The orchid family Orchidaceae is one of the largest angiosperm families, including many species of important economic value. While chloroplast genomes are very informative for systematics and species identification, there is very limited information available on chloroplast genomes in the Orchidaceae. Here, we report the complete chloroplast genomes of the medicinal plant Dendrobium officinale and the ornamental orchid Cypripedium macranthos, demonstrating their gene content and order and potential RNA editing sites. The chloroplast genomes of the above two species and five known photosynthetic orchids showed similarities in structure as well as gene order and content, but differences in the organization of the inverted repeat/small single-copy junction and ndh genes. The organization of the inverted repeat/small single-copy junctions in the chloroplast genomes of these orchids was classified into four types; we propose that inverted repeats flanking the small single-copy region underwent expansion or contraction among Orchidaceae. The AT-rich regions of the ycf1 gene in orchids could be linked to the recombination of inverted repeat/small single-copy junctions. Relative species in orchids displayed similar patterns of variation in ndh gene contents. Furthermore, fifteen highly divergent protein-coding genes were identified, which are useful for phylogenetic analyses in orchids. To test the efficiency of these genes serving as markers in phylogenetic analyses, coding regions of four genes (accD, ccsA, matK, and ycf1) were used as a case study to construct phylogenetic trees in the subfamily Epidendroideae. High support was obtained for placement of previously unlocated subtribes Collabiinae and Dendrobiinae in the subfamily Epidendroideae. Our findings expand understanding of the diversity of orchid chloroplast genomes and provide a reference for study of the molecular systematics of this family.

Orchidaceae (orchids) is the largest family in the monocots, including about 25,000 species in 880 genera and five subfamilies. Many orchids are highly valued for their beautiful and long-lasting flowers. However, the phylogenetic relationships among the five orchid subfamilies remain unresolved. The major dispute centers on whether the three one-stamened subfamilies, Epidendroideae, Orchidoideae, and Vanilloideae, are monophyletic or paraphyletic. Moreover, structural changes in the plastid genome (plastome) and the effective genetic loci at the species-level phylogenetics of orchids have rarely been documented. In this study, we compared 53 orchid plastomes, including four newly sequenced ones, that represent four remote genera: Dendrobium, Goodyera, Paphiopedilum, and Vanilla. These differ from one another not only in their lengths of inverted repeats and small single copy regions but also in their retention of ndh genes. Comparative analyses of the plastomes revealed that the expansion of inverted repeats in Paphiopedilum and Vanilla is associated with a loss of ndh genes. In orchid plastomes, mutational hotspots are genus specific. After having carefully examined the data, we propose that the three loci 5′trnK-rps16, trnS-trnG, and rps16-trnQ might be powerful markers for genera within Epidendroideae, and clpP-psbB and rps16-trnQ might be markers for genera within Cypripedioideae. After analyses of a partitioned dataset, we found that our plastid phylogenomic trees were congruent in a topology where two one-stamened subfamilies (i.e., Epidendroideae and Orchidoideae) were sisters to a multi-stamened subfamily (i.e., Cypripedioideae) rather than to the other one-stamened subfamily (Vanilloideae), suggesting that the living one-stamened orchids are paraphyletic.

Dendrobium is one of the largest genera in Orchidaceae, comprising about 800–1500 species mainly distributed in tropical Asia, Australasia, and Australia. There are 74 species and two varieties of this genus in China. Because of their ornamental and commercial value, Dendrobium orchids have been studied at low taxonomic levels. However, structural changes and effective mutational hotspots of Dendrobium plastomes have rarely been documented. Here, 30 Dendrobium plastomes were compared, comprising 25 newly sequenced in this study and five previously published. Except for their differences in NDH genes, these plastomes shared identical gene content and order. Comparative analyses revealed that the variation in size of Dendroubium plastomes was associated with dramatically changed length of InDels. Furthermore, ten loci were identified as the top-ten mutational hotspots, whose sequence variability was almost unchanged with more than 10 plastomes sampled, suggesting that they may be powerful markers for Dendrobium species. In addition, primer pairs of 47 polymorphic microsatellites were developed. After assessing the mean BS values of all combinations derived from the top-ten hotspots, we recommend that the combination of five hotspots—trnT-trnL, rpl32-trnL, clpP-psbB, trnL intron, and rps16-trnQ—should be used in the phylogenetic and identification studies of Dendrobium.

Dendrobium officinale , an important medicinal plant of the genus Dendrobium in Orchidaceae family, has been used as traditional Chinese medicine (TCM) for nearly thousands of years. Here, we report the first chromosome-level reference genome of D . officinale , based on PacBio long-reads, Illumina short-reads and Hi-C data. The high-quality assembled genome is 1.23 Gb long, with contig N50 of 1.44 Mb. A total of 93.53% genome sequences were assembled into 19 pseudochromosomes with a super scaffold N50 of 63.07 Mb. Through comparative genomic analysis, we explored the expanded gene families of D . officinale , and also their impact on environmental adaptation and biosynthesis of secondary metabolites. We further performed detailed transcriptional analysis of D. officinale , and identified the candidate genes involved in the biosynthesis of three main active ingredients, including polysaccharides, alkaloids and flavonoids. In addition, the MODIFYING WALL LIGNIN-1 ( MWL1 ) gene, which inferred from Genome-Wide Association Studies (GWAS) based on the resequencing date from D. officinale and five related species and their morphologic features, may contribute to the plant production (yield of stems) of D. officinale . Therefore, the high-quality reference genome reported in this study could benefits functional genomics research and molecular breeding of D. officinale .

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Owing to its great medicinal and ornamental values, Dendrobium officinale is frequently adulterated with other Dendrobium species on the market. Unfortunately, the utilization of the common DNA markers ITS, ITS2, and matK+rbcL is unable to distinguish D. officinale from 5 closely related species of it (D. tosaense, D. shixingense, D. flexicaule, D. scoriarum and D. aduncum). Here, we compared 63 Dendrobium plastomes comprising 40 newly sequenced plastomes of the 6 species and 23 previously published plastomes. The plastomes of D. officinale and its closely related species were shown to have conserved genome structure and gene content. Comparative analyses revealed that small single copy region contained higher variation than large single copy and inverted repeat regions, which was mainly attributed to the loss/retention of ndh genes. Furthermore, the intraspecific sequence variability among different Dendrobium species was shown to be diversified, which necessitates a cautious evaluation of genetic markers specific for different Dendrobium species. By evaluating the maximum likelihood trees inferred from different datasets, we found that the complete plastome sequence dataset had the highest discriminatory power for D. officinale and its closely related species, indicating that complete plastome sequences can be used to accurately authenticate Dendrobium species.

The genus Dendrobium Sw. is composed of 74 species and two varieties in China, and 32 species carry the name "Huangcao Shihu" on the herbal medicine market, making the identification of the origin of "Huangcao Shihu" difficult for consumers. Here, the ITS regions were sequenced and evaluated to differentiate the 18 Dendrobium species used as "Huangcao Shihu". Diversity in DNA sequences among various species was found with the inter-specific sequence divergence ranging from 3.2% to 37.9% in ITS1 and 5.0% to 26.6% in ITS2. Moreover, the variations within species were very low, ranging in sequence divergence from 0 to 3.0% in ITS1 and 0 to 4.0% in ITS2. Therefore, these species could be easily distinguished at the DNA level. Furthermore, based on the divergent ITS regions, five pairs of species-specific primers were designed and used for the rapid PCR identification of five Dendrobium species listed in the Chinese Pharmacopoeia.

Dendrobium officinale is a critically endangered perennial herb endemic to China. Determining the levels of genetic diversity and patterns of population genetic structure of this species would assist in its conservation and management. Data of 12 populations were used to assess its genetic diversity and population structure, employing the method of amplified fragment length polymorphism (AFLP). A high level of genetic diversity was detected (H (E) = 0.269) with POPGENE. As revealed by AMOVA analysis, there was moderate variation between pairs of populations with Phi(ST) values ranging from 0.047 to 0.578 and on average 26.97% of the genetic variation occurred among populations. Three main clusters were shown in UPGMA dendrogram using TFPGA, which is consistent with the result of principal coordinate ananlysis (PCO) using NTSYS. Keeping a stable environment is critical for the in situ conservation and management of this rare and endangered plant, and for ex situ conservation it is important to design an integrated germplasm bank.