*2.5. DNA Extraction and ISSR Analysis*

The donor plant and non-irradiated explants were used as controls. Leaf samples from the donor plant and ten randomly selected shoots per irradiation dose were used in all experiments. For DNA isolation, the Stewart and Via [34] method was performed. The amount and purity of the DNA was determined by a spectrophotometer (Nanodrop 2000, Thermo Scientific, Waltham, MA, USA). The DNA samples used were those obtained with an A260/230 ratio of 1.8–2.2. The DNA was verified on a 1.5% (*w*/*v*) agarose gel stained with 3 mg·mL−<sup>1</sup> ethidium bromide (Sigma-Aldrich) at a concentration of 0.1 mg·mL−<sup>1</sup> using TAE 1X as buffer. To detect the polymorphism of the *V. planifolia* genomic DNA, 14 ISSR primers were evaluated. Eight primers were selected based on its reproducibility of the banding patterns (Table 1).

The PCR reactions were brought to a volume of 25 μL, with 30 ng template DNA, 1.5 U GoTaq DNA Polymerase (Promega, Madison, WI, USA), 1X Buffer (10 mM Tris-HCL and 50 mM KCL), 2.5 mM MgCl2, 0.2 mM dNTPs and 0.5 μM primer. The products were amplified in an Engine System thermal cycler (PTC-200, BIO-RAD, Watertown, MA). A program starting with one cycle for 4 min at 94 ◦C was used, followed by 35 cycles for 50 s at 94 ◦C, 45–53 ◦C according to the primer for 50 s and 72 ◦C for 90 s. Finally, an extension at 72 ◦C for 10 min.


**Table 1.** Inter simple sequence repeat (ISSR) molecular markers selected to evaluate the somaclonal variation of vanilla (*Vanilla planifolia* Jacks. ex Andrews) at different doses of gamma irradiation with 60Co.

bp = base pair; Y = C or T.

The amplification fragments were separated by electrophoresis on 2.5% (*w*/*v*) agarose gels in a 1X TAE buffer solution at 90 V for 1.5 h. The gels were stained with 3 mg·mL−<sup>1</sup> ethidium bromide. A 1 kb Plus DNA ladder (Promega, Madison, WI, USA) was used. Finally, the gels were photographed using a gel documentation system (ChemiDocXRS, Bio-Rad, Hercules, CA, USA).
