Fragrance related candidate gene specific SSR based PCR optimization in landraces and improved varieties of aromatic rice

Abstract

PCR has been extensively used for the amplification of DNA sequences. We conducted a study to obtain the best amplification condition for detection of fragrance related candidate gene specific SSR polymorphism in aromatic rice. Using leaf samples collected from young seedlings, DNA was isolated from 18 genotypes including landraces and improved varieties of aromatic rice. The extracted DNA sample of any five genotypes was randomly taken for PCR optimization. The reaction condition optimized through this study included a combination of 5 X PCR buffer (3.0 µl), 10 mM MgCl2 (1.2 µl), 200 µM dNTP (3 µl), forward primer (1.2 µl), reverse primer (1.2 µl)), template DNA (1.5 μl) and 1 unit / ml Taq polymerase (0.5 μl). PCR was performed using initial denaturation for 5 minutes at 94 0C followed by 35 cycles of denaturation for 40 seconds at 94 0C, annealing for 1 min at 55-65 0C and extension for 2 minutes at 72 0C and final extension for 10 minutes at 72 0C. Utilizing 38 designed candidate gene-based SSR primer pairs, the optimized PCR condition produced sharp bands for molecular characterization of aromatic rice genotypes.