Development of One-Tube Multiplex Arbitrary (RAPD and ISSR) Marker-Based SCAR Assay for Simultaneous Detection and...

Indian senna (Senna alexandrina Mill.), a perennial medicinal species belonging to the family Fabaceae, holds significant therapeutic and commercial importance owing to its rich content of sennosides and rhein derivatives, which confer well-established laxative properties. Its high market demand, however, renders the species vulnerable to deliberate or inadvertent adulteration. While previous investigations have utilized functional marker systems such as SCoT (Start Codon Targeted Polymorphism)- and CBDP (CAAT Box Derived Polymorphism)-derived SCAR (Sequence Characterised Amplified Region) markers for genetic characterization, the present study is the first to report the development of sequence-specific RAPD- and ISSR-based SCAR markers consolidated into a single-tube multiplex PCR assay. Genomic DNA isolated from young leaves of S. alexandrina and its commonly encountered adulterant species was amplified using RAPD primer OPI-02 and ISSR primer UBC-835. Polymorphic amplicons were cloned, sequenced, and employed for the design of SCAR primers, which were rigorously validated for specificity. Species-specific SCAR markers were successfully integrated into a single multiplex reaction, enabling precise and unequivocal identification of S. alexandrina, Cassia fistula and Senna sophera. The multiplex amplification profiles were entirely consistent with corresponding uniplex assays, endorsing the method's robustness and reproducibility. This streamlined, one-tube multiplex SCAR-PCR system represents a significant advancement toward reliable, high-throughput molecular authentication of Indian senna and its closely related medicinal plant species (adulterants).