Development, optimization, and validation of a DNA barcoding–based multiplex PCR system for the molecular identification and taxonomic discrimination of Xanthium strumarium and Xanthium spinosum

Purpose: Accurate species identification is essential for the assessment of plant diversity, taxonomic classification, and the reliable evaluation of plant-derived materials. Molecular approaches have become increasingly important when morphological identification is limited by interspecific similarity, environmental variation, or the loss of diagnostic characteristics. The genus Xanthium, which is widely distributed throughout the flora of Türkiye, represents a suitable model taxon for molecular and taxonomic investigations due to its spiny, easily transferable fruits and pronounced phenotypic plasticity influenced by environmental conditions. These characteristics often limit the reliability of morphological identification and highlight the need for molecular-based approaches. The present study aimed to develop and validate a DNA barcoding based molecular identification method supported by multiplex PCR for the molecular characterization of Xanthium strumarium and Xanthium spinosum, with potential application in forensic botanical examinations.Method: A total of 50 Xanthium samples were analyzed using an optimized multiplex PCR system targeting five barcode loci: D35, psbA–trnH, trnL–trnF, ITS-2, and trnQ–rps16. The amplified products were evaluated by capillary electrophoresis. The method was validated in terms of analytical threshold, dynamic range, sensitivity, full-profile recovery, reproducibility, and specificity.Findings: Capillary electrophoresis results revealed consistent and reproducible electrophoretic profiles among the analyzed samples, with allele size ranges spanning 66–218 bp. Method validation established an analytical threshold of 93.39 RFU, a dynamic range of 0.1–3 ng/µL, and a minimum DNA concentration of 0.5 ng/µL for full-profile recovery. Specificity testing demonstrated no amplification in the taxonomically distant species Salvia officinalis and Ephedra major.Conclusion: The findings indicate that the proposed DNA barcoding and multiplex PCR–based approach represents a reliable, sensitive, and applicable molecular tool for the characterization of Xanthium strumarium and Xanthium spinosum. The method may complement morphological assessment and contribute to molecular identification studies involving morphologically similar plant taxa, including plant-derived evidence encountered in forensic investigations.