Genomic DNA was isolated using the DNeasy Plant DNA isolation kit (Qiagen, Valencia, CA) according to manufacturer guidelines.
PCR Protocol: AFLP analysis was conducted using a modified procedure based on Vos et al. (1995). The sequence of adapters and primers was described by Kim et al. (2004) with the exception of selective primers (Table 2), and was generated by Integrated DNA Technologies (Coralville, IA). EcoRI selective primers were labeled with WellRED D4, a blue phosphoramidite dye. In the restriction and ligation reaction, genomic DNA (500 ng) was incubated at 37 _C for 14 h with 1? T4 ligase buffer (with ATP), 50 mM NaCl, 5 U of MseI and EcoRI, 5 mMMseI adapter, 1 mMEcoRI adapter, and 1 U of T4 DNA ligase. Preamplification was performed using MseI+C and EcoRI+A primers. The reaction mixture contained 3 mL of 10? diluted restriction and ligation template DNA, 1? polymerase chain reaction (PCR) buffer (with 2.0 mM MgCl2), 0.2 mM each dNTPs, 0.3 mM each of two primers, and 0.5 U of Taq polymerase for a total volume of 13 mL. Preamplification cycle conditions were performed according to Kim et al. (2004). Selective amplification reactions (an 8-mL volume) contained 2 mL of 10? diluted preselective template DNA, 1? PCR buffer (with 2.0 mM MgCl2), 0.2 mM each dNTPs, 0.625 mM each of the two selective primers, and 0.2 U of Taq polymerase. Selective amplification PCR conditions consisted of an initial denaturation step of 2 min at 94 _C, 20 s at 94 _C, 30 s at 66 _C, and 2 min at 72 _C; the annealing temperature was then lowered 1 _C each cycle for the next nine cycles followed by 25 cycles at 94 _C for 30 s, 56 _C for 30 s, and 72 _C for 3 min, finishing with 60 _Cfor 30 min. After selective amplification, 1mLof reaction product was mixed with 35 mL of sample loading solution plus 0.5 mL of 600-bp size standard from Beckman Coulter (Fullerton, CA).
Excel file with AFLP data PDF file of the article Dendrogram of the data.