PCR has been extensively utilized for the amplification of DNA sequences. In our study, we aimed to identify the optimal amplification conditions for detecting specific SSR polymorphisms associated with heat stress in wheat. Leaf samples from young seedlings were collected, and DNA was isolated from 30 genotypes, representing both heat-tolerant and heat-susceptible types. The extracted DNA from these genotypes underwent PCR optimization. The optimized reaction conditions 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 µl), reverse primer (1 µl), template DNA (1.5 μl), and 1 unit/ml Taq polymerase (0.5 μl). The PCR protocol comprised an initial denaturation at 95 °C for 7 minutes, followed by 35 cycles of denaturation for 45 seconds at 94°C, annealing for 45 seconds at 48 to 65 °C, extension for 45 seconds at 72°C, and a final extension for 10 minutes at 72 °C. Employing 32 designed SSR primer pairs, the optimized PCR conditions produced well-defined bands for the molecular characterization of heat-tolerant and susceptible wheat genotypes.

PCR, SSR Primers, Optimization, Wheat, Heat Tolerant and Susceptible.

In conclusion, our study demonstrated consistent DNA purity across all samples, aligning with established absorption ratio ranges for satisfactory DNA quality. The quantity of DNA varied among 30 wheat genotypes, with the HS507 sample exhibiting the minimum and the RAJ4083 sample displaying the maximum DNA amount. Variations in DNA quantity were attributed to measurement errors and differential grinding of leaf samples. The absorbance ratios mostly fell within the range of 1.80 to 1.95, indicating superior DNA quality in samples from seedling stage leaves across diverse genotypes. To ensure successful PCR amplification and reproducible polymorphic bands, we optimized concentrations of various reagents. The PCR setup included specific concentrations for MgCl2, Taq polymerase, DNA template, and primers, while maintaining constant buffer and dNTPs concentrations. Optimal results were achieved with 1.2 μl of MgCl2, 0.1 μl of Taq polymerase, template DNA concentrations between 0.5 and 2 μl, and 1.0 μl for both forward and reverse primers. The chosen PCR reagent concentrations produced sharp bands, and the optimal annealing temperature for successful amplification of SSR primers based on heat stress-related candidate genes was determined to be 49-60°C. The primer pair Barc128 exhibited positive amplification in all genotypes, revealing polymorphic bands with distinct molecular weights. This comprehensive optimization process enhances the reliability of PCR results and contributes to the robustness of genomic studies in wheat genomics.

Prafulla Kumar, Ravindra Kumar, Ankit Agrawal and Neelesh Kapoor (2023). SSR based Heat Stress Related Primers Optimization for Heat Tolerant and Susceptible Genotypes of Wheat (Triticum aestivum L.). Biological Forum – An International Journal, 15(12): 300-308.