BIOTECHNOLOGY

TECHNIQUES
Volume 8 No.8 (August 1994) pp.557~560
Received as revtied 16th June

R4PD ANALYSIS
DIFFERENTIATED

OF WINE SACCHAROMYCES CEREvIsL4E

STRAINS

BY PULSED FIELD GEL ELECTROPHORESIS

M.S. Grando()*, J. Ubeda (2), A.I. Briones (2).

(1)

Laboratorio di genetica molecolare, Istituto Agrario di San Michele allAdige, Via

Mach 1, 38010 San Michele all Adige, Trento, Italy.

(2)

Departamento de Qufmica Analitica y Tecnologia de alimentos, Universidad de

Castilla - La Mancha, Campus Universitario, 13072 Ciudad Real, Espaiia.

SUMMARY
Enological yeast strains involved simultaneously during a fermentation can be identified
through the analysis of their electrophoretic karyotype. The right assignment of yeasts to
different strains has been checked by analysing the random amplified polymorphic DNA
(RAPD), using a simple minipreparation protocol to obtain the template DNA for the
polymerase chain reaction (PCR).
INTRODUCTION
Pulsed field gel elctrophoresis has revealed the existence of a wide variety of the molecular
karyotype within the enological strains of S. cerevisiue (Blondin and Vezinhet, 1988).
Recently, this technique has been used to control the effectiveness of seeding selected yeasts
into grape musts or to describe the distribution of different strains during wine making. In
ecological surveys of S. cerevisiae during spontaneous grape must fermentations, the number
of different electrophoretic karyotypes is very high and the assignment of a yeast to one
strain or another is not always easy.
To check whether yeast grouping based on electrophoretic karyotype analysis identifies the
clones of yeast present in an enological population, a batch of S. cerevisiae has been subjected
to the analysis of the random amplified polymorphic DNA (RAPD) (Welsh and McClelland,
1990; Williams et al., 1990), using a rapid minipreparation protocol to obtain the template
DNA for the polymerase chain reaction (PCR). With respect to a single comparison based
upon chromosomal patterns, the RAPD-PCR assay allows a genome scanning limited only by
the number of primers used, thus turning out to be more suitable for fingerprinting of yeast
genotypes.
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MATERIALS

AND METHODS

Sampling of S. cerevisiae: yeasts were collected during an ecological survey carried out on
grape musts fermentations of three cellars from the Valdepefias area in Spain. Through the
analysis of the electrophoretic karyotype of 392 clones, the yeasts have been classified in
several families corresponding to different patterns (strains) (Briones et al. 1994). A RAPD
analysis was then performed on three individuals, chosen at random for each of six strains.
Analysis of the molecular karyotypes - sample preparation: for yeast DNA preparation a
modification of the method of Schwartz and Cantor (1984) was used: 200 pl agarose insert
was obtained using the cells of a single colony grown after 48 h on YPD (2% glucose, 2%
bactopeptone, 1% yeast extract and 2% agar). Incubation time of the inserts in LET buffer
(0.45 M EDTA pH 8, 0.01 M Tris pH 7.5, 7.5% 2-mercaptoethanol) was reduced to
3-4 hours. The operations were performed on microtitulation plate volumes.
Pulsed field electrophoresis conditions: inserts (25 pl) were loaded onto wells of a 1%
agarose gel. The run was performed on a contour clamped homogeneous electric field system
(CHEF, Bio-Rad). Temperature of the migration buffer (0.5 x TBE: 45 mM Tris base, 45 mM
boric acid, 1 n&I EDTA) was maintained at 14C. Pulse time program was 60 s/15 h and 90
s/8 h , 200 V.
Analysis of RAPDs - sample preparation: yeast cells of one colony were added to 1.5 ml
of a 5% Chelex suspension in double distilled water (Chelex 100 resin, Bio-Rad). The
suspension was heated and frozen five times by alternating between 80C and -40C, 3 min
in each. After centrifuging for 5 min at 1500 g the resulting supernatant was used immediately
for PCR or stored at -20C. The method was adapted from Singer-Sam et al. (1992).
Amplification conditions: reaction volumes were 25 1.11
containing 5 pl of the template DNA
aqueous solution used as obtained or diluted 1:10, 10 mM Tris-HCl pH 9, 50 mM KCl, 2mM
MgCl,, 100 pM each of dATP, dCTP, dGTP, dTTP (Boehringer), 0.28 pM primer (from sets
A, U and J, Operon Technologies), 0.3 U of Taq DNA polymerase (Super Taq P.H. Stehelin
& Cie AG, Basel, Switzerland). Amplification was performed in a Perkin Elmer Cetus Gene
Amp PCR System 9600 programmed as follows (Koller et al., 1993): 2 cycles of 30 s at
94C 30 s at 36C 120 s at 72C; 20 cycles of 20 s at 94C 15 s at 36C 15 s at 45C,
90 s at 72C; 19 cycles of 20 s at 94C (increased 1 s/cycle), 15 s at 36C 15 s at 45C,
120 s at 72C (increased 3 s/cycle) followed by 10 min at 72C. Amplification products were
electrophoresed in 1.5% agarose gels with 0.5 x TBE and stained with ethidium bromide.
RESULTS AND DISCUSSION
392 colonies of S. cerevisiae subjected to electrophoretic karyotype analysis showed 174
different profiles. Yeast assignment to the different strains was complex because of that high
polymorphism and because of the difficulty

of comparing the numerous runs, although a

standard strain had been loaded into all gels. The comparison between some samples has made
necessary an electrophoretic re-run, with a different relative position of inserts in gels. The
18 yeasts subjected to the analysis of RAPD markers belonged to six different strains, the
electrophoretic karyotype of which are shown in Figure 1. The method used has made possible
to obtain with extreme easiness a DNA substrate, from few cells, suitable for PCR and able
to produce the same amplification fragment patterns even as late as six months after its first

558

preparation. 40 different oligonucleotides decamers, of arbitrary sequence, have been tested

for PCR, in order to identify the most proper primers for differentiating the 18 yeast samples.
In 80% of cases, amplification products of discrete size have been obtained. In the remaining
cases, it has been observed a trend of band consistence toward improvement, as far as the
ratio: ~1 DNA solution/TAQ polymerase units, in the reaction mixture, increased. Cases of
non-reproducibility

occurred when the different fragments of amplified DNA would come out

to be numerous but each present in a low number of copies. In general, a scanty

polymorphism of the DNA fragments which have generated the most intensive bands in gel
has been observed. Nevertheless, to perform R4PD analysis of the different yeast strains, it
has been possible to select some primers which generate simple profiles of consistent and
polymorphic bands.

Figure 1

Molecular karyotypes produced by pulsed field gel electrophoresisassigned 18 wine

yeasts to six different strains. Lane 1: yeasts 1,2,3 - strain a; lane 2: yeasts 4,5,6 strain b; lane 3: yeasts7,8,9 - strain c; lane 4: yeastslo,1 1,12 -strain d; lane 5: yeasts
13,14,15 - strain e; lane 6: yeasts 16,17,18- strain f.

The S. cerevisiae yeast triplettes belonging to each strain have always shown a similar profile
of RAPD markers (Figure 2). Six groups of yeasts, corresponding to the strains differentiated
according to their electrophoretic karyotype, have been identified using subsequently primers
Al 1, A13, A2, U5 and 520 (Figure 3). Each reaction has given as a result two types of
profiles, due to the alternate presence of a single band. These results then con&n

the right

assigmnent of yeasts to different strains by means of electrophoretic karyotype analysis and

at the same time they suggest a further possibility of analysing the genetic diversity of yeasts.

559

Figure 2

RAPD analysis of 18 wine yeast with primer A13. Lanes l-3: strain a; lanes 4-6:
strain b; lanes 7-9: strain c; lanes 10-12: strain d; lanes 13-15: strain e; lanes 16-l 8:
strain f; lane 19: molecular size marker.

Figure 3

Subdivision of different yeast triplettes (a,b,c,d,e,f) on the basis of the RAPDs

obtained with the primers Al 1, A13, A2, U5 and 520.

REFERENCES
Blondin, B., and Vezinhet, F. (1988). Rev. Fr. Oenol. 88, 7-l 1.
Briones, A.I., Ubeda, J., and Grando, M.S. (1994). Am. J. Enol. Vitic. (submitted)
Koller, B., Lehmann, A., McDermott, J.M., and Gessler, C. (1993). Theor. Appl. Genet. 85,
901-904.
Schwartz, D.C., and Cantor, C.R. (1984). Cell, 37, 67-75.
Singer-Sam, J., Tanguay, R.L., and Riggs, A.D. (1989). Amplzjkations, 3, 11.
Welsh, J., and McClelland, M. (1990). Nucleic Acids Res. 18, 7213-7218.
Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., and Tingey, S.V. (1990). Nucleic
Acids Res. 18, 6531-6535.

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