THE MOLECULAR AND PHYLOGENETIC CHARACTERIZATION OF THE TAGETES ERECTA L. AND COSMOS SULPHUREUS CAV. USING DNA BARCODING AND BIOINFORMATICS TECHNIQUES

DNA Barcoding and Bioinformatics Techniques

Abstract

The correct identification of key botanical specimens such as Tagetes erecta and Cosmos sulphureus Cav. is critical to ensure their safety, efficacy, and appropriate use in herbal medicines. DNA barcoding utilizes sequence variation within a specific and uniform region of the genome (referred as "barcode") in order to achieve precise species identification. The DNA barcodes "matK" and "rbcL" were employed in order to ascertain the phylogenetic relationships of Tagetes erecta and Cosmos sulphureus Cav. Once DNA extraction was completed, its amplification was done by Polymerase Chain Reaction (PCR). The determination of DNA quality was conducted using agarose gel electrophoresis. The PCR amplification was subsequently followed by BTseq, a Next Generation Sequencing Technique after which the length of DNA fragment of Tagetes erecta was found to range between 700 and 800 base pairs. The sequencing data obtained from the rbcL and matK primers was subjected to the BLAST search in the NCBI database. The closely related sequences corresponding to each plant sample were downloaded and utilized for further analysis. The calculation of pairwise nucleotide identity was performed using the MEGALIGN software, developed by DNA Star to conduct phylogenetic analysis. The MEGA X software was used in the construction of a maximum likelihood tree. The phylogram was cross-referenced with the existing data obtained from the fossil record of plant species. The results revealed that the rbcL primer was highly suitable for DNA barcoding of Tagetes erecta plants, whereas the matK primer was the most appropriate choice for identifying Cosmos sulphureus plants.