MOLECULAR DISCRIMINATION OF Rhinacanthus SPECIES USING rbcL MARKER

Abstract

Rhinacanthus is an important genus belonging to the family Acanthaceae and possesses valuable medicine properties used in traditional medicine for diverse therapeutic purposes. Some species of this genus are endemic to Sri Lanka, emphasizing the urgency of accurate identification for their conservation and future utilization. Therefore, this study aimed to categorize R. flavovirens from R. nasutus at the molecular level using the rbcL gene in the chloroplast. The genomic DNA from R. flavovirens leaves was extracted using optimized CTAB protocol and quality DNA was obtained for polymeric chain reaction (PCR). The PCR was performed using a universal primer targeting the chloroplast rbcL region (rbcL-BF and rbcL-724R) and the PCR product was subsequently subjected to Sanger sequencing. The obtained rbcL sequence for the R. flavovirens was deposited in GenBank under accession no. OQ181218. A comparative analysis was conducted by aligning the rbcL sequence of R. flavovirens with sequences of R. nasutus retrieved from GenBank (accessions GQ436493.1, LC461831.1, and KF381120.1). Results revealed only two variable sites differentiating these two species for rbcL marker. The smallest pairwise distance (0.0095) was observed between R. flavovirens and R. nasutus accessions GQ436493.1 and LC461831.1. While phylogenetic analysis indicated a sister relationship between R. flavovirens and R. nasutus vouchers, the complete phylogeny of Rhinacanthus species could not be fully resolved. Overall, our results indicated that the universal rbcL primer we used to be not enough to categorize R. flavovirens from its close relative R. nasutus. Future studies should explore additional molecular markers to achieve a more accurate delineation of Rhinacanthus species. These findings will contribute to understanding of the genetic diversity within this genus, aiding in effective conservation and sustainable utilization of these medicinally significant plant species.