Development and Validation of Species-Specific KASP and SCAR Markers for the Rapid Identification of the Endangered Orchid Calanthe aristulifera

Calanthe aristulifera is a critically endangered orchid species of profound horticultural and ecological significance. However, establishing its species integrity is complicated by frequent natural introgression with sympatric relatives, such as C. sieboldii and C. discolor. Because phenotypic plasticity and complex hybrid swarms often confound traditional floral phenotyping, establishing an accurate molecular diagnostic system is imperative for conservation. In this study, we developed and validated high-throughput Kompetitive Allele-Specific PCR (KASP) and rapid Sequence-Characterized Amplified Region (SCAR) markers utilizing Genotyping-by-Sequencing (GBS) data from 64 Calanthe individuals—the same dataset reported in a companion population genomic study—re-analyzed using a more stringent marker-development pipeline. From 853,301 SNPs and 55,857 InDels initially identified, we filtered 62,231 high-quality SNPs and 1271 InDels to mine fixed homozygous alleles specific to C. aristulifera. This process isolated 179 SNP and 107 InDel loci to design three KASP markers (Ca-KASP1–3) and two SCAR markers (Ca-SCAR1–2). The KASP assays demonstrated a concordance of 98.4% (63/64 individuals; 95% CI: 91.6–99.7%) with morphological pre-classification. The single discordant case (Sample 52) was independently confirmed as a heterozygous hybrid by GBS-based population genomic analysis of the same individuals, providing molecular ground truth entirely independent of morphological assessment. The combined SCAR marker system yielded 96.9% concordance (62/64; 95% CI: 89.3–99.1%). Our findings provide an essential molecular framework for assessing species integrity and guiding the restoration of endangered C. aristulifera populations.