Leaves of Vitis vinifera L cultivars were collected from the ampelographic collection of the Laboratory of Plant Physiology and Biotechnology, University of Crete and the Institute of Viticulture, Floriculture and Vegetable Crops of Heraklion (National Agronony Research Foundation; outdoors). Leaves were kept frozen at ' 80'C until 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. More specifically the extraction buffer consisted of: 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.|
|Nuclear microsatellite PCR|
Amplification primers sequences for nuclear microsatellite loci from Vitis riparia (Steinkellner et al., 1999), ssrVrZAG 21, ssrVrZAG 47, ssrVrZAG 62, ssrVrZAG 64, ssrVrZAG 79 ssrVrZAG 83 and from Vitis vinifera (Thomas and Scot, 1993; Bowers et al., 1996, Lefort et al., unpublished) VVS2, VMD5, VMD7, UCH2, UCH3, UCH11, UCH12, UCH17, UCH 18, UCH19, UCH29, UCH35, UCH40, UCH47 were used for DNA amplification. PCR amplifications were carried out in 96-wells propylene plates in 20 'l final volume reaction mixtures in a PTC-100 thermal cycler (M.J. Research Inc., Watertown, Ma.,USA). PCR reactions were as follows: 1 'M of each primer, 100 'M of each dNTPs (Biofinex, Praroman, Switzerland), 1.5 mM MgCl2 in the following buffer 75 mm Tris HCl (pH 9.0), 50 mM KCl, 20 mM (NH4)2SO4, 0.5 units Taq polymerase(Biotools, Madrid, Spain) and 50 ng DNA template. The forward primer was in each case labelled with the Licor IR800 fluorochrome. The following thermal cycling protocol was applied : 95 'C for 5 min, 10 cycles of 15 s at 51 'C, 15 s at 94 'C, followed by 23 cycles of 15 s at 51 'C, 15 s at 89 'C and terminated immediately at 4 'C. Amplification success was checked by electrophoresis in 2% agarose gel in 1xTBE buffer (Tris, boric acid, EDTA pH 8.0) and ethidium bromide staining. PCR products analysis was carried on in 2% polyacrylamide 7M urea 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).
|Chloroplast microsatellite PCR|
According to Echt
et al. (1998), the chloroplast genome, which does not exist in a heterozygous
state and does not genetically recombine, 'may be viewed as a single locus
and all sequence variation interpreted as giving rise to different haplotypes
of the genome'. An alternative approach may consider the chloroplast genome
as 'a circular haploid chromosome wherein sequence variation generates
different alleles within individual, non recombinant loci'. We used the
proposed terminology where the term 'locus' refers to 'a cpSSR site, defined
by the termini of a PCR primer pair', and 'alleles' to 'length variants
at a cpSSR site'. We used six primer pairs designed for dicotyledonous
angiosperms by Weising and Gardner (1999): ccmp2, ccmp3, ccmp4, ccmp6,
ccmp7 and ccmp10.
Identity 1.0 (Sefc and Wagner, 2000) was used for formatting the microsatellite data into the Microsat format. 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 then drawn by using Kitsch of the Phylip package (Feselstein, 1989) and Treeview (Page, 1996).