The evaluation of A. flaccida populations using ISSR markers was performed in two steps: Step 1: First, the best ISSR primers for genetic studies in A. flaccida were selected. The DNA of 20 genotypes of the population from Guarapuava, Brazil, was amplified using 42 ISSR primers from the UBC series (University of British Columbia) (Table 3). Step 2: Population genetic data were obtained from the seven populations of A. flaccida. After the analysis of the data obtained in Step 1, the best ISSR primers were identified (Table 3) and used to amplify DNA from 131 plants from the seven populations. In both steps, PCR amplification reactions were conducted in a final volume of 12.5 μL that contained 20 ng DNA, 0.2 μM each primer, 200 μM dNTPs, 1.5 mM MgCl2, 1 U Taq DNA Polymerase, 1X PCR buffer, and water to complete the volume. The thermocycler program consisted of an initial denaturation step at 94°C for 5 min, followed by 35 cycles of 90°C for 45 s, annealing temperature of the primer (Table 3) for 45 s, and 72°C for 60 s. At the end of the cycles, a final step of 72°C for 7 min was added for the complete extension of the fragments. The amplification products were separated by electrophoresis on 1.8% agarose gel at a constant voltage of 110 V for 2 h. The gels were stained with ethidium bromide and visualized under UV light. To determine the size of the amplified fragments, a 100-bp DNA ladder was used.