Comparison of RAPDs, AFLPs and SSR markers for the genetic ...

the most efficient markers in the study. This value may
rise even higher if attempts to optimize this technique
into a single multiple reaction involving all six microsatellites
prove successful.
Another important factor to consider when evaluating
marker efficiency is the ability to determine relationships
between yeast strains based on an estimation of
genetic similarity (Figs. 4a–c). Genetic similarity coefficients
were obtained (Table 3) for all three PCR-derived
techniques,reflecting the extreme variability and high
resolving power of these methods. These same results,
including lower similarity estimates for SSR analysis
than for both RAPDs and AFLPs,have been reported
in equivalent studies performed using other organisms
(in soybean, Powell et al.,1996; in maize, Pejic et al.,
1998; in Musa, Crouch et al.,1999). These findings
suggest that differences exist between a technique that
amplifies unique sequences,such as microsatellite
analysis,and other methods that amplify multiple
sequences,such as RAPD and AFLP techniques.
Poor correlation between estimates of genetic similarity
based on the three different techniques evaluated
in this study indicates that these methods may selectively
screen for different regions of the genome. To further
explore this issue,as well as the close similarity between
the strains assayed,a greater number of loci should be
analysed. In an effort to increase the integrity of our
analysis,a single dendrogram was constructed that
combines the information obtained from all three
marker types (Fig. 4d). This genetic tree revealed that
only the topology of some groupings is conserved,with
non-significant groups appearing random regardless of
the number of markers studied. Further physiological
experimentation is needed in order to determine if the
conserved groups are correlated with traits of interest.
Based on the results of this study,RAPD markers
were the least polymorphic markers of those evaluated,
and consequently had the least resolving power.
Although the amount of variability detected with
RAPD analysis is dependent upon the selection of
appropriate primers,this method has the advantage of
being inexpensive and simple to perform,and does not
require a previous knowledge of the genome. Problems
with reproducibility have plagued the use of this
technique in the past,due to the low temperature of
the hybridization of the primers. However,we were able
to limit these problems in this study,as evidenced by the
results of our duplicate analysis,by careful DNA
preparation,strict adherence to amplification protocols
and a rigorous interpretation of the results.
The AFLP technique resulted in high resolution and
good reproducibility in this study,a finding substantiated
by the recent use of AFLP markers in the
identification and intraspecific differentiation of S.
cerevisiae strains (deBarros Lopes et al.,1999). This
technique,however,is much more technically complex
ARTICLE IN PRESS
F. Javier Gallego et al. / Food Microbiology 22 (2005) 561–568 567
than the use of SSR markers,requiring numerous
experimental steps at a higher cost per informative
marker. Despite these limitations,the AFLP technique
has great value as a tool for use in genetic mapping and
evolutionary studies,as it can test a large number of loci
distributed randomly throughout a genome. Unlike
microsatellites,AFLP markers are not highly variable,
providing a less biased estimate of population variability.
SSR analysis revealed the highest genetic variability in
the microbial population studied,also achieving the
greatest discriminatory power. It also proved to be the
most efficient method,with the highest number of
effective alleles per assay. We also found the results of
SSR to be faster and easier to interpret than the other
techniques studied,making it an ideal technique for use
in the characterization and identification of S. cerevisiae
strains. Successful SSR analysis,however,depends on
the proper design and synthesis of primers,requiring a
considerable amount of effort in the selection of
polymorphic microsatellites that afford specific amplifications.
Our study benefited from an available panel of
six microsatellite loci and the appropriate primers
needed for its specific amplification (Pe´ rez et al.,2001).
In summary,SSR amplifications is a simple and
effective method,with a high degree of discrimination
and reproducibility,that can be used in the identification
and intraspecific differentiation of S. cerevisiae strains,a
species of great importance in industrial fermentations.
Acknowledgments
This research was supported by grants SC94-126 from
the Programa Sectorial I+D Agrario y Alimentario and
RM00-001 from the Accio´ n Estratégica ‘‘Conservacio´ n
de los recursos gene´ ticos de interés agroalimentario’’ del
Plan Nacional de Investigacio´ n Cientı´ fica,Desarrollo e
Innovacio´ n Tecnolo´ gica (I+D+I) from the Instituto
Nacional de Investigacio´ n y Tecnologı´ a Agraria y
Alimentaria (INIA) (Spain).
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