*2.3. PCR Amplification and Polyacrylamide Gel Electrophoresis*

PCR were carried out in the final volume of the 10 μL reaction mixture (1 μL DNA, 1 μL forward primer, 1 μL reverse primer (100 ng/μL), 5 μL of 2 × T5 Super PCR Mix, and 2 μL nuclease-free water) on a thermocycler (Applied Biosystems, StepOnePlus ABI7500) with the following reaction conditions: initial denaturation at 94 ◦C for 3 min, then 30 cycles at 94 ◦C for 30 s; annealing at 55 ◦C for 30 s, extension at 72 ◦C for 1 min; and final extension at 72 ◦C for 5 min. Electrophoretic analysis was completed in order to assess the PCR amplicons, employing 8% polyacrylamide gel and 0.5 × TBE buffer at a constant voltage of 180 V, 150 mA, and 50 W for three hours, along with a 100 bp DNA ladder. When the electrophoresis was completed, the gel was carefully retrieved, rinsed with sterile water, and kept incubated with 1% silver nitrate (AgNO3) (1 L) for 20 min under shaking conditions. Following the incubation, the gel was washed 3 times with sterile water and immersed in 1 L of developing solution (1.5% sodium hydroxide (NaOH) and 4 mL formaldehydes (CHHO)) until the bands were clearly visible (approximately 5 min). Then, based on the number of clearly visible bands, the alleles in each pepper variety were visually determined.
