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

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.

Keywords: Authentication; DNA; Food fraud; High-resolution melting; Plants; Species identification.

Fig. 1

Families, genera, species, sub-species, cultivars, and their relationships are presented. The order of angiosperm families reflects their phylogenetic relationship from Annonaceae to Euphorbiaceae (Davies et al., 2004). Arrows indicate if examination of melting curves was performed through a visual or a chemometric approach.

Fig. 2

Panel a) Simplexed melting curves from Thymus vulgaris. Panel b) Multiplexed melting profiles from T. vulgaris. For clarity, only the average curve or profile of a duplicate analysis is presented. NTC: Non-template control.

Fig. 3

Multiplexed melting profiles from 29 plant species representing 29 families. For clarity, only the average profile of a triplicate analysis for each species is presented. See Supplementary S4, for the full sized melting profiles.

Fig. 4

Multiplexed melting profiles of species from four plant genera within each of the Lamiaceae and Solanaceae families. For clarity, only the average profile of a triplicate analysis for each species is presented.

Fig. 5

Panel a) and Panel b) Multiplexed high-resolution melting and melting profiles, respectively, of five Capsicum species; Panel c) and Panel d) Multiplexed high-resolution melting and melting profiles, respectively, of three Thymus species. For clarity, only the average profile of a triplicate analysis for each species is presented.

Fig. 6

Panel a) Principal component analysis of five Capsicum species with 92% of the total variance explained by PC-1 and PC-3. Panel b) Sample grouping of the final PLS-DA with the species C. annuum and C. chinense. Panel c) A PCA of three Thymus species with 91% of the total variance explained by PC-1 and PC-2. Each data point represents a triplicate analysis.

Fig. 7

Panel a) Multiplexed high-resolution melting curves of three Capsicum baccatum sub-species in triplicate. Panel b) Multiplexed average melting profiles of a triplicate analysis for each sub-species.

Fig. 8

Panel a) Multiplexed high-resolution melting curves representing four cultivars of Capsicum annuum annuum in a triplicate analysis. Panel b) The multiplexed high-resolution melting curves, normalized around the 92 °C melting transition. Panel c) Multiplexed average melting profiles of a triplicate analysis. Panel d) PCA of the four cultivars with 98% of the total variance explained by PC-1 and PC-2. Each data point represents a triplicate analysis.