Developing A PCR-Based SNP Genotyping Method for the Tomato Self-Pruning Gene

Tomato farmers often prefer determinate tomatoes, as they are easier to harvest because of their compact size and their limited fruit production. Unlike determinate tomatoes, indeterminate tomato plants are much larger and can keep growing tomatoes until the plants die. A single-nucleotide morphism (SNP) at the self-pruning gene can cause an amino acid change which affects whether the tomato is indeterminate or determinate. To molecularly distinguish between the two types of tomato growth patterns, I designed two reverse primers that only annealed to either determinate or indeterminate DNA. To test these primers, a gradient PCR process was used that starts at 65°C and decreases by 0.5 degrees for six rounds and 24 cycles. After success with the gradient PCR, an optimum annealing temperature of 62° was identified. To refine any false bands that may have appeared, a touchdown PCR was used. We were able to visually distinguish indeterminate from determinate DNA on an agarose gel. Bands appear on indeterminate DNA only when it has C-reverse primers and on determinate DNA when it has the T-reverse primers. Multiple DNA concentrations were also tested other than 10 ng/µl of DNA. We tested 5 and 20 ng/µl successfully. When breeders cross pollinate an indeterminate and determinate plant, they will be able to tell what the growth pattern will be before the plant grows to maturity. This is helpful to people who only want to grow certain growth patterns