DNA was extracted from leaves and quantified as described by Mengoni et al. (2000b). PCR amplification reactions were performed in a 25-µl total volume containing 5 mM KCl, 1 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, 0.2 mM of each dNTP, 0.8 U of Taq DNA polymerase (Dynazyme II, Finnzyme, Espoo, Finland), 4 pmols of each primer, 10–25 ng of template DNA. The amplifications for the cpSSR haplotype definition were done in two separate multiplex reactions, one with primer pairs CCMP2, CCMP6 and CCMP10, the other one with primer pairs CCMP1 and CCMP7 (Weising & Gardner, 1999). Reactions were performed in a Perkin-Elmer 9600 thermal cycler (Perkin Elmer, Norwalk, CT, USA) programmed for an initial melting at 94°C for 5 min followed by 35 cycles at 94°C for 1 min, 50°C for 1 min (45°C for CCMP1 and CCMP7 primer pairs) and 72°C for 1 min. A final extension step at 72°C for 10 min was performed. Forward primers were labeled at the 5′ end with 6-FAM (6-carboxyfluorescein) for CCMP1, HEX (4,7,2′,4′,5′,7′-hexachloro-6-carboxyfluorescein) for CCMP2 and CCMP6, TET (4,7,2′,7′-tetrachloro-6-carboxyfluorescein) for CCMP7 and CCMP10. The amplification products of the two PCR reactions for each sample were mixed together and resolved in a Perkin-Elmer ABI 310 (PE Biosystems, Perkin Elmer, Norwalk, CT, USA) genetic analyzer by capillary electrophoresis. The capillary was filled with POP-4 (PE Biosystems, Perkin Elmer, Norwalk, CT, USA), and 3 µl of PCR product plus 0.5 µl of GenScan internal size standard TAMRA-500 (PE Biosystems, Perkin Elmer, Norwalk, CT, USA) were added to 11 µl of deionized formamide. Samples were denatured for 3 min at 95°C, chilled on ice, transferred into a sample tray, and injected at 15 KV for 3 s onto a 47-cm capillary and run at 15 kV for 30 min at 60°C. The data analysis was performed using ABI GenScan analysis software (PE Biosystems, Perkin Elmer, Norwalk, CT, USA) and size of the fragment was obtained by the comparison with an internal size standard (TAMRA-500, Perkin Elmer, Norwalk, CT, USA). The DNA fragments obtained from PCR amplification of single cpSSR loci were cloned into the pCR2.1 vector (Life Technologies-Invitrogen, Carlsbad, CA, USA) and sequenced using M13 universal primer with the BigDye system (Perkin Elmer, Norwalk, CT, USA) in a Perkin-Elmer ABI 310 genetic analyzer.