6 and OPY1; ITS1–5.8S–ITS2;
mitochondrial gene COX2
Industrial strains of
ITS1–5.8S–ITS2; microsatellite; minisatellite;
unspecified origin
flow cytometry
Italy
S. cerevisiae, S. uvarum
Double
Geographic origin
S6U
Putative donors
Double/triple/
multiple hybrid
Strain
Table 1. Natural and commercial interspecies Saccharomyces sensu stricto hybrids
Masneuf et al. (1998), Groth et al. (1999),
de Barros Lopes et al. (2002), Naumova
et al. (2005), Gonzalez et al. (2006)
Antunovics et al. (2005a)
Gonzalez et al. (2006)
Lopandic et al. (2007)
Lopandic et al. (2007)
Lopandic et al. (2007)
Lopandic et al. (2007)
Lopandic et al. (2007)
Bradbury et al. (2006)
Heinrich (2006)
Heinrich (2006)
Gonzalez et al. (2006)
Gonzalez et al. (2006)
Le Jeune et al. (2007)
Ciolfi (1994), Masneuf et al. (1998),
Johnston et al. (2000, 2005), Naumov
et al. (2000b), Azumi & Goto-Yamamoto
(2001), Gonzalez et al. (2006)
References
Interspecies hybridization in Saccharomyces
999
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1000
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 Gonzalez et al. (2006) are also
alloaneuploid-like because they do not have S. cerevisiaetype ITS1-5.8S-ITS2 regions. The industrial strains Lalvin
W46, Assmannshausen, DSM Fermicru VB1, Anchor Vin7
and the Austrian natural isolates HA1835 and HA1844 are
S. cerevisiae S. kudriavzevii hybrids of aneuploid genome
size. All have S. kudriavzevii-type ITS1-5.8S-ITS2 and probably 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 have S. cerevisiae-type ITS1–5.8S–ITS2. The
lack of this S. cerevisiae sequence 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 unequal 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. kudriavzevii ‘unequal hybrids’ in which
the S. kudriavzevii part 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 (Masneuf et al., 1998; Naumova et al., 2005;
Gonzalez et al., 2006). In the CID1 genome, no S. kudriavzevii-type MET2 allele has been detected and the S. cerevisiae-type and the S. kudriavzevii-type ITS1–5.8S–ITS2
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 (Naumova et al.,
2005). The other triple hybrid, CBS 2834, does not have S.
cerevisiae-type ITS1–5.8S–ITS2 and GSY1 sequences, and it
also lacks S. kudriavzevii-type ITS1–5.8S–ITS2.
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
Gonzalez et al. (2006), the S. cerevisiae S. uvarum strains
had either S. cerevisiae-like or S. uvarum-like COX2 alleles,
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M. Sipiczki
whereas all hybrids with S. kudriavzevii had S. kudriavzeviilike COX2 sequences. The COX2 sequence of CID1 clustered
only loosely with those of the type strain of S. 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 COX2 gene 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
(Marinoni et al., 1999).
The geographical paradox
The presence of S. kudriavzevii mosaics in certain hybrid
genomes is difficult to interpret because S. 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. kudriavzevii strains 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 perhaps other continents. In this context, it would be interesting 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 of Saccharomyces
sensu stricto (Greig et al., 2002). Hybrid construction
between wine strains of S. 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; Zambonelli et al., 1993, 1997; Kishimoto, 1994;
Giudici et al., 1998; Rainieri et al., 1998; Caridi et al., 2002;
Masneuf et al., 2002; Sato et al., 2002; Sebastiani et al., 2002;
Nakazawa & Iwano, 2004; Torriani et al., 2004; Antunovics
et al., 2005b). Hybrid wine yeasts have also been produced 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 haploid cells, by making use of ‘rare mating’ occurring between
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Interspecies hybridization in Saccharomyces
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; Coloretti et al., 2006): single spores or single haploid
cells of the strains to be hybridized are placed close to one
another 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; Sato et al., 2002; Nakazawa
& Iwano, 2004; Antunovics et al., 2005b). In this case, the
hybrids are identified as colonies produced under culturing
conditions restrictive for both parents (e.g. minimal medium if the partners are auxotrophic). ‘Rare mating’ (also
called ‘illegal mating’) means conjugation between cells
heterozygous for the mating-type alleles at the MAT 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).
et al., 1999; Sebastiani et al., 2002; Coloretti et 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 chromosomal bands missing in the karyotypes of the parents.
Genome structure of laboratory-bred
S. cerevisiae S. uvarum hybrids
Fertile 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 ascribable 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 absolute; 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
FEMS Yeast Res 8 (2008) 996–1007
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 homothallic, these allodiploids are unable to conjugate (heterozygosity at the MAT locus) and develop allodiploid F1 clones
incapable 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).
The hybrid and its filial generations produce viable spores
(fertile filial generations). In the few instances in which
genetic analysis was performed, tetraploid marker segregation 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 heterozygous 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 allotetraploid 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.
Antunovics et 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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1002
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. Other S. uvarum chromosomes underwent rearrangements in interactions with S. cerevisiae chromosomes, demonstrating that genetic recombination can
take place between nonhomologous genomes. The gradual
elimination 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-Pomarede et al. (2007) also observed the loss of
an S. uvarum chromosomal segment from a laboratorymade hybrid. Preferential elimination of S. uvarum chromosomes was also detected in triploid (S. cerevisiae S.
uvarum) S. cerevisiae sporulation (Sebastiani et 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 recombination can take place in alloploids. In addition to meiotic
changes, Sato et al. (2002) also detected mitotic instability in
one of their hybrids during prolonged vegetative propagation. It is pertinent to mention here that hybrids formed
between species of Saccharomyces sensu stricto and species of
the more distantly related Saccharomyces sensu lato also
tended to kick out most of the chromosomes from one of
the parents (Marinoni et al., 1999).
All artificial hybrids analysed were homoplasmic for the
mitochondrial genomes. They inherited their mtDNA either
from the S. uvarum or from the S. cerevisiae parent but never
from both (Masneuf et al., 2002; Antunovics et al., 2005b).
Oenological aspects
Interspecies hybridization of Saccharomyces wine strains
appears to have important biotechnological potentials in
winemaking. Numerous industrial strains (starters) developed 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. cerevisiae and
S. uvarum, has characteristic contribution to the composition of the wine, and distinct technological abilities that
make it better suited than the other species for fermentation
under particular conditions (see ‘Introduction’). Their hybrids, 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
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M. Sipiczki
high temperatures and produce minor fermentative compounds in intermediate quantities have been constructed,
and have intermediate ability to interact with phenolic
compounds, with respect to the individual species (Naumova et al., 1993; Zambonelli et al., 1993, 1997; Kishimoto,
1994; Caridi et al., 2002; Coloretti et al., 2006). Caridi et al.
(2002) observed low production of acetic acid and high
production of glycerol in a hybrid, two traits characteristic
of the cryotolerant S. uvarum parent. Masneuf et 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
parental S. uvarum strain (Serra et 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; Antunovics et al.,
2005b; Coloretti et al., 2006). Coloretti et al. (2006) constructed 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. cerevisiae with additional members of Saccharomyces sensu stricto
(http://www.awri.com.au/information_services/publications/
). The S. cerevisiae S. kudriavzevii hybrid AWRI 1503 has
retained the fermentation vigour of the S. cerevisiae parent
and is well suited for building aroma and palate complexity.
It shows high alcohol tolerance, low volatile acidity, moderate 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 phylogenetic
aspects
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)
(Greig et 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;
Sebastiani et al., 2002; Antunovics et al., 2005b).
This postzygotic barrier does not seem to be effective in
allotetraploids. Greig et al. (2002) produced allotetraploid
hybrids of Saccharomyces sensu stricto species in all possible
combinations and found 75–99% spore viability. The
FEMS Yeast Res 8 (2008) 996–1007
1003
Interspecies hybridization in Saccharomyces
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 possible (Greig et al., 2002; Antunovics et al., 2005b).
As shown above, the partner genomes are not isolated in
the hybrid cell and can interact. This interaction is manifested in recombination accompanied by extensive reduction 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 diploids. 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 consisting 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
Sp 2
(n)
Sp 1
(n)
spores can be sterile, most probably because of their inability
to fertilize each other. As shown for S. cerevisiae laboratory
strain, diploids heterozygous for the mating types do not
conjugate. But a diploid spore is not necessarily heterozygous at birth, and thus conjugation between allodiploid
spores may take place. This self-fertilization can restore
tetraploidy. The DNA content and marker composition of
the 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 taxonomic 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. However, when considering this possibility one has to bear in
Sp 1
(2n)
Mating
Hybridization
Rare mating
Rare
endomitosis
Allodiploid
Meiosis
Sp 2
(2n)
Mitosis
Allotetraploid
Meiosis
(Infertility)
Mitotic
recombination
Low spore
viability
Recombination
(+ loss of
chromosomes)
Mitosis
Mitotic
recombination
Genome
stabilization
High spore
viability
Loss of
chromosomes
Allodiploid
Loss of
chromosomes
Allotetraploid
Alloaneuploid
Stable
genome
Recombinant
haploid
Fig. 1. Postzygotic genetic events leading to genome stabilization in interspecies hybrids of Saccharomyces sensu stricto wine yeasts. Sp 1, species 1;
Sp 2, species 2.
FEMS Yeast Res 8 (2008) 996–1007
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c
1004
mind that the Saccharomyces sensu stricto species are supposed to have diverged from S. cerevisiae between 5 and 20
million years ago (Kurtzman & Piskur, 2006) and are
regarded as still being in the early stages of species differentiation (Delneri et al., 2003). Nevertheless, interspecies
hybridization may play an important role in the evolution of
the existing species. The extensive reduction of one partner
genome 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
stricto species. 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 Saccharomyces interspecies 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 environmental changes lethal to vegetative cells. Allodiploid and alloaneuploid 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) on S. cerevisiae S. uvarum hybrids, 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 Saccharomyces 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
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M. Sipiczki
interpreted as demonstrating that interspecies hybridization
is also a mode of horizontal gene transfer. Because the
hybrids usually possess oenological properties of the parental strains in new combinations, interspecies hybridization 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 directed
and 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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