Lefort,
F., Laboratory of Plant Physiology and Biotechnology, Department of Biology,
University of Crete, P.O. Box 2208, 71 409 Heraklion, Crete, Greece.
Anzidei, M., 1Roubelakis-Angelakis, K.A & 2Vendramin, G.G., Istituto
Miglioramento Genetico Piante Forestali, Consiglio Nazionale Delle Ricerche,
Via Atto Vannucci 13, 50134 Firenze, Italy.
| 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) |
|
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