Optimization of DNA Isolation and PCR Protocol for ISSR Analysis of Species of Spilanthes - A Medicinal Herb from Peninsular India

Spilanthes of Asteraceae with over 140 species is distributed in the warm regions of both hemispheres. Most species of this genus are medicinally important and are widely used in folklore medicines. Genetic analysis of plants rely on high yields of pure DNA samples. The molecular characterization of Spilanthes species has not been reported in the literature. In the present study, attempts were made to optimize DNA isolation using the CTAB method and characterize six species of Spilanthes. The DNA yield from leaf tissue was 1-2 gl-1 and the purity ranged between 1.8-2.0 indicating minimal levels of contaminating metabolites. The modified technique was found to be ideal for PCR amplification of pure DNA from Spilanthes. The ISSR protocol was optimized for template DNA, dNTPs, MgCl2, Taq DNA polymerase, primer concentration and annealing temperature for each species and primer. The reproducible amplifiable products were obtained in all PCR reactions. The protocol established in the present study could be employed in determining the diversity of Spilanthes species and studies relating to barcoding and phylogeny.