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75
RESTRICTION PROFILES OF 26S rDNA AS A MOLECULAR APPROACH FOR
WINE YEASTS IDENTIFICATION
IDENTIFICAÇÃO DE LEVEDURAS DE INTERESSE ENOLÓGICO POR PERFIS DE RESTRIÇÃO
DO ADNr 26S
Geni C. Zanol *, M. Margarida Baleiras-Couto, Filomena L. Duarte
Instituto Nacional de Recursos Biológicos, I.P./ INIA Dois Portos, Quinta d’Almoínha 2565-191, Dois Portos, Portugal
*Corresponding author: geni.zanol@inrb.pt
(Manuscrito recebido em 30.11.10 . Aceite para publicação em 23.12.10)
SUMMARY
The complex microbial ecosystem existing in grape, must and wine comprises a wide diversity of yeast species. The knowledge of composition
and dynamics of yeast biota occurring along vinifi cation process would provide a better control of wine quality. The sequence of D1/D2 domain
of 26S ribosomal DNA (rDNA), refl ects ascomycetous yeast phylogenetic relationships and enables their separation at the species level. A region
of the 26S rDNA, with around 1100 bp comprising domain D1/D2, was amplifi ed by PCR and then digested with restriction endonucleases
(ApaI, HinfI, MseI, HaeIII and CfoI) in order to differentiate yeast species frequently isolated from grape surfaces, wine and cellar equipments.
A total of 78 yeast strains (including 36 type strains) belonging to 53 species were used to generate the restriction profi les. Numerical analysis
of the profi les generated by the fi ve restriction enzymes enabled to group the strains in 47 different clusters and 42 of them clearly correspon-
ded to different yeast species. The remaining groups comprise closely related species. The enzymes MseI, HaeIII and CfoI revealed a high
discrimination power and the restriction profi les generated were suffi cient to clearly identify the 42 species mentioned above. Despite one of
the clusters included different yeast genera, with different wine characteristics, the common wine spoilage yeasts Zygosaccharomyces bailii
and Z. lentus could be separated to one distinctive cluster through the use of ApaI restriction profi les. Since the analysis of restriction profi les
of amplifi ed 26S rDNA showed to be a valuable method to identify oenological yeast species, a database comprising the majority of wine yeast
biota was created to be applied both at research and industrial environment.
RESUMO
O ecossistema microbiano existente nas uvas, no mosto e no vinho é composto por uma grande diversidade de espécies de leveduras. O conhe-
cimento deste biota de leveduras ao longo do processo de vinifi cação permite um melhor controlo da qualidade do vinho. Para a identifi cação de
leveduras o ADN ribossómico (ADNr) tem-se revelado muito adequado para estimar relações fi logenéticas, consideradas pelas correntes mais
actuais da taxonomia como estando na base da classifi cação taxonómica. No presente trabalho, avaliou-se um método baseado na amplifi cação do
ADNr 26S, compreendendo a região D1/D2, seguido de digestão por enzimas de restrição - Perfi s de Restrição - para a identifi cação de espécies
de leveduras envolvidas no processo de produção de vinho. Esta avaliação foi efectuada através do uso de 78 estirpes pertencentes a 53 espécies
(incluindo 36 estirpes tipo). Utilizaram-se as enzimas de restrição ApaI, HinfI, MseI, HaeIII e CfoI e, análise numérica dos perfi s de restrição
gerados permitiu agrupar as espécies estudadas em 47 grupos, 42 dos quais correspondendo a uma única espécie. As enzimas de restrição MseI,
HaeIII e CfoI foram as que apresentaram maior poder discriminante ao nível da espécie, permitindo a identifi cação das mesmas 42 espécies.
Apesar da enzyma ApaI ter apresentado o mais baixo grau de polimorfi smo, esta enzima poderá ser útil para medidas de controlo uma vez que
seu perfi l de restrição pôde agrupar em um grupo distinto as leveduras Zygosaccharomyces bailii e Z. Lentus. O método desenvolvido revelou
efi cácia, rapidez e facilidade de aplicação na identifi cação de leveduras de interesse enológico. Com o presente trabalho iniciou-se a construção
de uma base de dados de perfi s de restrição para posterior aplicação em condições industriais e de investigação.
Key words: 26S rDNA, endonucleases, non-Saccharomyces yeasts, restriction profi les, wine yeasts
Palavras-Chave: ADNr 26S, endonucleases, leveduras enológicas, leveduras não-Saccharomyces, perfi s de restrição
INTRODUCTION
The art of wine making represents one of the oldest
technological uses of yeast by man. Only during the
last century the scientifi c knowledge of wine has
signifi cantly increased, assisted by newly developed
techniques that permitted deeper investigation into
the biological and physiological diversity of yeast
species associated to the process (Pretorius, 2000).
The grape has an incontestable infl uence in aroma
and fl avour of wines leading to the creation of distinct
products. However, wine has more fl avour than the
grape juice which is fermented from (Romano et al.,
2003) and it is the metabolism of grape constituents
by yeast that is essential to the development of wine
fl avour (Bartowsky and Pretorius, 2009).
The wine fermentation is a complex ecological and
biochemical process involving the sequential deve-
lopment of different microbiota such as non-Saccha-
romyces yeasts, Saccharomyces yeasts and lactic acid
bacteria present in must and on surface of cellar equi-
Ciência Téc. Vitiv. 25 (2) 75-85. 2010
76
pments (Fleet, 2003). The non-Saccharomyces yeasts
can produce a diversity of enzymatic activities and
fermentation metabolites of oenological importance
and may interfere with the growth and/or change the
fermentation behaviour of the starter Saccharomyces
cerevisiae yeast, thus ultimately influence wine
quality (Cabrera et al. 1988; Romano et al., 1997;
Ciani and Ferraro, 1998; Ciani and Maccarelli, 1998;
Ferreira et al., 2001; Romano et al., 2003; Ciani et
al., 2006; Domizio et al., 2007; Bely et al., 2008;
Romano et al., 2008). The use of selected strains of
S. cerevisiae as starters became a widespread practice
in wineries. Nevertheless, wine makers have recently
returned to spontaneous fermentation as well as to the
use of non-Saccharomyces in order to obtain wine of
distinctive quality and diversifi ed products.
On the other hand, yeasts can negatively affect wine
quality. Spoilage yeasts such as Brettanomyces/
Dekkera produce volatile phenols and acetic acid
that under uncontrolled conditions can lead to sen-
sorial defects (Renouf and Lonvaud-Funel, 2007).
Zygosaccharomyces is another yeast genus that is
often regarded as synonymous of food spoilage due
to their osmotolerance and resistance to food pre-
servatives (Loureiro and Malfeito-Ferreira, 2003).
Therefore, the analysis and identifi cation of yeast
biota throughout wine fermentation and conservation
are currently important driving forces for innovation
in wine technology.
Traditionally, yeast taxonomy has been based on mor-
phological, physiological and biochemical charac-
teristics of species and genera which ambiguity due
to strain variability has led to errors in classifi cation
(Martini, 1992; Kurtzman and Robnett, 1994; Kurt-
zman and Fell, 1998). Isoenzymes electrophoretic
profi les have also been applied and prove to refl ect
DNA based yeast species delimitation (Smith et
al., 1990; Duarte et al., 1999; Sampaio et al., 2001;
Naumova et al., 2003; Duarte et al., 2004). However,
this technique is highly time-consuming.
Several approaches based on nucleic acids poly-
morphisms have been developed in an attempt to
simplify yeast identifi cation, such as electrophoretic
karyotyping, temperature gradient gel electrophoresis
(TGGE), microsatellite PCR fingerprinting, ran-
dom amplifi ed polymorphic DNA, ribosomal DNA
(rDNA) restriction profi les and partial rDNA sequen-
cing (Török et al.,1993; Baleiras-Couto et al., 1995;
Baleiras-Couto et al., 1996; Guillamón et al., 1998;
Kurtzman and Robnett, 1998; Esteve-Zarzoso et al.,
1999; Hernán-Gómez et al., 2000; Esteve-Zarzoso
et al., 2003; Baleiras-Couto et al., 2005; Rodriguez
et al., 2010).
Nowadays, innovative wine yeast identifi cation te-
chniques such as DGGE (Denaturing Gradient Gel
Electrophoresis) on PCR amplifi ed rRNA genes,
FISH (Fluorescence in situ Hybridization), real time
quantitative PCR (qPCR) and next-generation DNA
sequencing can enable the quantifi cation and/or to
monitor yeast dynamics throughout the fermentation
process (Hierro et al., 2007; Mardis, 2008; Salinas
et al., 2009; Tessonniere et al., 2009; Zott et al.,
2010). However, these techniques need sophisticated
and expensive equipments which are not commonly
available.
The ribosomal genes (5.8S, 18S and 26S), which
have as ultimate function the protein synthesis, are
grouped in tandem forming transcription units that
are repeated in the genome (Fernández-Espinar et al.,
2006). rRNA genes have a common origin, are present
in all cellular organisms and have proved to be ade-
quate to establish taxonomic relationships, namely on
yeasts, as it is present in all cellular organisms, have a
common origin and are easy to sequence (Kurtzman
and Piškur, 2005). Nucleotide sequences of the D1/
D2 domains of the large subunit (26S) of rDNA are
suffi ciently substituted to allow recognition of most
individual yeast species. Kurtzman and Robnett
(1998) have sequenced D1/D2 domains for all kno-
wn ascomycetous yeasts thus, initiating a universal
database for rapid identifi cation.
Simpler identifi cation methods were developed ba-
sed on the amplifi cation of specifi c regions of rDNA
followed by restriction of the amplifi ed fragment.
The digested fragments are then separated by elec-
trophoresis in agarose gels and their sizes determined
by comparison with appropriate markers. White et al.
(1990) used this methodology to amplify the riboso-
mal gene 5.8S and the adjacent intergenic regions
ITS1, ITS2 and further to digest with restriction en-
zymes. Another ribosomal region that is very useful
to differentiate at species level is the one that includes
18S gene and the intergenic region ITS1 (Baleiras-
Couto et al., 1996; Dlauchy et al., 1999). Since then,
this approach has been used for identifying yeast
species mainly associated alcoholic beverages and
soft drinks (Guillamón et al., 1998; Esteve-Zarzoso
et al., 1999; Arias et al., 2002; Ferreira et al., 2009).
Restriction profi les generated have been considered
reproducible, cheaper, a less-laborious method and
frequently used for yeast identifi cation (Fernández-
Espinar et al., 2006).
Baleiras-Couto et al. (2005) started to evaluate the
restriction profi les of a PCR amplicon of the large
subunit of rDNA (26S rDNA), comprising the D1/
D2 region, as a routine methodology to examine wine
yeast species. In the present study, we extended the
restriction profi les, originated through digestion with
fi ve restriction enzymes (ApaI, HinfI, MseI, HaeIII
and CfoI), of the same PCR amplicon, in order to
develop an effi cient and rapid methodology for oe-
nological yeasts genotyping. The aim of this work
was to create a database of restriction profi les, based
on certifi ed yeast strains, to be used in wine related
yeast identifi cation carried out both at research and
industrial level.
77
MATERIAL AND METHODS
Microorganisms
A total of 78 yeast isolates, comprising 53 species
belonging to 22 genera, included in the Colecção de
Microrganismos EVN (INRB/INIA Dois Portos),
were used in the present study (Table I). Thirty eight
strains were originated from other culture collec-
tions, 36 of which are type strains. The remaining
40 strains were isolated from grapes, wine and cellar
equipments in our Laboratory and identifi ed by DNA
sequencing of D1/D2 region of rDNA.
Yeast cells were grown on YPD medium (20 g/L D-
glucose, 10 g/L bacto-peptone, 5 g/L yeast extract and
20 g/L agar) for 48 to 72 hours at 25ºC. Two to three
loops of yeast culture (from fresh YPD agar plates)
were resuspended in 500 L of ultrapure sterilised
water. Yeast cells lysate was obtained by disrupting
cells through freezing of cell suspension in liquid
nitrogen for 5 min, followed by incubation at 95 ºC
for 5 min, accordingly to Baleiras-Couto et al. (2005).
The cell lysate containing DNA was then used for
PCR amplifi cation purposes. When the ribosomal
DNA amplifi cation by PCR was not successful, the
cells lysate was obtained by cell disruption using
glass beads (0.5 mm Ø) in 500 L lyses buffer (50
mM Tris-HCl, 250 mM NaCl, 50 mM EDTA and 0.3
% SDS). The cell lysate solution was appropriately
diluted and then used for PCR amplifi cation.
Amplifi cation of the ribosomal DNA D1/D2 region
Primer sequences for the amplifi cation of 26S rDNA
fragments were as follows: NL1 (5’-GCATATCAA-
TAAGCGGAGGAAAAG-3’) and LR6 (5’-CGC-
CAGTTCTGCTTACC-3’). Reactions were perfor-
med in a fi nal volume of 50 L containing 10 mM taq
buffer (MBI Fermentas, Vilnius, Lithuania), 2.5 mM
MgCl2, 250 M dNTPs, 0.75 M of each primer, 2 U
taq polymerase (MBI Fermentas) and 2 L of DNA
solution. PCR was performed on a thermocycler (T
Gradient 96 cycler, Whatman-Biometra, Gottingen,
Germany) with an initial denaturation at 94ºC for 3
min, followed by 36 cycles of 94 ºC at 1 min, 58 ºC
for 1 min and 72 ºC for 1.5 min. The fi nal extension
was done at 72 ºC for 5 min. Visualization of the PCR
amplifi ed fragments was performed by electropho-
resis in 1.2 % of agarose in 0.5 X TBE (0.45 M Tris-
HCL, 0.45 M boric acid and 10 mM EDTA, pH 8.0)
and staining with ethidium bromide (0.5 M/mL).
The amplifi cation effi ciency was visualised under
UV light and digital images were acquired through
a Kodak 290C camera and processed by Kodak 1D
Image Analysis software.
Restriction analysis
Aliquots (3-10 L according to the band intensity) of
PCR products were digested with 3 U and 5 U, res-
pectively, of restriction enzymes MseI, HinfI and ApaI
(MBI Fermentas) and HaeIII and CfoI (Promega,
Madison, WI) in a fi nal volume of 20 L, following
manufacture’s instructions. The resulting fragments
were separated by 2% agarose gel electrophoresis
followed by ethidium bromide staining, as referred
above. A standard DNA marker (100 bp DNA Ladder,
MBI Fermentas) was used as a reference to determine
the size of digested fragments. Restriction fragments
were visualised under UV light and digital images
were acquired through a Kodak 290C camera and
processed as referred above. All restriction profi les
obtained were analysed using GelCompar II software,
version 5.1 (Applied Maths, Saint-Martens-Latem,
Belgium) which determined the molecular sizes of
restriction products. Fragments smaller than 100 bp
were not included on the analysis because of their
low reproducibility. Similarities among banding
profi les of the strains in study were based on Dice
coeffi cient and dendrograms were generated by the
Unweighted Pair Group Method using Arithmetic
Average (UPGMA) clustering algorithm.
RESULTS AND DISCUSSION
Several molecular methods are presently being
applied for microbiological identification and
classifi cation. Each method has its advantages and
disadvantages according to the convenience of appli-
cability, reproducibility, availability of equipments,
and resolution level.
In this study, analysis of restriction profi les of NL1-
LR6 region of 26S rDNA was used to differentiate
wine yeast species associated to wine production. In
a total of 78 strains comprising 53 species, the PCR
amplifi cation yielded a fragment size of around 1100-
1150 bp. The amplifi ed fragment was then digested
with fi ve endonucleases (ApaI, HinfI, MseI, HaeIII
and CfoI) and the restriction products were separa-
ted by agarose gel electrophoresis. Representative
restriction profi les presented by the 53 yeast species
analysed, are shown in Figure 1.
Each restriction enzyme generated a large number of
digested fragments (19 or 20), with exception of ApaI
which originated only 10 band classes, allowing the
discrimination of only four species (Table II). Indeed,
for most analysed yeast species (36), this enzyme
was not able to digest the PCR amplifi ed fragment,
a fact that was already reported by Baleiras-Couto et
al. (2005). The profi les generated after digestion with
ApaI enzyme presented the lowest polymorphism and
discrimination power.
On the other hand, the digestion with restriction
enzymes HaeIII and CfoI produced higher number
of well-developed bands and higher degree of poly-
morphism (with 22 and 24 distinct restriction profi les,
respectively). The discrimination power of HaeIII
and CfoI was also higher as many restriction profi les
were species specifi c (16 and 14 respectively). The
remaining enzymes HinfI and MseI despite the high
78
TABLE I
Strains used in the present study, their collection number, geographical origin and sources of isolation (when available).
Estirpes de leveduras utilizadas no presente trabalho e respectivos números de colecção, origem geográfi ca e fonte de isolamento (quando
disponíveis).
a Only for type strains; b yeast-like fungus
EVN-Colecção de Microrganismos EVN, INRB/INIA Dois Portos, Portugal; CBS-Centraalbureau voor Schimmelcultures,
Utrecht, The Netherlands; PYCC – Portuguese Yeast Culture Collection, Caparica, Portugal
T Type strain, NT Neotype strain, LT Lectotype strain
79
Figure 1 - 53 yeast species representative restriction profi les obtained after digestion with ApaI, HinfI, MseI, HaeIII
and CfoI enzymes of the 26S rDNA region. The number following each species corresponds to the access number of Co-
lecção de Microrganismos EVN (INRB/INIA Dois Portos); (T), (NT) and (LT) mean type, neotype and lectotype yeasts,
respectively.
Perfi s de restrição representativos das espécies de 53 espécies de leveduras obtidos após digestão de uma região do
ADNr 26S com as enzimas ApaI, HinfI, MseI, HaeIII and CfoI. O número que segue a espécie de cada estirpe correspon-
de ao número de entrada na Colecção de Microrganismos EVN (INRB/ INIA Dois Portos); (T), (NT) e (LT) signifi cam
leveduras tipo, neotipo e lectótipo, respectivamente.
TABLE I
Characteristics of the restriction fragment length polymorphism profi les of the PCR amplifi ed 26S rDNA region corresponding to each re-
striction enzymes ApaI, HinfI, MseI, HaeIII and CfoI.
Características dos perfi s de restrição gerados após digestão com cada uma das enzimas ApaI, HinfI, MseI, HaeIII and CfoI do produto
amplifi cado por PCR da região 26S do ADNr.
80
degree of polymorphism (with 22 and 16 restriction
profi les, respectively) showed an intermediate discri-
mination power presenting high number of profi les
shared by many of the studied species.
Cluster analysis of the strains in study were performed
considering the fi ngerprints of all restriction enzymes,
their relationship was calculated by applying the Dice
coeffi cient, and a dendrogram was generated using
UPGMA clustering algorithm. The 26S rDNA-based
restriction analysis generated 47 clusters 42 of them
corresponding to a single yeast species and only fi ve
clusters not species-specifi c (Figure 2). The calcula-
ted cophenetic correlation coeffi cient (0.83) indicates
a good fi t for the cluster analysis. The species-specifi c
restriction profi les generated by the fi ve endonuclea-
ses used in this study allowed the identifi cation of the
most predominant non-Saccharomyces yeast genus
found in grape surfaces or winery environments such
as Hanseniaspora, Candida, Pichia, Rhodotorula,
and Kluyveromyces (Longo et al., 1991; Fleet and
Heard, 1993; Schütz and Gafner, 1993; Torija et al.,
2001; Clemente-Jimenez et al., 2004; Zott et al.,
2008). This identifi cation is of major importance
as non-Sacharomyces yeasts might infl uence wine
fermentations both directly, through production of
off-fl avors, and indirectly by modulating the growth
Figure 2 - Dendrogram of restriction profi les fi ngerprint, obtained after digestion with HinfI, MseI, ApaI, HaeIII and CfoI enzymes,
presented by the 78 yeast strains. Dendrogram was generated by the Unweighted Pair Group Method using Arithmetic Average (UPGMA)
clustering algorithm, calculated by using GelCompar II (version 5.1), cophenetic correlation coeffi cient = 0.83. The fi ve clusters that could
not be solved at species level are shown in dotted lines. The number following each species corresponds to the access number of Colecção de
Microrganismos EVN (INRB/INIA Dois Portos); (T), (NT) and (LT) mean type, neotype and lectotype yeasts, respectively.
Dendrograma representando a semelhança entre as 78 estirpes com base nos perfi s de restrição de ApaI, HinfI, MseI, HaeIII and CfoI obti-
dos de uma região do ADNr 26S. O dendrograma foi criado usando o coefi ciente de Dice pelo método de agrupamento UPGMA (GelCom-
par II, versão 5.1), coefi ciente de correlação cofenética = 0,83. Os cinco grupos em que não foi possível a identifi cação ao nível da espécie
estão indicados por linhas pontilhadas. O número que segue a espécie de cada estirpe corresponde ao número de entrada na Colecção de
Microrganismos EVN (INRB/INIA Dois Portos); (T), (NT) e (LT) signifi cam leveduras tipo, neotipo e lectótipo, respectivamente.
81
or metabolism of the dominant Saccharomyces po-
pulation (Fleet, 2003).
The non-Saccharomyces yeast species belonging
to Metschnikowia, Kluyveromyces, Cryptococcus,
Rhodotorula, Aureobasidium, Issatchenkia, Deba-
ryomyces, Lachancea, Zygoascus and Saccharomyco-
des genera were all well assigned by presenting
distinctive restriction profi les (Figure 2). These yeast
species although in a lower extent, are normally
present during wine fermentation (Mills et al., 2002;
Baleiras-Couto et al., 2005; Nisiotou et al., 2007;
Bisson and Joseph, 2009).
Schizosaccharomyces pombe, characterized by its
special mode of vegetative reproduction and a cer-
tain degree of osmophily, can cause food spoilage
(Esteve-Zarzoso et al., 1999). This species presented
a unique restriction profi le and, therefore, could be
clearly separated.
In the present study, unique species-specifi c restric-
tion profi les for the fi ve studied Pichia species were
obtained (Figure 2). Some Pichia species are present
at high levels at the beginning of fermentations and
have been associated with the development of surface
fl ora in wines exposed to air or incompletely fi lled
tanks or barrels (Fleet, 1993). The P. membranifa-
ciens species may also present killer property by
producing toxins that could inhibit the growth of some
spoilage yeast such as Brettanomyces bruxellensis
(Santos et al., 2009).
The very heterogeneous genus Candida includes all
yeast species that cannot be classifi ed in any other
assexual ascomycetous yeast genera (Esteve-Zarzoso
et al., 1999). Some Candida species have become
very interesting for oenology due to their highly
fructophilic nature allowing their use along with S.
cerevisiae which is highly glucophilic (Mills et al.,
2002). In this study, we analysed six Candida species
that are frequently isolated in food and beverages.
Through the restriction profi les generated with the
fi ve endonucleases, all these species could be clearly
assigned (Figure 2). In some cases, Candida species
have been shown to be able to complete the alcoholic
fermentations (Clemente-Jimenez et al., 2004). The
species C. stellata was found to be present at high
level in musts (Hierro et al., 2006; González et al.,
2007). However, in a recent work Csoma and Sipiczki
(2008) have proposed that most isolates from grapes
and wine are C. zemplinina rather than C. stellata.
In this work, both species were evidently separated
(Figure 2).
The unsolved group, constituted by Dekkera ano-
mala and D. bruxellensis, which presented identical
restriction profi les, was clearly separated from all
other studied species constituting a reliable approach
for Dekkera genus identifi cation (cluster number
1, Figure 2). While Esteve-Zarzoso and co-authors
(1999) clearly separated these two species using the
5.8S-ITS region restriction profi les, these authors
could not separate D. anomala from H. uvarum and
H. guilliermondii. In an industrial perspective, the
methodology under study enabled the identifi cation
of the genus Dekkera which includes dangerous
wine spoilers as they negatively modify physical and
sensorial properties of wine provoking severe econo-
mical losses (Loureiro and Malfeito-Ferreira, 2003).
The closer Brettanomyces species (B. naardenensis
and B. custersianus) were also separated from each
other and from Dekkera species.
The species Torulaspora delbrueckii, Saccharomyces
bayanus and S. pastorianus were grouped in one
cluster whereas S. cerevisiae and S. paradoxus were
separated from them forming another cluster (clusters
number 2 and 3, Figure 2). T. delbrueckii can produ-
ce positive effects on the taste and aroma of wines
(Ciani and Maccarelli, 1998) whilst Saccharomyces
complex (S. bayanus, S. cerevisiae, S. paradoxus and
S. pastorianus) is the most strongly fermenting and
ethanol-tolerant yeast group which takes over the
wine fermentation (Fleet and Heard, 1993). In an
early study, James and co-authors (1997) reported that
the four species of the Saccharomyces sensu stricto
were found to be closely related, displaying sequences
similarity of the 18S rDNA higher than 99.9 %. Inde-
ed, formerly the separation of Saccharomyces sensu
stricto species could be achieved through isoenzyme
analysis (Duarte et al., 1999) and more recently by
an extensive and combined gene analysis (Kurtzman
and Robnett, 2003). The restriction profi le of the 26S
rDNA enabled the separation of the Kazachstania
exigua (formerly named as Saccharomyces exiguus),
a species member of Saccharomycetaceae family.
The grouping of Zygosaccharomyces bailii and Z.
lentus, in one cluster allowed separating these spe-
cies which can be very important for quality control
purposes (cluster number 4, Figure 2). According to
phylogenetic data of the 18S rRNA gene and the ITS
region some strains that were previously identifi ed
as Z. bailii were reclassifi ed as new species Z. lentus
(Steels et al., 1999). This new species also showed
some physiological differences when compared to
Z. bailii. The remaining studied Zygosaccharomyces
species (Z. bisporus and Z. mellis) and Zygotorulas-
pora fl orentinus (formerly named as Z. fl orentinus)
presented species-specifi c restriction profi les.
Hanseniaspora species (anamorph Kloeckera sp.)
are common yeast constituents on grapes and often
dominate the early stages of wine fermentations
(Romano et al., 1993). Growth of these apiculate
yeasts may contribute to the fi nal wine quality through
production of esters, glycerol and acetoin (Gil et al.,
1996). On the other hand, Hanseniaspora sp. may
also negatively affect wine fermentations (du Toit
and Pretorius, 2000). High levels of this yeast have
been found in damaged grapes and might be asso-
ciated with stuck fermentations (Bisson, 1999). The
last unsolved cluster was constituted by H. uvarum
and H. guilliermondii which present a very close
82
relatedness (cluster number 5, Figure 2). These two
species showed an insignifi cant D1/D2 sequence
divergence which did not exceed 1% (Kurtzman
and Robnett, 1998; Cadez et al., 2003), a value that
is considered the borderline of species separation
(Kurtzman and Robnett, 1998). Indeed, recent re-
sults have showed that D. anomala presented a high
similarity with these two Hanseniaspora species in
restriction profi le 5.8S-ITS region, after the digestion
with HinfI, HaeIII and CfoI enzymes (Barata et al.,
2008). These authors only achieved the differentia-
tion of H. uvarum from H. guilliermondii and D.
anomala by using physiological and biochemical
tests. In this work, a separation of Hanseniaspora
and Dekkera genus was achieved, highlighting the
advantage of using 26S rDNA instead of 5.8 S- ITS
region. Nonetheless, for an accurate identifi cation
of Hanseniaspora species, sequencing of the ITS
regions might be needed (Cadez et al., 2003). In this
study, Hanseniaspora occidentalis and H. osmophila
presented species-specifi c restriction profi les.
In order to simplify wine yeast identifi cation using the
generated restriction profi les database, cluster analy-
sis was also performed to all possible combinations
of three restriction enzymes. The combination of the
profi les obtained with the restriction enzymes MseI,
HaeIII and CfoI revealed the highest discrimination
power. A total of 46 distinct clusters were formed,
from which 42 were assigned to a single species
(Figure 3). The main difference from the separation
achieved with the fi ve restriction enzymes is that S.
bayanus, S. pastorianus, T. delbrueckii were grouped
together with Z. bailii and Z. lentus. The very close
relationship between Zygosaccharomyces, Saccha-
romyces and Torulaspora genera has already been
suggested based on the phylogenetic trees deduced
from 18S rDNA (James et al., 1996; 1997) and 26S
rDNA (Kurtzman and Robnett, 1998). The closeness
between these three genera regarding their response
similarity to several physiological tests has also been
reported (Esteve-Zarzoso et al., 2003). However, the
ApaI enzyme enabled the generation of a distinctive
profile for the two Zygosaccharomyces species,
therefore allowing their separation from S. bayanus,
S. pastorianus and T. delbrueckii (Figure 4). This
additional restriction enzyme would be used only if
it is necessary to clarify this situation. For example,
in wine quality control might be necessary to identify
Zygosaccharomyces species which are considered
dangerous wine spoilage yeasts as they can produce
off-fl avors, are osmotolerant, fructophiles, highly-
fermentative, tolerant to high ethanol levels and
extremely preservative-resistant (Steels et al., 2000;
Loureiro and Malfeito-Ferreira, 2003).
CONCLUSIONS
The analysis of the restriction profi les obtained from
the PCR amplifi ed NL1-LR6 region of the 26S rDNA
allowed the discrimination of 42 species among the
53 yeast species analyzed in this study. The remai-
ning groups comprise closely related species both
at taxonomic and wine making levels. The method
pointed out in this study represents a fast, less labo-
rious and less expensive technique when compared to
sequencing besides it does not require sophisticated
equipment. This method is a very useful tool when
there is a large number of isolates to be identifi ed.
Another practical applicability of the method relies
on the capacity to clearly assign the common wine
spoilage yeasts D. anomala and D. bruxellensis to
one cluster and Z. bailii and Z. lentus to another dis-
tinctive cluster. This is an important result in terms
of the applicability of the method for quality control
purposes. This study allowed the establishment of a
restriction profi le database based on certifi ed yeast
strains that can be used in yeast identifi cation carried
out both at research and industrial level.
ACKNOWLEDGEMENTS
The authors thank M. Filomena Alemão for technical
assistance. This research was partially supported
by the program POCI 2010 (FEDER/FCT, POCTI/
AGR/56102/2004).
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