Curr Microbiol (2015) 70:441–449 DOI 10.1007/s00284-014-0741-2

Study of Saccharomyces cerevisiae Wine Strains for Breeding Through Fermentation Efficiency and Tetrad Analysis ´ beda Mo´nica Ferna´ndez-Gonza´lez • Juan F. U Ana I. Briones

•

Received: 28 March 2014 / Accepted: 28 October 2014 / Published online: 2 December 2014 Ó Springer Science+Business Media New York 2014

Abstract One of the issues that most concerns to both winemakers and producers of active dry yeasts is the stuck and sluggish fermentations of grape musts with high levels of sugar, reflecting the inability of inoculated yeast strain to complete the fermentation process. It is difficult to obtain a wine strain that possesses both adequate oenological and technological properties; thus, the correct approach to solving these problems is the application of breeding programs primarily focused on both properties. The first step toward this process is to characterize the phenotypic diversity between potential parental strains. In the present study, we have analyzed the fermentative behavior of 26 Saccharomyces cerevisiae wine strains in high-sugar conditions at 20 °C, using a range of tests, such as sporulation ability, spore viability, and tetrad analysis to determine the tolerance of these yeasts to several stress conditions. Most tested strains were homothallic and heterozygous for more than one character. Two auxotrophic derivatives with defects in amino acid or nucleic acid metabolism were obtained, and these strains could potentially be used for the development of hybridization techniques without using laboratory strains.

Electronic supplementary material The online version of this article (doi:10.1007/s00284-014-0741-2) contains supplementary material, which is available to authorized users. ´ beda
A. I. Briones M. Ferna´ndez-Gonza´lez (&)
J. F. U Regional Institute of Scientific Applied Research (IRICA), University of Castilla-La Mancha, Edificio Marie Curie, Avda. Camilo Jose´ Cela, s/n, 13071 Ciudad Real, Spain e-mail: [email protected] M. Ferna´ndez-Gonza´lez Albacete Scientific and Technology Park, Paseo de la Innovacio´n, 1, 02006 Albacete, Spain

Introduction During the alcoholic fermentation of wine, yeasts are primarily responsible for transforming the glucose and fructose in the juice into mainly ethanol and CO2 . Musts contain equal fructose and glucose amounts, ranging from 160 to 300 g/L [19]. Castilla La Mancha is the largest wine-producing region worldwide, and frequently as a consequence of climate change [23], an increasing amount of sugar has been observed in musts, which is affecting the overall quality of the final product. Although Saccharomyces strains generally consume both glucose and fructose during fermentation, they consume glucose faster than fructose. This glucophilic behavior reduces the glucose/ fructose ratio, leading residual fructose amounts at the end of fermentation [5]. During this phase of fermentation, when the nitrogen sources have been completely consumed and the ethanol concentration is high, some strains have difficulties fermenting the residual fructose, resulting in a sluggish or stuck fermentation [2]. The wines obtained from stuck fermentations are more susceptible to microbial spoilage, generating undesirable metabolites responsible for off-flavors. Although fermentation can be rescued through re-inoculation with a strong fermenting, alcohol tolerant yeast strain that utilizes fructose and aeration, or metabolizes adjuvants such as ‘yeast cell walls’, the additional operation time is a burdensome cost to the winery and the active dry yeast earns a poor reputation, leading to significant losses for the sector. In recent years, the demand for yeast strains with an optimal and heterogeneous enological and technological profile, for use as starter culture, has increased. Strain improvement strategies are numerous and often complementary, but because the use of recombinant yeasts has not been readily accepted, only classical techniques, such as clonal selection of variants, mutation

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and selection, and mating/hybridization, are currently used to produce food-grade starter cultures [7]. Wine yeast strains are primarily diploid, aneuploid, or polyploid, homothallic [33], highly heterozygous [12, 34], and sporulate poorly, thus the few spores that are generated have low viability [29], making it difficult to improve these strains using sexual hybridization techniques and genetic analyses. However, improvements have been made through the use of classical genetics [17] to study the regulation of of various properties, including improved dryness, thermal and osmotic stress, ethanol tolerance, and the extraction of color compounds from the fruit skins using cryotolerant strains [10]. Flocculation has also been successfully introduced [32]. In addition, undesirable properties, such as the reduced production of H2S, have been eliminated [7], and a wide range of growth temperatures [27], increased glycerol production or desirable volatiles [4, 18] have been achieved. The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection, and tetrad analysis, form the basis of yeast genetics [36].The aim of the present work was to select wine yeast strains as candidates for inbreeding program. For this purpose, we characterized 26 Saccharomyces wine strains and their monosporic clones with regards to a panel of significant oenological traits, including the total sugar consumption at low temperatures in high-sugar musts, and adequate sporulation rates for the hybridization of the strain or strains derived from this study, with other strains previously shown to produce relatively high concentrations of positive flavor compounds [8].

Materials and Methods Strains The strains used in this study are listed in Table 1. These strains were all previously isolated from wine fermentations and stored in 15 % glycerol at -80 °C in the University of Castilla La Mancha (UCLM) yeast collection. Three commercial Saccharomyces cerevisiae strains were also used (encoded to maintain confidentiality). ADY1 exhibits excellent fermentative skills and is resistant to extreme fermentation conditions, and ADY2 and ADY3 produce relatively high concentrations of positive flavor compounds. The strains were identified based on fragment obtained from the restriction digest of the ITS-5.8S rDNA region [14]. To characterize the 26 S. cerevisiae strains, repeated interspersed delta sequences were PCR amplified using primers d12 and d21 according to Legras and Karst [25].

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Fermentation of High-Sugar Musts Fermentation was performed in duplicate 125 mL Erlenmeyer flasks containing 75 mL of a white must containing 127 g/L of glucose and 133 g/L of fructose at pH 3.7 (Mostos Espan˜oles, SA). The musts were individually inoculated with each of the 26 yeast strains at 2 9 106 cells per mL. The flasks were maintained at 20 °C without agitation and plugged with Mu¨ller valves to facilitate the evolution of only CO2 from the system. The fermentation evolution was assessed two to three times a day to determine weight loss [31] and time required for constant weight was 28 days. The maximum fermentation rate (lmax) (h-1) was calculated using a modified Gompertz equation [37] with Origin8.0 software. The concentrations of glucose, fructose, glycerol, and ethanol were measured in the fermentation products through HPLC using a Biorad Aminex HPX-87H (300 9 7.8 mm) column and a refractive index detector. Sulfuric acid (0.245 g/L) was used as the mobile phase and the oven temperature was 25 °C [13]. The concentrations of glucose, fructose, and glycerol were also measured by enzymatic analysis (Chemelex, Canovelles). Analysis of Yeast Tolerance to Several Stress Conditions As one of the key features in wine fermentation, we examined the ability of the yeast strains to grow under different stress conditions, such as SO2 (250 and 500 mg/ L), ethanol (12 and 16 % w/v), sucrose (200, 250, and 300 g/L), pH (2.5, 2.7, and 3), and temperature (15, 28, and 37 °C). The media were prepared using YPD agar plates (1 % yeast extract, 2 % peptone, 2 % dextrose, and 2 % agar), containing the appropriate stress, and there was an YPD plate that was included as a no-stress control. The inability of yeast to grow on a non-fermentable carbon source was tested on YPG medium (1 % yeast extract, 2 % peptone, 3 % glycerol, 1 % ethanol [added after autoclaving), and 2 % agar]. Auxotrophic cells were detected on minimal media lacking amino acids (SD). To determine the characteristics of natural (non mutagenized) auxotrophic variants obtained after tetrad dissection (see below), drop-out medium (DO) supplemented with the appropriate auxotrophic requirements [30] was used, and synthetic complete medium was used as a control (SC). Copper resistance (CUP) was considered as growth on SC plates containing 60 mg/L copper ion, and hydrogen sulfide production was assayed based on the color (cream to brown) of the colonies grown on Biggy agar (Oxoid company, England) [29]. The strains were spotted onto solid media after overnight growth in YPD broth, washed with water, and adjusted to a concentration of 3 9 103 cells

M. Ferna´ndez-Gonza´lez et al.: Study of Saccharomyces cerevisiae Wine Strains Table 1 Maximum specific fermentation rate (lmax), glucose (G), fructose (F), residual sugar (RS), glycerol (Gly), and alcohol degree for 26 wine yeast strains

Strains

lmax (h-1)

G (g/L)

UCLMS-3

0.40 ± 0.05

0.4 ± 0.3

UCLMS-4

0.29 ± 0.01

1.4 ± 0.1

UCLMS-31

0.35 ± 0.04

0.6 ± 0.2

UCLMS-33

0.43 ± 0.12

UCLMS-38

443

Gly (g/L)

°Alc (%v/v)

6.7 ± 1.2

7.3 ± 0.0

14.8 ± 0.4

18.0 ± 3.2

7.1 ± 0.1

13.6 ± 0.2

7.7 ± 0.8

7.8 ± 0.2

14.2 ± 0.3

0.2 ± 0.1

3.0 ± 0.5

7.8 ± 0.4

14.1 ± 0.3

0.26 ± 0.03

6.7 ± 0.6

37.9 ± 0.9

7.2 ± 0.1

12.4 ± 0.2

UCLMS-59

0.31 ± 0.01

0.2 ± 0.0

4.5 ± 0.5

7.9 ± 0.1

15.1 ± 0.3

UCLMS-71

0.26 ± 0.02

1.9 ± 0.2

28.4 ± 1.7

7.2 ± 0.0

13.8 ± 0.0

UCLMS-75

0.37 ± 0.05

0.3 ± 0.1

5.4 ± 0.3

7.8 ± 0.3

15.0 ± 0.1

UCLMS-147

0.41 ± 0.04

0.2 ± 0.0

1.8 ± 0.4

8.8 ± 0.1

14.4 ± 0.0

UCLMS-202

0.52 ± 0.03

0.1 ± 0.1

3.7 ± 0.2

7.8 ± 0.3

15.2 ± 0.3

UCLMS-218

0.34 ± 0.08

0.2 ± 0.1

14.9 ± 2.3

7.9 ± 0.3

14.4 ± 0.4

UCLMS-220

0.30 ± 0.01

0.6 ± 0.0

7.8 ± 0.0

6.7 ± 0.1

15.4 ± 0.4

UCLMS-227

0.31 ± 0.07

1.3 ± 0.2

21.2 ± 0.4

7.4 ± 0.1

14.0 ± 0.7

UCLMS-228

0.36 ± 0.01

0.4 ± 0.0

2.5 ± 0.2

7.9 ± 0.3

14.5 ± 0.3

UCLMS-234 UCLMS-239

0.32 ± 0.01 0.38 ± 0.04

0.5 ± 0.1 0.1 ± 0.0

9.0 ± 1.2 3.4 ± 0.2

8.1 ± 0.1 7.3 ± 0.2

13.2 ± 0.3 14.3 ± 0.5

UCLMS-241

0.54 ± 0.09

0.1 ± 0.0

4.1 ± 1.0

8.2 ± 0.3

14.9 ± 0.1

UCLMS-263

0.60 ± 0.10

0.3 ± 0.0

3.9 ± 0.7

7.8 ± 0.0

14.6 ± 0.4

UCLMS-267

0.42 ± 0.02

0.1 ± 0.0

1.6 ± 0.3

8.2 ± 0.2

14.6 ± 0.3

Fermentation was performed in duplicate using grape musts containing 260 g/L of sugar at 20 °C. The concentration of the final wine product was determined

UCLMS-273

0.37 ± 0.03

0.3 ± 0.0

7.0 ± 0.5

8.2 ± 0.1

14.7 ± 0.0

UCLMS-277

0.44 ± 0.04

0.1 ± 0.0

1.5 ± 0.2

7.9 ± 0.0

14.9 ± 0.2

UCLMS-280

0.32 ± 0.04

1.2 ± 0.3

23.4 ± 3.4

7.7 ± 0.0

13.7 ± 0.3

UCLMS-287

0.37 ± 0.06

0.3 ± 0.0

11.4 ± 1.4

7.8 ± 0.4

14.6 ± 0.4

ADY1

0.58 ± 0.01

0.0 ± 0.0

1.3 ± 0.0

8.2 ± 0.2

15.2 ± 0.1

UCLMS University of Castilla La Mancha, collection number, ADY active dry yeasts

ADY2

0.28 ± 0.03

0.3 ± 0.0

14.2 ± 0.6

7.6 ± 0.2

14.6 ± 0.3

ADY3

0.24 ± 0.01

3.3 ± 1.0

28.3 ± 1.6

8.3 ± 0.1

13.1 ± 0.3

per spot. Except for temperature stress, all plates were incubated aerobically at 28 °C and examined 1–5 consecutive days for colony development. All the tests were performed in triplicate. Tolerance to each stress condition was indicated with numbers from 0 (absence of growth) to 3 (complete growth) for each strain (Supplementary Fig S1). Sporulation, Tetrad Dissection, and Spore Viability After optimizing the pre sporulation and sporulation conditions, including liquid or solid medium, and temperature and incubation time for wine strains (data not shown), the yeasts were grown on solid pre sporulation medium (PRE5) for 24 h, subsequently streaked onto solid sporulation medium (SPO1) [12], and incubated at 28 °C for 7 days until asci were observed microscopically. The sporulation frequency was microscopically determined after counting the number of asci (dyads, triads, and tetrads) in a total cell population of at least 300 (total asci scored divided by the sum of cells and asci counted). The sporulation efficiency was calculated as the sum of four-

F (g/L)

and three-spored asci divided by the total number of asci [9]. Approximately, ten asci from each isolate were dissected using a micromanipulator (MSM System 400, Singer Instruments), plated onto YPD medium and incubated at 28 °C for 3–5 days. The spores that formed visible colonies were designated viable. Spore viability was determined as the number of viable spores divided by the total number of spores seeded. Tetrad Analysis The ability of the single spore progeny from asci with four or three viable meiotic products to ferment sucrose (SUC), raffinose (RAF), maltose (MAL), and galactose (GAL) was examined on yeast extract-peptone plates containing the corresponding sugars and the pH indicator bromothymol blue (BTBL agar) [29]. Fermentation-positive cultures turned the agar beneath the strain from blue to yellow. Yellow (Y) colonies were also observed on sugar media. Copper resistance (CUP), hydrogen sulfide production (H2S), non-fermentable carbon source assimilation (YPG),

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and auxotrophy (SD) were detected as described above. Homothallism was microscopically determined as the ability of the meiotic derivatives from an ascus to sporulate [26], and mating type (MAT) was analyzed through PCR using MATa- and MATa-specific primers [21].

In the present study, the inter-delta sequence analysis generated strain-specific banding patterns. The combination of primers d12 and d21 [25] showed high discriminatory power to differentiate the 26 S. cerevisiae strains. Fermentations

Statistical Analysis The data obtained from the alcoholic fermentation trial were statistically analyzed with SPSS 17.0 software for Windows, using one-factor ANOVA. The differences were evaluated using Duncan’s test at P B 0.05 (Tukey´s test).

Results and Discussion Identification of Isolates To identify the strains belonging to S. cerevisiae PCR– RFLP analysis of the 5.8S-ITS rRNA was performed. In the present study, two different restriction patterns for the 5.8S-ITS region were obtained. A total of 25 yeasts showed typical S. cerevisiae restriction patterns using the endonucleases HaeIII, HinfI, and CfoI as previously described [14]; However, strain UCLMS-147, isolated from a refermented wine, showed a different PCR–RFLP pattern using HaeIII (Fig. 1). Indeed, the restriction pattern of this strain was 320, 230, 160, 155 (bp), whereas the normal S. cerevisiae profile was 320, 230, 180, and 150 (bp). Tofalo et al. recently published a similar result [35].

Fig. 1 Restriction fragment length polymorphism in the 5.8S-ITS regions of S.cerevisiae strains through HaeIII digestion. Lane M molecular ladder 100 bp (biotools). Lanes 1, 3, 4, 5, and 6 typical restriction profiles and lane 2 atypical restriction profile obtained for strain UCLMS-147

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Small-scale fermentations were performed on high-sugar musts at 20 °C to study the fermentation of the 26 selected strains. The maximum fermentation rate (lmax) and glucose, fructose, ethanol, and glycerol concentrations were measured at the end of fermentation. The results are shown in Table 1. Regarding the maximum rate of fermentation at 20 °C, ADY3 was the slowest of all (0.24 h-1) followed by four strains with a rate of B0.30 h-1. However, there were no significant differences between the yeasts which were observed when the lmax ranged from 0.24 to 0.36 h-1. Strain UCLMS-263 exhibited the fastest fermentation rate and three other strains showed values above 0.50 (ADY1, UCLMS-202 and -241). Most of the strains showed intermediate rates of fermentation, with values between 0.30 and 0.44 h-1. The 26 yeast strains displayed considerable differences in fermentation behavior. After fermentation for 28 days, the residual sugar (RS) levels, expressed as the sum of glucose and fructose in the growth medium of 14 strains, were higher than 5 g/L. Only four strains (ADY1, UCLMS-147, UCLMS-277, and UCLMS-267) showed RS values of B2 g/L, and eight strains showed RS values between 2 and 5 g/L. However, no statistically significant differences between the yeasts with RS values ranged from 1.9 to 4.2 g/L. In general, the lmax was associated with the RS content at the end of fermentation. The yeasts generating the highest concentration of total sugars in the medium generally had a lower lmax, and the four strains generating a sugar content of less than 2 g/L had a lmax greater than 0.40 h-1. However, UCLMS-59 and -227, which had a lmax of 0.31 h-1, showed highly variable RS values of 4.7 and 22.4 g/L, respectively. Almost all yeasts strains were able to deplete medium glucose, to less than 1 g/L, and six strains achieved higher levels of depletion, while the strains ADY3 and UCLMS38, exhibited significantly more glucose content than the other strains. The fructose values were much more variable, ranging between 44.6 and 1.3 g/L. The studied yeasts differed in fructose consumption, indicating potentially problematic musts. Fortunately, the S. cerevisiae wine yeast strains differed in glucose and fructose utilization [5], thus the appropriate yeast choice would reduce the risk of stuck or sluggish fermentations. The concentration of glycerol in the wines varied between 6.7 and 8.8 g/L, and

M. Ferna´ndez-Gonza´lez et al.: Study of Saccharomyces cerevisiae Wine Strains

strain UCLMS-147 produced the highest concentration of glycerol (8.8 g/L), Therefore, this strain might be an ideal candidate to meet recent market demands for wines with a higher glycerol content and lower ethanol concentration [1]. The ethanol production was between 15.4° and 12.4°, and there were no statistically significant differences in the ethanol content among the 11 strains, ranging from 14.6° to 15.4°. However, the UCLMS-38 strain, which had a higher RS value than other strains, produced significantly less ethanol (12.4°).

strains. The assayed strains showed wide variation in growth at the extreme pH of 2.5 and a high ethanol content of 16 % w/v. Other factors, such as high and low temperatures greatly affected the growth of some of the yeasts tested. At 37 °C, the majority of strains of S. cerevisiae grew after 24 h of incubation, except strain UCLMS-147 (data not shown). This result is consistent with that of Belloch et al. (2008) [3]; however, yeast growth was highly variable at 15 °C. For copper resistance, half of the tested strains grew in this medium, thus this parameter could be used as a phenotypic marker for the selection of future hybrids. Regarding H2S production, there was much variability. A total of eight cream or pale colonies were observed on Biggy agar and seven high H2S producers (dark hazel color) were identified. Although many of the phenotypic differences observed were likely neutral, providing no benefit or disadvantage to the tested strains, some differences were more likely to confer a selective advantage. Copper sulfate resistance in

Phenotypic Analysis of the Parental Strains Table 2 shows the stress conditions tested among the yeasts. Little variation in growth on minimal medium lacking supplemental amino acids and containing non-fermentable glycerol and high concentrations of sucrose and SO2 was observed. Presumably, auxotrophy, defects in respiration, and no growth under must conditions would represent a significant selective disadvantage for wine

Table 2 Tolerance to several stress factors, sporulation percentage (ST), spore viability (SV), sporulation efficiency (SE), and percentage of asci with 4, 3, 2, 1, or no viable spores in the 26 yeast strains

Tolerance to different stress factors is indicated by numbers from 0 (absence of growth) to 3 (maximum colony development). Growth at low pH was observed at 24 h of incubation at 28 °C. Growth at 15 °C was recorded after 72 h of incubation. Growth at 16 % of ethanol and CUP resistance were observed at 72 h of incubation at 28 °C. Quantitative variation for production of H2S on BIGGY media were recorded after 5 days of incubation at 28 °C a Ten tetrads were dissected for all sporulated strains except 40 to UCLMS-202, and only eight viable colonies were obtained

Strains

445

Stress factor

ST

SV

15 °C

PH2.5

16 %alc

CUP

H2S

UCLMS-3

2

2

3

1

3

0

–

UCLMS-4

2

1

1

0

0

31.3

82.5

UCLMS-31

3

2

3

0

0

28.5

UCLMS-33

3

2

3

3

2

UCLMS-38

2

3

3

3

UCLMS-59

2

2

3

0

UCLMS-71

1

3

1

UCLMS-75

3

2

UCLMS-147

1

1

2 1

UCLMS-220 UCLMS-227

SE

Viable spore/ascus 4

3

2

1

0

–

–

–

–

–

–

80.0

30

50

10

10

0

80.0

71.9

20

80

0

0

0

4.8

92.5

100.0

80

0

10

10

0

1

61.5

22.5

8.2

0

0

10

90

0

1

43.3

47.5

76.9

10

40

10

20

20

0

0

0

–

–

–

–

–

–

–

2

0

0

0

–

–

–

–

–

–

–

3

0

0

41.8

77.5

87.0

0

70

20

10

0

3 2

3 2

0 0

0 3

24.5 0

5.0 –

42.1 –

0 –

2.5 –

2.5 –

10 –

85 –

3

3

3

1

1

44.2

47.5

26.3

0

20

50

30

0

1

2

2

2

0

73.3

95.0

42.4

80

20

0

0

0

UCLMS-228

2

2

2

3

3

79.2

72.5

45.2

10

70

20

0

0

UCLMS-234

1

3

2

3

3

48.4

90.0

33.3

70

20

10

0

0

UCLMS-239

2

3

1

3

3

0

–

–

–

–

–

–

–

UCLMS-241

2

2

1

0

0

0

–

–

–

–

–

–

–

UCLMS-263

2

2

1

0

1

56.6

65.0

56.7

0

60

40

0

0

UCLMS-267

2

1

2

3

1

56.6

97.5

91.2

90

10

0

0

0

UCLMS-273

3

3

2

3

3

66.7

100

85.3

100

0

0

0

0

UCLMS-277

2

2

1

2

2

47.2

45.0

76.5

40

0

40

0

20

UCLMS-280

1

2

1

1

3

64.7

70.0

59.1

20

50

20

10

0

UCLMS-287

2

3

1

3

2

68.3

45.0

20.9

0

0

80

20

0

ADY1

3

1

3

0

1

11.4

37.5

12.5

0

10

30

50

10

ADY2 ADY3

2 3

2 3

3 1

0 0

2 1

23.4 17.4

65.0 77.5

69.2 12.5

10 30

50 50

30 10

10 10

0 0

UCLMS-202 UCLMS-218

a

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European vineyard strains might have been derived through positive selection, as copper has long been used as an antimicrobial agent in vineyards and orchards [24, 28]. After incubation for 5 days, the growth of all yeasts under all tested conditions except in the presence of copper was observed. Sporulation and Spore Viability Table 2 shows the percentage and efficiency of sporulation, the average viability of the spores, and the viability of the asci. Six of the tested strains were unable to sporulate in the media tested, even when the incubation time was increased to 20 days. The sporulation levels were partially genetically determined, but also widely varied according to the medium used and other parameters such as temperature [6]. The percentage of sporulation ranged from 79.2 % for strain UCLMS-228 to 4.8 % for UCLMS-33. These results are consistent with those obtained for the 43 strains of Saccharomyces strains used in natural Italian wine fermentation [29], and 12 commercial yeast strains [22]. The sporulation efficiency varied between 100 and 8.2 %, and all strains were able to form asci with four or three spores, although in some cases, the percentage of asci with two spores (dyads) was relatively high. According to Gerke et al. [16], many wine strains produce larger numbers of two-spored (dyads) and three-spored (triads) asci and produce tetrads and triads earlier in sporulation than dyads. Spore viability ranged from 100 % for UCLMS-273 to 5 % for UCLMS-202. For strain UCLMS-202, it was necessary to dissect a larger number of tetrads in order to obtain at least eight viable spores and these spores were generally slow growing and only produced visible colonies after 5–8 days. Viability is generally defined as the ability to produce colony spore visible after 2–5 days of incubation, typically at 30 °C [22]. In some strains, spore colonies are visible only after prolonged incubation and their extremely slow growth rates make further testing either difficult or impractical [22]. UCLMS-33 presented a low percentage of sporulation but had high spore viability (higher than 90 %), whereas UCLMS-38 showed a high percentage of sporulation (more than 60 %) and low viability (under 25 %) and dyads were predominantly formed. In general, there was no relationship between these two parameters. Phenotypic Analysis of Single Spore Progeny Table 3 shows the results of the phenotypic analysis of the spore cultures tested, illustrating whether a strain is homozygous (?/? or -/-) or heterozygous (±) for a particular trait. Of the 20 strains genetically examined through tetrad analysis, 19 strains had one or more

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heterozygosities and only one strain appeared to be completely homozygous. These results are inconsistent with those obtained by Mortimer et al. [29] who demonstrated that 35 % of strains were completely homozygous. Most natural strains were heterozygous [34] for one or more traits, suggesting the occurrence of mutations occurring during the life of the organism. Among the 20 strain analyzed, 14 strains were heterozygous for mutations affecting sugar metabolism [i.e., galactose (GAL), maltose (MAL), and raffinose (RAF)]. All studied strains were homozygous for the fermentation of sucrose (SUC), and approximately 60 % of the strains were homozygous for the fermentation of maltose and raffinose. Galactose fermentation showed the largest discrepancy, with only 35 % heterozygous strains and 30 % homozygous strains, presenting the -/- phenotype. Half of the spores produced from the four strains were unable to grow using galactose as a sole carbon source and therefore, greatly affect the metabolism of this sugar. All of the spores for strain UCLMS-147 were unable to grow in the presence of galactose. Copper resistance (CUP) was equally divided into different types of segregants. The recessive allele that is expressed as copper sensitivity confers definite selective advantage, as copper resistance involves synthesis of a metallothionein from a complex gene and that in the absence of copper it is advantageous to cells not to synthesize this complex molecule [28]. The qualitative assessment of H2S production on Biggy agar confirmed the increased potential for H2S production in UCLMS-33, -234, and -267 as these strains were homozygous and displayed a dark brown phenotype in this medium, while although strains, UCLMS-202 and UCLMS-227 were also homozygous, these yeasts showed a white phenotype. The remaining strains were heterozygous for this trait. Regarding the sporulation capacity of the spores (SPO), more than half of the strains were heterozygous for this trait. In addition, six to eight spore colonies were randomly selected from each strain, and the mating type was assessed [21]. A single band showed that the spore clones were haploid (a or a) and if it two bands illustrated that the spore clones were diploid (a/a) and therefore homothallic (Fig. 2). Among the 20 strains examined, 19 strains were homothallic; 18 strains were HO/HO, and one strain was HO/ho (ADY1). For ADY1, the coexistence of homothallic and heterothallic spores was interpreted as a consequence of heterozygosity for some of the genes involved in the homothallism of the parental strain [11]. Only strain UCLMS-202 was likely heterothallic (ho/ho). However, this condition was uncertain, as only a small number of viable spores was available for testing (only eight viable spores from 40 dissected asci).

M. Ferna´ndez-Gonza´lez et al.: Study of Saccharomyces cerevisiae Wine Strains

447

Table 3 Phenotypes of the parental strains using tetrad analysis Traitsa

Yeast

SUC

MAL

GALb

RAF

CUP

H2Sc

SD

SLO

YPG

Y/W

SPO

MAT

Het

UCLMS-4

?/?

±

1/1

±

-/-

3/0

?/?

±

?/?

±

±

?/?

6

UCLMS-31

?/?

±

?/?

?/?

±

3/0

?/?

?/?

?/?

±

±

?/?

5

UCLMS-33

?/?

?/?

?/?

?/?

±

3/2

?/?

?/?

?/?

?/?

±

?/?

2

d,e

UCLMS-38

?/?

±

?/?

±

?/?

3/1

±

?/?

?/?

?/?

±

?/?

5

UCLMS-59

?/?

?/?

-/-/0

±

-/-

3/0

?/?

±

±

±

-/-

?/?

5

UCLMS-147

?/?

±

-/-/0

±

-/-

3/0

?/?

±

±

±

±

?/?

6

UCLMS-202f UCLMS-220

?/? ?/?

± ?/?

-/-/-

± ?/?

-/±

2/0 3/0

± ?/?

± ±

± ±

± ±

-/±

-/?/?

6 6

UCLMS-227

?/?

?/?

?/?

?/?

?/?

2/2

?/?

?/?

?/?

?/?

?/?

?/?

0

UCLMS-228

?/?

±

?/?

?/?

±

3/0

?/?

?/?

?/?

?/?

±

?/?

2

UCLMS-234

?/?

?/?

?/?

?/?

?/?

3/3

?/?

?/?

?/?

?/?

±

?/?

1

UCLMS-263

?/?

?/?

±/0

?/?

±

3/1

?/?

?/?

?/?

?/?

?/?

?/?

2

UCLMS-267

?/?

?/?

±

?/?

?/?

3/2

?/?

?/?

?/?

?/?

?/?

?/?

1

UCLMS-273

?/?

?/?

±

?/?

?/?

3/1

?/?

?/?

?/?

?/?

±

?/?

3

UCLMS-277

?/?

?/?

±

±

±

3/1

?/?

?/?

?/?

?/?

?/?

?/?

4

UCLMS-280

?/?

?/?

±

±

±

1/0

?/?

?/?

?/?

?/?

?/?

?/?

4

UCLMS-287d

?/?

?/?

?/?

±

?/?

2/0

?/?

?/?

?/?

?/?

±

?/?

3

ADY1

?/?

?/?

-/-/0

?/?

-/-

2/0

?/?

±

±

±

-/-

±

5

ADY2

?/?

±

±

?/?

-/-

3/0

?/?

?/?

?/?

±

?/?

?/?

4

ADY3

?/?

?/?

-/-

?/?

-/-

3/1

?/?

?/?

?/?

?/?

±

?/?

2

a

Symbols denote phenotypes as follows: SUC, MAL, GAL, RAF are fermentation of sucrose, maltose, galactose, and raffinose respectively; CUP is resistance/sensitivity to copper ion; H2S is sulfide production as pigment intensity on BIGGY medium; SD growth on minimal media; SLO is slow growth rate; YPG contains glycerol and detects respiratory deficiency (petite); Y/W. ? = W (white), - = Y (yellow). This color is a pigment inside the yeast cells on BTBL agar; SPO is the ability of individual spore clones to sporulate; MAT. Mating behavior detected by PCR. a/a (?); a or a (-). Het is number of heterozygosities

b

0 No growth on galactose media

c

Includes quantitative variation for production of H2S

d

Only two spore viable by tetrad was obtained so they have a putative recessive lethal mutation, and probably had more heterozigosities than described

e f

Four colonies were methionine auxotrophs Four colonies were tryptophan auxotrophs and three of them in addition to uracil and histidine

From a breeding perspective, we favored strains with mating behaviors corresponding to the MAT locus composition [21], as other studies [15] have demonstrated that larger spore clones were heterozygous at the MAT locus, but showed a specific mating behavior with tester strains. If an HO/HO strain has heterozygosities and sporulates, then the haploid spores from such a strain will represent all possible combinations of these heterozygosities. The HO gene induces mating type, switching; thus some of the descendants of these haploid spores will change mating type, mate, and form diploid spores [28]. These diploid spores will be completely homozygous and will compete with each other and with the original diploid parent strains. Only strain UCLMS-227 was homozygous diploid,

reflecting genome renewal from a formerly heterozygous diploid strain. Although 12 of the 20 strains produced four viable and uniformly large colonies, six strains showed a 2:2 ratio for two large and two very small colonies, and these yeast grew slowly on YPD media (SLO), i.e., UCLMS-4 and UCLMS-59, and most of these strains were heterozygous for glycerol growth (YPG) and designated as petite i.e., UCLMS-59. Both UCLMS-38 and UCLMS-287 showed the putative presence of a recessive lethal mutation (inviability of at least two spores/tetrad), heterozygosity for other traits associated with growth as yellow pigment (in colonies) on sugar media (Y/W), slow growth on glucose, and no growth on glycerol (non-fermentable).

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448

M. Ferna´ndez-Gonza´lez et al.: Study of Saccharomyces cerevisiae Wine Strains

analyzed S. cerevisiae strains have increased the current knowledge of the natural yeasts isolated from wine ecosystems, suggesting the potential use of these strains in hybridization programs. Specifically, we obtained stable haploid spores from strains ADY1, UCLMS-202, and natural auxotrophs from strains UCLMS-202 and UCLMS38, that might be useful as selection markers for future hybrids. Fig. 2 Assessment of yeast strain mating type by colony PCR, using three primers to amplify either a MATa- or MATa-specific bands. Strains with both MAT loci were considered diploid. Whenever a single band was observed in the mating type PCR, it corresponded with the mating phenotype. M molecular ladder 100 bp (Biotools). Lines 1–12: 8f (MAT a), 8i (MATa/a), 7b (MATa), and 7i (MATa/a) are spore colonies derived from strain ADY1; 4a (MAT a/a), 4b (MATa/a), 4c (MATa/a), and 4d (MATa/a) are spore colonies derived from strain ADY2; and 8b (MATa), 8d (MATa), 9a (MATa), and 9c (MATa) are spore colonies derived from strain UCLMS-202

The mutations that caused the yeast cells to turn yellow on bromothymol blue agar were very pleiotropic and caused slow growth and/or sporulation defects [28]. These mutations that appeared in 40 % of the studied population and were not observed under homozygous conditions. Growth rate mutations were also selected against. Table 3 lists a series of auxotrophic derivatives (SD) with defects in amino acid or nucleotide metabolism obtained from UCLMS-202 (tryptophan a/o histidine and uracil) and UCLMS-38 (methionine), and these derivatives are of interest for the development of hybridization techniques that use laboratory strains. To our knowledge, however, auxotrophic mutants of industrial yeasts have not been routinely isolated, as these yeasts are diploid or polyploid. Thus, the isolation of auxotrophic mutants of industrial yeasts has been thought to be difficult without the use of special selection methods, such as 5-fluoroorotic acid (5-FOA) or a-aminoadipic acid, or after UV mutagenesis [20]. However, in the present study, natural auxotrophic haploid and diploid wine yeasts were obtained through tetrad dissection.

Conclusions In the present study, we identified yeasts suitable for use in breeding programs, with good sporulation rates, such as ADY1, UCLMS-267, and UCLMS-277, which are capable of completely consuming sugars in problematic musts under low temperatures, and UCLMS-147, with a more moderate fermentative performance in terms of ethanol yield and high glycerol production. The strains ADY2 and ADY3, with adequate sensorial properties, were also able to sporulate and viable spores were isolated from these yeasts. The results of the tetrad analysis of 20 of the 26

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Acknowledgments Mo´nica Ferna´ndez Gonza´lez would like to thank the Albacete Science & Technology Park for an award from INCRECYT co-financed by the European Social Fund.

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