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

<i>Calanthe aristulifera</i> 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 <i>C. sieboldii</i> and <i>C. discolor</i>. 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 <i>Calanthe</i> 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 <i>C. aristulifera</i>. 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 <i>C. aristulifera</i> populations.