Assembly and comparative analysis of the first complete mitochondrial genome of a traditional Chinese medicine Bletilla striata (Thunb. ex A. Murray) Rchb. f.
The plant materials of Bletilla striata were collected from the germplasm resource nursery in Lijiang City, Yunnan Province, China (coordinates: 26 56' N, 100 11' E). The species identification was conducted by Professor Tao Aien from Lijiang Culture and Tourism College. The voucher specimen of this sample has been deposited in the herbarium of Lijiang Culture and Tourism College with the specimen number TC11. Fresh leaves were collected, washed with ultrapure water, immediately flash-frozen in liquid nitrogen, and stored at -80 C in a deep freezer to ensure integrity. For simultaneous extraction of intact genomic DNA and RNA, the leaves were mixed and ground, and high-quality genomic DNA was extracted from the leaves using a modified CTAB method. Subsequently, the library was constructed using SQK-LSK109 and DNA sequencing was performed on the Nanopore PromethION platform (Nanodrop Technologies, Wilmington, USA). For the assembly of the Bletilla striata mitochondrial genome, Ratatosk v0.9.0 was first used to correct the ONT third-generation sequencing data and second-generation sequencing data. The corrected data was then treated as HiFi data and used as input for PMAT. PMAT v2.1.0 (autoMito -t hifi) was employed to assemble the mitochondrial genome using third-generation HiFi data (or the corrected third-generation data). Bandage was subsequently utilized for visual analysis and manual adjustment of the assembly results. The mitochondrial genome of Bletilla striata was annotated using GeSeq with reference genomes of closely related species: Apostasia fujianica (PP724664), Danxiaorchis yangii (PQ306624), Dendrobium hancockii (LC771085), Dracaena cambodiana (PQ279227), Dracaena cochinchinensis (PP704687), Polygonatum kingianum (PQ374235), and Polygonatum sibiricum (PQ932596). The OGDRAW software was employed to visualize the mitochondrial genome map of Bletilla striata. The assembled and annotated complete mitochondrial genome sequence of Bletilla striata has been submitted to the NCBI GenBank database and is publicly available under the accession number XXX. The results demonstrate that the mitochondrial genome of Bletilla striata exhibits a complex (multi-branched/multi-circular) structure, composed of 14 chromosomes with a total length of 517169 bp and a GC content of 43.52%. A total of 77 genes were annotated, including 41 protein-coding genes (PCGs), 33 tRNA genes, and 3 rRNA genes. Additionally, 46 simple sequence repeats (SSRs), 24 tandem repeats, and 0 pairs of dispersed repeats were identified. Analysis of codon usage in Bletilla striata mitochondria revealed 31 codons with RSCU>1, where leucine (Leu) showed the highest occurrence frequency at 11.51%, while methionine (Met) had the lowest at only 2.16%. The codon TTT (encoding Phe) was the most frequently used, with a relative synonymous codon usage (RSCU) value of 1.034. Additionally, the study identified 79 homologous fragments transferred from the chloroplast to the mitochondrial genome, with a total length of 73818 bp (accounting for 14.27% of the mitochondrial genome), and predicted 6089 C-to-U type RNA editing sites in mitochondrial protein-coding genes. Phylogenetic analysis revealed that Bletilla striata is most closely related to Danxiaorchis yangii. Collinearity analysis demonstrated significant rearrangements in the mitochondrial genome of Bletilla striata. Selection pressure (Ka/Ks) analysis indicated that over half of the protein-coding genes exhibited Ka/Ks ratios greater than 1, suggesting these genes have undergone positive selection during evolution, with functional optimization potentially related to the adaptive evolution of the species to specific environments. This study presents the first description of the complete mitochondrial genome of Bletilla striata, laying a foundation for future research on genetic variation, evolutionary relationships, and breeding in the Bletilla genus.