DNA metabarcoding of pollen mixtures for environmental monitoring: qualitative and quantitative robustness based on mock mixtures and honeybee-collected samples from across Europe

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Semi-automated sequence curation for reliable reference datasets in ITS2 vascular plant DNA (meta-)barcoding

Publication . Quaresma, Andreia; Ankenbrand, Markus J.; Garcia, Carlos Ariel Yadró; Rufino, José; Honrado, Mónica; Amaral, Joana S.; Brodschneider, Robert; Brusbardis, Valters; Gratzer, Kristina; Hatjina, Fani; Kilpinen, Ole; Pietropaoli, Marco; Roessink, Ivo; Steen, Jozef van der; Vejsnæs, Flemming; Pinto, M. Alice; Keller, Alexander

One of the most critical steps for accurate taxonomic identification in DNA (meta)-barcoding is to have an accurate DNA reference sequence dataset for the marker of choice. Therefore, developing such a dataset has been a long-term ambition, especially in the Viridiplantae kingdom. Typically, reference datasets are constructed with sequences downloaded from general public databases, which can carry taxonomic and other relevant errors. Herein, we constructed a curated (i) global dataset, (ii) European crop dataset, and (iii) 27 datasets for the EU countries for the ITS2 barcoding marker of vascular plants. To that end, we first developed a pipeline script that entails (i) an automated curation stage comprising five filters, (ii) manual taxonomic correction for misclassified taxa, and (iii) manual addition of newly sequenced species. The pipeline allows easy updating of the curated datasets. With this approach, 13% of the sequences, corresponding to 7% of species originally imported from GenBank, were discarded. Further, 259 sequences were manually added to the curated global dataset, which now comprises 307,977 sequences of 111,382 plant species.

2024Journal article

Open access

Note: Cytonuclear patterns of a honey bee population from the Azores show a stable population at the nuclear but not at the mitochondrial DNA level

Publication . Henriques, Dora; Lopes, Ana; Costa, Maíra; Quaresma, Andreia; Doblas-Bajo, Mónica; Pinto, M. Alice

The Azores archipelago has been the stage for multiple introductions of Apis mellifera from varying origins, which have led to widespread admixture and the existence of phenotypically and genotypically heterogeneous populations. This is evident on the São Miguel Island, where the historically introduced black phenotype of A. m. iberiensis (lineage M) co-exists with the contemporaneously introduced yellow phenotype of C-lineage ancestry. Interestingly, the cytonuclear markers used herein revealed that C-lineage ancestry is residual at the nuclear level for both the black (5.82 ± 1.66%) and yellow (5.91 ± 1.85%) phenotypes, although this is more pronounced at the mitochondrial level (27.27% for black and 14.74% for yellow). While the C-lineage contribution has remained stable at the nuclear level for over 20 years, there has been a recent decrease in the proportion of C-derived mitotypes.

2025Journal article

Open access

Identification of the entomological origin of European honey by high resolution melting analysis of a COI mini-barcode

Publication . Honrado, Mónica; Lopes, Ana; Pinto, M. Alice; Amaral, Joana S.

Honey is widely consumed worldwide and highly appreciated for its organoleptic, nutritional and health properties. Honey is also considered one of the foods most prone to be adulterated, either by admixing of honey with lower quality, by sugars’ addition, or by origin mislabelling, among other possible frauds. Recently, great attention has been paid to the development of techniques for authenticating honey through its entomological origin, which is also related to its geographical origin, since bees carrying mitochondrial DNA (mtDNA) from distinct ancestries can be found throughout Europe. Moreover, consumers are increasingly concerned with ethical and environmental issues, paying attention to issues such as the protection of biodiversity and the mode of production. For these reasons, the development of methodologies to authenticate the entomological origin of honey contributes not only to assure consumers rights and avoid unfair competition by the identification of frauds, but also to promote and valorise autochthonous honeybee subspecies. In this work, a one-step approach based on HRM analysis of a 150 bp fragment of the COI gene was developed to establish the entomological origin of honey by discriminating A, M and C mtDNA lineages and differentiating a SNP associated with a high frequency of C1 or C2 mitotypes in the Italian honey bee A. m. ligustica and the Carniolan honey bee A. m. carnica. The method showed high analytical performance and was able to successfully identify the entomological origin of honeys of known origin obtained from research apiaries/beekeepers. Therefore, it was applied to 44 commercial honeys from different countries. It confirmed the entomological authenticity of French PDO honeys that should be produced by the Corse ecotype A. m. mellifera. For the remaining honeys, the results were also in good agreement with the declared geographical origin. This method is also capable of indicating the mixture of honeys produced by honey bees of different lineages, although not allowing to identify the lineages or mitotypes in the mixture. This was the case of three honeys from Slovenia that did not cluster with C2 mitotype A. m. carnica as expected, suggesting the mixture of honeys produced by honeybees of different mitotypes.

2022Conference object

Open access