Development of ISSR-derived SCAR markers for precise identification and conservation of Rhynchostylis gigantea and its variety rubrum in Thailand

• ISSR markers were successfully converted into reliable SCAR markers for Rhynchostylis identification. • Developed SCAR markers enable precise discrimination of R. gigantea and its variety rubrum . • SCAR markers showed high reproducibility and specificity across tested orchid accessions. • The markers provide an effective molecular tool for conservation and germplasm management in Thailand. Rhynchostylis orchids, endemic to Thailand, possess significant commercial and cultural value but face escalating threats from illegal trade. Frequently misrepresented as hybrids in online markets and local nurseries to evade regulation, such practices have accelerated the decline of wild populations, pushing some taxa toward extinction. Although protected under CITES Appendix II, the accurate identification of Rhynchostylis species remains challenging due to their high morphological similarity, particularly during non-flowering stages. Therefore, rapid and accurate species- or variety-specific identification tools are urgently needed to support conservation, legal enforcement, and sustainable use. This study utilized ISSR, SCoT, and SRAP markers to assess genetic diversity and develop reliable SCAR markers for species- and variety-level identification in Rhynchostylis orchids. Among the three marker systems, ISSR demonstrated the highest discriminatory power, revealing greater polymorphism compared to SCoT and SRAP. Genetic similarity coefficients and UPGMA dendrograms derived from all marker types consistently revealed two distinct clusters, confirming the genetic cohesion of the R. gigantea complex and its clear divergence from allied genera such as Vanda and Aerides . Among the DNA profiles generated, two ISSR primers, UBC817 and UBC827, amplified distinct ∼500 bp and ∼700 bp fragments, respectively. Sequencing of these fragments enabled the development of specific SCAR primers: RG817_f/RG817_r amplified a 202 bp fragment unique to R. gigantea , while RGR827_f/RGR827_r produced a 424 bp band specific to R. gigantea var. rubrum . These markers were absent in all other tested accessions. Multiplex PCR validation confirmed their high specificity and reproducibility for genotype identification. This study reports the first ISSR-derived SCAR markers for accurate differentiation of R. gigantea and its variety rubrum , offering practical tools for species authentication. These markers offer practical tools for eliminating illegal trade, supporting biodiversity conservation efforts, and promote sustainable cultivation of Thailand's endemic Rhynchostylis orchids, which are important both ecologically and economically.