Background A preliminary assessment of genetic diversity is an essential step toward elucidating the evolutionary relationships and identifying valuable germplasm resources within Acanthophyllum species .In this study, genetic diversity was explored in different species of the genus Acanthophyllum using SCoT and SRAP molecular markers for the first time. Methods A total of 71 accessions from seven Acanthophyllum species ( A. bracteatum, A. glandulosum, A. korshinskyi, A. microcephalum, A. sordidum, A. spinosum, and A. squarrosum) were collected from natural habitats of Iran. The genetic diversity was analyzed using SCoT (12) and SRAP (20) molecular markers. Genetic diversity parameters, indices, and population differentiation were evaluated. Population structure was investigated using AMOVA, PCoA, UPGMA- based clustering, and Bayesian analysis (STRUCTURE). Results Both marker systems exhibited high informativeness, generating 200 (SCoT) and 242 (SRAP) fragments. Polymorphic information content (PIC = 0.37), Shannon’s information index (I = 0.41), and average expected heterozygosity (He = 0.27) generated by SCoT primers were higher than those obtained from SRAP analysis (PIC = 0.34, I = 0.34, and He = 0.22). A. glandulosum, A. sordidum, and A. microcephalum displayed the highest levels of genetic diversity, while A. korshinskyi showed consistently low variation. The results of the STRUCTURE analysis indicated that the Acanthophyllum species could be grouped into four genetically distinct subpopulations. AMOVA indicated that most genetic variation (~86%) occurred within populations, a pattern supported by high gene flow estimates (Nm > 2.3), which reflects considerable genetic connectivity across geographic regions. Widespread admixture, especially in A. bracteatum, A. korshinskyi, and A. spinosum, points to historical hybridization events and shared ancestry. Conclusion Both SCoT and SRAP markers proved highly efficient in revealing inter- and intra-specific genetic diversity within the Acanthophyllum genus. These results provide a critical foundation for identifying the diversity of Acanthophyllum germplasm. They highlight A. glandulosum as a genetically rich resource, due to its high genetic diversity revealed by both marker systems, and identify A. korshinskyi as a conservation priority. Subsequent genome-wide studies are recommended to build upon these findings and to explore the adaptive genetic variation associated with environmental resilience.

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