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Keywords:

  • Wine;
  • Molecular typing;
  • Genetic variability;
  • Delta element;
  • Saccharomyces cerevisiae

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgments
  8. References

A new primer pair (delta12–delta21) for polymerase chain reaction-based yeast typing was designed using the yeast genome sequence. The specificity of this primer pair was checked by the comparison of the electrophoresis pattern with a virtual profile calculated from Blast data. The analysis of 53 commercial and laboratory Saccharomyces cerevisiae yeast strains showed a clear improvement of interdelta analysis using the newly designed primers.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgments
  8. References

Numerous studies have been conducted on wine yeast since the early work of Pasteur. Yeast strains have specific enzymatic activities necessary for the production of key aromatic compounds during alcoholic fermentation. Enzymes appear to be required for the release of bound compounds synthesised from grapes such as mercaptopentanone in Sauvignon wines [1] or volatile phenols in Gewurztraminer wines [2]. Such uncoverings led to the technological success of tested commercial dry-wine yeast strains in the process of wine making. However, these important enzymatic differences are laborious to analyse in different strains and a more rapid but just as definitive method is necessary. Recent molecular biological techniques have allowed the characterisation of yeast strains [3,4], leading to a new era for ecological surveys. These techniques have enabled the population dynamics of Saccharomyces cerevisiae strains in vineyards or wineries to be studied [5–10], as well as the control of industrially dried yeast production. They also proved extremely beneficial for yeast laboratories testing strains for their enological properties in order to optimise wild-strain isolates collections. Since the first application of mitochondrial DNA restriction profiling to brewing yeast [11], several techniques have been developed. Pulsed-field electrophoresis karyotyping applied to enological yeast strains [12] is often regarded as a time-consuming procedure although it is highly efficient [13]. Other polymerase chain reaction (PCR) techniques were developed with randomly amplified polymorphic DNA (RAPD) primers for wine yeast [14,15], but poor discrimination of strains was obtained with each primer set. De Barros Lopez et al. [16] developed an interesting technique using primers based on intron splicing sites.

More recently, a powerful microsatellite-based technique has been developed [17]. It differentiates yeast from various origins (French enological, medical isolates, Asian yeast, etc.) through the analysis of seven loci. Similar techniques were developed [18,19], but as they were based on one locus analysis only, these approaches were, however, not powerful enough for an accurate characterisation of numerous yeast strains. The potential of such a technique for enological strains has still to be evaluated. An AFLP-based analysis of yeast strains, clustering beer, whisky, bakery, and sake yeast, was also described [20]. However, these two methods require DNA sequence analysis for accurate determination.

The use of a rapid PCR-based protocol, relying on the amplification of interdelta regions, was initially proposed in 1993 [21]. Delta elements form the LTR flanking retrotransposons TY1 and TY2 in yeast, but can also be found separate from these retrotransposons and are called solo delta elements. About 300 such delta elements are described in the genome of S288C and are therefore good candidate targets for identification of polymorphisms. This interdelta method is now often used for routine analysis of yeast strains [22], but is less discriminatory than the previously mentioned pulsed-field electrophoresis [13].

In this report, the interdelta method has been improved utilising the now complete yeast genome sequence database. Analysis of the database allowed the design of the interdelta primers to be optimised, as well as the development of a new method compatible with numeric profiling.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgments
  8. References

2.1Yeast strains

Laboratory and enological yeast strains used in this work are listed in Table 1.

Table 1. Saccharomyces strains used in the study
StrainOrigin – Commercial name
S. carlsbergensis Clib176Clib laboratory strain
S. exiguus Clib179Clib laboratory strain
S. bayanus Clib181Clib laboratory strain
S. kluyveri Clib182Clib laboratory strain
S. paradoxus Clib228Clib laboratory strain
S. uvarum Clib533Clib laboratory strain
S. uvarum D24Wild-type isolate, INRA Colmar
S. bayanus Clib181Clib laboratory strain
S. servazzii Clib187Clib laboratory strain
Saccharomyces cerevisiae laboratory strains
SC288CLaboratory strain
FL100Laboratory strain
FL200Laboratory strain
SW303Laboratory strain
FY1679Laboratory strain
Clib176Clib brewery strain
Clib227Clib laboratory strain
Clib319Bakery yeast
Saccharomyces cerevisiae enological yeast
182007IOEC 18-2007
1M8Wild-type isolate, INRA Colmar
1N1dWild-type isolate, INRA Colmar
522DDavis Montrachet
58W3Alsaflore (INRA Colmar)
595 DaviesVitilevure ‘B’
67JEnolevure OV
7013Enolevure SL
70S1Vitilevure Albaflore
71B71B (INRA Narbonne)
7303Enolevure CR
AWRI350AWRI350
AWRI796AWRI796
B10GE7 (INRA Colmar)
B94/201Ceres (INRA Colmar)
C19C19 (INRA Colmar)
CH158SIHA4
CIVC8130CHP
CY3079CY3079
D1Wild-type isolate, ULP Strasbourg
D576SIHA7
DV10DV10
E51SIHA2
Eg8/136ALS (INRA Colmar)
K1Enolevure K34 (INRA Montpellier)
L13LAl13
L1414L1414
L2056Lalvin 2056
LW128-91Hefix 1000
LW 185-25Hefix 2000
LW317-29Oenoferm Klosterneubourg
MBZ1levuline MBZ1
MV94017Vitilevure ‘Sauvignon’
R2KD
RC212Lavin RC212
UP3OY5BRG
RCA17Lalvin RCA17
RHSTRHST
ST-Clib2026Zymaflore ST
Vl1Zymaflore VL1
Vl3Zymaflore VL3
WET 136SIHA3
xxSpindal Aromatic
yyVitilevure Rubiflore
zzZymasil

2.2DNA extraction

DNA was prepared from 10 ml YPD cultures (Yeast Extract (Difco) 1% w/v, Bactopeptone (Difco) 1% w/v, glucose 2% w/v) agitated for 24 h at 28°C.

2 ml of the culture was centrifuged in an Eppendorf tube (5000 rpm, 5 min). 400 μl of lysis buffer (Tris 10 mM, pH 7.6, EDTA 1 mM, NaCl 100 mM, Triton X-100, 2% w/v, sodium dodecyl sulphate (SDS) 1% w/v), 400 μl of phenol/chloroform/iso-amyl alcohol (25/24/1 v/v), and 600 μl of glass beads were added to the pellet. The mixture was vortexed for 4 min. Then 200 μl of Tris EDTA (pH 7.6) buffer was added, and the mixture centrifuged for 5 min at 6000 rpm. 500 μl of chloroform/iso-amyl alcohol (98/2 v/v) was added to the upper phase and after gentle agitation, the mixture was submitted to centrifugation (14 000 rpm for 2 min). Two volumes of ethanol were added to the aqueous phase. After centrifugation (14 000 rpm, 5 min), the nucleic acid pellet was dissolved in 10 mM TE buffer pH 8.0.

DNA from commercial dried yeast was prepared in the same way after rehydrating 50 mg of yeast powder in 500 μl 50 mM EDTA, for 15 min.

2.3DNA amplification

PCR amplifications were carried out in 25 μl reaction volumes containing 5–20 ng yeast DNA, 10 mM Tris pH 9.0, 50 mM KCl, 0.1% Triton X-100, 0.2 mg ml−1 gelatin, 200 mM of each dNTP, 2.5 mM of MgCl2 and 1 μM for each oligonucleotide primer of the delta1 family and delta2 family. Primer sequences for delta 1 and delta 2 are those described previously [21] and newly designed primers are delta12 (5′-TCAACAATGGAATCCCAAC-3′) and delta21 (5′-CATCTTAACACCGTATATGA-3′).

Amplification reactions were performed with a Stratagene thermal cycler using the following programme: 4 min at 95°C followed by 35 cycles of 30 s at 95°C, 30 s at 46°C and 90 s at 72°C and a finishing step of 10 min at 72°C. For the delta1/delta2 primer pair, the annealing temperature was 42°C for the first five cycles and 45°C for the following cycles as described in [21].

2.4Electrophoresis

Amplification products were separated by electrophoresis on 10 or 15 cm 2% agarose gels submitted to 100 V for 1 h in 1×TBE buffer. Nusieve 3:1 agarose (BMA) was used for newly designed primers, since better resolution was required.

2.5Numerical analysis

After staining with ethidium bromide (10 μg ml−1), gels were scanned with a Gel Doc 1000 apparatus (Bio-Rad), and compared with Molecular Analyst Fingerprinting plus (Bio-Rad) after normalisation of the profiles. Clustering of profiles was done using the UGMPA calculation methods based on Dice coefficient. Gel images and dendrograms were processed with Adobe Photoshop and CorelDraw.

2.6Sequence alignment tools

Blast searches were performed with the ‘nearly exact match option’ at NCBII. As differences were noticed for chromosome III, between NCBI and SGD data, only coordinates obtained from SGD were kept for that chromosome.

The statistical significance threshold for reporting matches against database sequences was 1000, and word size 7. Sequences and coordinates were treated in a 4D® database, and band-size calculation was established using an Excel® data sheet. Possible amplification sites were selected from nine base-length cohesive 3′ end sequences. Possible bands were determined from the coordinates given by the Blast procedure; one possible band corresponds to two primers on different strands with opposite coordinates (5′ 3′). Band weight was calculated from the distance between 3′ ends of each primer increased by the size of the respective primers. Melting temperature was calculated for the complementary sequences from the approximate model of Bolton and McCarthy [23]. As the low melting temperature was unlikely to give small bands under our amplification conditions only those possibilities corresponding to a calculated Tm greater than 25°C were retained.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgments
  8. References

3.1Optimisation of the interdelta primers

The location of the two primers delta1 and delta2 given previously [21] on the delta element no. YOLC delta3 is indicated in Fig. 1.

image

Figure 1. Location of the different primers on YOLC delta3. Underlined letters correspond to conserved zones [25]. Primers designed by Ness et al. [21] are written with italics, delta12 and delta21 primers are written with bold letters.

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We compared the homology of both delta1 and delta2 primers to the whole S. cerevisiae genome. Resulting Blast searches showed that primer delta1 (78 hits) gave poor homology. However, primer delta2 (303 hits) displayed a high consensus with the delta elements dispersed within the genome with an uneven matching close to the 5′ end. The low Tm of these primers [21] could be responsible for less stable patterns after amplification [24]. Comparison, using the sequence of delta element YOLC delta3, enabled the design of two new primers: delta12 and delta21. Primer delta12 is close to the primer delta1 in the well-conserved ATG region in TYA/GAG open reading frame [25]. Primer delta21 was chosen 60 bp upstream of primer delta2 in a 22-bp well-conserved region [25]. It has a higher Tm.

Data from the Blast searches made for each primer were then compared with the location of the 305 delta elements that we located on the S. cerevisiae genome (SGD, 2002). The different delta elements that are potential amplification start points are indicated for each chromosome in Table 2. The results showed the major improvement when primer delta12 was used instead of primer delta1. Indeed, primer delta1 gave only 34 hybridisation sites whereas delta12 gave 180 sites. However, primers delta2 and delta 21 showed similar efficiency.

Table 2.  Theoretical number of delta elements giving amplificationa
  1. aTheoretical number of delta elements was determined by Blast searches as described in Section 2.

  2. bNumber of delta elements identified on S. cerevisiae genome.

  3. cPCR primers are described in Section 2.

Chromosome no.Delta elementsbPrimerc
  Delta1Delta2Delta12Delta21
182554
2193101011
3172111110
4344262525
5263111212
6101255
7322191918
8150576
960423
1020012910
11121665
12272162019
13211141412
1492788
15252151411
16249161313
Total30534179180172

Fig. 2 displays electrophoresis patterns obtained with different combinations of these four primers. As expected, profiles showing more bands were obtained with delta12–delta21 and delta12–delta2 primer pair combinations. Only three fragments were amplified with delta1–delta2 for strain S288C, whereas eight and 11 fragments were amplified with primer combinations delta12–delta2 and delta12–delta21. The whole gel including seven strains gave 15 or 27 or 28 different bands with primer pairs delta1–delta2, delta12–delta2, and delta12–delta21, respectively. Since a better association (Tm and amplification patterns) was achieved with primer pair delta12–delta21, we focused our study on this primer pair. However, the delta12–delta2 primer combination gave similar results.

image

Figure 2. Comparison of electrophoresis gels obtained for different yeast strains with delta1–delta2, delta12–delta2 and delta12–delta21. Lanes 1–10 of each gel: 1, molecular mass markers φX174 digested by HaeIII; 2, S288C; 3, FL100; 4, W303; 5, FY1689; 6, AWRI350; 7, AWRI750; 8, Clib319.

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3.2Theoretical analysis of possible amplification products

The specificity of a method is a key point for its reproducibility, which is the drawback of RAPD techniques. We therefore checked if delta elements were the only source of amplification using primers delta12–delta21. We determined first the annealing sites of each primer by performing Blast searches on the S288C genome and then predicted the number of fragments that could be amplified.

Table 3 lists all the expected amplification fragments deduced from the Blast data. A great number of bands were predicted from 70 bp to 4 kb or higher; however, we chose to focus only on fragments <1.5 kb. Tm was determined for each homologous sequence found for each primer by Blast in order to assess the likelihood of amplification. Therefore, theoretical fragments obtained from sites with low homology (Tm lower than 25°C) were excluded. This reduced the number of virtual bands to 25 (Table 3); 24 of the virtual bands arose between two delta elements and the last additional virtual band was instead due to the presence of a partially homologous region adjacent to a bonafide delta element. This virtual profile also shows an uneven representation of chromosomes: two-thirds of the possible fragments are obtained from chromosomes 3, 4, 7 and 16; coincidentally, no amplification was forecasted for chromosomes 1, 2, 6, 8, 9, 11, 12, 13, 14 even if a high number of delta elements are possible amplification start points (i.e. chromosome 12, see Table 2). This result shows that only a limited number of delta elements is involved in the amplification pattern.

Table 3.  Theoretical fragments and size obtained from Blast analysis of each primera
  1. Lines in bold letters correspond to bands confirmed after purification and sequencing.

  2. aFragments are determined by Blast searches as described in Section 2.

  3. bTm are calculated as described in Section 2.

Chromosome number5′ Primer5′ CoordinateTmb3′ Primer3′ CoordinateTmbFragment size
3D1283 10041.6D1282 98941.6115
3D2184 36628.6D1282 98941.61385
3D2184 14028.6D1282 98941.61159
3D2184 88228.6D1284 58429.3310
3D21169 27640.5D21168 85426.3430
4D12513 72429.3D21513 50740.5225
4D21520 54328.6D21519 56928.6986
4D12651 70742.9D21651 34028.6376
4D12878 34141.6D12877 72835.7620
4D12878 34141.6D21877 96428.6385
4D12987 18741.6D12987 07735.7117
4D21992 67440.5D21992 56128.6120
4D121 151 38535.7D121 151 31535.780
5D21449 36728.6D21449 24428.6135
7D21535 83140.5D12535 49041.6344
7D12568 77835.7D21567 68228.61107
7D12817 43341.6D12817 35829.384
10D21203 83940.5D21203 74328.6103
15D21664 88428.6D12664 42934.7465
15D12976 29034.0D12976 19335.7108
16D2162 46128.6D1262 32941.6140
16D2162 74728.6D1262 67735.781
16D2162 74728.6D1262 32941.6426
16D12804 68641.6D12804 60940.682
16D12850 67041.6D12850 28941.6385

Experimental data show that among the 25 expected fragments, only 11 intense bands are obtained for S288C (Fig. 2). Furthermore, these bands were purified, cloned, sequenced and compared with databank sequences. Every single one of the 11 determined sequences matched one of the 25 predicted fragments (Table 3, in bold letters). Among the 14 virtual bands that were not amplified, 10 had the same primer at both ends. It has been described that this situation leads to a ‘PCR-suppressive effect’[26,27]. According to this theory, the fragments with two complementary ends will form a ‘panhandle-like structure’ during the PCR, thus preventing further annealing and amplification. It should, however, be noticed that the fragment at 620 bp arose from a region located between two delta12 primers; it might have been obtained because of the simultaneous amplification of a 385-bp fragment at the same site that had primed the extension of the DNA.

Two forecasted fragments with asymmetrical primers at 1385 and 1159 were only weakly detected. Several attempts were made to increase the amplification level of these faint bands, without noticeable success. One explanation for this could be that amplification from multiple sites could reduce the level of individual signals. Nevertheless, this hypothesis is not valid for the three bands at 81, 140 and 426 bp obtained for chromosome 16 (Fig. 2).

In conclusion, all of the detected bands corresponded to one calculated possibility, and the differences between the virtual profile and the electrophoretical pattern can be explained mainly by a PCR-suppressive effect.

3.3Evaluation of the technique with strains of the Saccharomyces genera

Sequences homologous to parts of TY retrotransposons have been found in various other yeast genomes [28], but the level of homology varies greatly among the species: The ‘Genolevure Program’ revealed that S. bayanus var. uvarum, S. exiguus and S. kluyveri show significant homology with S. cerevisiae TY1 or TY2, and S. servazzii show little homology. Fig. 3 presents amplification profiles obtained for these species. Only S. cerevisiae and S. carlsbergensis strains gave rich profiles, whereas other yeasts gave no signal. Amplification obtained from S. carlsbergensis is consistent with the hybrid nature of that species described elsewhere [29].

image

Figure 3. Electrophoretical patterns obtained for different yeasts with delta12–delta21 primers. Lanes 1–11 of each gel: 1, molecular mass markers φX174 digested by HaeIII; 2,3, S. cerevisiae S288C and Clib227; 4, S. carlsbergensis Clib176; 5, S. exiguus Clib179; 6, S. bayanus Clib181; 7, S. kluyveri Clib182; 8, S. servazzii Clib187; 9, S. paradoxus Clib228; 10, S. uvarum D24; 11, molecular mass markers.

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3.4Characterisation of different industrial laboratory and wild-type yeast strains

As the final aim of the technique is to differentiate the highest number of strains, 53 preparations of industrial yeast, laboratory strains or wild-type isolates were evaluated (Fig. 4).

image

Figure 4. Electrophoretical patterns and dendrogram showing the clustering of the 53 yeast strains used in the study. Calculated percentages of homology are given on the ruler on the left of the dendrogram.

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Numerical data processing of the obtained profiles led to a rapid and easy classification. As expected, S. carlsbergensis strain Clib176 gave a pattern differing from the other yeast strains.

Some clustering can also be easily noticed: laboratory strains FL100, S288C, W303, and FY1679 are gathered in a separate branch of the tree, which can easily be understood from the history of laboratory strains. Indeed, these strains arose from a limited set of laboratories and have been extensively crossed and genetically engineered in order to obtain stable mating types or multiple auxotrophic markers. Some strains gave identical patterns DV10 – CIVC8130, 7013-ST, or UP3OY5 isolated from Burgundy in 1987 and 1N1d isolated in 1996 from Alsatian grapes. The identities of those profiles were confirmed by pulsed-field electrophoresis (data not shown). Furthermore, some close profiles, L1414 and B94/201 or CIVC8130 and C19, were also confirmed by pulsed-field electrophoresis, showing the coherence of both techniques. Different samples of DNA from the same yeast (laboratory strain culture, dried yeast) were analysed and showed identical profiles, demonstrating the reliability of this technique.

Comparable work performed on this yeast set with the former version of primer (delta1–delta2) gave poor clustering and led to numerous erroneous associations (data not shown).

4Conclusion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgments
  8. References

The S. cerevisiae genome sequence was used to design two new primers for interdelta typing. One primer, delta21, shows similar or slightly better results on gels than the delta2 primer proposed formerly [21], but the second primer, delta12, is far more efficient than delta1. The specificity of this method was checked by comparison of the electrophoretic pattern of S288C with the virtual profile calculated from Blast data. With primer pair delta12–delta21, we were able to differentiate unequivocally between 53 industrial, laboratory and wild-type yeast strains. Results are in accordance with data acquired from pulsed-field electrophoresis, thus highlighting the importance of re-analysing yeast strains identified by the sole use of the former delta typing method [21].

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgments
  8. References

The authors would like to thank Luc Baudrey for his helpful technical assistance, Dr C. Jackson and Dr Chan Ka Nin for critical discussion of the manuscript.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusion
  7. Acknowledgments
  8. References
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