DNA purifi cation and AFLP analysis -DNA for all samples was purifi ed using the DNeasy Plant Minikit (QIAGEN, Doncaster, Victoria, Australia) and quantifi ed using a Picofl uor model 8000 -003 fl uorometer (Turner Designs, Sunnyvale, California, USA) with Quant iT PicoGreen DNA assay kit (Invitrogen, Eugene, Oregon, USA). The AFLP method was adapted from Vos et al. (1995) using simultaneous restriction and ligation of DNA samples. All eucalypt DNA samples were pretested to ensure full

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... digestion under the conditions used for restriction -ligation reactions. Reproducibility of AFLP profi les was confi rmed experimentally for replicate DNA purifi cations and restriction -ligation reactions containing 10, 20, 50, 100 or 200 ng of genomic DNA. Final restriction -ligation reactions (30 µ L) for all samples contained 50 -200 ng genomic DNA, 1 × T4 DNA-ligase buffer (Promega, Madison, Wisconsin, USA), 50 mM NaCl, 50 ng/ µ L bovine serum albumin, 5 U Mse I, 16 U Eco RI, 2 Weiss U T4 DNA ligase, 3 pmol Eco RI adapter and 30 pmol Mse I adapter. Restriction -ligation reactions were incubated for 16 h at 37 ° C. Preselective PCRs (30 µ L) contained 1 × Taq DNA polymerase buffer [67 mM Tris-HCl pH 8.8, 16.6 mM (NH 4 ) 2 SO 4 , 0.45% Triton X-100, 0.2 mg/mL gelatin], 1.2 U Taq DNA polymerase, 1.5 mM MgCl 2 , 0.2 mM of each dNTP, 45 ng EcoR I-A primer, 45 ng Mse I-C primer, and 2 µ L of restriction -ligation reaction as template. Thermocycler conditions were: 94 ° C for 30 s, followed by 28 cycles of 94 ° C for 30 s, 60 ° C for 30 s, and 72 ° C for 1 min; followed by 72 ° C for 5 min. Selective amplifi cation reactions (20 µ L) contained 1 × Taq DNA polymerase buffer as described earlier, 0.8 U Taq DNA polymerase, 1.5 mM MgCl 2 , 0.2 mM of each dNTP, 0.25 µ M of each selective primer, and 5 µ L of diluted (1 in 10) preselective PCR as template. Thermocycler conditions were: 94 ° C for 1 min, followed by 11 cycles of 94 ° C for 30 s, 65 ° C decreasing by 0.7 ° C per cycle for 30 s, and 72 ° C for 1 min; then 23 cycles of 94 ° C for 30 s, 56 ° C for 30 s and 72 ° C for 1 min; followed by 72 ° C for 5 min. EcoRI selective primers were labeled using fl uorescent WellRED dyes (Sigma Proligo,