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.

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.

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