Optimization of DNA extraction and PCR protocols for plants with high Phenolics: Bael, Mango, Pomegranate as examples

Abstract

The genetic studies of tropical tree species containing high amount of phenols are greatly hampered by the inability to extract sufficient quantities of high quality DNA and the pres­ence of PCR inhibitors in extracted DNA samples. While there are some DNA extraction and PCR protocols for such species, no consistency in results. Hence, optimization of such protocols is a prerequisite for conducting research studies on tropical flora. This study reports the optimization of cheaper DNA extraction and PCR procedures for plants having high amount of phenol­ic compounds and PCR inhibitors. Three fruit crop species, Bael (Aegle marmelos L. Correa), Pomegranate (Punica granatum L.) and Mango (Mangifera indica L.), with distinctly diverse sec­ondary metabolic profiles, were used as examples. The CTAB DNA extraction method, with some modifications, was compared with two commercially available DNA extraction kits namely; Promega Wizard® Genomic DNA purification kit and QIAGEN DNeasy® Plant Mini kit. DNA from three to five genotypes from each species was extracted from each method and the quality and quantity were assessed. Spermidine was added to the PCR mix at the rate of 0.8 μM to block the PCR inhibitors and the DNA samples were amplified using universal plant barcoding primer pair rbcL, and SSR or ISSR primers. The modified CTAB method resulted significantly higher quantity of quality DNA in all sam­ples compared to two commercial kits. Henceforth DNA extract­ed from CTAB method, and the two commercial kits were used to precede PCR. However, expected bands were not generated in regular PCR. Interestingly, the inclusion of spermidine amplified the relevant band/s in relatively easy PCR reactions such as rbcL, as well as trickier reactions such as SSR and ISSR. These results suggest that cheaper alternative procedures used in this research study could be used successfully for the range of applications in plants with array of secondary metabolic profiles.

Keywords

• aegle marmelos l. correa, punica granatum l., mangifera indica l., dna extraction, spermidine, pcr inhibitors, phenolic compounds