Optimization of the DNA Barcoding Protocol and the Evolutionary History of Botanic Garden Plants

Abstract

DNA barcoding uses the sequences of a small section of DNA that is universally present and sufficiently varied to identify cryptic species, discover species, revise taxonomic schemes, and unravel food webs. The use of rbcLa and matK genes provides a method to test for species richness with genes that are sufficiently differentiated and universal. The aims of the study included: i) verification of identification of plants collected from the Tulsa Botanic Garden; ii) lab process analysis using two DNA extraction methods; iii) and evaluation of phylogenetic divergence using freely available software. DNA was extracted from the plant tissue using two methods, and the two genes were amplified using published primers and spectrophotometric data. Products were verified by fragment size on an agarose gel with a molecular mass ruler. Amplicons were purified using a spin column, diluted, and sent for sequencing. Freely available software tools were used for phylogenetic analysis. Sequencing showed a 29% success rate of amplicon production. Three out of twelve samples were positive for both matK and rbcLa identification, one sample was positive for only rbcLa identification. Twenty-five percent of our positive specimens had a 260/230 ratio less than 0.40. Fifty percent of our successful samples used less than the recommended 0.25ug of DNA for each 25ul reaction. There was no clear divergence between Angiosperm and non-flowering plants.