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.

Introduction: Changnienia amoena is an endangered orchid endemic to China, with notable medicinal and ornamental significance. Like other orchids, it relies heavily on microbial symbionts for germination, growth, and survival; however, its associated microbiomes remain poorly understood. Methods: This study employed high-throughput amplicon sequencing targeting bacterial 16S rRNA and fungal ITS regions and provided an in-depth characterization of the root-associated microbiomes of C. amoena across its native range. Results: Both bacterial and fungal communities exhibited significant biogeographic patterns, but only fungal Shannon-Wiener diversity showed a decreasing trend with increasing latitude. The neutral model revealed predominantly stochastic assembly of bacterial communities. In contrast, fungal communities exhibited a lower estimated migration rate, indicating a relatively stronger influence of non-neutral processes on their assembly. Soil nutrients emerged as key environmental drivers shaping microbial communities. Functional predictions highlighted multiple bacterial functions related to nitrogen cycling and the enrichment of mycorrhizal fungi, suggesting the functional potential of root-associated microbes in promoting plant nutrient acquisition, growth, and environmental adaptation. Co-occurrence network analysis further revealed positive associations between mycorrhizal fungi and potentially beneficial bacteria based on taxonomic inference. Discussion: This study reveals distinct biogeographic patterns, assembly processes, and functional potentials of root-associated microbiomes of C. amoena , which not only expands the understanding of the plant–soil-microbe interactions, but also provides crucial insights for manipulating them for the conservation of this endangered orchid.