Development of SSR Markers and Genetic Diversity Analysis of Using Transcriptome Sequencing.

, the underground rhizome of Siebold et Zucc., serves as a traditional medicinal resource among the Miao ethnic group in Guizhou, China. This study aimed to develop SSR molecular markers from the transcriptome to assess the genetic diversity of its germplasm across diverse regions. A transcriptome database was constructed using high-throughput sequencing, followed by the extraction of SSR loci with MISA software. Polymorphic SSR markers were identified and utilized to evaluate the genetic diversity of 45 germplasm resources. In total, 38,806 Unigenes were obtained, with an average length of 993.00 bp. Comparative annotation against the GO, KEGG, EggNOG, NR, Swiss-prot, and Pfam databases yielded 23,558, 12,412, 25,448, 28,101, 20,056, and 18,460 annotated Unigenes, respectively. A total of 7501 SSR loci were identified, with trinucleotide repeats constituting the predominant class (44.03%), and ACC/GGT being the most frequent motif. Ten pairs of polymorphic primers were developed, all exhibiting a PIC value greater than 0.5. The 45 spp. accessions were classified into three distinct groups using the UPGMA method. The transcriptome harbors abundant polymorphic SSRs. The developed primers enable rapid molecular identification of germplasm and provide insights into its genetic diversity, which may underlie variations in medicinal properties and environmental adaptation. These findings support applications in germplasm conservation, marker-assisted breeding, and quality control of medicinal resources.