DNA extraction of Catasetum gladiatorium K.G.Lacerda (Orchidaceae) for use in molecular studies

Abstract

Catasetum gladiatorium is endemic to Brazil, being usually found in areas subjected to environmental disturbances that may affect its genetic diversity. Analyses of genetic diversity require extraction of pure DNA through fast and efficient protocols. We propose here an efficient protocol to extract the DNA of C. gladiatorium for use in molecular analyses. DNA was extracted from leaf tissue using eight protocols based on the CTAB method. We tested: (1) two types of plant material maceration (using STE buffer and liquid nitrogen); (2) two CTAB concentrations (2% and 5%); and (3) two concentrations of β-mercaptoethanol (0% and 2%) in the extraction buffer. We performed amplification reactions with the extracted DNA using two ISSR primers. The methods of plant material maceration influenced the quality and amount of DNA extracted from C. gladiatorium. Maceration with liquid nitrogen allowed us to obtain high-quality DNA in all other variations of the tested protocols. On the other hand, maceration with STE yielded DNA at low concentrations and highly degraded. The best results were obtained using 5% CTAB. The absence of β-mercaptoethanol did not influence the quality of the extracted DNA. PCR demonstrated that DNA extraction using the eight tested protocols allowed for amplification with both ISSR markers. The eight protocols were able to extract amplifiable C. gladiatorium DNA, and may thus all be indicated for future molecular studies on the species.