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  • Molecular detection of the botanical origin of pollen in honey bee-collected pellets: a comparison of methods
    Publication . Haba, Eya; Pinto, M. Alice; Amaral, Joana S.; Hamda, Cherif Ben
    Identification of botanical origin of mixed pollen samples has several applications, including assessment of plant-pollinator interactions, botanical origin of honey, monitoring of pesticide use, monitoring of allergy-related airborne pollen sources, among others. Such applications, however, have previously been limited to conventional pollen identification via light microscopy, which usually has low taxonomic resolution and requires expert knowledge. One alternative for botanical identification of mixed pollen samples is to use of DNA metabarcoding high throughput sequencing (HTS), which could overcome these drawbacks. Recent studies demonstrate that the nuclear barcoding marker ITS2 (internal transcribed spacer 2 region of nuclear ribosomal DNA) can be amplified from DNA extracted from mixed pollen samples. The aim of this study was to compare a variety of methods of storage/transportation and DNA extraction that ensure good DNA yield and quality appropriate for botanical identification of mixed pollen samples by means of a DNA metabarcoding approach, combining the amplification of ITS2 with HTS. In the context of the international project “INSIGNIA: environmental monitoring of pesticide use through honeybees”, mixed pollen samples were collected from traps set up in apiaries from several European countries, stored by beekeepers and later transported to the laboratory of CIMO for identification of plant taxa and inference of relative abundances. Four methods of genomic DNA isolation (NucleoSpin Food kit, GF-1 Plant kit, HigherPuritykit, and CTAB- PVP) were compared regarding DNA yield and purity by means of spectrophotometry and standard gel electrophoresis. Additionally, four storage/transportation methods of trap- collected pollen samples (freezing at -20 °C, drying at 25°C for 2 days, drying with silica, and placing in ethanol) were compared to assess their impact on the quality and quantity of extracted DNA. The results demonstrated the superior efficacy of the NucleoSpin DNA extraction method. The different storage/transportation conditions of pollen samples were compared for their impact on DNA quality and quantity using the NucleoSpin as the DNA extraction method. The results showed that the DNA extracted from the pollen samples placed in ethanol had the best quality/yield compared to the DNA extracted from the other samples with different storage conditions. Two primer pairs targeting ITS2 region ITS- S2F/ITS4R and ITS-u3/ITS-u4, were employed to identify plant taxa via metabarcoding HTS. The number of taxa identified in common using these two primers were 48 families, 118 genera, and 204 species, corresponding to 87.2 % , 79.5%, and 68.7%, respectively.The results of identification of taxa we present very similar results, making comparisons difficult, with a slight difference in the number of taxa (ITS-u3/ITS-u4 with higher number of identified taxa) and the abundance (ITS-S2F/ITS4R with higher abundance of taxa identified). This study thus offers improvements in the laboratory workflow ensuring a good DNA quantity and quality for downstream HTS applications.
    2019Dissertação de mestrado Acesso aberto Ver mais