Illumina Sequencing Data Research Articles

Characterisation of the complete mitochondrial genome of Taraxacum mongolicum revealed five repeat-mediated recombinations.

We reported the mitochondrial genome of Taraxacum mongolicum for the first time. Five pairs of repeats that can mediate recombination were validated, leading to multiple conformations of genome. Taraxacum mongolicum belongs to the Asteraceae family and has important pharmaceutical value. To explore the possible interaction between the organelle genomes, we assembled the complete mitochondrial genome (mitogenome) of T. mongolicum using Illumina and Oxford Nanopore sequencing data. This genome corresponded to a circular molecule 304,467bp long. It encodes 52 unique genes including 31 protein-coding, 3 ribosomal RNA (rRNA) and 18 transfer RNA (tRNA) genes. In addition to the single circular conformation, the existence of alternative conformations mediated by five repetitive sequences in the mitogenome was identified and validated. Recombination mediated by the inverted repeats resulted in two conformations. Conversely, recombination mediated by the two direct repeats broke one large circular molecule into two subgenomic circular molecules. Furthermore, we identified 12 homologous fragments by comparing the sequences of mitogenome and plastome, including eight complete tRNA genes. Lastly, we identified a total of 278 RNA-editing sites in protein-coding sequences based on RNA-seq data. Among them, cox1 and nad5 gene has the most sites (21), followed by the nad2 gene with 19 sites. We successfully validated 213 predicted RNA-editing sites using PCR amplification and Sanger sequencing. This project reported the first mitogenome of T. mongolicum and demonstrated its multiple conformations generated by repeat-mediated recombination. This genome could provide critical information for the molecular breeding of T. mongolicum, and also be used as a reference genome for other species of the genus Taraxacum.

Tomato Brown Rugose Fruit Virus Nextstrain Build Version 3: Rise of a Novel Clade

In the Netherlands, tomato brown rugose fruit virus (ToBRFV; genus Tobamovirus) was first identified in tomato crops in 2019. Since then, the National Plant Protection Organization (NPPO-NL) has performed surveys to track and trace this regulated virus aiming for its eradication. To gain more insight in the epidemiology of ToBRFV, genomes were assembled from Illumina sequence data. Whole-genome phylogenetics was integrated with epidemiological metadata in a Nextstrain build. Two new clades were defined, one of which displayed a rapid increase in comparison to the previous version of the Nextstrain build. This rapid increase could be attributed to the unauthorized application of an isolate of ToBRFV as a cross-protection product. Further analysis of the test results of positive samples from tomato production sites suggests that both deliberate application and accidental introduction had occurred. This report introduces the inclusion of 61 new (near) complete ToBRFV genomes in version three of the Nextstrain build, available from https://nextstrain.nrcnvwa.nl/ToBRFV/20220412 . [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

The complete chloroplast genome of Senna alata (L.) Roxb., an important medicinal plant from the Philippines

Senna alata, a flowering shrub, is widely cultivated in the Philippines for its anti-fungal properties. Despite this, its chloroplast genome is not yet established. We assembled and annotated the complete chloroplast genome of accession from the germplasm collection of the Institute of Crop Science, University of the Philippines, Los Baños, using Illumina sequencing data. The complete cp genome was 159,176-bp long characterized by a large single copy of 88,769 bp, short single-copy of 18,301 bp and a pair of inverted repeat regions of 26,053 bp each. The overall GC content of the chloroplast genome was 36.4%. The plastome comprised 37 tRNA genes, 8 rRNA genes and 78 mRNA genes. Phylogenetic analysis showed that S. alata is closely related to S. siamea.

The complete mitochondrial genome of Cladosporium anthropophilum (cladosporiaceae, dothideomycetes)

The genus Cladosporium (Cladosporiaceae, Capnodiales) is a large genus of Ascomycota. Although the genus is mostly reported as saprobes from a wide range of substrates with a worldwide distribution, members of this genus comprise infectious agents in animals and plants. Of those, Cladosporium anthropophilum is a common saprophytic fungus and has been found to be a human opportunistic pathogen and plant pathogen. The complete mitochondrial genome of C. anthropophilum is characterized through the de novo assembly of Illumina sequencing data. The mitochondrial genome is a circular molecule of 35,937 bp with 30.23% GC content and has a total of 47 genes including 16 protein-coding genes, 29 transfer RNA genes, and two ribosomal RNA genes. Based on protein-coding sequences of the mitochondrial genome sequence, a phylogenetic tree was constructed to demonstrate the phylogenetic relationship of C. anthropophilum and its related genera.

Identification of Cry toxin receptor genes homologs in a de novo transcriptome of Premnotrypes vorax (Coleoptera: Curculionidae).

The white potato worm Premnotrypes vorax (Hustache) (Coleoptera: Curculionidae) is one of the most destructive insect pests of potato crops in South America. Like many coleopteran insects, P. vorax shows low susceptibility to Cry insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). However, the presence of Cry toxin receptors in the midgut of this this insect has never been studied. The main Cry-binding proteins described in other insect species are cadherin (CAD), aminopeptidase N (APN), alkaline phosphatase (ALP) and ATP-binding cassette (ABC) transporters. In this study, we analyzed and validated a de novo assembled transcriptome of Illumina sequencing data to identify and to characterize homologs of Cry toxin receptors. We identified the protein sequences in P. vorax that show high identity with their orthologous sequences of the Cry toxin binding proteins in other coleopteran larvae such as APN, ALP, CAD and ABC transporter. This study provides preliminary identification of putative receptor genes of Cry proteins that would be useful for future studies involving biocontrol of this important potato crop pest.

Comparative analysis of mitochondrial genomes of Broussonetia spp. (Moraceae) reveals heterogeneity in structure, synteny, intercellular gene transfer, and RNA editing.

The genus Broussonetia (Moraceae) is comprised of three non-hybrid recognized species that all produce high quality fiber essential in the development of papermaking and barkcloth-making technology. In addition, these species also have medicinal value in several countries. Despite their important economical, medicinal, and ecological values, the complete mitogenome of Broussonetia has not been reported and investigated, which would greatly facilitate molecular phylogenetics, species identification and understanding evolutionary processes. Here, we assembled the first-reported three complete Broussonetia (B. papyrifera, B. kaempferi, and B. monoica) mitochondrial genomes (mitogenome) based on a hybrid strategy using Illumina and Oxford Nanopore Technology sequencing data, and performed comprehensive comparisons in terms of their structure, gene content, synteny, intercellular gene transfer, phylogeny, and RNA editing. Our results showed their huge heterogeneities among the three species. Interestingly, the mitogenomes of B. monoica and B. papyrifera consisted of a single circular structure, whereas the B. kaempferi mitogenome was unique and consisted of a double circular structure. Gene content was consistent except for a few transfer RNA (tRNA) genes. The Broussonetia spp. mitogenomes had high sequence conservation but B. monoica with B. kaempferi contained more synteny blocks and were more related, a finding that was well-supported in organellar phylogeny. Fragments that had been transferred between mitogenomes were detected at plastome hotspots that had integrated under potential mediation of tRNA genes. In addition, RNA editing sites showed great differences in abundance, type, location and efficiency among species and tissues. The availability of these complete gap-free mitogenomes of Broussonetia spp. will provide a valuable genetic resource for evolutionary research and understanding the communications between the two organelle genomes.

The complete chloroplast genome of Korean bred apple ‘Kamhong’ (Malus domestica Borkh.)

Apple (Malus domestica (Suckow) Borkh., 1803) is economically important horticultural fruit crop in the world. In this study, the complete chloroplast genome of Korean apple cultivar ‘Kamhong’ was characterized through the de novo assembly of Illumina sequencing data. The chloroplast genome is a circular molecule of 161,069 bp length with 36.55% GC content and has a total of 112 genes including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. Phylogenetic analysis with protein-coding sequences of chloroplast genome revealed that ‘Kamhong’ was closely grouped with M. domestica cultivars reported in China. The genomic data generated in this study can extend a molecular basis for phylogenetic relationships of Korean cultivar ‘Kamhong’ with other M. domestica cultivars bred in other countries.

Benchmarking taxonomic classifiers with Illumina and Nanopore sequence data for clinical metagenomic diagnostic applications.

Culture-independent metagenomic detection of microbial species has the potential to provide rapid and precise real-time diagnostic results. However, it is potentially limited by sequencing and taxonomic classification errors. We use simulated and real-world data to benchmark rates of species misclassification using 100 reference genomes for each of the ten common bloodstream pathogens and six frequent blood-culture contaminants (n=1568, only 68 genomes were available for Micrococcus luteus). Simulating both with and without sequencing error for both the Illumina and Oxford Nanopore platforms, we evaluated commonly used classification tools including Kraken2, Bracken and Centrifuge, utilizing mini (8 GB) and standard (30-50 GB) databases. Bracken with the standard database performed best, the median percentage of reads across both sequencing platforms identified correctly to the species level was 97.8% (IQR 92.7:99.0) [range 5:100]. For Kraken2 with a mini database, a commonly used combination, median species-level identification was 86.4% (IQR 50.5:93.7) [range 4.3:100]. Classification performance varied by species, with Escherichia coli being more challenging to classify correctly (probability of reads being assigned to the correct species: 56.1-96.0%, varying by tool used). Human read misclassification was negligible. By filtering out shorter Nanopore reads we found performance similar or superior to Illumina sequencing, despite higher sequencing error rates. Misclassification was more common when the misclassified species had a higher average nucleotide identity to the true species. Our findings highlight taxonomic misclassification of sequencing data occurs and varies by sequencing and analysis workflow. To account for 'bioinformatic contamination' we present a contamination catalogue that can be used in metagenomic pipelines to ensure accurate results that can support clinical decision making.

A high-quality genome assembly of the Laotian shad (Tenualosa thibaudeaui), an endemic species of the Mekong River Basin.

The Laotian shad (Tenualosa thibaudeaui) belongs to the family Clupeidae and is mainly distributed across Lao PRD, Cambodia, and northern Thailand. Due to overfishing and dam reconstruction, the Laotian shad is on the verge of extinction and currently listed as vulnerable by the IUCN. Nanopore and Illumina sequencing data were integrated to generate the first high-quality genome assembly for T. thibaudeaui. The assembled genome was 638 Mb in size, including 228 scaffolds with a N50 value of 16.6 Mb. BUSCO analysis revealed the completeness of the assembly to be more than 96%. A total of 24,810 protein-coding genes were predicted. According to the pairwise sequentially Markovian coalescent analysis, the effective population size of the Laotian shad sharply declined from 3 Mya to 20 Kya. We found a significant ratio in contraction of gene families that may reflect secondary gene loss. Our high-quality genome assembly of the Laotian shad will provide a valuable resource for future research in conservation genetics, as well as for investigating the phylogenetics and comparative genomics of shads.

The complete chloroplast genome of Korean cultivar ‘Harmony’ (Prunus salicina × Prunus armeniaca)

Prunus Linnaeus 1753 species include many economically important fruit crops, of which plumcot is generated by inter-species crossing between Prunus salicina Lindley 1830 × Prunus armeniaca Linnaeus 1753. In this study, the complete chloroplast genome of Korean plumcot cultivar ‘Harmony’ was characterized through the de novo assembly of Nanopore and Illumina sequencing data. The chloroplast genome is a circular molecule of 157,916 bp length with 36.75% GC content and has a total of 113 genes including 79 protein-coding genes, 30 transfer RNA genes, and four ribosomal RNA genes. Phylogenetic analysis with protein-coding sequences of chloroplast genome revealed that ‘Harmony’ showed closer to P. salicina than P. armeniaca.

S5.3a Unraveling the genetic determinants of virulence in Cryptococcus neoformans

S5.3 Cellular pleomorphism and fungal virulence, September 22, 2022, 3:00 PM - 4:30 PM Cryptococcus neoformans is a human pathogenic basidiomycete yeast that can cause cryptococcal meningitis (CM), predominantly in immunocompromised individuals. The patient outcome depends on both host and pathogen-specific factors, including C. neoformans genetics. A groundbreaking 2012 study was the first to show that patient outcome is associated with genetic differences between C. neoformans isolates. Subsequent population-wide sequencing studies have revealed over 100 sequence types (ST) of C. neoformans that are associated with both geographic location and clinical outcome. All these studies have been broad, examining the severity of disease cryptococcal phenotypes in a collection of highly diverse strains. We chose a narrow focus and collected various genotypic and phenotypic data from a single ST: ST93. ST93 is a common sequence type isolated from patients globally and is the most common clinical isolate found in the sub-Saharan African country of Uganda. Previously, we performed whole genome sequencing on 38 ST93 Ugandan clinical isolates. We identified 652 unique SNPs in this ST93 population compared to the H99 reference genome. We also showed that ST93 contained two subpopulations: ST9A and ST93B. In the current study, we further characterized the genotypic, phenotypic, and virulence differences between these 38 clinical isolates. Using Illumina sequence data, we identified a pattern of linkage disequilibrium that suggested that ST93A and ST93B are evolving separately. We performed long-read sequencing on each isolate to investigate chromosomal changes and large structural variations, allowing us to identify a chromosomal translocation event wherein parts of chromosome 11 had recombined with chromosome 3. Additionally, we characterized several in vitro phenotypes for each isolate and identified three distinct phenotypic clusters based on cell wall challenge and growth experiments. Next, we infected mice with 35 isolates and observed eight different disease manifestations, including isolates that caused non-CNS infections. Overall, by working within a single sequence type, we can gain a deeper understanding of how some small genetic changes can impact strain-specific phenotypes while others have no discernable effect. Eventually, these data can be used to provide valuable information about how each clinical isolate impacts patient outcomes.

Genome-wide association analysis of 101 accessions dissects the genetic basis of shell thickness for genetic improvement in Persian walnut (Juglans regia L.)

BackgroundUnderstanding the underlying genetic mechanisms that drive phenotypic variations is essential for enhancing the efficacy of crop improvement. Persian walnut (Juglans regia L.), which is grown extensively worldwide, is an important economic tree fruit due to its horticultural, medicinal, and material value. The quality of the walnut fruit is related to the selection of traits such as thinner shells, larger filling rates, and better taste, which is very important for breeding in China. The complex quantitative fruit-related traits are influenced by a variety of physiological and environmental factors, which can vary widely between walnut genotypes.ResultsFor this study, a set of 101 Persian walnut accessions were re-sequenced, which generated a total of 906.2 Gb of Illumina sequence data with an average read depth of 13.8× for each accession. We performed the genome-wide association study (GWAS) using 10.9 Mb of high-quality single-nucleotide polymorphisms (SNPs) and 10 agronomic traits to explore the underlying genetic basis of the walnut fruit. Several candidate genes are proposed to be involved in walnut characteristics, including JrPXC1, JrWAKL8, JrGAMYB, and JrFRK1. Specifically, the JrPXC1 gene was confirmed to participate in the regulation of secondary wall cellulose thickening in the walnut shell.ConclusionIn addition to providing considerable available genetic resources for walnut trees, this study revealed the underlying genetic basis involved in important walnut agronomic traits, particularly shell thickness, as well as providing clues for the improvement of genetic breeding and domestication in other perennial economic crops.

Single-molecule Real-time (SMRT) Sequencing Facilitates Transcriptome Research and Genome Annotation of the Fish Sillago sinica.

As a newly described Sillaginidae species, Chinese sillago (Sillago sinica) needs a better understanding of gene annotation information. In this study, we reported the first full-length transcriptome data of S. sinica using the PacBio isoform sequencing Iso-seq and a description of transcriptome structure analysis. A total of 454,979 high-quality full-length transcripts were obtained by single-molecule real-time (SMRT) sequencing, which was corrected by Illumina sequencing data. After that, 66,948 non-redundant full-length transcripts were generated after mapping to the reference genome of S. sinica, including 49 fusion isoforms and 9,250 novel isoforms. 63,459 isoforms were successfully annotated by one of the Nr, Nt, SwissProt, Pfam, KOG, GO, and KEGG databases. Additionally, 30,987 alternative polyadenylation (APA) sites, 451,867 alternative splicing (AS) events, 21,928 long non-coding RNAs (lncRNAs) and 12,911 transcription factors (TFs) were identified. The full-length transcripts of S. sinica would provide a precious resource for characterizing the transcriptome of S. sinica and for the further study of gene function and regulatory mechanism of this species.

Novel Megaplasmid Driving NDM-1-Mediated Carbapenem Resistance in Klebsiella pneumoniae ST1588 in South America.

Carbapenem-resistant Enterobacterales (CRE) is a critical public health problem in South America, where the prevalence of NDM metallo-betalactamases has increased substantially in recent years. In this study, we used whole genome sequencing to characterize a multidrug-resistant (MDR) Klebsiella pneumoniae (UCO-361 strain) clinical isolate from a teaching hospital in Chile. Using long-read (Nanopore) and short-read (Illumina) sequence data, we identified a novel un-typeable megaplasmid (314,976 kb, pNDM-1_UCO-361) carrying the blaNDM-1 carbapenem resistance gene within a Tn3000 transposon. Strikingly, conjugal transfer of pNDM-1_UCO-361 plasmid only occurs at low temperatures with a high frequency of 4.3 × 10−6 transconjugants/receptors at 27 °C. UCO-361 belonged to the ST1588 clone, previously identified in Latin America, and harbored aminoglycoside, extended-spectrum β-lactamases (ESBLs), carbapenem, and quinolone-resistance determinants. These findings suggest that blaNDM-1-bearing megaplasmids can be adapted to carriage by some K. pneumoniae lineages, whereas its conjugation at low temperatures could contribute to rapid dissemination at the human–environmental interface.

Sex-based de novo transcriptome assemblies of the parasitoid wasp Encarsia suzannae, a host of the manipulative heritable symbiont Cardinium hertigii.

Parasitoid wasps in the genus Encarsia are commonly used as biological pest control agents of whiteflies and armored scale insects in greenhouses or the field. They are also hosts of the bacterial endosymbiont Cardinium hertigii, which can cause reproductive manipulation phenotypes, including parthenogenesis, feminization, and cytoplasmic incompatibility (the last is mainly studied in Encarsia suzannae). Despite their biological and economic importance, there are no published Encarsia genomes and only one public transcriptome. Here, we applied a mapping-and-removal approach to eliminate known contaminants from previously-obtained Illumina sequencing data. We generated de novo transcriptome assemblies for both female and male E.suzannae which contain 45,986 and 54,762 final coding sequences, respectively. Benchmarking Single-Copy Orthologs results indicate both assemblies are highly complete. Preliminary analyses revealed the presence of homologs of sex-determination genes characterized in other insects and putative venom proteins. Our male and female transcriptomes will be valuable tools to better understand the biology of Encarsia and their evolutionary relatives, particularly in studies involving insects of only one sex.

Making the Most of Its Short Reads: A Bioinformatics Workflow for Analysing the Short-Read-Only Data of Leishmania orientalis (Formerly Named Leishmania siamensis) Isolate PCM2 in Thailand.

Simple SummaryLeishmaniasis is a parasitic disease caused by flagellated protozoa of the genus Leishmania. Multiple genome sequencing platforms have been employed to complete Leishmania genomes at the expense of high cost. This study proposes an integrative bioinformatic workflow for assembling only the short-read data of Leishmania orientalis isolate PCM2 from Thailand and produce an acceptable-quality genome for further genomic analysis. This workflow gives extensive information required for identifying strain-specific markers and virulence-associated genes useful for drug and vaccine development before a more exhaustive and expensive investigation.Background: Leishmania orientalis (formerly named Leishmania siamensis) has been neglected for years in Thailand. The genomic study of L. orientalis has gained much attention recently after the release of the first high-quality reference genome of the isolate LSCM4. The integrative approach of multiple sequencing platforms for whole-genome sequencing has proven effective at the expense of considerably expensive costs. This study presents a preliminary bioinformatic workflow including the use of multi-step de novo assembly coupled with the reference-based assembly method to produce high-quality genomic drafts from the short-read Illumina sequence data of L. orientalis isolate PCM2. Results: The integrating multi-step de novo assembly by MEGAHIT and SPAdes with the reference-based method using the L. enriettii genome and salvaging the unmapped reads resulted in the 30.27 Mb genomic draft of L. orientalis isolate PCM2 with 3367 contigs and 8887 predicted genes. The results from the integrated approach showed the best integrity, coverage, and contig alignment when compared to the genome of L. orientalis isolate LSCM4 collected from the northern province of Thailand. Similar patterns of gene ratios and frequency were observed from the GO biological process annotation. Fifty GO terms were assigned to the assembled genomes, and 23 of these (accounting for 61.6% of the annotated genes) showed higher gene counts and ratios when results from our workflow were compared to those of the LSCM4 isolate. Conclusions: These results indicated that our proposed bioinformatic workflow produced an acceptable-quality genome of L. orientalis strain PCM2 for functional genomic analysis, maximising the usage of the short-read data. This workflow would give extensive information required for identifying strain-specific markers and virulence-associated genes useful for drug and vaccine development before a more exhaustive and expensive investigation.

Identifying a novel serum microRNA biomarker panel for the diagnosis of childhood asthma.

The pathological mechanism of childhood asthma is complex, and timely diagnosis is the key to effective prevention and control of childhood asthma. We collected 170 serum samples from 95 children with asthma and 75 healthy children. Serum miRNA biomarkers were analyzed by Illumina sequencing for childhood asthma. Differentially serum miRNAs were confirmed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. The Illumina sequencing data showed the differential expression of 111 serum miRNAs among asthmatic and healthy children. After confirmation of miRNAs expression through qRT-PCR, four of them (namely hsa-miR-106a-5p, hsa-miR-18a-5p, hsa-miR-144-3p, and hsa-miR-375) manifested significant differential expression between asthmatic children and healthy controls. The biomarkers classification tree model created with these four miRNAs using the Biomarker Patterns Software could effectively separate childhood asthma and healthy children, with a specificity of 88.3%, a sensitivity of 95.0%, and an area under the curve (AUC) value of 0.942. The regulatory networks containing miRNAs and their gene targets suggested that the four miRNAs might have gene targets implicated in inflammation, immunity, and transcriptional efficiency. Taken together, this four-serum-miRNA panel is a promising biomarker to diagnose childhood asthma noninvasively.

Comparative analysis of the plastid and mitochondrial genomes of Artemisia giraldii Pamp.

Artemisia giraldii Pamp. is an herbaceous plant distributed only in some areas in China. To understand the evolutionary relationship between plastid and mitochondria in A. giraldii, we sequenced and analysed the plastome and mitogenome of A. giraldii on the basis of Illumina and Nanopore DNA sequencing data. The mitogenome was 194,298 bp long, and the plastome was 151,072 bp long. The mitogenome encoded 56 genes, and the overall GC content was 45.66%. Phylogenetic analysis of the two organelle genomes revealed that A. giraldii is located in the same branching position. We found 13 pairs of homologous sequences between the plastome and mitogenome, and only one of them might have transferred from the plastid to the mitochondria. Gene selection pressure analysis in the mitogenome showed that ccmFc, nad1, nad6, atp9, atp1 and rps12 may undergo positive selection. According to the 18 available plastome sequences, we found 17 variant sites in two hypervariable regions that can be used in completely distinguishing 18 Artemisia species. The most interesting discovery was that the mitogenome of A. giraldii was only 43,226 bp larger than the plastome. To the best of our knowledge, this study represented one of the smallest differences between all sequenced mitogenomes and plastomes from vascular plants. The above results can provide a reference for future taxonomic and molecular evolution studies of Asteraceae species.

Chromosome-level assembly of Gymnocypris eckloni genome

Gymnocypris eckloni is widely distributed in isolated lakes and the upper reaches of the Yellow River and play significant roles in the trophic web of freshwater communities. In this study, we generated a chromosome-level genome of G. eckloni using PacBio, Illumina and Hi-C sequencing data. The genome consists of 23 pseudo-chromosomes that contain 918.68 Mb of sequence, with a scaffold N50 length of 43.54 Mb. In total, 23,157 genes were annotated, representing 94.80% of the total predicted protein-coding genes. The phylogenetic analysis showed that G. eckloni was most closely related to C. carpio with an estimated divergence time of ~34.8 million years ago. For G. eckloni, we identified a high-quality genome at the chromosome level. This genome will serve as a valuable genomic resource for future research on the evolution and ecology of the schizothoracine fish in the Qinghai-Tibetan Plateau.

The complete chloroplast genome sequence of Habenaria dentata (Orchidaceae)

Habenaria dentata is a rare species with high ornamental value in China. In this study, we report the complete chloroplast (cp) genome of H. dentata using the Illumina sequencing data. The total genome of H. dentata is 153,682 bp in length and the GC content is 36.62%, with a pair of inverted repeats (IRs) regions of 26,339 bp each, a large single-copy (LSC) region of 83,963 bp and a small single-copy (SSC) region of 17,041 bp. The cp genome encoded 133 genes, including 87 protein-coding genes (PCG), eight rRNA genes, and 38 tRNA genes. The maximum-likelihood phylogenetic analysis based on 12 cp genomes showed that H. dentata was sister to Habenaria chejuensis and Habenaria ciliolaris. This work will be valuable for genetic and phylogenetic studies on H. dentata.