FEMS Yeast Research 4 (2004) 711–719
www.fems-microbiology.org
Inheritable nature of enological quantitative traits is demonstrated
by meiotic segregation of industrial wine yeast strains
P. Marullo
a
a,b,*
, M. Bely a, I. Masneuf-Pomarede
a,c
, M. Aigle d, D. Dubourdieu
a
Laboratoire d’Œnologie Generale, Faculte d’Œnologie de Bordeaux, University of Bordeaux, 351 cours de la Liberation, 33400 Talence, France
b
Laboratoire de recherche SARCO, Z.A. la Jacquotte rue Aristide Berges, 33370 Floirac, France
c
Ecole Nationale Ingenieur Travaux Agricoles de Bordeaux, 1 cours du General de Gaulle, 33175 Gradignan, France
d
Institut de Biochimie et Genetique Cellulaires, 1, Rue Camille Saint-Sa€ens, 33077 Bordeaux Cedex, France
Received 24 September 2003; received in revised form 2 December 2003; accepted 6 January 2004
First published online 20 February 2004
Abstract
Wine yeast strains exhibit a wide variability in their technological properties. The large number of allelic variants and the high
degree of heterozygosity explain this genetic variability found among the yeast flora. Furthermore, most enological traits are
controlled by polygenic systems presenting complex interactions between the alleles. Taking this into account, we hypothesized that
the meiotic segregation of such alleles from a given strain might generate a progeny population with very different technological
properties.
In this work, a population of 50 progeny clones derived from four industrial wine strains of Saccharomyces cerevisiae was
characterized for three major enological traits: ethanol tolerance, volatile-acidity production and hydrogen sulphide production. For
this purpose, reliable laboratory fermentation tests were developed in accordance with enological practice. A wide variability in the
values of the various parameters was found among spore clones obtained after sporulation. Many clones presenting better aptitudes
than the parental strains were obtained. Moreover, analysis of the progeny demonstrated that: (1) traits are in part inheritable; (2)
traits are clearly polygenic; (3) broad relations of dominance/recessivity can be established. All these findings constitute an initial
step for establishing breeding strategies for wine yeast improvement.
Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Wine yeast improvement; Quantitative traits; Sporulation; Enology
1. Introduction
Wine fermentation is traditionally performed by a
yeast flora, mainly Saccharomyces cerevisiae, present on
grapes and wine equipment [1,2]. Spontaneous fermentation is still used in numerous wineries, but in the last
twenty years, yeast-manufacturing companies have developed selected starter cultures. These strains are now
widely used to reduce the risk of wine spoilage, prevent
stuck fermentation and improve wine quality.
*
Corresponding author. Tel.: +33-5-40-00-89-43;
fax: +33-5-40-00-64-68.
E-mail address: [email protected] (P. Marullo).
At present, four main strategies can be used to obtain such optimized strains. Firstly, pure strain clones
may be isolated from spontaneous fermenting must.
This is the main strategy used at present [3–5]. Most
commercialized wine yeasts are isolated through
screening based on technological parameters which
ensure quality winemaking, industrial growth and dry
survival of cells. This strategy is based on the traditional microbiological techniques, which assimilate
yeasts to bacteria. Moreover, the wide genetic polymorphism of yeasts no doubt contributes to the efficiency of the method [4]. Secondly, gene transfer has
been used to add, modify or destroy specific genes encoding enzymatic or other activities [6]. Although very
efficient, the low level of acceptance of this technology
1567-1356/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsyr.2004.01.006
712
P. Marullo et al. / FEMS Yeast Research 4 (2004) 711–719
by consumers has limited its impact [7]. Thirdly, mutagenesis may be used to widen the natural genetic
polymorphism. This strategy is an essential approach to
optimize bacteria and fungi, and has proved to be very
efficient for industrial purposes. As wine yeast strains
are not haploid and since most mutations are recessive,
it is not likely to be efficient in the short term, but efforts in this direction have been made. Fourthly, as
yeasts are eukaryotes with standard Mendelian genetic
behavior, the strategies which have been successfully
developed for plants and animals could be applied
to yeasts. Specifically, crosses and progeny analysis
could theoretically be used to improve genotypes,
thereby accumulating general and specific properties in
a strain. Unfortunately many wine yeasts are homothallic. Homothallic haploid cells (HO) are able to
switch their mating type and to conjugate with cells of
the same single spore colony. Consequently, hybridization techniques for developing new strains have
proved elusive and have required the use of a micromanipulator to achieve direct spore-to-spore mating [7].
Several attempts have already been made to this end
[8–11]. To rationalize the latter strategy, the first requirement is to try to establish the importance of the
genetic determinism of the enological parameters of
yeast.
The availability of relevant and reliable phenotypic
tests to screen a large population of yeast strains in
laboratory conditions is the prerequisite condition to
appreciate the contribution of genetics in different
characters. Simple phenotypes like hydrogen sulphide
production, flocculation or killer activity [4,7,12] can be
screened reliably. However, more complex traits such as
ethanol production [3,11], release of metabolic byproducts [5,13–16] or aromatic properties [17] are difficult to measure in enological conditions.
Low volatile-acidity production and high ethanol
tolerance are highly desirable properties for yeast selection by winemakers. The continuous quantitative
variation of these traits within wine yeast populations
can in part be tentatively explained by a polygenic determinism. However, the environmental conditions of
fermentation (i.e. sugar concentration, temperature,
grape juice composition, turbidity) also have a major
impact on these traits [18].
In this paper, we tested the inheritable nature of some
enological quantitative traits. For this purpose, reliable
and relevant laboratory fermentations measuring volatile-acidity production and ethanol tolerance in enological conditions were established. We also showed
how the meiotic segregation of industrial yeast strains
can provide new and interesting genetic variants without
screening natural isolates. These strains presenting unique karyotypes could be readily commercialized or used
in breeding programs.
2. Materials and methods
2.1. Strains, media and growth conditions
All yeast strains are referenced as Saccharomyces
cerevisiae (Table 1). Laboratory strains X2180-1A
(MAT a) and X-2180-1B (MAT a) were used as tester of
mating-type activity a and a respectively. The wine
yeasts were strains isolated from native microflora of
spontaneous fermentations. Yeast was grown at 30 °C
on complete YPD medium (1% yeast extract, 1% peptone, 2% dextrose) solidified with 2% agar when required.
2.2. Sporulation, isolation of spores and determination of
mating type
Sporulation was induced on acetate medium (1%
potassium acetate, 2% agar) after three days at 24 °C.
Ascospores were isolated by a micromanipulator Singer
MSM Manual on YPD-agar. Ascus wall was digested
using cytohelicase (Sigma) adjusted to 2 mg ml1 . Germination efficiency was expressed as the percentage of
isolated spores forming a colony after three days at
30 °C. Mating types were defined by microscopic observation of zygote formation with either tester strain
X2180 1A or X2180 1B. For this purpose, cells of
analyzed strains were mixed with each tester and then
incubated on YPD-agar for 6–18 h.
Table 1
Strains of Saccharomyces cerevisiae used in this work
Strains
Origin
Descriptive
X2180-1B
X2180-1A
SAP
L43
VL1b
VL3cb
ISS
YGSCa
YGSCa
Faculte d’nologie de Bordeaux
Inter Rh^
one
Faculte d’nologie de Bordeaux
Faculte d’nologie de Bordeaux
Enological strain derived from a natural isolate of Sancerre
Haploid laboratory strain
Haploid laboratory strain
Dry yeast, (not commercialized)
Lalvin 43, LallemandÓ
Zymaflor VL1, LaffortÓ
Zymaflor VL3c, LaffortÓ
Monosporic clones
a
b
Yeast Genetic Stock Center (Berkley).
VL1 and VL3c were respectively referenced as no. 2015 and 2016 at CLIB (Collection de Levures d’Inter^et Biotechnologique, Thiverval-Grignon).
P. Marullo et al. / FEMS Yeast Research 4 (2004) 711–719
2.3. Pulse-field gel electrophoresis
Chromosomal DNA was prepared from overnight
cultures of yeasts in agarose plugs as described by Bellis
et al. [19]. Chromosomes were separated with a CHEF
DRII apparatus (Bio-Rad, Richmond, California) on a
1% agarose gel (Qbiogene, Carlsbad, CA, USA).
Electrophoresis was carried out at 200 V and 14 °C for
16.5 h with a switching time of 60 s, and then for 10 h
with a switching time of 90 s. Chromosomal DNA of
X2180-1A was used as S. cerevisiae standard karyotype.
2.4. Determination of hydrogen sulphide production
Production of hydrogen sulphide was estimated by
the blackening of a yeast culture on BIGGY agar
(Difco) after three days of culture as described by
Mortimer [4]. Five levels of color were used: 1 – white, 2
– light brown, 3 – brown, 4 – dark brown, 5 – black. The
determination was done twice.
2.5. Composition of model synthetic medium
Model synthetic medium (MSM) (pH 3.3) simulated
a standard grape juice and contained the following
components (expressed in g l1 ): glucose (100 or 150 g),
fructose (100 or 150 g), tartaric acid (3 g), citric acid (0.3
g), L -malic acid (0.3 g), MgSO4 (0.2 g), KH2 PO4 (2 g).
Nitrogen sources were adjusted to 190 mg total N l1 as
(NH4 )2 SO4 (0.3 g) and asparagine (0.6 g). Mineral
salts (mg l1 ): MnSO4 H2 O (4), ZnSO4 7H2 O (4),
CuSO4 5H2 O (1), KI (1), CoCl2 6H2 O (0.4), (NH4 )6 Mo7 O24 4H2 O (1), H3 BO3 (1). Vitamins (mg l1 ): mesoinositol (300), biotin (0.04), thiamin (1), pyridoxine (1),
nicotinic acid (1), pantothenic acid (1), p-amino benzoic
acid (1). Fatty acids (mg l1 ): palmitic acid (1), palmitoleic acid (0.2), stearic acid (3), oleic acid (0.5), linoleic
acid (0.5), linolenic acid (0.2).
Before yeast inoculation, the medium was sterilized
by filtration (nitrate cellulose membrane, 0.45 lm, Millipore, France) and supplemented with sulfur dioxide
(20 mg l1 ) in accordance with enological treatments.
The fatty-acid mixture [20] was prepared in ethanol solution and fixed by drying on cellulose (0.5 g l1 ) in order to obtain 200 NTU (nephelometric turbidity units).
2.6. Grape must
Three Vitis vinifera cv. Sauvignon musts, collected
from Bordeaux cellars during 2001 and 2002 harvests,
were used. Must turbidity and sulphur dioxide content
were adjusted to 200 NTU (with natural must solids)
and 20 mg l1 , respectively. To preserve their nutritive
properties and to mimic cellar conditions as closely as
possible, musts were not sterilized. Initial sugar concentrations ranging between 180 and 195 g l1 were
713
adjusted to 200 or 300 g l1 with sucrose. (NH)2 SO4 was
added to obtain a concentration of 190 mg l1 nitrogen
corresponding to the MSM composition.
2.7. Fermentation procedures
Yeast pre-culture of 24 h was obtained in MSM or
must diluted 1:1 with milli-Q water. Fermentations were
carried out in 100-ml Erlenmeyer flasks containing 80 ml
of MSM or musts inoculated with 3.5 106 cells/ml.
Cultures were incubated at 24 °C and shaken at 75 rev
min1 . After three days of fermentation, 6 mg l1 of
oxygen were added by air bubbling.
2.8. Wine analysis
For natural must fermentations, implanted strains
were identified by analysis of Delta-PCR-amplified
DNA patterns obtained from total biomass [21]. Ethanol produced (%w/v) was measured by infrared reflectance (Infra-Analyzer 450, Technicon, Trappes, France).
Volatile acidity expressed in g l1 of acetic acid was
determined chemically after distillation by a colorimetric
method (460 nm) in continuous flux (ICA instrument,
Rocquencourt, France).
2.9. Statistical processing of results
Each fermentation experiment was done in triplicate.
Seven series of fermentations were carried out, to test a
large number of strains. To compare strains tested in
different series, we used an internal standard strain (ISS)
in all fermentation series. One-way analysis of variance
(a ¼ 0:05) showed no significant difference in fermentative properties of ISS between all series (n ¼ 7). However, to normalize phenotypic values measured, data
values were corrected as follows:
YN ¼ Yi ðXi X Þ;
where YN is the normalized phenotypic value of a tested
strain, Yi is the measured phenotypic value of the strain
in the i series, Xi is the measured phenotypic value of the
ISS strain in the i series and X is the mean phenotypic
value of ISS measured across all series. All data were
normalized by this method. To determine the presence
of significant differences among the population tested, a
Newman–Keuls test was performed (a ¼ 0:05) using the
StatBoxProÒ software (Montpellier, France).
A principal components analysis (PCA) was performed by using the StatBoxPro software. The technique can be summarized as a factor analysis method
that can be used to simplify the data matrix by identifying the factors making the greatest contribution to the
variance in the data. PCA generates components that
can be used to represent the main differences between
strains tested.
714
P. Marullo et al. / FEMS Yeast Research 4 (2004) 711–719
3. Results
3.1. Progeny constitution
3.1.1. Sporulation and spore viability
Four S. cerevisiae strains used in wine proved to
sporulate efficiently. About ten tetrads of each were
micro-dissected and spore viability, as scored by clone
formation, was measured (Table 2). Although heterogeneous viability was obtained in accordance with previous studies [22], all strains gave viable spores in a
range between 37% and 76%. The homo- or heterothallic status of these clones was checked by their
sporulation ability, and status of HO locus of parental
strains was deduced (Table 2). Two of them which are
known to be diploid (VL3c and VL1) proved to be (HO/
HO), one (L43) heterozygous (HO/ho) and the last
(SAP) (ho/ho). The (ho) status was confirmed by establishing the mating type of the relevant strains. HO
spore clones sporulated easily and we checked the viability of their progeny. Tetrads were micro-dissected and
a high percentage of viable spores was obtained in all
cases (>95%, see Table 2).
The presence of the (ho) allele in a portion of the
progeny tested led us to compare the quantitative traits
between spore clones having a different ploidy. However, by analyzing the L43 progeny, no correlation was
observed between the ploidy and the measured phenotype of spore clones. Moreover, using a disruption
strategy, our team had recently obtained an isogenic ho
clone from an HO/HO monosporic clone. No significant
differences were found between these two strains for any
of the traits analyzed in that study (Marullo, personal
communication). Finally, concerning the global expression of the whole genome, very few differences have been
found in isogenic strains harboring different ploidy [23].
All these data suggest that ploidy had little influence on
the traits analyzed.
3.1.2. Karyotypes of industrial strain progeny
The development of new strains for industrial purposes required clones with a stable, unique and identifiable genetic pattern. Chromosomal profiles obtained by
pulsed-field gel electrophoresis are a powerful technique
to identify enological yeast strains [24]. The karyotypic
patterns of five spore clones derived from each industrial
strain were analyzed. All spore clones showed a pattern
different from the parental strain. Moreover, in many
cases sister spore clones presented different profiles. For
example, Fig. 1 shows karyotypic variability for spore
clones derived from the VL1 strain. As expected, the
great majority of bands observed in the progeny patterns
were inherited as such from the parental strain. Segregation of homologous chromosomes with a different size
was frequent. Although the spore clones analyzed came
from different meiotic products, it is probable that the
new chromosomal bands visible were due to rearrangements during meiosis. Chromosomal patterns obtained
from other strains and their derived spore clones led
to the same kind of segregations (data not shown).
Fig. 1. Electrophoretic karyotypes of VL1 strain and five derived spore
clones. Chromosomal patterns of VL1 parental strain (PS) and its
progeny (1–5) were obtained by contour-clamped homogeneous electric field. DNA of laboratory strain (LS) X2180-1A was used as reference to identify chromosome bands. Principal polymorphic bands
between parental strain and progeny are indicated by *.
Table 2
Homothallism characterization of wine strains used in this work
Strain
SAP
L43
VL1
VL3c
Viable spores/ascus (ascus number)
4
3
2
1
0
4
0
2
2
3
1
0
0
2
4
6
9
1
4
3
0
3
1
0
0
Percent of viability
HO
Viabilities clone progenya
76%
37%
55%
63%
ho/ho
HO/ho
HO/HO
HO/HO
NR
97%
95%
99%
a
Viability of spores derived from HO clones was measured by microdissection. Data presented are the mean of the percent of viability for 7 tetrads
per each homothallic clone dissected. NR: not relevant.
P. Marullo et al. / FEMS Yeast Research 4 (2004) 711–719
3.2. Analysis of enological traits
3.2.1. Development of reliable procedures to study
important enological traits in wine yeast
Model synthetic medium and microvinification procedures were developed to screen enologically relevant
yeast strains by using laboratory tests. Firstly, the production of hydrogen sulphide was measured by the
colorimetric test previously described [4]. Secondly, the
production of volatile acidity during fermentation was
scored in a MSM. This medium represented standard
grape juice with a sugar concentration of 200 g l1 .
Thirdly, the maximum capacity of yeast to ferment at a
non-limited sugar concentration was scored in the same
medium with 300 g l1 of sugar. The quantity of ethanol
produced on this medium was taken to represent the
ethanol tolerance of wine yeast in enological conditions.
As reviewed by d’Amore [25], many methods can be
used to evaluate ethanol tolerance. However, the ability
of a strain to achieve fermentation at high sugar concentrations is a combination of various parameters such
as osmotic stress tolerance, fermentation rate and cell
viability in the presence of ethanol. Consequently, a
fermentative test taking these parameters globally into
account appears to be highly relevant and close to
winemaking practices.
To test the relevance of MSM compared to natural
white grape musts, fermentation experiments were carried out using three natural musts (Vitis vinifera cv.
Sauvignon) from a Bordeaux vineyard. For this purpose, ethanol tolerance and volatile-acidity production
of three strains were compared after fermentation in
MSM or natural musts (Table 3). These strains were
chosen for their very different properties. For natural
must fermentation, implantation of the selected strain
was controlled analyzing Delta-PCR amplified DNA
polymorphisms [21] (data not shown). As shown in
Table 3, strains 1, 2 and 3 appeared to be significantly
(a ¼ 0:05) different with regard to their volatile-acidity
production, both in MSM and natural must tests. Regarding ethanol tolerance, MSM and natural musts gave
only partly similar results. In fact, strains 1 and 3 gave
715
similar values in MSM and in the three musts fermented,
with respectively low and high levels of ethanol tolerance. However, strain 2 presented lower ethanol tolerance in musts than in MSM, but these results are to be
considered in the light of the growth difficulties discussed below.
3.2.2. Distribution of quantitative traits among industrial
wine yeast progeny
About ten single-spore cultures obtained from each
industrial strain were tested for three relevant enological
criteria: ethanol tolerance, volatile acidity and H2 S
production (Table 4). For the majority of the populations analyzed, single-spore clones exhibited significant
differences from their parental strains. As expected for
polygenic determined quantitative traits, the distribution of technological values in a population of spore
clones derived from a parental strain did not show a 2:2
segregation. To illustrate these results, the distribution
of volatile-acidity production for the entire population
tested is presented in Fig. 2. The levels of acetic acid
(volatile acidity) formed by spore clones compared to
those obtained by each parental strain were higher,
lower or not significantly different. Other traits gave a
similar distribution.
Different patterns of distribution of the values were
obtained depending on the parental strain and the trait.
For example, the distribution patterns of volatile acidity
values differed in two ways. Firstly, comparison of parental versus progeny trait levels indicated various degrees of amplitude. We established the ratio of each
spore clone value/parental strain value. Taking arbitrarily into account ratios of <0.5 or >2 for volatile
acidity, strain VL1 gave only one spore clone with very
different values compared to the parental strain. On the
contrary, strains L43 and SAP gave numerous spore
clones with very different values (respectively 5 and 12
spore clones) (Fig. 2). Similar results were also observed
for ethanol tolerance and H2 S production (Table 4).
Thus, the amplitude of distribution was typical of one
trait in one strain, a result probably due to the different
degree of heterozygosity for loci involved in a given trait
Table 3
Technological properties of enological strains in a model synthetic medium (MSM) and in three natural wine musts
Strain
Strain 1
Strain 2
Strain 3
Acetic-acid production (g l1 )
Ethanol production (% w/v)
MSMa
MSMb
0.45 b
0.55 a
0.19 c
Sauvignon mustsa
1
2
3
Mean
0.39
NI
0.22
0.33
0.48
0.21
0.40
NI
0.18
0.37 b
0.48 a
0.20 c
16.80 b
16.5 b
14.5 a
Sauvignon mustsb
1
2
3
Mean
15.43
NI
13.48
15.98
14.23
13.92
14.77
NI
14.05
15.54 b
14.23 a
13.82 a
Strains 1, 2 and 3 are derived from the industrial wine yeast strains presented in Table 1. Each value represents the mean of three replicates.
Within the column, means followed by a different letter are significantly different (Newman–Keuls test, a ¼ 0:05).
a
Sugar concentration adjusted up to 200 g l1 .
b
Sugar concentration adjusted to 300 g l1 . NI: not implanted.
716
P. Marullo et al. / FEMS Yeast Research 4 (2004) 711–719
Table 4
Technological properties of four industrial strains and their progenya
Strain
H2 S production (colorimetric scale 1–5)
Volatile acidity production (g l1 of acetate) Ethanol tolerance (Ethanol% w/v)
Parent
Parent Derived spore clones
Derived spore clones
Mean Range Different Better
(%)
(%)
SAP
L43
VL1
VL3c
5.0
3.0
3.0
3.5
4.2
2.0
3.0
3.3
3–5
1–4
2–4
2–5
57
77
28
33
57
66
14
11
0.12
0.75
0.24
0.34
Parent Derived spore clones
Mean Range
Different Better
(%)
(%)
0.39
0.40
0.28
0.45
100
100
57
55
0.12–0.83
0.09–0.75
0.18–0.52
0.20–0.69
0
100
28
11
12.6
15.0
14.5
14.2
Mean
Range
Different Better
(%)
(%)
11.8
15.0
14.0
14.3
10.2–14.6
13.9–16.8
11.2–15.5
13.4–15.2
64
66
14
0
7
55
7
0
a
Newman–Keuls analysis was performed for each parental strain and its progeny (a ¼ 0:05) to determine the proportion of spore clones different
from and better than parental strains. Each value represents the mean of three triplicates.
Fig. 2. Volatile-acidity production for four parental strains and their derived spore clones. Bar graphs indicate level of production of volatile acidity
(g l1 of acetate) for each parental strain (hatched area) and derived spore clone. Progeny presenting higher, lower or non-significant differences from
their parental strain are, respectively, shown in black, white or grey. Statistical analysis was carried out by Newman–Keuls test (a ¼ 0:05). A dotted
line indicates mean of volatile-acidity production for spore clone population.
among the parental strains analyzed. Consequently, trait
segregation of various characters can be obtained.
Secondly, the respective positions of parental strain
values among the spore clone values were different. For
example, regarding volatile-acidity production, values of
L43, SAP and VL1 occupied, respectively, the highest,
the lowest and the median position in a population
constituted by their progeny (Fig. 2). Therefore, the
relations of dominance/recessivity between alleles involved in the control of a particular trait are different
from one strain to another.
As a direct consequence of these findings, spore
clones presenting better technological properties than
those of the parent were frequently obtained for all the
traits measured. For the entire population tested (46
spore clones derived from four industrial strains), eighteen were significantly better than their own parental
strain for one trait, five were significantly better for two
traits and two for all traits.
3.2.3. Analysis of heritability of technological traits in
wine yeast
The heritability of ethanol tolerance and volatileacidity production was estimated as (rP rE Þ=rP . The
variance of each progeny population tested, rP , explained the genetic + environmental variance, whereas
the variance of the ISS strain in the different batch series, rE , explained only the environmental fraction of
the phenotypic variance. For each genetic background
and each trait analyzed, this ratio reached 0.8, indicating
the high degree of heritability of these traits.
Principal-component analysis of the three technological properties was carried out on the population
tested to illustrate whether the distribution of trait val-
P. Marullo et al. / FEMS Yeast Research 4 (2004) 711–719
717
Fig. 3. Principal component analysis (PCA) of 48 yeast strains for three enological traits. PCA was carried out with the entire population studied in
this work. For three traits analyzed (ethanol tolerance, volatile acidity or H2 S production), maximal projection conserves 83% of the information
with two components axis, 1 and 2, that explain 50% and 33% of the total inertia, respectively. Industrial strains and their progeny are noted in the
legend.
ues could demonstrate inheritance from a determined
parental strain. The projection in Fig. 3 conserves 83%
of the information with two components, explaining
respectively 50% and 33% of the total inertia. Despite
the strong amplitude in phenotypic values frequently
found between a parental strain and its progeny, spore
clone values were clustered around their parental strain.
Axis 1 relates to discrimination between SAP and L43
clusters and this axis is strongly correlated with two
variables: ethanol tolerance and H2 S production. Axis 2
clearly separates the VL1 and VL3c clusters. The most
explanatory variable correlated with axis 2 is volatileacidity production. The visual clustering of values
presented in Fig. 3 was confirmed by a DFA (discriminating factorial analysis) (data not shown).
In other words, the L43 cluster presents a relatively
strong ethanol tolerance, a wide range of volatile-acidity
production and a low level of H2 S production. In contrast, SAP exhibits a very low ability to tolerate ethanol, a
high level of H2 S production and a very variable volatileacidity production. Finally, the VL1 and VL3c clusters
are principally separated by volatile-acidity production.
quality due to different vintage and geographic areas
makes it impossible to compare strains and studies over
many years. Model synthetic media, which provide
better reproducibility [26], often lack numerous nutritive
elements naturally present in musts. For example, the
lipids contained in vegetal lees play an important role in
the regulation of volatile-acidity production [27].
Moreover, must turbidity improves the speed of fermentation [28]. Oxygenation of the medium at exponential phase is also an important parameter for
performing fermentation [18,29,30].
The MSM medium developed here takes these metabolic requirements into account. Indeed, one series of
tests accomplished with the same strain, in the same
batch of medium and at the same time, gave very similar
results. Nevertheless, strain ISS taken as the standard
gave slightly but not significantly different results with
different batches of medium. To take these slight interbatch differences into account, values obtained with the
strains tested were corrected by using the actual ISS
value for the particular batch medium, thus leading to
more accurate results. Nevertheless, the results obtained
in Figs. 2 and 3 and Table 4 are globally similar even
without using this correction.
4. Discussion
4.1. Development of model synthetic medium and fermentation procedure to test wine yeast traits
4.1.1. Reproducibility of the test
Most parts of yeast selection programs are carried
out with grape musts. However, the variability of grape
4.1.2. Enological relevance of the tests
First, the parental values we obtained are in good
agreement with the known practical properties of the
commercialized strains used. This empirical observation,
although not rationalized, is very encouraging. Second,
the relevance of the tests is demonstrated by the good
correlation between the ranking obtained in MSM and
718
P. Marullo et al. / FEMS Yeast Research 4 (2004) 711–719
in natural juice (Table 3). Nevertheless, an important
enological parameter is the competition of inoculated
strains against indigenous flora. This parameter, which
is partly linked to growth rate and length of lag phase,
was not tested in our procedure and can sometimes alter
the enological values of the strain. As shown in Table 3,
strain 2 did not finish fermentation in non-sterile musts.
This difficulty is probably due to the well-known slow
growth rate of this strain (data not shown).
4.2. Heritability of enological properties
4.2.1. Improvement of wine yeast strain by meiosis
Wine yeasts are frequently heterozygous for alleles
controlling complex traits like the production of H2 S,
acetic acid, acetaldehyde and other by-products [4,5,14]
(and this study). This can be due to the accumulation of
mutational events during propagation of a given yeast in
its natural environment [4]. Thus, the industrial strains
commonly selected from indigenous flora might have
some genetic faults that can be eliminated by sporulation [31]. Our work demonstrates the effectiveness of
meiosis in providing clones with different and frequently
better properties than their parental strain. In fact,
among the populations tested, almost 50% of spore
clones were better than the parental strain for one or
more traits.
The karyotypic pattern of spore clones derived from
industrial strains was controlled by pulsed-field electrophoresis. The karyotypes showed distinct patterns
between industrial strains and derived spore clones. As
a result, the spore clones generated by meiosis presented
a distinct genetic pattern and could be directly used as a
new strain for commercial purposes. Furthermore, aneuplo€ıd wine yeasts contain extra-numerary chromosomes or portions of them. These atypical karyotypes
are also obtained in derived spore clones as expected
[32–34]. Thus, extra-numerary copies of genes involved
in quantitative traits could be present in some progeny
populations. Some of the strong variation found among
industrial strain progeny may be due to this phenomenon.
4.2.2. Heritability of some quantitative traits as an asset
for wine yeast improvement
The PCA demonstrates the inheritable nature of some
enological traits. Spore clones derived from an industrial
strain formed a cluster that globally presented the
‘‘technological profile’’ of the parental strain (Fig. 3).
This finding emphasizes the strong contribution of genetic determinism in the enological quantitative traits of
yeast. In practice, the selection of new strains focusing
on a particular trait should be carried out on the progeny of strains already well defined technologically for
this trait, instead of screening less characterized populations of natural isolates. This strategy could rapidly
lead to effective results because industrial strains are
readily available and their performances are well documented. Moreover, strains used as parents should be
suitable in industrial processes. Thus, some spore clones
derived from such parents probably should present industrial aptitudes similar to those of their parental strain
for industrial growth and the dry survival of cells.
Our results offer two main perspectives that could
form the basis of breeding strategies. Firstly, the degree
of heterozygosity for alleles controlling a given trait
differs from one strain to another. In fact, some industrial strains provide spore clones with very different
values compared to themselves (Fig. 2, SAP and L43).
In other cases, a non-significant difference was found
between the parental strain and the progeny (Table 4,
VL3c for ethanol tolerance). Therefore, the improvement of a homozygote strain for such alleles cannot be
performed by simple sporulation but requires the introduction of external alleles (by breeding) conferring
better properties.
It is interesting to note that the two parental strains
bearing the (ho) locus seem to be more heterozygous than
the homothallic strains, so their progeny is more variable.
One explanation might be the possibility for such strains
to generate by sporulation spore clones with a stable
sexual form and able to mate with strains from other
genetic backgrounds. On the other hand, spore clones
derived from homothallic strains are likely to undergo
self-diploidization and to have a low level of heterozygosity [4]. Consequently, this strategy seems to be more
effective with heterothallic strains or with homothallic
strains which have accumulated many mutations.
Secondly, the dominance/recessivity relationship between alleles involved in the control of a particular trait
differs from one strain to another, a finding illustrated
by the position of the parental strain value among the
progeny values (Fig. 2 and Table 4). With a view to
improving yeast strains by breeding methods, the
knowledge of dominance/recessive relationships between
such alleles could help in choosing appropriate candidates to be crossed.
Taken together these results constitute an initial step
in establishing the rational basis for improvement of
enological yeast strains through a breeding strategy.
Acknowledgements
The authors gratefully acknowledge Olivier Lavialle
(Ecole Nationale d’Ingenieur des Travaux Agricoles de
Bordeaux) for assistance in the statistical study. Wine
analyses were carried out with the technical collaboration of SARCO Laboratory, France.
This work was supported by a CNRS (Centre National de Recherche Scientifique) grant: ‘‘puces a
ADN’’.
P. Marullo et al. / FEMS Yeast Research 4 (2004) 711–719
References
[1] Mortimer, R. and Polsinelli, M. (1999) On the origins of wine
yeast. Res. Microbiol. 150, 199–204.
[2] Peynaud, E. and Domercq, S. (1953) Ann. Technol. Agric. 4, 265–
300.
[3] Benıtez, T., del Castillo, L., Aguilera, A., Conde, J. and CerdaOlmedo, E. (1983) Selection of wine yeast for growth and
fermentation in the presence of ethanol and sucrose. Appl.
Environ. Microbiol. 45, 1429–1436.
[4] Mortimer, R.K., Romano, P., Suzzi, G. and Polsinelli, M. (1994)
Genome renewal: a new phenomenon revealed from a genetic
study of 43 strains of Saccharomyces cerevisiae derived from
natural fermentation of grape musts. Yeast 10, 1543–1552.
[5] Sipiczki, M., Romano, P., Lipani, G., Miklos, I. and Antunovics,
Z. (2001) Analysis of yeasts derived from natural fermentation in
Tokaj winery. Antonie van Leeuwenhoek 79, 97–105.
[6] Volschenk, H., Viljoen, M., Grobler, J., Petzold, B., Bauer, F.,
Subden, R.E., Young, R.A., Lonvaud, A., Denayrolles, M. and
van Vuuren, H.J. (1997) Engineering pathways for malate
degradation in Saccharomyces cerevisiae. Nat. Biotechnol. 15,
253–257.
[7] Pretorius, I.S. (2000) Tailoring wine yeast for the new millennium;
novel approaches to the ancient art of winemaking. Yeast 16, 675–
729.
[8] Dubourdieu, D., Tominaga, T., Masneuf, I., Peyrot des Gachons,
C. and Murat, M.L. (2000). The role of yeasts in grape flavour
development during fermentation: the example of Sauvignon
blanc. In: Proceedings of the ASEV 50th anniversary annual
meeting, Seattle (Rantz, J.A.M., Ed.), Washington, pp.
196–203.
[9] Masneuf, I., Murat, M.L., Naumov, G.I., Tominaga, T. and
Dubourdieu, D. (2003) Hybrids S. cerevisiae X S. bayanus var.
uvarum having a high liberating ability of some sulfur varietal
aromas of Vitis vinifera Sauvignon Blanc wines. J. Int. Sci. Vigne
Vin. 36, 1–8.
[10] Prior, B.A., Baccari, C. and Mortimer, R.K. (2000) Selective
breeding of Saccharomyces cerevisiae to increase glycerol levels in
wine. J. Int. Sci. Vigne Vin. 33, 57–65.
[11] Romano, P., Suzzi, M.G., Grazia, G. and Zambonelli, C. (1985)
Improvement of a wine Saccharomyces cerevisiae strain by a
breeding program. Appl. Environ. Microbiol. 50, 1064–1067.
[12] Barre, P. (1982) Group determination of killer wine yeasts. Sci.
Aliment 2, 125–130.
[13] Romano, P., Suzzi, G., Turbanti, L. and Polsinelli, M. (1994)
Acetaldehyde production in Saccharomyces cerevisiae wine yeasts.
FEMS Microbiol. Lett. 118, 213–218.
[14] Romano, P., Suzzi, G., Mortimer, R. and Polsinelli, M. (1995)
Production of high levels of acetoin in Saccharomyces cerevisiae
wine yeasts is a recessive trait. J. Appl. Microbiol. 78,
169–174.
[15] Romano, P., Parraggio, M. and Turbandi, L. (1998) Stability in
by-product formation as a selection tool of Saccharomyces
cerevisiae wine yeasts. J. Appl. Microbiol. 84, 336–341.
[16] Soles, R.M., Ough, C.S. and Kunkee, R.E. (1982) Ester concentration differences in wine fermented by various species and strains
of yeasts. Am. J. Enol. Vitic. 33, 94–98.
719
[17] Murat, M.L., Masneuf, I., Darriet, P., Lavigne, V., Tominaga, T.
and Dubourdieu, D. (2001) Effect of Saccharomyces cerevisiae
yeast strains on the liberation of volatile thiols in Sauvignon blanc
wine. Am. J. Enol. Vitic. 52, 136–139.
[18] Ribereau-Gayon, P., Dubourdieu, D., Doneche, B. and Lonvaud,
A. (2000) In: Handbook of Enology, vol. 1. John Wiley & Sons,
New York.
[19] Bellis, M., Pages, M. and Roizes, G. (1987) A simple and rapid
method for preparing yeast chromosomes for pulsed field gel
electrophoresis. Nucl. Acids Res. 15, 6749.
[20] Lavigne, V. (1995). Origine du methionol dans les vins blancs secs.
In: Lavoisier TEC & DOC 5th International Symposium of
Enology, Bordeaux, France, pp. 251–255.
[21] Masneuf, I. and Dubourdieu, D. (1994) Comparaison de deux
techniques d’identification des souches de levures de vinification
basee sur le polymorphisme de l’ADN genomique: reaction de
polymerisation en cha^ine et analyse des caryotypes. J. Int. Sci.
Vigne Vin. 28, 153–160.
[22] Johnston, J.R., Baccari, C. and Mortimer, R.K. (2000) Genotypic
characterization of strains of commercial wine yeasts by tetrad
analysis. Res. Microbiol. 151, 583–590.
[23] Galitski, T., Saldanha, A.J., Styles, C.A., Lander, E.S. and Fink,
G.R. (1999) Ploidy regulation of gene expression. Science 285,
251–254.
[24] Frezier, V. and Dubourdieu, D. (1992) Ecology of yeast strains
Saccharomyces cerevisiae during spontaneous fermentation in a
Bordeaux winery. Am. J. Enol. Vitic. 43, 375–380.
[25] D’Amore, T. and Stewart, G. (1987) Ethanol tolerance of yeast.
Enzyme Microb. Technol. 9, 322–330.
[26] Giudici, P. and Zambonelli, C. (1992) Biometric and genetic study
on acetic acid production for breeding of wine yeast. Am. J. Enol.
Vitic. 43, 370–374.
[27] Delfini, C., Pessione, E., Garcia Moruno, E. and Giunta, C. (1992)
Localization of volatile acidity reducing factors in grape. J. Ind.
Microbiol. 11, 19–22.
[28] Ollivier, C., Stonestreet, T., Larue, F. and Dubourdieu, D. (1987)
Incidence de la composition collo€ıdale des mo^
uts blancs sur leur
fermentescibilite. Conn. Vigne Vin. 21, 59–70.
[29] Bely, M., Sabblayroles, J.M. and Barre, P. (1990) Automatic
detection of assimilable nitrogen deficiencies during alcoholic
fermentation in enological conditions. J. Ferment. Bioeng. 70,
246–252.
[30] Sablayrolles, J.M., Dubois, C., Manginot, C., Roustan, J.L. and
Barre, P. (1996) Effectiveness of combined ammoniacal nitrogen
and oxygen additions for completion of sluggish and stuck wine
fermentations. J. Ferment. Bioeng. 82, 377–381.
[31] Ramirez, M., Regodon, J.A., Perez, F. and Rebollo, J.E. (1999)
Wine yeast fermentation vigor may be improved by elimination of
recessive growth-retarding alleles. Biotechnol. Bioeng. 65, 212–218.
[32] Bakalinsky, A.T. and Snow, R. (1990) The chromosomal constitution of wine strains of Saccharomyces cerevisiae. Yeast 6, 367–382.
[33] Bidenne, C., Blondin, B., Dequin, S. and Vezinhet, F. (1992)
Analysis of the chromosomal DNA polymorphism of wine strains
of Saccharomyces cerevisiae. Curr. Genet. 22, 1–7.
[34] Miklos, I., Vargas, T., Nagy, A. and Sipiczki, M. (1997) Genome
instability and chromosomal rearrangements in a heterothallic
wine yeast. J. Basic Microbiol. 37, 345–354.