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in southern Dalmatian and on the islands that morphologically resembled Zinfandel and
could thus be potential Croatian Zinfandel counterparts. I performed genetic profiling of all
the varieties using microsatellite markers and compared the profiles with that of Zinfandel.
I compiled the results of this research in my Ph.D (which resulted in the finding of
Zinfandel in Croatia). dissertation defended at the University of Zagreb and entitled
'
Investigation of relatedness between Zinfandel and autochthonous Croatian grape
varieties (Vitis vinifera L.)
'.
Microsatellite repeat region
(AG)
9
Forward primer
AGAGAGAGAGAGAGAGAG
TCTCTCTCTCTCTCTCTC
UNIQUE
UNIQUE
Reverse primer
Figure 1.
A schematic representation of a microsatellite repeat.
Delseney et al. (1983) first demonstrated the existence of simple sequence motifs in
plant nuclear DNA.
Simple sequence repeat
(SSR
)
markers are di-, tri- or tetra-nucleotide
repeats numbering in thousands in every eukaryotic genome (Zietkiewicz et al. 1994,
Goldstein and Schlötterer 1999). They are locus-specific and codominant, making
complicated parentage relationships resolvable (Bowers and Meredith 1997, Meredith et al.
1999) and enabling the reconstruction of grapevine pedigrees (Sefc et al. 1998b). They also
proved to be useful in resolving dilemmas regarding induced crosses - e.g. Müller Thurgau
(Regner et al. 1996, Dettweiler et al. 2000a) and confirming synonyms (Cipriani et al.
1994, Botta et al. 1995, Bowers et al. 1996, Lopes et al. 1999, Maletiü et al. 1999, Lefort et
al. 2000). Further advantages of SSR markers in identification and parentage analyses are:
(i) their reproducibility - consistent microsatellite profiles were obtained for the same
cultivars in different years (Botta et al. 1995) and different laboratories (Grando and
Frisinghelli 1998, Lefort et al. 2000), (ii) high degree of polymorphism observed (between
5-10 alleles per marker) enabling differentiation of clones (Vignani et al. 1996, Regner et
al. 2000), and (iii) objectiveness in comparison to ampelographic or isozyme methods
alone. Each simple sequence repeat region is flanked by unique sequences, and PCR
primers complementary to the flanking sequences can uniquely detect each SSR. The PCR
primers that are used to detect an SSR locus are so specific that they recognize only a single
location in the plant DNA. All the fragments amplified by a pair of primers represent alleles
of a single locus. Two bands at the same position represent identical DNA sequences. The
banding patterns of each cultivar are easy to distinguish, and the information (expressed as
the length, in nucleotides, of each band) may be communicated to other research groups for
comparison. This makes SSRs the method of choice in cultivar identification.
Since Thomas and Scott (1993) published the first grape microsatellite markers,
many additional polymorphic markers have been developed (Bowers et al. 1996, 1999a,
Sefc et al. 1999). Because international cooperators can easily use the same SSR markers
and because results obtained can easily be shared electronically, it is possible to compare
the SSR profiles of varieties grown in different countries without exchanging gel images or
importing cuttings or DNA. Six markers are in most cases sufficient to differentiate
