Paphiopedilum delenatii is a native orchid of Vietnam with highly attractive floral traits. Unfortunately, it is now listed as a critically endangered species with a few hundred individuals remaining in nature. In this study, we performed next-generation sequencing of P. delenatii and assembled its complete chloroplast genome. The whole chloroplast genome of P. delenatii was 160,955 bp in size, 35.6% of which was GC content, and exhibited typical quadripartite structure of plastid genomes with four distinct regions, including the large and small single-copy regions and a pair of inverted repeat regions. There were, in total, 130 genes annotated in the genome: 77 coding genes, 39 tRNA genes, 8 rRNA genes, and 6 pseudogenes. The loss of ndh genes and variation in inverted repeat (IR) boundaries as well as data of simple sequence repeats (SSRs) and divergent hotspots provided useful information for identification applications and phylogenetic studies of Paphiopedilum species. Whole chloroplast genomes could be used as an effective super barcode for species identification or for developing other identification markers, which subsequently serves the conservation of Paphiopedilum species.

Paphiopedilum is among the most popular ornamental orchid genera due to its unique slipper flowers and attractive leaf coloration. Most of the Paphiopedilum species are in critical danger due to over-exploitation. They were listed in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora, which prevents their being traded across borders. While most Paphiopedilum species are distinctive, owing to their respective flowers, their vegetative features are more similar and undistinguished. Hence, the conservation of these species is challenging, as most traded specimins are immature and non-flowered. An urgent need exists for effective identification methods to prevent further illegal trading of Paphiopedilum species. DNA barcoding is a rapid and sensitive method for species identification, at any developmental stage, using short DNA sequences. In this study, eight loci, i.e., ITS, LEAFY, ACO, matK, trnL, rpoB, rpoC1, and trnH-psbA, were screened for potential barcode sequences on the Vietnamese Paphiopedilum species. In total, 17 out of 22 Paphiopedilum species were well identified. The studied DNA sequences were deposited to GenBank, in which Paphiopedilum dalatense accessions were introduced for the first time. ACO, LEAFY, and trnH-psbA were limited in amplification rate for Paphiopedilum. ITS was the best single barcode. Single ITS could be used along with nucleotide polymorphism characteristics for species discrimination. The combination of ITS + matK was the most efficient identification barcode for Vietnamese Paphiopedilum species. This barcode also succeeded in recognizing misidentified or wrongly-named traded samples. Different bioinformatics programs and algorithms for establishing phylogenetic trees were also compared in the study to propose quick, simple, and effective tools for practical use. It was proved that both the Bayesian Inference method in the MRBAYES program and the neighbor-joining method in the MEGA software met the criteria. Our study provides a barcoding database of Vietnamese Paphiopedilum which may significantly contribute to the control and conservation of these valuable species.

Dendrobium has been widely used not only as ornamental plants but also as food and medicines. The identification and evaluation of the genetic diversity of Dendrobium species support the conservation of genetic resources of endemic Dendrobium species. Uniquely identifying Dendrobium species used as medicines helps avoid misuse of medicinal herbs. However, it is challenging to identify Dendrobium species morphologically during their immature stage. Based on the DNA barcoding method, it is now possible to efficiently identify species in a shorter time. In this study, the genetic diversity of 76 Dendrobium samples from Southern Vietnam was investigated based on the ITS (Internal transcribed spacer), ITS2, matK (Maturase_K), rbcL (ribulose-bisphosphate carboxylase large subunit) and trnH-psbA (the internal space of the gene coding histidine transfer RNA (trnH) and gene coding protein D1, a polypeptide of the photosystem I reaction center (psaB)) regions. The ITS region was found to have the best identification potential. Nineteen out of 24 Dendrobium species were identified based on phylogenetic tree and Indel information of this region. Among these, seven identified species were used as medicinal herbs. The results of this research contributed to the conservation, propagation, and hybridization of indigenous Dendrobium species in Southern Vietnam.

Background: Discrimination of Paphiopedilum species using floral morphology has been used effectively and commonly due to distinct features of their reproductive parts. Most classification references also focus on description of flower morphology and structure. Nevertheless species preservation meets great problems when illegal trading plants are mostly at non-flowered or plantlet stages. Molecular approaches have been applied and obtained highly precise results but spent time and could not rate 100% of resolution over the genus. Thus, quickly identification using only vegetative characteristics would be an efficient support for other distinguishing methods and a remarkable contribution to the detection and prevention of Paphiopedilum ilicit sales. Aims: In this research we aimed to describe in details leaf features of Paphiopedilum species in Vietnam serving for quick and easy discriminating species without flower present or laboratory techniques. Study Design:  Observed variables based on qualitative features of leaf shape, color, vein feature, thickness, toughness and quantitative features of leaf size were saved and organized using Microsoft Excel 2010. Place and Duration of Study: Tay Nguyen Institute for Scientific Research, Agricultural Genetics Institute, between November 2018 and November 2019. Methodology: We minutely analyzed the leaf morphology of nineteen species and two variants which belongs to Vietnamese Paphiopedilum population in an order manner from parameters that easiest to be realized to the less clearly traits for the most efficient recognition even to the one who is not botanist. Results: 16 out of 20 species were identified using leaf morphology. This rate was up to 100% when combining morphological and molecular methods without the present of their flowers. An artificial key to genus Paphiopedilum using the vegetative details was first time established. Conclusion: Leaf morphology can be effectively used as the first step for identifying Paphiopedilum species. Our results provided useful tool in severally or in combination with molecular methods in biodiversity and commercial management of these valuable species.