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Inheritable nature of enological quantitative traits is demonstrated by meiotic segregation of industrial wine yeast strains
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
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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).
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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
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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.
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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.
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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
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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.
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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-
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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
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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’’.
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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.
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[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.
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
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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).
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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
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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.
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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
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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.
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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-
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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
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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’’.
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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.