Orchidinae-205: A new genome-wide custom bait set for studying the evolution, systematics, and trade of terrestrial orchids.

Terrestrial orchids are a group of genetically understudied, yet culturally and economically important plants. The Orchidinae tribe contains many species that produce edible tubers that are used for the production of traditional delicacies collectively called 'salep'. Overexploitation of wild orchids in the Eastern Mediterranean and Western Asia threatens to drive many of these species to extinction, but cost-effective tools for monitoring their trade are currently lacking. Here we present a custom bait kit for target enrichment and sequencing of 205 novel genetic markers that are tailored to phylogenomic applications in Orchidinae s.l. A subset of 31 markers capture genes putatively involved in the production of glucomannan, a water-soluble polysaccharide that gives salep its distinctive properties. We tested the kit on 73 taxa native to the area, demonstrating universally high locus recovery irrespective of species identity, that exceeds the total sequence length obtained with alternative kits currently available. Phylogenetic inference with concatenation and coalescent approaches was robust and showed high levels of support for most clades, including some which were previously unresolved. Resolution for hybridizing and recently radiated lineages remains difficult, but could be further improved by analysing multiple haplotypes and the non-exonic sequences captured by our kit, with the promise to shed new light on the evolution of enigmatic taxa with a complex speciation history. Offering a step-up from traditional barcoding and universal markers, the genome-wide custom loci targeted by Orchidinae-205 are a valuable new resource to study the evolution, systematics and trade of terrestrial orchids.

Barcoding High Resolution Melting (Bar-HRM) enables the discrimination between toxic plants and edible vegetables prior to consumption and after digestion.

The consumption of poisonous plants can lead to serious health problems or even casualties due to various factors, including easy access to poisonous plants due to their common distribution, co-occurrence and resemblance with edible plants, and the lack of regulation in the food product supply chain. Clinical diagnosis of intoxications usually relies on the availability of the plant consumed by the patient and on the morphology of the plant parts found in the patient's stomach. Therefore, given the fragmented nature of ingested plant material, species identification may face serious difficulties, can be inaccurate, and time-consuming. This highlights the need for rapid and reliable tools to identify toxic species. In the present study, we developed an ITS2-high-resolution melting (HRM) assay for: (1) the discrimination of common toxic plants and their edible lookalikes, and (2) the detection of toxic plants in digested samples. More specifically, we designed species-specific ITS2 primers for the authentication of poisonous species in simulated mixtures and verified them with Bar-HRM. Moreover, the developed HRM-based molecular tool was capable of quantifying the toxic species Datura stramonium in simulated mixtures with the edible Amaranthus retroflexus down to at least 0.5% v/v. This study shows that species-specific ITS2 primers can amplify the DNA from fragmented and/or artificially digested samples and that Bar-HRM is capable of detecting poisonous plant species in digested samples even after 4 h. The developed Bar-HRM protocol has important implications for application in medicine, forensics, and the agricultural industry, either to accurately detect the cause of plant intoxications or as a tool for quality control in the supply chain. PRACTICAL APPLICATION: In this work, we established a high-resolution melting DNA-based protocol capable of discriminating between phenotypically similar common toxic and edible plant species in mixtures, even at very low quantities. This technology also proved efficient in detecting the toxic species in mixtures digested in artificial gastric acid, as it would be the case after accidental ingestion. This work is expected to have important implications for application in medicine, forensics, and the agricultural industry, either for identifying the cause of plant intoxications or as a tool for quality control in different steps of the supply chain.