| Microsatellite sequences represent ideal markers for providing an accurate molecular identification of cultivars or indivuals and consequently for solving wrong synonymies and homonymies, whatever it is in germplasm repositories or in the vineyard itself. Furthermore microsatellites allow to reach discrete genetic information such as pedigree and thus to trace back to very ancient crossing events Such a genotyping method provides genetic profiles, consisting of di-allelic information at each locus. Such profiles may be compared by the use of population genetic programmes for diploid markers or specific to microsatellites, and genetic relationships between cultivars can be shown. The Greek genetic resources of grapevine account for about 670 cultivars, among which 300 are maybe still be cultivated, and very little is known about their origins and their genetic relationships. On the other hand it is supposed, from the ancient literature and history, that part of the modern Western European cultivars could originate from Greek cultivars. But it is also true that Western cultivars have been imported in Greece at different periods. What has been the real influence of Greek cultivars on Western European cultivars? What has been the importance of exchanges between Greek and Italian cultivars, between Greece and other regions, including Near East, the putative domestication site of grapevine? These are, for instance the questions, that will be addressed in with microsatellite markers in a very next future, provided an exhaustive molecular survey is carried out with standardised markers. |
| MATERIAL AND METHODS |
| Plant material |
| Leaves of 254 cultivars of Vitis vinifera L were collected from the glasshouse collection of the Laboratory of Plant Physiology and Biotechnology at the University of Crete, the Institute of Viticulture, Floriculture and Vegetable Crops of Heraklion (National Agricultural Research Foundation; outdoors). |
| Transliteration |
| Names of Greek cultivars are given according the ISO 843 scheme transliteration (Information and documentation - Conversion of Greek characters into Latin characters, ISO843:1997(E), IOS, Geneva, Switzerland). |
| DNA extraction |
| DNA was extracted from 100-150 mg fresh weight of leaf tissue according to a micro-method of DNA purification (Lefort and Douglas, 1999) developed for hardwood species and modified for Vitis species. |
| Microsatellite profiling |
| Amplification primers sequences for nuclear microsatellite loci from Vitis riparia (Sefc et al., 1999a), ssrVrZAG 21, ssrVrZAG 47, ssrVrZAG 62, ssrVrZAG 64, ssrVrZAG 79 ssrVrZAG 83, and from Vitis vinifera, VVS2 (Thomas et al. 1994), UCH11, UCH29 (Lefort et al., unpublished) were used for DNA amplification. PCR products analysis was carried on 6% polyacrylamide, 7M urea, 1xTBE sequencing gels in a Licor 2400 DNA Sequencer (Licor, Lincoln, Nebraska, USA) and alleles were sized with the software GENE PROFILER v3.54 (Scanalytics, Fairfax, Va., USA). Sizing with GENE PROFILER was standardised with sizing with GENESCAN in a Pharmacia ALFexpress DNA sequencer by Kristina Sefc and Herta Steinkellner at VVS2, ssrVrZAG 21, ssrVrZAG 47, ssrVrZAG 62, ssrVrZAG 64, ssrVrZAG 79 and ssrVrZAG 83. The phenogram in Figure 1 was constructed by using the software MICROSAT (Minch, 1997) for calculating genetic distance in [-log(proportion of shared alleles)]. The distance matrix obtained from MICROSAT was processed with Kitsch from the PHYLIP package (Felsenstein, 1989) and TREEVIEW (Page, 1996). Probability of identity [PI = Spi4 + SS (2pipj)2 ] and probability of null alleles [ r = (He-Ho) (1 + He) ] were calculated with IDENTITY 1.0 (Wagner and Sefc, 1999), Austria). |
| RESULTS AND DISCUSSION |
| The present study was carried out on 254 cultivars, of which 235 were Greek and thus representing 35% of the total Greek germplasm. Genotyping these 254 cultivars with nine microsatellite markers provided 219 single profiles, while 195 cultivars had a unique profile. The observed heterozygosity was high at all loci ranging from 0.67 to 0.87 and the probability of null alleles was not significant at any locus. The nine markers yielded a total of 96 alleles, i.e a mean number of alleles per locus of 10.66. The probability of two cultivars to share the same genetic profile without being related is given by the total probability of identity which was very low at 1,19.10-8. Genetic diversity and genetic relationships between these cultivars are presented in a phenogramme (Fig. 1) based on their proportion of shared alleles. Genetic synonymy, where a same profile is shared by more than one cultivar, accounted for 15 pairs, 9 triplets and a group of 4 cultivars. In some cases, synonymy was expected as it is the case for Italia and Razaki moschato but was unexpected in numerous cases such as between Chakiki oporiko and Aetonychi kokkino or Syriki and Provatina. On the other hand genotyping the synonyms Tsardana and Romeiko mavro at more loci showed that the latter was an offspring of Tsardana (results not shown). Inside groups of cultivars with homologous names, either a close genetic similarity or a synonymy was confirmed (Fileri cultivars, Fraoula cultivars, Liatiko cultivars, Moschato cultivars, Razaki cultivars), or these cultivars seemed to be very different of each other (Aeonychi cultivars, Nychato cultivars). A few cultivars share very little alleles with the group of Greek cultivars: for instance Tressallier, a minor cultivar from Central France was the more distant, Pafsanias, a cultivar known to be a Vitis sylvestris, Fegi, Karnalachades, Malvasia del Chianti, Cabernet Franc, Sauvignon Blanc and their offspring Cabernet Sauvignon. This could suggest that these cultivars appeared locally in their regions of origin from wild grapevine stock. It also seems in other cases that a close genetic relationship is in agreement with a common geographic origin, such is the case of Mavro Kyprou and Kypreiko (Cyprus) or Psilomavro Kalavryton and Mavro kalavrytiko (Kalavryta, Peloponnese). The present data represent a helpful milestone for understanding the relationships within the Greek germplasm and between grapevine from other regions, and more results can be now expected from further genotyping of possible pedigrees. |
| Note: genetic profiles and regions of cultivation are viewable in the Greek Vitis Database at http://www.biology.uch.gr/gvd |
| Figure 1: Phenogramme of 254 Greek and foreign cultivars at 9 nSSR loci |
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| REFERENCES
Felsenstein J (1989) Phylogeny inference package. Cladistics 5:164-166. Lefort F, Douglas GC (1999) An efficient micro-method of DNA isolation from mature leaves of four hardwood tree species Acer, Fraxinus, Prunus and Quercus. Annals of Forest Science (formerly Annales des Sciences Forestieres), 56:259-263. Lefort F, Kyvelos CJ, Poisse E, Edwards KJ, Roubelakis-Angelakis KA (2000) New microsatellite markers for Vitis vinifera and their conservation in Vitis spp. and hybrids (unpublished) Minch E, Ruiz-Linares A, Goldstein D, Feldman M, Cavalli-Sforza LL (1997) Microsat v.1.5d : a computer program for calculating various statistics on microsatellite allele data (http://lotka.stanford.edu/microsat/microsat.html) Sefc KM, Regner F, Turetschek E, Glossl J, Steinkellner H (1999) Identification of microsatellite sequences in Vitis riparia and their applicability for genotyping of different Vitis species. Genome 42:1-7. Thomas MR, Scott NS (1993) Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs). Theor Appl Genet, 86:985-990 Wagner HW, Sefc KM (1999) IDENTITY 1.0. Centre for Applied Genetics, University of Agricultural Sciences Vienna, Austria. |