DNA mini-barcodes coupled to high resolution melting (hrm) analysis for the botanical authentication of rosemary honey
Conference Paper
Overview
Overview
abstract
Honey is a natural product highly consumed for its taste, nutritional value and health benefits. Monofloral
honeys are the most appreciated by consumers and frequently attain high market values, thus being prone
to fraudulent practices. Therefore, the development of methodologies to assess and authenticate the
botanical origin of honey is of utmost importance. For this purpose, traditional methods based on pollen
identification by microscopic analysis are still being used, but they are time‐consuming and greatly
dependent on the experience/skill of trained analysts. As an alternative, the use of DNA markers represents
promising approach for the identification of botanical species in honey. Currently, DNA barcoding has been
regarded with increasing interest for the taxonomic identification of plants, with two plastidial genes (matK
and rbcL) being proposed for their differentiation (Bruni et al., 2012). Thus, the objective of this work was
to identify the botanical species in rosemary honey using mini‐barcode regions coupled to high resolution
melting (HRM) analysis. For this purpose, different plant species (Lavandula spp.) and ten mono‐ and
multifloral honeys were used. Three DNA barcoding loci, namely the plastidial coding genes rbcL and matK
and the noncoding intergenic trnH‐psbA region, were used to design primers targeting Lavandula spp.
(GenBank Z37408.1, KJ196360.1 and HQ902822.1). DNA from plants and honeys was extracted with
NucleoSpin Plant II kit (method A), according to Soares et al. (2015). The specificity and sensitivity of the
designed primers were assayed by qualitative polymerase chain reaction (PCR) and real‐time PCR. Prior to
the specific amplifications, DNA extracts were positively tested targeting a universal eukaryotic sequence
(18S rRNA gene). Results from specific PCR assays were further confirmed by real‐time PCR amplification
using EvaGreen fluore scence dye. The application of HRM analysis allowed discriminating Lavandula spp.
into distinct clusters with high level of confidence. When applying the developed methodology to rosemary
honey, samples were classified on the same cluster of Lavandula stoechas (endemic species in Portugal),
therefore confirming its botanical origin. To our knowledge, this is the first study using HRM analysis for the
rapid discrimination of plant species in honey.
This work was supported by FCT grant no. LAQV UID/QUI/50006/2013. Joana Costa and
Sónia Soares are grateful to FCT grants (SFRH/BPD/102404/2014 and SFRH/BD/75091/2010, respectively)
financed by POPH‐QREN (subsidized by FSE and MCTES). The authors are grateful for the kind supply of
samples from Bank of Plant Germplasm of Tucson, USA and from Jardim de Serralves, Porto.
