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DNA material
The non-carbonised grape pips from the Neolithic pile-dwelling site ofHočevarica were preserved by waterlogging, but after sieving they were dried, which probably caused further DNA degradation. Therefore, we decided to take grape pips from another pile-dwelling settlement, Stare Gmajne in Ljubljansko barje, which was excavated in 2006 and 2007 (see archaeobotanical methods) for the DNA extraction. These seeds were found and preserved in a waterlogged state. Seeds with various morphological characteristics, of rounded and pyriform shape, were taken for the preliminary DNA analysis.
After their excavation in 2005, the Roman grape pips from Vrhnika were originally preserved waterlogged, but later they were dried. Excavation of new material was not possible, so the fragile, dried seeds were used for the analysis. Morphologically rounded and pyriform seeds were taken, as well.
For the purpose of comparison between the DNA extracted from the archaeological grape pips and the DNA from the modern cultivated Vitis viniferassp. viniferapips were included in our analysis. Pips from each of the four varieties used for wine making in Slovenia were chosen (see morphological material; Fig. 3, without Vitis labrusca).
Since there are no more pure, natural wild populations of Vitis vinifera ssp. sylvestris in Slovenia, we included young green sprouts of three presumably wild plants that have only recently been found in the middle of the woods in the village of Vinje, Slovenia (Piltaver 2007). These were all phenotypically characterized as male plants.
DNA extraction and manipulation
To avoid potential contamination of samples with exogenous DNA in the molecular laboratory, several precautions were taken: all glassware used was extensively machine washed and autoclaved several times. The common CTAB method (Doyle and Doyle 1987) was employed for the DNA isolation. Briefly, three seeds of each sample were processed together or 2 cm2 of leaf material was ground in the presence of prewarmed (68°C) CTAB buffer [2% CTAB, 1.4 M NaCl , 20 mM EDTA, 100 mM Tris-HCl (pH 8.0), 0.2% -mercaptoethanol] and incubated at 68°C for 60 min. The samples were extracted with a chloroform:isoamylalcohol (24:1) mixture, centrifuged for 10 min at 12,000 rpm (4°C), and the DNA was precipitated with the addition of 1/10 vol of 3M sodium acetate (pH 5.2) and 1 vol of ice-cold isopropanol. The samples were kept for an hour at –20 °C and centrifuged for 10 min at 12,000 rpm (4°C). The DNA pellet was briefly washed in 70 % ethanol and resuspended in 50 μl of TE buffer. The seed extracted DNA was further purified using a glass milk procedure with the DNA extraction kit (Fermentas) according to the manufacturer’s recommendation. Final DNA was eluted in 30 μl of TE buffer. DNA extracted from the leaves was quantified by fluorimetry (DyNA Quant 200 fluorometer, Amersham Biosciences), and dilutions of 4 ng/μl were made in sterile water.
PCR analysis
Amplification of chloroplast DNA was performed using the specific primers C and F (C: 5’ CGAAATCGGTAGACGCTACG 3’, F: 5’ ATTTGAACTGGTGACACGAG 3’) amplifying the chloroplast region trnL-trnF (Taberlet et al. 1991). PCR reaction was performed in 25 μl with the following components: 5 μl of DNA, 1×PCR buffer [10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, Promega], 2 mM MgCl2, 0.5 μM of each primer pair, and 0.625U of Taq polymerase (Promega). Cycling conditions were as follows: initial denaturation 94°C 2 min and 32 cycles of 94°C 1 min, 58°C 1 min and 72°C 2 min ended by 8 min at 72 °C. Amplification was checked on 1.8 % agarose gels.
References
Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for smallquantities of fresh leaf tissue. Phytochem Bull 19:11–15
Piltaver A (2007) O stari trti iz Vinj (About old grapevine from Vinj, 693in Slovenian). Proteus, Prirodoslovno drusˇtvo Slovenije, Ljubljana 69:390–399
Taberlet P, Gielly Ludovic, Pautou G, Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol Biol 17:1105–1109