High-resolution melting of multiple barcode amplicons for plant species authentication

Nicolai Zederkopff Ballin,Jone Omar Onaindia,Hadeel Jawad,Rafael Fernandez-Carazo,Alain Maquet

doi:10.1016/j.foodcont.2019.05.022

Nicolai Zederkopff Ballin, Jone Omar Onaindia + Show 3 more

Open Access

https://doi.org/10.1016/j.foodcont.2019.05.022

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Journal: Food Control	Publication Date: May 20, 2019
Citations: 19	License type: cc-by

Affiliation: European Commission

Abstract

In recent years, species identification in herbs has attracted considerable attention due to several cases of fraud; hence inexpensive high-throughput authentication methods are highly welcomed. Species authentication is often performed through DNA analysis and several specific regions (barcodes) are considered suitable. Each barcode (Bar) possesses different qualities in terms of universality and discrimination power. A multiplexed format where information can be extracted simultaneously from several barcode regions is seemingly appropriate to ensure the power of both universality and discrimination. In this approach, we amplified DNA from five different barcode regions in a multiplexed PCR format followed by high-resolution melting (HRM). This multiplexed Bar-HRM approach was first applied to plants spanning the plant kingdom and then gradually narrowing down the genetic variability within the Lamiaceae and the Solanaceae families to finally reach closely related cultivars. Universality was demonstrated through distinct melting profiles obtained for species originating from 29 different families spanning the angiosperms, gymnosperm, mosses, and liverwort (Marchantiophyta). Discrimination power was retained for species, sub-species, and a few cultivars through the application of multivariate statistics to the high-resolution melting profiles. This preliminary investigation has shown the potential to discriminate a vast amount of species within the whole plant kingdom. It requires no a priori knowledge of the species' DNA sequence and occurs in a closed system within 2.5 h at a reduced cost per sample compared to other DNA based approaches.

Highlights

Authentication of plant species is important in a variety of different areas such as the trade of illegal and endangered species, herbal medicine, and food authentication, where one species is replaced with a cheaper one
Since the approach targets a broad range of species, an indepth validation and intraspecific variability of individual species was out of scope; the intention was merely to provide a proof of concept for this cheap and high-throughput authentication approach
It should be stressed that the creation of a library is necessary to identify a truly unknown species, or that authentic material should be used in a comparison to verify whether a claim made on a product is valid

Summary

Introduction

Authentication of plant species is important in a variety of different areas such as the trade of illegal and endangered species, herbal medicine, and food authentication, where one species is replaced with a cheaper one. Authentication of plant species can be performed through microscopy (Matias et al, 2016), detection of metabolites (Wielogorska, Chevallier, Black, Galvin-King, Delêtre, Kelleher, et al, 2018), speciesspecific peptides (Li et al, 2018), and DNA based techniques (Bosmali, Ordoudi, Tsimidou, & Madesis, 2017). Several DNA markers exist, including satellites, insertions and deletions (InDels), single nucleotide polymorphisms (SNPs), and barcode regions. Barcode regions are popular because of a high variability flanked by conserved regions suitable for primer design. This allows discrimination of a large number of species using the same primer pair

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Conclusion