Genome Skimming Reveals Plastome Conservation, Phylogenetic Structure, and Novel Molecular Markers in Valuable Orchid Changnienia amoena

Background/Objectives: Changnienia amoena is a rare and endangered terrestrial orchid endemic to China, valued for its ornamental and medicinal properties. However, limited genomic resources hinder its effective conservation strategies and sustainable utilization. This study aimed to generate comprehensive plastome resources and develop molecular markers to support the phylogenetics, identification, and conservation management of C. amoena. Methods: Genome skimming was employed to assemble and annotate the complete plastomes of seven geographically distinct C. amoena accessions. Comparative analyses were conducted to assess structural features and sequence divergence within C. amoena and across related species in the Calypsoinae subtribe. Phylogenetic relationships were inferred from protein-coding genes. Simple sequence repeats (SSRs), dispersed repeats, and hypervariable regions were identified from the plastomes, while nuclear SSRs were developed from assembled nuclear sequences. Results: All seven plastomes exhibited a conserved quadripartite structure with identical gene content and order, showing only minor variations in genome size. Sequence divergence was mainly confined to non-coding regions. Across Calypsoinae species, mycoheterotrophic taxa exhibited reduced plastomes. Phylogenetic analyses resolved four well-supported intergeneric clades within Calypsoinae and revealed a notable divergence between the HuNGZ accession and other C. amoena accessions, which otherwise showed low plastome-level differentiation. We also identified 69–74 plastome-derived SSRs, 22–25 dispersed repeats, and three hypervariable regions that may serve as informative molecular markers for C. amoena. Additionally, 16 polymorphic nuclear SSRs were developed from assembled nuclear sequences. Conclusions: These findings significantly expand the genomic resources available for C. amoena and provide essential insights for its phylogeny, molecular identification, conservation management, and future breeding efforts.