Transcriptome-Wide Identification and Development of SSR Markers for Genetic Diversity Studies in Medicinal Polygonatum Species

The genus Polygonatum encompasses numerous species with complex phenotypes, necessitating robust molecular markers for accurate species identification and superior germplasm screening. This study identified and developed SSR markers based on transcriptome analysis of three Polygonatum species to assess the genetic diversity of Polygonatum resources. The results showed that a total of 43,217 SSR loci were detected, and 31,703 primer pairs were successfully designed. Characterization of SSR motifs revealed mono-nucleotide repeats (SNRs) were the most frequent (59.45%). Unigenes containing SSRs were annotated across seven databases. In KEGG, 222 pathways were assigned, with genes annotated to carbohydrate metabolism being the most abundant. To validate and apply these markers, 100 primer pairs covering all eight SSR locus types were tested across 21 Polygonatum accessions. Of these, 49 polymorphic markers were identified, revealing high genetic diversity, with average expected heterozygosity (He) and polymorphism information content (PIC) values of 0.763 and 0.718, respectively, alongside significant population differentiation (Fst = 0.307). Cluster analysis grouped 21 accessions into three groups, which correlated with certain agronomic traits. Nine core markers were selected that effectively distinguished six species and intraspecific groups. Notably, the FB-9 marker, associated with polysaccharide biosynthesis, effectively discriminated among six Polygonatum species and also distinguished distinct germplasm resources within P. kingianum var. grandifolium. Overall, the transcriptome-derived SSR markers validated in this study constitute valuable resources for gene function analysis, population genetics research, and variety identification and genetic improvement of Polygonatum.