First report of the complete mitochondrial genome of 3 beetles (Coleoptera: Scarabaeidae) harming Gastrodia elata (Asparagales: Orchidaceae).

Gastrodia elata Blume, a valuable traditional Chinese medicine with significant clinical and nutritional importance, is a fungal heterotrophic orchid. We present the first report of the mitochondrial genome structure and characteristics of 3 Scarabaeidae pests affecting G. elata: Sophrops peronosporus Gu & Zhang, Anomala rufiventris Kollar & Redtenbacher, and Callistethus plagiicollis Fairmaire. Each mitogenome contained 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and a control region, with no gene rearrangements observed. All 21 tRNAs, except trnS1 that lacks a dihydrouridine, had a stable cloverleaf secondary structure. Maximum likelihood and Bayesian inference analyses based on the 13 PCGs produced 2 topologically similar phylogenetic trees, both of with high nodal support. Larvae of these Scarabaeidae pests cause substantial damage by gnawing on the tubers and roots of G. elata, leading to reduced yield and compromised quality. These findings contribute to phylogenetic studies of Scarabaeidae, expand knowledge of G. elata pests, and offer valuable reference materials for their identification and control.

Utilizing epoxy Bletilla striata polysaccharide collagen sponge for hemostatic care and wound healing.

Hemostatic materials that are lightweight and possess good blood absorption performance have been widely considered for use in modern wound care. Natural hemostatic ingredients derived from traditional Chinese medicine have also received extensive attention. Bletilla polysaccharides are valued by researchers for their excellent hemostatic performance and good reactivity. Collagen is favored by researchers due to its high biocompatibility and low immunogenicity. In this study, Bletilla striata polysaccharide, the main hemostatic component of Bletilla striata, was activated by epoxy groups, and epoxidized Bletilla striata polysaccharide (EBSP) was prepared. Then, EBSP was crosslinked with collagen under alkaline conditions, and a new hemostatic material that was an epoxidized Bletilla polysaccharide crosslinked collagen hemostatic sponge was prepared. We demonstrated that endowing collagen with better hemostatic performance, cytocompatibility, and blood compatibility does not destroy its original three-stranded helical structure. Compared with the medical gauze, hemostasis time was shorter (26.75 ±â€¯2.38 s), and blood loss was lower (0.088 ±â€¯0.051 g) in the rat liver injury hemostasis model. In the rat model of severed tail hemostasis, hemostasis time was also shorter (47.33 ±â€¯2.05 s), and the amount of blood loss was lower (0.330 ±â€¯0.122 g). The sponge possessed good hemostatic and healing performance.

Bletilla Striata polysaccharides thermosensitive gel for photothermal treatment of bacterial infection.

Skin is the most important defense shield which touched external environment directly. Effectively clearing microbes in infected wound via non-antibiotic therapy is crucial for the promotion of recovery in complex biological environments, and the wound healing is a crucial process after sterilization to avoid superinfection. Herein, a kind of Prussian blue-based photothermal responsive gel, Bletilla striata polysaccharide-mingled, isatin-functionalized Prussian blue gel (PB-ISA/BSP gel) was reported for effective treatment of bacterial infection and wound healing. The introduction of effective components of traditional Chinese medicine (TCM), isatin (ISA), enhanced the efficiency of sterilization synergistically. Furthermore, the process of wound healing was promoted by Bletilla striata polysaccharides (BSP). PB-ISA@BSP had a considerable antibacterial rate with 98.5 % under an 808 nm laser for 10 min in vitro. Besides, PB-ISA/BSP gel showed an effective antibacterial efficacy in vivo and a fast wound healing rate as well. The as-prepared functional particles can invade and destroy bacteria membrane to kill microbes. This work highlights that PB-ISA/BSP gel is a promising antibacterial agent based on synergistically enhanced photothermal effect and wound healing promotion ability and provides inspiration for future therapy based on the synergy between photothermal agent and active components in TCM.

Neuroprotective neolignan glycosides from the pseudobulbs of Bletilla striata.

Four undescribed neolignan glycosides, bletineosides A-D (1-4) were isolated from the pseudobulbs of Bletilla striata. Their structures with absolute configurations were elucidated on the basis of spectroscopic analyses, along with acidic hydrolysis reactions and ECD experiments. All isolates were evaluated for their neuroprotective activities against glutamate-induced PC12 cell injury. Compound 3 and 4 showed significantly neuroprotective effects at the concentration of 10 µM when compared with the model group. Compounds 1-4 represented the first examples of neolignan glycosides from the genus Bletilla. This study disclosed the potency of Bletilla striata as a new source of anti-neurodegenerative agents.

Extraction, structural-activity relationships, bioactivities, and application prospects of Bletilla striata polysaccharides as ingredients for functional products: A review.

Bletilla striata is a well-known medicinal plant with high pharmaceutical and ornamental values. Polysaccharide is the most important bioactive ingredient in B. striata and has various health benefits. Recently, B. striata polysaccharides (BSPs) have attracted much attention from industries and researchers due to their remarkable immunomodulatory, antioxidant, anti-cancer, hemostatic, anti-inflammatory, anti-microbial, gastroprotective, and liver protective effects. Despite the successful isolation and characterization of BSPs, there is still limited knowledge regarding their structure-activity relationships (SARs), safety concerns, and applications, which hinders their full utilization and development. Herein, we provided an overview of the extraction, purification, and structural features, as well as the effects of different influencing factors on the components and structures of BSPs. We also highlighted and summarized the diversity of chemistry and structure, specificity of biological activity, and SARs of BSP. The challenges and opportunities of BSPs in the food, pharmaceutical, and cosmeceutical fields are discussed, and the potential development and future study direction are scrutinized. This article provides comprehensive knowledge and underpinnings for further research and application of BSPs as therapeutic agents and multifunctional biomaterials.

Phenotype-fitness relationships and pollen-transfer efficiency of five orchid species with different pollination strategies.

PREMISE: Deceptive pollination, a fascinating mechanism that independently originated in several plant families for benefiting from pollinators without providing any reward, is particularly widespread among orchids. Pollination efficiency is crucial in orchids due to the aggregated pollen in a pollinarium, which facilitates pollen transfer and promotes cross-pollination as pollinators leave after being deceived. METHODS: In this study, we compiled data on reproductive ecology from five orchid species with different pollination strategies: three deceptive-strategy species (shelter imitation, food deception, sexual deception), one nectar-rewarding species, and one shelter-imitation but spontaneously selfing species. We aimed to compare the reproductive success (female fitness: fruit set; male fitness: pollinarium removal) and pollination efficiency of species representing these strategies. We also investigated pollen limitation and inbreeding depression among the pollination strategies. RESULTS: Male and female fitness were strongly correlated in all species but the spontaneously selfing species, which had high fruit set and low pollinarium removal. As expected, pollination efficiency was highest for the rewarding species and the sexually deceptive species. Rewarding species had no pollen limitation but did have high cumulative inbreeding depression; deceptive species had high pollen limitation and moderate inbreeding depression; and spontaneously selfing species did not have pollen limitation or inbreeding depression. CONCLUSIONS: Pollinator response to deception is critical to maintain reproductive success and avoid inbreeding in orchid species with non-rewarding pollination strategies. Our findings contribute to a better understanding of the trade-offs associated with different pollination strategies in orchids and highlight the importance of pollination efficiency in orchids due to the pollinarium.

Comparative analyses and phylogenetic relationships of thirteen Pholidota species (Orchidaceae) inferred from complete chloroplast genomes.

BACKGROUND: The orchid genus Pholidota Lindl. ex Hook. is economically important as some species has long been used in traditional medicine. However, the systematic status of the genus and intergeneric relationships inferred from previous molecular studies are unclear due to insufficient sampling and lack of informative sites. So far, only limited genomic information has been available. The taxonomy of Pholidota remains unresolved and somewhat controversial. In this study, the complete chloroplast (cp.) genomes of thirteen Pholidota species were sequenced and analyzed to gain insight into the phylogeny of Pholidota and mutation patterns in their cp. genomes. RESULTS: All examined thirteen Pholidota cp. genomes exhibited typical quadripartite circular structures, with the size ranging from 158,786 to 159,781 bp. The annotation contained a total of 135 genes in each cp. genome, i.e., 89 protein-coding genes, 38 tRNA genes, and eight rRNA genes. The codon usage analysis indicated the preference of A/U-ending codons. Repeat sequence analysis identified 444 tandem repeats, 322 palindromic repeats and 189 dispersed repeats. A total of 525 SSRs, 13,834 SNPs and 8,630 InDels were detected. Six mutational hotspots were identified as potential molecular markers. These molecular markers and highly variable regions are expected to facilitate future genetic and genomic studies. Our phylogenetic analyses confirmed the polyphyletic status of the genus Pholidota, with species grouped into four main clades: Pholidota s.s. was resolved as the sister to a clade containing species of Coelogyne; the other two clades clustered together with species of Bulleyia and Panisea, respectively; species P. ventricosa was placed at the basal position, deviated from all other species. CONCLUSION: This is the first study to comprehensively examine the genetic variations and systematically analyze the phylogeny and evolution of Pholidota based on plastid genomic data. These findings contribute to a better understanding of plastid genome evolution of Pholidota and provide new insights into the phylogeny of Pholidota and its closely related genera within the subtribe Coelogyninae. Our research has laid the foundation for future studies on the evolutionary mechanisms and classification of this economically and medicinally important genus.

Non-species-specific pollen transfer and double-reward production in euglossine-pollinated Vanilla.

Commonly attributed to orchids, the pollen movement in Vanilla has been associated with food deception and specific plant-pollinator relationships. This study investigated the role of flower rewards and pollinator specificity in the pollen transfer of a widely distributed member to the euglossinophilous Vanilla clade, V. pompona Schiede using data collected from Brazilian populations. These included investigations on morphology, light microscopy and histochemistry, and analysis of flowers scent using GC-MS. The pollinators and the mechanisms of pollination were recorded through focal observations. The yellow flowers of V. pompona are fragrant and offer nectar as reward. The major volatile compound of the V. pompona scent, carvone oxide, shows convergent evolution in Eulaema-pollinated Angiosperms. The pollination system of V. pompona is not species-specific, but its flowers are strongly adapted to pollination by large Eulaema males. Pollination mechanism is based in a combination of perfume collection and nectar seeking. The dogma of a species-specific pollination system based on food deception in Vanilla has been broken with the increase in studies on this Pantropical orchid genus. Here, least three bee species and dual reward-offering are involved in pollen transfer in V. pompona. Visitation frequency of bees collecting perfumes, used in courtship by male euglossines, is higher than in searching for food, as short-lived young euglossine males seem to be more interested in sex than food. A pollination system based on offering both nectar and perfumes as resources is described for the first time in orchids.

Effects of Different Drying Methods on the Quality of Bletilla striata Scented Tea.

Flower tea is widely loved as a drink, especially for the beautiful and rich flowers of the orchid family, and the drying method for different flowers is also unique. GC-MS is widely used to study volatile substances to determine the quality of flower teas. The findings show that the freeze-drying method can retain the original aroma and flavor of Bletilla striata has the highest sensory evaluation score, with the key flavor substances ethyl caproate and N-heptanal containing 1.14% and 6.28%, respectively, and their ROAV values reaching 54.46 and 100.00. Additionally, the freeze-drying method can well retain flavonoids, polysaccharides, and phenolic components, while providing better antioxidant and antibacterial properties. The stove-drying method would make Bletilla striata slightly burnt and less flavorful and efficacious than freeze-drying; the air-drying method is difficult to retain the special odor and fragrance of Bletilla striata flowers and has the lowest sensory evaluation score, with the presence of volatile components with irritating and unpleasant odors such as pyrazine and 2-pentylfuran, while not showing better efficacy. In addition, steam fixation would destroy the morphology and flavor of Bletilla striata, lose polysaccharide and phenolic components, and reduce the efficacy of Bletilla striata scented tea, but could retain the flavonoid components well. In summary, direct freeze-drying without steam fixation is the best process for drying Bletilla striata scented tea, stove-drying without steam fixation is more economical and convenient in actual production and application, steam fixation and air-drying are not suitable as drying processes for Bletilla striata scented tea. This study analyzed the quality of Bletilla striata scented tea under different drying methods, promoted the further processing of Bletilla striata scented tea, and provided a reference for the comprehensive utilization of Bletilla striata scented tea.

Isolation, structural and bioactivities of polysaccharides from Anoectochilus roxburghii (Wall.) Lindl.: A review.

Anoectochilus roxburghii (Wall.) Lindl. (A. roxburghii), a valuable herbal medicine in China, has great medicinal and edible value. Polysaccharides, as one of the main active components of A. roxburghii, comprise glucose, arabinose, xylose, galactose, rhamnose, and mannose in different molar ratios and glycosidic bond types. By varying the sources and extraction methods of A. roxburghii polysaccharides (ARPS), different structural characteristics and pharmacological activities can be elucidated. ARPS has been reported to exhibit antidiabetic, hepatoprotective, anti-inflammatory, antioxidant, antitumor, and immune regulation activities. This review summarizes the available literature on the extraction and purification methods, structural features, biological activities, and applications of ARPS. The shortcomings of the current research and potential focus in future studies are also highlighted. This review provides systematic and current information on ARPS to promote their further exploitation and application.

Active Compounds with Medicinal Potential Found in Maxillariinae Benth. (Orchidaceae Juss.) Representatives-A Review.

Orchids are widely used in traditional medicine for the treatment of a whole range of different health conditions, and representatives of the Neotropical subtribe Maxillariinae are not an exception. They are utilized, for instance, for their spasmolytic and anti-inflammatory activities. In this work, we analyze the literature concerning the chemical composition of the plant extracts and secretions of this subtribe's representatives published between 1991 and 2022. Maxillariinae is one of the biggest taxa within the orchid family; however, to date, only 19 species have been investigated in this regard and, as we report, they produce 62 semiochemicals of medical potential. The presented review is the first summary of biologically active compounds found in Maxillariinae.

Analysis of trait-performance-fitness relationships reveals pollinator-mediated selection on orchid pollination traits.

PREMISE: The role of pollinators in evolutionary floral divergence has spurred substantial effort into measuring pollinator-mediated phenotypic selection and its variation in space and time. For such estimates, the fitness consequences of pollination processes must be separated from other factors affecting fitness. METHODS: We built a fitness function linking phenotypic traits of food-deceptive orchids to female reproductive success by including pollinator visitation and pollen deposition as intermediate performance components and used the fitness function to estimate the strength of pollinator-mediated selection through female reproductive success. We also quantified male performance as pollinarium removal and assessed similarity in trait effects on male and female performance. RESULTS: The proportion of plants visited at least once by an effective pollinator was moderate to high, ranging from 53.7% to 85.1%. Tall, many-flowered plants were often more likely to be visited and pollinated. Given effective pollination, pollen deposition onto stigmas tended to be more likely for taller plants. Pollen deposition further depended on traits affecting the physical fit of pollinators to flowers (flower size, spur length), though the exact relationships varied in time and space. Using the fitness function to assess pollinator-mediated selection through female reproductive success acting on multiple traits, we found that selection varied detectably among taxa after accounting for sampling uncertainty. Across taxa, selection on most traits was stronger on average and more variable when pollination was less reliable. CONCLUSIONS: These results support pollination-related trait-performance-fitness relationships and thus pollinator-mediated selection on traits functionally involved in the pollination process.

Sugars and sucrose transporters in pollinia of Phalaenopsis aphrodite (Orchidaceae).

The pollen grains of Phalaenopsis orchids are clumped tightly together, packed in pollen dispersal units called pollinia. In this study, the morphology, cytology, biochemistry, and sucrose transporters in pollinia of Phalaenopsis orchids were investigated. Histochemical detection was used to characterize the distribution of sugars and callose at the different development stages of pollinia. Ultra-performance liquid chromatography-high resolution-tandem mass spectrometry data indicated that P. aphrodite accumulated abundant saccharides such as sucrose, galactinol, myo-inositol, and glucose, and trace amounts of raffinose and trehalose in mature pollinia. We found that galactinol synthase (PAXXG304680) and trehalose-6-phosphate phosphatase (PAXXG016120) genes were preferentially expressed in mature pollinia. The P. aphrodite genome was identified as having 11 sucrose transporters (SUTs). Our qRT-PCR confirmed that two SUTs (PAXXG030250 and PAXXG195390) were preferentially expressed in the pollinia. Pollinia germinated in pollen germination media (PGM) supplemented with 10% sucrose showed increased callose production and enhanced pollinia germination, but there was no callose or germination in PGM without sucrose. We show that P. aphrodite accumulates high levels of sugars in mature pollinia, providing nutrients and enhanced SUT gene expression for pollinia germination and tube growth.

Gymnadenia conopsea (L.) R. Br.: Comprehensive review of propagation and breeding, traditional uses, chemical composition, pharmacology, quality control, and processing.

ETHNOPHARMACOLOGICAL RELEVANCE: Gymnadenia conopsea, a perennial herbaceous flowering plant that belongs to the family of Orchidaceae, sporadic distributed in the altitudes of 200-4700 m across northern Europe and, temperate and subtropical Asia region. The dried tubers of G. conopsea have been used to treat cough, asthma, and their syndromes, and also as a tonic in China and surrounding countries for a long history. G. conopsea is often processed deeply processed before use to enhance its efficacy. In recent years, because of its remarkable pharmacological activity and health care function, G. conopsea has been used more and more widely. Due to its extensive application and bad growth environment, the wild distribution of G. conopsea is decreasing and it has been listed as an endangered plant. AIM OF THE REVIEW: This review aims to summarize the propagation and breeding, traditional uses, chemical composition, pharmacology, quality control, and processing of G. conopsea. Moreover, it also provides suggestions for the future high-value utilization of G. conopsea. MATERIALS AND METHODS: A literature search on Gymnadenia genus and G. conopsea was performed using scientific databases including SciFinder, ACS, Web of Science, Springer, ScienceDirect, PubMed, and CNKI. Information was also collected from classic books of Chinese herbal medicine, official websites, Ph.D. and M.Sc. Dissertations, and so on. Structures of chemical compounds were drawn by ChemDraw software. RESULTS: As of submission date of this manuscript, total 170 natural compounds have been isolated and characterized from G. conopsea, and all of the compounds were isolated from the tubers. The isolated compounds including benzylester glucosides, dihydrostilbenes, phenanthrenes, phenolic compounds, alkaloids, polysaccharide, lignans, flavones, triterpenoids, steroids, and other compounds. Some of these compounds and active extracts exhibited a wide range of pharmacological activities, in which, the tonifying, anti-fatigue, anti-oxidant, anti-viral, sedative and hypnotic activities are consistent with the traditional uses for the treatment of diseases. In addition, a variety of new pharmacological activities, such as preventing and treating gastric ulcers, immunoregulatory, anti-hyperlipidemia, anti-anaphylaxis, anti-silicosis, anti-cancer and neuroprotective activities have also been reported. However, the bioactive compounds responsible for most of the above pharmacological effects have not been well summarised till now. In this manuscript, analysis, speculation and summary of compounds that responsible for pharmacological effects were conducted. CONCLUSIONS: The chemical constituents and pharmacological activities studies of G. conopsea extract have been summarised in this context, the isolated compounds responsible for the pharmacological activities were also analyzed and deduced according to the publications, all above led to suggestions for the future high-value utilization of G. conopsea.

Effect of Flowering Stages on the Content of Active Ingredients and Antioxidant Capability of Bletilla striata Flowers.

Bletilla striata (Thunb.) Reichb.f. is a perennial herb with abundant active ingredients. Previous research mainly focused on its tubers, however, the study on flowers, especially the variation of active ingredient contents at different flowering stages, was rarely seen. This study analyzed the total phenols, flavonoids, polysaccharides, anthocyanins, and cyanidin-3-O-glucoside content of B. striata flowers which were in cultivated in Herb Garden of Zhejiang A&F University and collected in May, 2019, in order to investigate the changes in active ingredients and antioxidant capacity among different flowering stages (bud, initial, and full bloom). Changes in radical scavenging capability of DPPH (1,1-Diphenyl-2-picrylhydrazyl radical), ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate)), and hydroxy were analyzed. Significant differences in active ingredient content of flowers were detected among different flowering stages. The total phenolic content increased continuously during the entire flowering stage. The contents of total flavonoid, total polysaccharide, and cyanidin-3-O-glucoside reached peaks at the initial blooming stage and then fell as the flowering process continued. The antioxidant activity in initial stage was the highest than in any other flowering stages. Therefore, we conclude that the initial blooming stage is the best harvesting stage of B. striata flowers. This study provides a robust basis for the harvest and utilization of B. striata flowers in food, medical, and cosmetic industries.

Evaluation of whitening and antimicrobial activity of two strains of Bletilla striata WT and HL20.

ETHNOPHARMACOLOGICAL RELEVANCE: Bletilla striata (Thunb.) Reixchb.f. is a perennial herb of the Orchid-aceae Bletilla and have various ethnopharmacological uses. As a traditional astringent hemostatic Chinese herbal medicine, B. striata has been widely used in the treatment of 127 different kinds of hemorrhagic diseases. Moreover, B. striata has been a beauty medicine since ancient times, with the first ancient records dating back to 2000 years ago, traditionally used to removing freckle and smooth the skin. Because of the high content of polysaccharides, which is considered the primary active substance of B. striata and having anti-aging, whitening, and anti-oxidation functions, this is also widely used in the cosmetics industry. AIM: We screened the germplasm resources of B. striata in the early stage and the superior HL20 strain was obtained. Our research aims to analyze and compare the whitening and antimicrobial activities of different extracts (aqueous extract, ethanol extract, and aqueous extract from ethanol extract filter residue) of the selected superior varieties (HL20) and the control (WT). MATERIALS AND METHODS: L-tyrosine and L-dopa were used as substrates to establish a tyrosinase inhibition system with arbutin as the positive control and the whitening activity was measured by the inhibition rate of TYR-M and TYR-D. Besides, an in vitro antimicrobial susceptibility test was performed to assess the antimicrobial activity of the B. striata extracts. In a nutshell, the method of punching diffusion was used to thoroughly examine the effects of three extracts from two strains on the antimicrobial activity of five types of microorganism in cosmetics microbiological testing products. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of different extracts were also assessed. RESULTS: Results showed that the whitening and antimicrobial properties of the HL20 strain were found to be more potent than those of the WT strain. Compared with the other two extraction methods, the aqueous extract from ethanol extract filter residue of B. striata exhibited better inhibition of tyrosinase activity. The antimicrobial assay manifested that only the ethanol extract of B. striata had an inhibitory effect and had a potent antimicrobial impact on E. faecalis. CONCLUSIONS: In summary, we evaluated the pharmacological activity of the pre-selected excellent variety (HL20) in terms of whitening and antimicrobial activity. Our results reveal that the selected strain (HL20) has certain advantages over the control (WT). These characteristics make it a candidate additive for whitening cosmetics. Our study also provides a further contribution to the product application of B. striata in cosmetics and antimicrobial agents and the selected HL20 also lays a foundation for the breeding of superior B. striata varieties.

Two new bibenzyls from Pleione grandiflora (Rolfe) Rolfe and their antioxidant activity.

Two new bibenzyls (1 and 2) were isolated from the pseudobulbs of Pleione grandiflora (Rolfe) Rolfe along with six known compounds, including isoarundinin I (3), isoarundinin II (4), bulbocodin D (5), batatasin III (6), 5,3'-dihydroxy- 4-(p-hydroxybenzyl)-3-methoxybibenzyl (7) and shancigusin F (8). Their structures were established on the basis of spectroscopic methods. These compounds showed potent DPPH free radical scavenging effects with IC50 values ranging from 49.72 ± 0.35 µM to 65.41 ± 0.49 µM.

Characterization of Angraecum (Angraecinae, Orchidaceae) Plastomes and Utility of Sequence Variability Hotspots.

Angraecum, commonly known as Darwin's orchid, is the largest genus of Angraecinae (Orchidaceae). This genus exhibits a high morphological diversity, making it as a good candidate for macroevolutionary studies. In this study, four complete plastomes of Angraecum were firstly reported and the potential variability hotspots were explored. The plastomes possessed the typical quadripartite structure and ranged from 150,743 to 151,818 base pair (bp), with a guanine-cytosine (GC) content of 36.6-36.9%. The plastomes all contained 120 genes, consisting of 74 protein-coding genes (CDS), 38 transfer RNA (tRNA) genes and 8 ribosomal RNA (rRNA) genes; all ndh genes were pseudogenized or lost. A total of 30 to 46 long repeats and 55 to 63 SSRs were identified. Relative synonymous codon usage (RSCU) analysis indicated a high degree of conservation in codon usage bias. The Ka/Ks ratios of most genes were lower than 1, indicating that they have undergone purifying selection. Based on the ranking of Pi (nucleotide diversity) values, five regions (trnSGCU-trnGGCC, ycf1-trnNGGU, trnNGUU-rpl32, psaC-ndhE and trnSGCU-trnGGCC) and five protein-coding genes (rpl32, rps16, psbK, rps8, and ycf1) were identified. The consistent and robust phylogenetic relationships of Angraecum were established based on a total of 40 plastomes from the Epidendroideae subfamily. The genus Angraecum was strongly supported as a monophyletic group and sister to Aeridinae. Our study provides an ideal system for investigating molecular identification, plastome evolution and DNA barcoding for Angraecum.

Transcriptome analysis and development of EST-SSR markers in Anoectochilus emeiensis (Orchidaceae).

Anoectochilus emeiensis K. Y. Lang, together with other Anoectochilus species, has long been used as the main source of many traditional Chinese medicines. Owing to the shortcomings of molecular markers, the study of the genetic diversity and medicinal component synthesis mechanism of the endemic Anoectochilus species has been delayed. In this study, I carried out a transcriptome analysis of A. emeiensis. A total of 78,381 unigenes were assembled from 64.2 million reads, and 47,541 (60.65%) unigenes were matched to known proteins in the public databases. Then, 9284 expressed sequence tag-derived simple sequence repeats (EST-SSRs) were identified, and the frequency of SSRs in the A. emeiensis transcriptome was 9.88%. Trinucleotide repeats (3699, 39.84%) were the most common type, followed by dinucleotide (3251, 35.02%) and mononucleotide (1750, 18.85%) repeats. Based on the SSR sequence, 6683 primer pairs were successfully designed, 40 primer pairs were randomly selected, and 10 primer pairs were identified as polymorphic loci from 186 individuals of A. emeiensis. The EST-SSR markers examined in this study will be informative for future population genetic studies of A. emeiensis.