Development and application of universal SSR molecular markers in Paphiopedilum

In this study, a multi-species transcriptome-based homologous sequence approach was employed to improve the efficiency and transferability of simple sequence repeat (SSR) molecular marker development in Paphiopedilum, replacing conventional single-species strategies. Homologous transcriptomic sequences from P. concolor (Bateman) Pfitz. and P. hirsutissimum (Lindl. ex Hook. f.) Stein were used to identify SSR loci and design primers, followed by empirical validation across diverse Paphiopedilum taxa. Results identified a total of 7 627 SSR loci, dominated by mononucleotide repeats (47.66%), with dinucleotide (37.35%) and trinucleotide (14.10%) repeats comprising the remainder. From these loci, 5 098 primer pairs were generated, of which 30 were selected, synthesized, and tested. Nineteen primer pairs achieved a 100% amplification rate across the 27 surveyed Paphiopedilum species and cultivars. These validated markers exhibited high levels of polymorphism, with the number of effective alleles (Ne) ranging from 4 to 14, observed heterozygosity (Ho) between 0.074 and 0.592, expected heterozygosity (He) between 0.443 and 0.890, and polymorphism information content (PIC) between 0.406 and 0.873. Notably, 89.47% of the SSR markers showed PIC values exceeding 0.5. Phylogenetic reconstruction based on these markers resolved all 27 Paphiopedilum species/cultivars into three major clades at a genetic distance of 0.74, with clear species- and cultivar-level discrimination. In conclusion, these broadly transferable, highly polymorphic SSR markers establish a robust molecular framework for Paphiopedilum, enabling precise germplasm discrimination, rigorous genetic analysis, and informed conservation management across the genus.