Economizing marker assisted selection through cost-effective assay of sd1 gene in rice (Oryza sativa)

Abstract

PCR amplification and detection of GC rich sequences in DNA is a challenge due to formation of secondary structures which resist denaturation, thereby stalling Taq DNA polymerases as well as affecting primer annealing. Presently, high fidelity Taq DNA polymerase is used for amplifying long and GC-rich fragments, while dimethyl sulfoxide (DMSO) has also been suggested as an additive in Polymerase Chain Reaction (PCR) mix to avoid formation of secondary structures in templates containing high GC content. In the present study, the amplification efficiency of normal Taq DNA polymerase with 5% DMSO as compared to high fidelity Taq DNA polymerase has been applied for better amplification of fragments with high GC content of sd1 gene in rice. Normal Taq DNA polymerase with 5% DMSO displayed better and reproducible results as compared to platinum DNA polymerase for the amplification of gene sd1, thereby successfully detecting the dominant wild type allele governing tall plant height from the recessive semi-dwarf allele. There were no adverse effects of 5% DMSO in PCR amplification and the amplified fragments improved significantly indicating the improvement in amplification efficiency. The modified PCR protocol including DMSO provides a cost effective and cheaper alternative for reliable assessment of the genomic differences in GCrich regions of the gene. This will help in eliminating the need for costly high fidelity Taq DNA polymerase, thereby helping in economizing marker assisted selection.