Breeding strategies for combining fermentative qualities and
reducing o¡-£avor production in a wine yeast model
Philippe Marullo1,2, Marina Bely1, Isabelle Masneuf-Pomarède1,3, Monique Pons1, Michel Aigle4 &
Denis Dubourdieu1
1
UMR1219, Faculté d’Œnologie de Bordeaux, Talence, France; 2Laboratoire de recherche SARCO, Z.A. la Jacquotte, Floirac, France; 3Ecole Nationale
Ingénieur Travaux Agricole de Bordeaux, Gradignan, France; and 4Institut de Biochimie et Génétique Cellulaires, Bordeaux Cedex, France
Correspondence: Philippe Marullo,
Laboratoire d’Œnologie Générale, Faculté
d’Œnologie de Bordeaux, 351 cours de la
Libération, F-33405 Talence, France. Tel.: 133
5 40 00 89 43; fax: 133 5 40 00 64 68;
e-mail: [email protected]
Received 7 June 2005; revised 7 October 2005;
accepted 25 October 2005.
First published online 18 January 2006.
doi:10.1111/j.1567-1364.2006.00034.x
Editor: Monique Bolotin-Fukuhara
Abstract
In agricultural sciences, breeding strategies have historically been used to select
new, optimized plant varieties or animal breeds. Similar strategies are possible for
genetic improvement of wine yeasts. We optimized 11 relevant enological traits in a
single clone using successive hybridization and segregation steps. A hybrid
obtained by crossing two parent strains derived from commercial wine yeasts
showed that some of the traits were readily optimized. Dominance/recessivity,
heterosis and transgression were observed among 51 segregating progeny. On the
basis of this information, all the optimal characters from both parents were
combined in a single strain following two targeted sexual crosses. This article
presents a powerful methodology for obtaining a single wine strain with numerous
fermentative qualities that does not produce off-flavors.
Keywords
breeding; fermentation kinetics; off-flavor
production; quantitative distribution; wine
yeasts.
Introduction
Saccharomyces cerevisiae is predominantly involved in the
transformation of numerous foods and beverages, such as
bread, beer and wine (Oliver, 1991). Yeast strains are
genetically diverse (Bidenne et al., 1992; Hennequin et al.,
2001; Winzeler et al., 2003), with some genetic polymorphisms of industrial importance.
Wine yeasts form a particularly heterogeneous group,
both genetically and technologically (Mortimer, 2000). The
characteristics of wine yeasts are numerous and vary according to the wine type desired. Two broad parameters are
essential for wine quality: fermentation properties and
absence of organoleptic defects (Pretorius, 2000). The most
important fermentation properties are ethanol tolerance
and the ability to produce wine without residual sugars
(except in the case of sweet wines) (D’Amore et al., 1990).
Kinetic properties, such as a short lag phase, high fermentation rate and the capacity to complete fermentation quickly
are also important (Bely et al., 1990). Yeast strains compromise wine quality by producing organoleptic defects, such
as acetate (volatile acidity) (Giudici & Zambonelli, 1992;
Marullo et al., 2004), hydrogen sulfide (Giudici, 1994;
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Jiranek et al., 1995) and phenol compounds (Shinohara
et al., 2000). Yeast strains also have a positive effect by
producing esters (Soles et al., 1982) and volatile thiols
(Murat et al., 2001).
At present, wine yeast selection is based mainly on
screening wild yeast populations. However, the likelihood
of identifying a strain expressing all of the optimal properties for winemaking is very low. An alternative approach to
obtaining a strain with numerous qualities is to use breeding
strategies, such as those developed for domesticated plants
and animals (Crow, 1998; Howe, 2002; Giudici et al., 2005).
Surprisingly, few breeding programs have worked with wine
yeasts and each of them have studied only a few parameters
(Romano et al., 1985; Loiez et al., 1992; Prior et al., 1999).
The lack of progress in this field is partly due to the
quantitative genetic nature of most enological traits and the
difficulty associated with genetically manipulating the relevant strains, which may be both homothallic and aneuploid.
In a previous work, homozygous progeny clones presenting different, complementary traits were obtained from
meiotic segregation of commercial yeasts (Marullo et al.,
2004). As these traits are genetically determined, a careful
selection of hybridization partners followed by meiotic
FEMS Yeast Res 6 (2006) 268–279
269
Breeding strategies for wine yeast improvement
Materials and methods
procedure adapted for wine yeast (Puig et al., 1998). The
resulting SB ho::KanMX4 strain (ho SB) was crossed by cellto-spore pairing with spores from the GN strain, to obtain
the BN hybrid (HO/ho::KanMX4).
Progeny clones of BN were obtained by tetrad microdissection. The haploid or diploid status of these progenies
was tested both by mating and sporulation tests (Marullo
et al., 2004). Fifty-one progenies carrying the ho::KanMX4
allele were further analyzed for 11 enological traits. Hybrids
H6097 and H35 were isolated using a micromanipulator
from the mass-mating of their haploid parents.
Yeast strain construction and culture conditions
Pulsed-field gel electrophoresis
Yeasts were grown at 30 1C on complete YPD medium (1%
yeast extract, 1% peptone, 2% dextrose) solidified with 2%
agar as necessary. G418 (100 mg mL1) was added to selected
strains carrying the ho::KanMX4 allele. Sporulation was
induced on acetate medium (1% potassium acetate, 2%
agar) after 3 days at 24 1C.
The strains listed in the text are shown in Table 1. Two
parent strains, GN and SB, were obtained by tetrad microdissection of VL1 and Bo213 commercial wine yeasts,
respectively (Laffort, France). These monosporic diploid
clones (HO/HO) were assumed to be totally homozygous
genetically, as they were obtained by tetrad microdissection
(Marullo et al., 2004).
The HO gene was disrupted in strain SB to obtain a
haploid clone for the crossing experiments. The ho::KanMX4 cassette, which confers resistance to G418, was
obtained by PCR with primers p25: 5 0 -TGGTTTACGAAATGATCCACG-3 0 and P26: 5 0 -AAATCGAAGACCCATCTGCT-3 0 and genomic DNA from strain BY4741 carrying the
ho::KanMX4 allele (Euroscarf collection). This cassette was
used to disrupt the HO gene in the SB strain, using a
Chromosomal DNA was prepared from overnight yeast
cultures in agarose plugs, as previously described (Marullo
et al., 2004). Chromosomes were separated on 1% agarose
gel (Q-biogen, Illkirch, France) using a CHEF DRII apparatus (Biorad, Richmond, CA). Electrophoresis was carried
out at 200 V and 14 1C for 15 h, with a switching time of 60 s,
then for 9 h with a switching time of 90 s. Chromosomal
DNA of X2180-1A was used as Saccharomyces cerevisiae
standard karyotype.
segregation should provide an efficient mean of optimizing these parameters. We investigated the phenotypic
distribution of 11 enological traits in a large population
of progenies obtained from an heterozygous hybrid.
Once we had evaluated these distributions, we crossed
appropriate partners to combine fermentation qualities
in a unique model strain while eliminating off-flavor
characteristics.
Media and culture conditions for kinetic
parameter measurements
Kinetic parameters were measured in batch fermentations
carried out at 24 1C in 1.2 L bioreactors, locked to maintain
anaerobiosis, with permanent stirring. The Kinetic Parameter Medium (KP Medium) was modified from (Bely
et al., 1990a, b). This medium was strongly buffered to pH
3.3 and contained (g L1): glucose (105), fructose (105), L1
tartaric acid (3), citric acid (0.3), and L-malic acid (0.3);
nitrogen source: 190 mg L1 available nitrogen provided by
Table 1. Yeast strains used
Strain
Genetic characteristics
Origin
X2180-1A
VL1
Bo213
SB
GN
S9
ho SB
BN
G1-97
G1-60
H6097
G2-3
G2-5
H35
Laboratory strain
HO/HO diploid commercial strain
HO/HO diploid commercial strain
HO/HO, monosporic clone of Bo213
HO/HO, monosporic clone of VL1
HO/ho::kanR isogenic of SB
ho::kanR, mat a segregant of S9
HO/ho::kanR cross of GN X ho SB
Clone selected from BN segregants ho::kanR, mat a
Clone selected from BN segregants ho::kanR, mat
Cross of G1-60 and G1-97
Clone selected from H6097 segregants ho::kanR, mat
Clone selected from H6097 segregants ho::kanR, mat a
Cross of G2-3 and G2-5
YGS
Laffort
Laffort
Marullo et al. (2004)
Marullo et al. (2004)
This work
This work
This work
This work
This work
This work
This work
This work
This work
Yeast Genetic Stock (Berkley).
FEMS Yeast Res 6 (2006) 268–279
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270
300 mg L1 (NH4)2SO4 (corresponding to 63.6 mg nitrogen)
and a mixture of 18 amino acids (mg L1): L-glutamine
(247), L-arginine (183), L-tryptophan (87.7), L-alanine (71),
L-glutamic acid (58.9), L-serine (38.4), L-threonine (37.1),
L-leucine (23.7), L-aspartic acid (21.8), L-valine (21.8),
L-phenylalanine (18.6), L-isoleucine (16), L-histidine (16),
L-methionine (15.4), L-tyrosine (9), L-glycine (9), L-lysine
(8.3), and L-cysteine (6.4), corresponding to 126.4 mg L1
nitrogen; mineral salts (mg L1): KH2PO4 (2000),
MgSO4 7H20 (200), MnSO4 H2O (4), ZnSO4 7H2O
(4), CuSO4 5H2O (1), KI (1), CoCl2 6H2O (0.4),
(NH4)6Mo7O24 4H2O (1), and H3BO3 (1); vitamins (mg):
myo-inositol (300), biotin (0.04), thiamin HCl (1), pyridoxine HCl (1), nicotinic acid (1), calcium panthothenate
(1), and para-amino benzoic acid (1); anaerobic growth
factors: ergosterol (15 mg L1), sodium oleate (5 mg L1),
and 1 mL Tween 80/ethanol (1 : 1, v/v).
Before yeast inoculation, the medium was sterilized by
filtration through a 0.45 mm nitrate–cellulose membrane
and supplemented with sulfur dioxide (20 mg L1) corresponding to enological treatments. Cells were precultured
in diluted half-synthetic medium at 24 1C in flasks with
agitation for 24 h. Inoculation was standardized at
106 cells mL1.
Kinetic parameter measurements
The amount of CO2 released was determined by automatic
measurement of bioreactor weight loss every 20 min (Bezenger et al., 1985; El Haloui et al., 1988). The CO2
production rate (dCO2 dt1) was calculated by polynomial
smoothing of the last 11 bioreactor weight-loss measurements. The numerous data points and their accuracy
(10 mg) made it possible to calculate the five following
kinetic parameters very accurately: (1) lag phase (h) was the
time between inoculation and the beginning of CO2 release.
This parameter reflects the time necessary for a specific
strain to adapt to the must. (2) Amax (g L1 h 2) was the
maximal acceleration of CO2 release. This parameter occurred at the beginning of CO2 production, during cell
growth. Amax is the maximal fermentation capacity of a
strain and was explained jointly by the specific fermentation
activity and growth rate of each strain. (3) Vmax (g L1 h1)
was the maximal CO2 production rate. This parameter was
reached when nitrogen was depleted in the medium and was
strongly correlated to strain nitrogen demand. (4) The V50/
Vmax ratio corresponded to the relative CO2 production rate
in the middle stage of fermentation (50% of CO2 produced)
divided by Vmax. This parameter reflects the relative drop in
CO2 production rate after nitrogen depletion and is a good
indicator of the second part of fermentation, during the
stationary growth phase. (5) AF time (h) was the time
necessary to ferment all the sugars in the medium, excluding
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P. Marullo et al.
the lag phase. The percentage coefficients of variation were:
lag phase (10.6), Amax (3.8), Vmax (2.1), V50/Vmax (1.4), and
AF time (1.5), respectively.
Fermentation completion parameters
The maximal fermentation ability of each strains was
estimated by 80 mL Erlenmeyer fermentations under drastic
conditions. The model synthetic medium (MSM) and the
entire procedure used have been described previously (Marullo et al., 2004). This medium contained a nonlimiting
sugar concentration (150 g L1 glucose and 150 g L1 fructose). Three parameters were measured: (1) ethanol production (vol%) and (2) residual sugars (g L1) were the amount
of ethanol and residual sugars, respectively, in the medium
at the end of CO2 release; (3) S/E yield (g per vol%) was the
conversion efficacy of sugar into ethanol, an important
parameter in chaptalization (Ribéreau-Gayon et al., 2000).
The high sugar content of this experiment makes it possible
to assess relevant differences between yeast strains.
Phenolic off-flavor test
The ability to release volatile phenols, e.g. vinyl-4-phenol,
was evaluated as previously described (Chatonnet et al.,
1993). Yeast strains were cultivated in 10 mL YPD containing
1 mg L1 para-coumaric acid for 48 h without shaking. The
phenolic aroma was extracted by vortexing with 3 mL ether.
Volatile phenols were identified after injection in splitless
mode (230 1C; injection volume 3 mL; purge rate
55 mL min1) on a Carbowax 20 M capillary column (50 m 0.25 mm; particle size 0.25 mm; SGE, Melbourne, Australia)
He N55 at 121 MPa, installed on a gas chromatograph
(HP5890, Hewlett Packard). The temperature gradient from
45 to 230 1C at 3 1C min1, with a final isotherm for 30 min.
Aroma molecules were detected by a mass spectrometer
(HP5970, Hewlett Packard) in electron impact mode (ionization energy 70 eV, source 250 1C). Vinyl phenol production
ratio of phenolic off-flavor (POF)1/POF strain values was
about 1000-fold; consequently, the POF phenotype was
considered a qualitative variable (1/ ). Low POF activity is
desirable in white-wine yeasts, as POF activity can be used to
reduce the pharmaceutical taste of phenolic compounds
(Chatonnet et al., 1993).
H2S production
Hydrogen sulfide production was estimated by the blackening of the yeast culture on BIGGY agar (Difco, Detroit,
MI) after 3 days in culture (Marullo et al., 2004). Five color
levels were distinguished: 1, white; 2, light brown; 3, brown;
4, dark brown; 5, black. The determination was carried out
in duplicate.
FEMS Yeast Res 6 (2006) 268–279
271
Breeding strategies for wine yeast improvement
Table 2. Technological properties of commercial strains, parents and hybrid
Lag
phase
(h)
VL1
(diploid)
Bo213
(diploid)
Parent GN
(diploid)
Parent SB
(diploid)
Parent ho SB
(haploid)
Hybrid BN
Mean 13.00
(n = 2)
SD
0.01
Mean 13.67
(n = 2)
SD
0.01
Mean 11.50
(n = 10)
SD
0.70
Mean 20.67
(n = 3)
SD
3.51
Mean 19.30
(n = 10)
SD
3.50
Mean 12.33
(n = 2)
SD
0.90
V50/Vmax
Vmax
AF
Amax
(g L1 h2) (g L1 h1) time (h)
Ethanol
production
(vol%)
Residual
sugar
(g L1)
Acetate
production
(g L1)
S/E yield
(g per
vol%)
H2S
(BIGGY) POF
0.52
0.165
1.52
170.0
14.55
39.3
0.21
17.9
3
0.00
0.75
0.00
0.183
0.03
1.52
1.0
98.0
0.8
15.5
14.0
16.0
0.02
0.19
0.1
18.3
3.5
1
0.00
0.36
0.02
0.19
0.05
1.48
1.3
206.3
0.0
15.6
7.0
38.4
0.02
0.24
0.4
16.7
2
0.03
0.62
0.01
0.13
0.04
1.36
7.9
119.7
0.5
16.2
8.7
4.2
0.05
0.45
0.2
18.3
4
1
0.03
0.70
0.00
0.15
0.02
1.54
2.1
100.7
0.2
16.2
2.0
4.1
0.06
0.35
0.2
17.9
4
1
0.03
0.52
0.00
0.21
0.02
1.53
2.1
130.3
0.2
16.9
2.0
5.9
0.06
0.27
0.2
17.4
3
1
0.03
0.01
0.02
2.0
0.3
2.0
0.06
0.2
POF, phenolic off-flavor.
Results
Fermented medium analysis
1
Ethanol concentration (vol%) and residual sugars (g L )
were measured at the end of fermentation in an Erlenmeyer
flask (i.e. 300 g L1 initial sugars). These parameters were
determined by infrared reflectance (Infra-Analyzer 450,
Technicon, France) and colorimetry (A460 nm) in continuous
flux (Sanimat, France), respectively.
Acetic acid production (g L1) (i.e. volatile acidity) was
measured by colorimetry (A460 nm) in continuous flux
(Sanimat, St Nazaire, France) at the end of fermentation in
the bioreactor (i.e. 210 g L1 residual sugars).
Genetic variance and heritability estimates
Heritability was estimated for each phenotype as (s2P–s2E)/
s2P, where s2P is the variance of progeny population,
explaining both the genetic and environmental variance of
the phenotype measured, whereas s2E is the mean of parent
strain variance calculated on 10 measurements per strain,
explaining only the environmental fraction of phenotypic
variance.
Transgression estimate
The transgression level was calculated for each phenotype as
the percentage of progeny clones presenting a trait value
above mM12sM or below mm 2sm, where mM, mm, sM and
sm were the means and standard deviations of higher and
lower parent strains, respectively.
FEMS Yeast Res 6 (2006) 268–279
Parent strains
To achieve this breeding program, two meiotic clones (SB
and GN) were obtained by microdissecting two commercial
wine yeasts (Bo213 and VL1, respectively). These industrial
strains were homothallic (HO/HO), so their monosporic
clones were completely homozygous, i.e. autozygous, after
self-diploidization. Both SB and GN were able to sporulate
efficiently, giving morphologically homogenous progenies
with a germination rate of nearly 100%. The karyotype
patterns (obtained by CHEF analysis) of a tetrad derived
from either GN or SB had the same profile as the parent
(data not shown). This control indicates that the chromosomal contents of these strains are stable.
Technological values for 11 traits of the VL1 and Bo213
commercial strains and their derivative meiotic clones, GN
and SB, and ho SB, are presented in Table 2. For each trait, a
significant strain effect was found between the SB and GN
background (ANOVA) (a = 0.05). Both SB and GN strains
showed technological properties similar to their ancestors
(Bo213 and VL1). Like Bo213, SB was a very good strain in
terms of fermentation kinetics, completing an ordinary
alcoholic fermentation (210 g L1 sugars) in 5 days. The SB
strain was also particularly tolerant of high ethanol content
(up to 16.2 vol%), representing a significant improvement
over its ancestor (15.5 vol%). However, unlike the Bo213
industrial strain, this monosporic clone had a long lag phase
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272
P. Marullo et al.
Fig. 1. Kinetic properties of SB and GN parent strains. The CO2 production rates are presented for SB (gray circles) and GN (black circles) parent strains
in function of fermentation time (a) and CO2 produced (b). The lag phase, Vmax, AF time and V50/Vmax points for both strains are indicated by arrows.
in KP-Medium (about 20 h). This would be a disadvantage
when colonizing a natural must containing a large number
of indigenous microorganisms. Moreover, unlike Bo213, SB
had a high acetate production level (0.45 g L1). These
phenotypes are probably due to recessive and deleterious
mutations, masked by functional alleles in the Bo213 background. Finally, both SB and Bo213 produced significant
off-flavors, such as POF1 character, and high hydrogen
sulfide (H2S) (level 4 on BIGGY scale).
The other parent strain, GN, did not produce off-flavors (i.e.
low production of acetate (0.24 g L1), level 2 on BIGGY scale,
no POF activity). However, this strain had low ethanol tolerance
and completed an ordinary fermentation slowly (about 10 days
for 210 g L1 sugars). These characteristics are also found in the
VL1 industrial strain, well known for its aromatic neutrality and
modest fermentative ability. Despite some differences, the global
technological values of monosporic clones and their ancestors
are very similar, illustrating the heritable nature of both traits.
In terms of enological performance, the initial GN and SB
parent strains had complementary, extreme properties,
especially regarding their fermentative kinetic profiles (Fig.
1). The rapid fermentation kinetics of SB and the aromatic
neutrality of GN are particularly relevant for winemaking
and their ancestors (Bo213 and VL1) are widely appreciated
for their respective characteristics.
Impact of ploidy on kinetic parameters
To simplify crossing procedures, the SB strain was disrupted
at the HO locus by integrating a ho::KanMX4 cassette by
homologous recombination. Correct integration was verified by PCR using external primers. The resulting diploid, S9
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(HO/ho::KanMX4), had the same phenotype for all of the
traits analyzed in this study as SB (HO/HO) (data not
shown). The ho SB strain was obtained following meiotic
segregation of S9 and its ploidy was verified by flow
cytometry. Diploid/haploid status and HO/ho::KanR alleles
cosegregated as expected (data not shown). Due to the
monosporic nature of the SB strain (resulting from selfdiploidization of a spore), SB, S9 and ho SB can be
considered isogenic.
We compared the kinetic properties of the SB diploid
strain to those of the ho SB haploid. The haploid strain had
relatively higher Vmax (113%) and V50/Vmax (112%) values
than the diploid and a relatively shorter ( 19%) AF time.
These phenomena were previously reported by J. M. Salmon
and are thought to be due to the relative differences in
membrane surface area/volume ratio between ploidy series
(Salmon, 1997). In fact, these parameters are strongly
dependent on membrane transporters (nitrogen transporters for Vmax and sugar transporters for V50/Vmax) that are
related to membrane surface. Consequently, for these three
traits, haploid progeny were compared to the haploid ho SB
strain. Strains SB and ho SB did not show any significant
differences for any other traits: ANOVA (a = 0.05).
Genetic and karyotype analysis of the BN hybrid
The BN strain was obtained by pairing GN spores and ho SB
vegetative cells. This hybrid sporulated efficiently and had a
germination rate of 53%. Tetrad analysis of BN is presented
in Table 3. Only 16 out of 95 dissected tetrads germinated
completely and most of them (58) only produced two viable
spores.
FEMS Yeast Res 6 (2006) 268–279
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Breeding strategies for wine yeast improvement
Table 3. Tetrad analysis of BN progeny
Viable spore clones per tetrad
No. of tetrads
dissected
No. of viable
progeny clones
Total no. of
spores dissected
Germination rate
(4)
(3)
(1)
(0)
Total
16
4
(2)
58
11
6
95
64
12
116
11
0
203
64
16
232
44
24
380
–
–
–
–
–
53%
The nature of the hybrid was confirmed by contourclamped homogeneous electric field gel electrophoresis
(CHEF) analysis (Fig. 2). Some chromosomal bands identified as III, IX, V–VIII and XI were twins in hybrid BN. These
bands segregated in 2 : 2 fashion in two complete tetrads
derived from BN (data not shown). However, abnormal
chromosome patterns were observed in a few progeny clones
(PC). Figure 2 shows a probable translocation for PC1 at the
level of chromosome XI. Moreover, some aneuploidies were
detected for chromosome III and IX, as shown for PC3 (2
bands of chromosome IX). Although few progeny karyotypes (15) were analyzed, these results and the tetrad
analysis presented in Table 3 are consistent with a translocation between two chromosomes of parent strains. More
generally, the complex chromosomal organization of some
progeny clones of BN may have a considerable impact on
their phenotypic response. In fact translocation (PerezOrtin et al., 2002) or aneuploidy (Dunham et al., 2002)
were found to have major adaptive effects in S. cerevisiae
strains. However, for BN progeny, we did not find any
correlation between aneuploidy or translocation and extreme phenotype values.
Dominance, codominance and heterosis effects in
the BN hybrid
The technological values of the BN hybrid (GN ho SB) are
summarized in Table 2. The relative dominant and recessive
effects for each trait are also graphically presented in Fig. 3
(dots on top borders). The BN hybrid had similar phenotypic values to the best parent for residual sugars, acetate
production and lag phase (Figs 3a–c). This suggests that loci
determining suitable aptitudes for these parameters are
dominant in this genetic background. This may be explained
by recessive deleterious alleles present in the defective parent
that are easily counterbalanced by a simple cross with the
efficient parent. More interestingly, heterosis effects were
observed for three parameters: Amax, Vmax and ethanol
production (Figs 3g–i). The substantial, significant gain in
ethanol production was amplified comparing the VL1 (14.5
FEMS Yeast Res 6 (2006) 268–279
Fig. 2. Karyotype analysis of BN hybrid and some progeny clones. The
hybrid nature of BN was confirmed by pulsed-field electrophoresis. The
BN strain presented both ho SB and GN bands. Twin bands were
observed for small and medium chromosomes corresponding to Chr. III,
IX, V–VIII and XI. Some Progeny Clones (PC) derived from this strain
presented abnormal chromosome patterns. PC1 shows a probable
translocation at the level of chromosome XI () and PC3 shows an
aneuploidy for IX (two bands indicated by ‰). DNA of laboratory strain
X2180-1A (LS) was used as reference to identify chromosome bands.
vol%) and Bo213 (15.5 vol%) commercial strains with the
BN hybrid (16.9 vol%). This result suggests that these
commercial strains carried deleterious mutations, some of
which were removed from SB and GN by sporulation (see
Parent strains, above). The final superiority of the hybrid
(comparing SB and GN) probably resulted from the combination of positive alleles by the effect of dominance (Zeyl &
Bell, 1997) or overdominance (Hall & Wills, 1987). The
reduction in S/E yield for the hybrid (Fig. 3f) may be one
cause of this phenomenon. Although very small differences
in ethanol yield are usually described between S. cerevisiae
strains, SB and GN were substantially different on this point.
This difference was partially explained by the drastic conditions of this test (300 g L1 sugars) and the substantial
difference (30%) in growth-rate between SB and GN on
MS Medium. Cell maintenance or final biomass production
may explain these phenomena and may thus be of particular
interest for controlling the sugar to ethanol conversion,
especially in high-sugar musts.
For other traits, hybrid BN exhibited intermediate properties compared to the parent strains (SB and BN). Thus,
fermentative properties such as AF time and V50/Vmax were
not optimized by the simple addition of two heterogeneous
genomes. In the same way, desirable aromatic properties,
such as low hydrogen-sulfide production and POF character, were additive and recessive, respectively, leading to
nonoptimal performances. The recessive character of POF
has already been proved (Goodey & Tubb, 1982).
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274
P. Marullo et al.
Fig. 3. Distribution of quantitative traits and dominance/recessivity relations for the BN hybrid. Bar graphs show the frequency of spore clones in
function of trait values. (a) residual sugars (g L1); (b) acetate production (g L1); (c) lag phase (h); (d) V50/Vmax (ratio); (e) AF time (h); (f) S/E yield (g per
vol%); (g) Amax (g L1 h2); (h) Vmax (g L1 h2); (i) ethanol production (vol%). The technological values of the SB, GN, and BN strains are shown on the
top of each bar graph by open cirles, black circles and grey circles, respectively.
In summary, for this specific cross, seven of the eleven
best traits were easily combined in the hybrid. Genetic effects
such as dominance and heterosis explain why good values
from both parents are found in the hybrid. However, the BN
hybrid also showed some unsuitable values, especially offflavor production.
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
Distribution and genetic dissection of enological
quantitative traits in BN strain progeny
Progeny derived from BN (G1 population) included both
(HO/HO) diploid (not analyzed) and (ho::KanMx4) haploid
clones. As only ho::KanMx4 clones were tested for enological
FEMS Yeast Res 6 (2006) 268–279
275
Breeding strategies for wine yeast improvement
traits, the spore clones analyzed were from independent
tetrads. In this way, allele segregation effects on phenotypic
distribution were not biased by analyzing clones derived
from the same meiosis.
As a primary result, the heritability of each trait analyzed
was over 65% (Table 4), showing the importance of genetic
determinism under our conditions. The transgression level
of progeny for each trait, ranging from 0% to 38%, is
indicated in Table 4. Continuous distributions of progeny
values for nine parameters (n = 51) are shown in Fig. 3.
Distributions were quite different depending on the trait.
For example, S/E Yield, Vmax and Amax showed the Gaussian
distribution frequently found for quantitative traits (Figs
3f–h). In contrast, residual sugars, acetate production, and
lag phase (Figs 3a–c) showed an L-shaped distribution, with
about 50% of the progeny in the same extreme phenotypic
class. These descriptive results are informative concerning
the genetic framework of these traits and, consequently,
contribute to developing a rationalized selection program
from the BN background.
Two illustrative cases with practical implications for
strain selection are described below. Time parameters for
AF are apparently difficult to optimize, as few progeny
clones (13%) presented as good a trait value as the best
parent (ho SB). In this case, the absence of transgressive
progeny values indicated that all of the enhancer loci were
present in the best parental strain (ho SB) and silencer loci
in the worst one. On the contrary, acetate production was
easy to optimize: 50% of the progeny revealed a phenotypic
value similar to that of the best parent. The percentage
transgression (20%) indicated that both parents had favorable alleles for this trait. The L-shaped distribution also
found for residual sugars (Fig. 3a) and lag phase (Fig. 3c)
indicates that a major epistatic allele controls 50% of the
population phenotype. Statistical tests for epistasis developed in Lynch & Walsh (1998) were positive in all cases (G.
Yvert pers. comm.).
To test this hypothesis experimentally, parameters with an
L-shaped distribution were measured for four complete
tetrads derived from BN (Table 5). Both HO/HO diploids
and ho::KanMx4 haploids were tested. However, values for
these parameters were comparable as ploidy had no effect
(see Impact of ploidy on kinetic parameters, above). There
was a significant 2 : 2 segregation for the lag phase parameter, indicating that it is controlled by a major locus. For
both tetrads, two spore clones presented short lag phases
(under 15 h), while the other two had long lag phases (over
15 h). The monogenetic determinism of this trait was
confirmed using a QTL-mapping approach (P. Marullo, D.
Dubourdieu and M. Aigle, pers. commun.). For residual
sugars or acetate production, tetrad analysis gave less
information concerning a possible locus with a major effect
on these phenotypes. Although a 2 : 2 segregation appeared
in some tetrads (i.e. tetrad 2 for residual sugars), the
continuous distribution of values in other tetrads indicated
probable polygenetic control.
A Pearson correlation test was performed on all parameters studied. For our data, a significant threshold of 0.585
was found using a permutation test (a = 0.01). Most of the
parameters were independent. Only three trivial correlations
were found: (1) ethanol production and residual sugar
(r = 0.867); (2) S/E yield and residual sugars (r = 0.679) and
(3) V50/Vmax and AF time (0.790). Considering that most
traits analyzed were genetically and physiologically independent, it should be possible to obtain a strain exhibiting all
the desirable properties.
Fixing alleles with a favorable effect on
fermentation properties and discarding those
that produce off-flavors
Two spore clones with opposite mating types were selected
from the BN progeny. G1-60 and G1-97 both presented
optimal values for kinetic properties, such as a short lag
phase, a short AF time, and a high V50/Vmax. Moreover, both
clones had low H2S production and very good fermentation
completion parameters, producing up to 17 vol% ethanol
and leaving less than 4 g L1 residual sugars). These spore
clones were chosen on the basis of phenotypic distribution
(represented in Fig. 3), weighted by the enological relevance
Table 4. Phenotypic values, heritability and transgression rate of progeny clones
Progeny
(haploid) (n = 51)
Mean
Range
Heritability (%)
Transgression (%)
Lag
phase (h) V50/Vmax
Amax
Vmax
(g L1 h2) (g L1 h1)
AF
time
(h)
Ethanol
Acetate
production Residual
production S/E yield
H2 S
(vol%)
sugar (g L1) (g L1)
(g vol%1) (BIGGY) POF
15.90
9.33–38
65.2
8
0.15
0.1–0.21
66.6
10
142.3
103–238
94.4
2
16.2
14.5–17.2
74.7
38
0.53
0.33–0.68
83.7
0
1.51
1.29–1.89
87.1
35
17.7
0.5–51.5
88.8
0
0.37
0.15–0.79
86.5
20
17.4
16.3–18.6
86.0
0
2.8
1–4.5
100
nd
26(1)
25( )
nd
nd
nd, not determined; POF, phenolic off-flavor.
FEMS Yeast Res 6 (2006) 268–279
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
276
P. Marullo et al.
Industrial yeast strains or relevant isolate
(Well known technological potential)
Monosporic clone collection
genetically characterized:
Homothallism
Chromosomal stability
Crossing monosporic
clones with
complementary
properties
Sporulation
Need the
input of new
alleles
Hybrid (zygote)
Phenotypic tests
Yes
No
Segregation
of traits ?
Desirable
improvement
?
Yes
No
Dominance
for all traits ?
No
Sporulation and analysis
of trait distribution
Yes
Homozygoties for
genes of interest
No
New genetic
profiles ?
(karyotypes)
Fixing all the optimal
properties step-by-step
by crossing selected
segregants.
Yes
Yeast strain
Fig. 4. Yeast selection and breeding flow chart.
of the traits analyzed. Therefore, priority was given to
selecting optimal clones for traits with a Gaussian distribution and a low transgression percentage, due to the difficulty
in optimizing them. Thus, G1-60 was chosen despite its
undesirable POF character, and G1-97 in spite of its
excessive acetate production. These strains were crossed to
obtain the H6097 hybrid that presented optimal values for
selected traits (Table 6). The effect of ploidy on V50/Vmax, AF
time and Vmax, discussed above, was confirmed here. As
expected POF1character was a dominant trait (Goodey &
Tubb, 1982; Clausen et al., 1994). Moreover, the contribution of the high acetate producer, G1-97, was still present in
the hybrid (0.34 g L1), making H6097 unsuitable for fermenting quality white wines.
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
A small population of G2 progeny (four complete tetrads
of H6097) was analyzed for acetate and H2S production,
POF character and fermentation completion parameters. As
described for BN progeny, acetate production exhibited a
high percentage transgression (28%), with values ranging
from 0.09 to 0.95 g L1. Among this population, four clones
with low acetate production and negative POF activity were
selected. These four G2 progeny clones were finally tested for
all traits presented in this study. As shown in Table 6, only
small differences between these clones and their haploid
parents G1-60 and G1-97 were observed for previously
selected traits (i.e. H2S production, fermentation completion and kinetic parameters). This shows that these trait
values had already been successfully fixed in the H6097
FEMS Yeast Res 6 (2006) 268–279
277
Breeding strategies for wine yeast improvement
hybrid. The hybridization of G2-3 and G2-5 produced the
H35 diploid strain, which had nearly all of the desirable
properties of both parents (SB and GN) (Table 6). As
expected, POF character was negative and acetate production reduced to 0.31 g L1.
Discussion
In this study, we propose a generic breeding model for
industrial yeast selection (Fig. 4). Several stages will be
required to develop improved yeast strains.
The first stage consists of starting a genetic program with
completely homozygous clones. In such strains, the phenotype reflects the subjacent genotype. Although this step is
Table 5. Tetrad analysis of three parameters presenting L-shaped
distributions
Tetrads
Lag phase (h)
Acetate
production (g L1)
Residual
sugars (g L1)
2A
2B
2C
2D
4A
4B
4C
4D
5A
5B
5C
5D
14A
14B
14C
14D
12.70
11.30
18.00
21.30
39.30
28.70
12.60
15.00
12.30
18.70
34.70
12.70
13.30
27.00
20.30
14.70
0.13
0.35
0.46
1.08
0.33
0.60
0.09
0.46
0.57
0.26
0.33
0.25
0.41
0.26
0.12
0.35
62.0
60.7
0.7
12.0
12.7
25.0
41.3
35.0
46.0
23.0
20.0
41.0
13.9
18.8
37.3
12.0
Mean of triplicates.
not theoretically necessary, it facilitates subsequent choices.
As natural yeast flora and commercial yeasts are frequently
aneuploid and heterozygous, it is useful to work with
monosporic clones. In a previous study, we demonstrated
that spore clones from specific industrial strains frequently
exhibited the same technological profiles as their ancestor
(Marullo et al., 2004). Consequently, a vast collection of
monosporic clones from divergent, well-documented strains
is a prerequisite for large breeding programs. This collection
constitutes relevant genetic material for introducing specific
properties into new strains. Moreover, in some cases, this
first sporulation step leads to the elimination of deleterious
mutations, readily improving the specific strain (Mortimer
et al., 1994; Regodon et al., 1997).
As many qualities are required, it is necessary to combine
alleles by successive crosses. Following the example of this
investigation, it is possible to optimize many complex traits
by mating strains from divergent genetic backgrounds with
extreme, opposite characteristics. This improvement is
explained by complementation and can show heterosis
effects, e.g. ethanol production, in our experiments. Hybrids
of this type contain all desirable alleles of both parents.
However, if the desirable alleles are recessive, some optimal
traits are masked and must be ‘recovered’ by meiotic
segregation.
It would, theoretically, be possible to obtain a spore clone
containing all optimal alleles. However, the high number of
traits analyzed makes this task difficult. We demonstrated
that many traits could be fixed step-by-step, using appropriate crosses with segregants from the BN hybrid. Indeed,
when the two copies of the main ‘enhancer alleles’, controlling a specific trait, are present in a hybrid, the trait is
considered to be ‘fixed’ and should not segregate widely in
the related hybrid progeny. Other remaining unsuitable
traits can be optimized in the next step, if the resulting
Table 6. Fixing aromatic and fermentation parameters by two successive crosses
SB
GN
G1-60
G1-97
H6097
H6097
progeny
(four tetrads)
G2-3
G2-5
H35
V50/Vmax
Amax
(g L1 h2)
Vmax
(g L1 h1)
Ethanol
Residual
production sugar
AF time (h) (vol%)
(g L1)
Acetate
production S/E yield
(g L1)
(g vol%1)
H2S
(BIGGY)
POF
20.6
11.5
13.3
12.3
13.0
11–14w
0.62
0.36
0.62
0.65
0.58
0.55–0.61w
0.13
0.19
0.16
0.14
0.20
0.14–0.16w
1.36
1.48
1.52
1.58
1.52
1.36–1.53w
119.6
206.3
118.0
110.6
128.0
120–135w
16.2
15.6
17.1
17.0
16.4
16.2–16.9
0.37
0.24
0.31
0.48
0.34
0.09–0.95
18.3
16.7
17.3
17.7
18.0
17.5–18.1
4
2
1
2
2
1–2
1
1
1
/1
12.3
11.0
12.6
0.61
0.55
0.53
0.16
0.15
0.17
1.44
1.53
1.43
128.0
128.0
128.0
16.9
16.2
16.7
0.29
0.20
0.31
17.5
18.1
17.7
Lag
phase
(h)
4.2
38.4
3.87
0.43
5.3
4.3–7.4
4.3
7.4
4.0
1
1
1
–
–
–
Values of the optimal parent (SB or GN) that were optimized in H35. wOnly four POF negative clones with low acetate production levels were analyzed
for kinetic parameters. POF, phenolic off-flavor.
FEMS Yeast Res 6 (2006) 268–279
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Published by Blackwell Publishing Ltd. All rights reserved
c
278
hybrid carries one copy of each of the desirable recessive
alleles. Consequently, this method is useful for optimizing
most traits by successive crosses and segregation cycles.
The technological properties of the H35 model strain
obtained are far from trivial. In fact, this strain combines
the optimal qualities of two parental strains previously
selected from a population of spore clones of industrial
(selected) strains.
Breeding strategies are among the most pragmatic applications of genetics, and have been used empirically in
agriculture for many centuries. Complex traits, such as
maize yield, have increased sixfold since breeding programs
began 90 years ago (Crow, 1998). This improvement was
possible without any molecular knowledge, simply by crossing genetically-chosen inbred lines (Howe, 2002). Twenty
years ago, Romano et al. (1985) ran a pioneer program to
improve S. cerevisiae wine yeast strains. Their program
focused on four enological traits, two of which were monogenetically determined. They concluded that: ‘many winemaking traits can be determined quantitatively so that
genetic improvement with breeding program is achievable’.
However, quantitative traits such as fermentation vigor or
ethanol production have not been completely optimized.
Our research takes this approach further and confirms that
breeding strategies are an excellent way of combining
numerous (11) technological properties in a single strain.
We have shown that complex, polygenetic traits, such as
ethanol tolerance and kinetic properties, also present a
strong genetic determinism and can be improved in the
same way as traits determined by a single locus. Priority
must be given to traits with Gaussian distributions by
analyzing a large number of progenies. This study demonstrated that a small number of rationalized crosses (two in
our case) was effective in improving several technological
properties. If HO alleles are present in both parent strains, as
is usually the case, they must be crossed by spore-to-spore
pairing; a time-consuming procedure. In this project, the
high number of quantitative traits led us to foresee several
steps of progeny inter-crosses. Therefore, we decided to
disrupt one of the HO alleles in this model study. Surprisingly, our results showed that only two inter-progeny crosses
were sufficient, probably due to the L-shaped distribution of
some traits. Consequently, the same strategy can be achieved
readily using spore-to-spore crosses of homothallic strains
avoiding the introduction of GM yeasts for commercial
purposes.
Acknowledgements
The authors would like to thank G. Yvert for his comments
on the manuscript and his help with Pearson and epistatic
tests. We also thank SARCO laboratory for its technical
assistance in wine analysis.
2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
P. Marullo et al.
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