| We present here the first attempt of assessing the genetic diversity of the chloroplast genome of Vitis vinifera. Six universal microsatellites primers developed recently were tested in 77 grapevine cultivars from Greece and other geographic origins. All six SSR markers amplified and five gave reproducible results. Four of these latter markers were polymorphic. One locus ccmp2 displayed a large insertion/deletion in 18 out of 77 cultivars. Such a mutational event is very rare in the chloroplast genome, which is known for its high conservation through taxa. Given the modern areas of cultivation and the number of cultivars per region, which displayed this insertion deletion, this event showed to be preferentially distributed in Southern Greece and Southern Aegean with predominance in Peloponnese. A di-allelic pattern was revealed at locus ccmp3 and was present in most cultivars. Since both bands were of equal intensity and polymorphic, they were considered as a di-allelic compound. This di-allelic pattern could have been caused by duplication at this locus, or could also be explained by heteroplasmy. The total diversity found in 77 cultivars was high since 17 haplotypes were found. This high diversity suggests a maternal type of inheritance, which has to be confirmed by genotyping more known parentages. On the other hand, no clear geographic structure was made visible at the scale of the Country, with this set of universal cp markers. That could be explained by a high level of exchanges between viticultural regions, without prejudging about the times and the directions in which they occured. |
| Material and Methods |
| Plant material |
| Leaves of Vitis vinifera L cultivars were collected from the collections of the Laboratory of Plant Physiology and Biotechnology at the University of Crete (green house), the Institute of Viticulture, Floriculture and Vegetable Crops of Heraklion (National Agronomy Research Foundation; outdoors). |
| DNA extraction |
| DNA was extracted from 100-150 mg fresh weight of leaf tissue according a micro-method of DNA purification (Lefort and Douglas, 1999) developed for hardwood species and modified for Vitis species. The modification refers to the extraction buffer which included : 50 mM Tris, pH 8.0, 50 mM EDTA, pH 8.0, 1.1 M NaCl, 0.4 M LiCl, 1% CTAB, 2% PVP (MW = 25,000), 0.5% Tween 20. |
| Microsatellite PCR and analysis of PCR products |
| We used six primer pairs for universal chloroplast microsatellite markers designed for dicotyledonous angiosperms by Weising and Gardner (1999): ccmp2, ccmp3, ccmp4, ccmp6, ccmp7 and ccmp10. PCR amplifications and electrophoresis analyses in sequencing gels (6% polyacrylamide (Pharmacia), 7M urea, TBE 1X) were repeated four times at all loci for all DNA samples. A distance matrix was constructed from microsatellite data with MICROSAT (Minch et al., 1997). The distance used was [-Log (proportion of shared alleles)]. A phenogramme was drawn by using KITSCH of the PHYLIP package (Felsenstein, 1989) and TREEVIEW (Page, 1996). Gene diversity (Nei, 1973) was calculated with POPGENE (Yeh and Boyle, 1997). |
| Results and Discussion |
| The 5 cp SSRs
loci yielded a total of 13 alleles whose combinations yielded 17
chloroplast haplotypes (Figure 1). As shown in Table 1, alleles
sizes at each locus were found in the range of previously reported
sizes for 27 angiosperm species (Weising and Gardner, 1999). Four
out of 5 loci were polymorphici, and at each polymorphic locus,
a main allele was visible with frequencies ranging from 0.51 to
0.76, which according to current theories about microsatellites,
would mean that other alleles originated from the main allele by
subsequent mutations. The locus ccmp2 displayed a large and polymorphic
insertion/deletion, which was present in 18 (23.3%) of the cultivars.
Two alleles were of the short form (208 bp; 209 bp) and one of the
large form (228 bp). A large insertion/deletion is known to be a
rare mutational event in nuclear microsatellite sequences and is
expected to be even rarer in chloroplast microsatellites since the
cp genome does not recombine and is highly conserved. At our knowledge,
this finding is the first report of a microsatellite insertion/deletion
in chloroplasts. Map 1 shows the geographic distribution of the
cultivars harbouring the large insertion/deletion, based on modern
areas of cultivation of these cultivars. Although the cultivation
of grapevine in Greece is very ancient and exchanges of cultivars
between regions of viticulture have certainly occurred, the deletion/insertion
was found to be still preferentially clustered in Southern Greece:
its main occurrences were found in Peloponnese (10 cultivars), Crete
(7 cultivars), the Ionian islands (7 cultivars), Euboea (5 cultivars),
Attica (4 cultivars), the Cyclades (4 cultivars), Dodecanese (4
cultivars), with only minor occurrences in other parts of Greece
(Central Greece, Epirus, Macedonia and Thrace). Locus ccmp3 showed surprisingly a two peaks pattern. One peak appeared under one allelic form (95 bp), present in all cultivars, while a second peak of equal intensity with 2 alleles (106 bp, 107 bp) was present in 63 (81%) cultivars Although other bands of minor intensity have been reported in a few species at several loci by Weising and Gardner (1999) or Vendramin (unpublished results), they did not ever find any bands of equal intensity. For haplotypes, we considered the di-allelic pattern as one allele, so that locus ccmp3 had 3 alleles (95bp, 95bp + 106bp and 95bp + 107bp). Genotyping reproducibility was shown by repeating 4 times PCR amplifications and electrophoretic analyses for all loci and all cultivars. Genotyping different plants from different ampelographic collections for a same cultivar (Italia, Muscat de Hambourg) also confirmed reproducibility. Contamination can be excluded since nuclear microsatellite profiling was carried on duplicates of the same DNA samples and did not report any contamination. The pedigree of the French cultivar Cabernet Sauvignon was established by Bowers and Meredith (1997) with the use of nSSR as being Cabernet Franc and Sauvignon Blanc. Genotyping with cp SSR enabled to precise the sense of this supposedly spontaneous cross: since Cabernet sauvignon and Sauvignon Blanc shared the same haplotype (Figure 1), Sauvignon Blanc could then be the chloroplast donor. The type of inheritability is inferred to be maternal in grapevine and genotyping 2 cultivars members of another pedigree supported this hypothesis: Italia was obtained by crossing Muscat de Hambourg as a male parent and Bicane as a female parent (Ambrosi et al., 1999). Bicane is only female at the contrary of most cultivated grapevine which are hermaphrodites. Genotyping showed that Muscat de Hambourg did not give its chloroplast to Italia (Figure 1) and consequently the type of inheritability of the chloroplast in grapevine should be maternal. Genotyping more fully known pedigrees is needed for confirming this observation. Despite a likewise preferential distribution of the deletion/insertion at ccmp2, globally no clear genetic structure was made visible by cpSSR profiling of 77 Greek grapevine cultivars. That can be seen as a consequence of extensive exchanges of cultivars between Greek regions. On the other hand grapevine cultivars retain a high chloroplastic diversity with 17 haplotypes for 77 grapevine cultivars studied at 5 cpSSR loci. Such a high diversity is generally a characteristic of maternally inherited plastids. |
| Note : genetic profiles and regions of cultivation are viewable in the Greek Vitis Database, http://www.biology.uch.gr/gvd |
| Table
1: Characterisation of 5 universal cpSSR markers in grapevine
Figure 1: Phenogram of grapevine cultivars profiled at 5 universal cp SSRs Map 1: Geographic distribution of the insertion/deletion at locus ccmp2 (one dot per cultivar occurence) |
|
|
| 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, 56:259-263. 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). Page RDM (1996) Weising K, Gardner RC (1999) A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms. Genome 42(1):9-19 Yeh FC, Boyle TJB (1997) Population genetic analysis of co-dominant and dominant markers and quantitative traits. Belg J Bot, 129:157. |