Abstract
Metabarcoding is a promising DNA-based method for identifying airborne pollen from environmental samples with advantages over microscopic methods. Sample preparation and DNA extraction are of fundamental importance for obtaining an optimal DNA yield. Currently, there is no standard procedure for these steps, especially for gravimetric pollen samplers. Therefore, the aim of this study was to develop protocols for processing environmental samples for pollen DNA extraction and for metabarcoding analysis and to assess the efficacy of these protocols for the taxonomic assignment of airborne pollen collected by gravimetric (Tauber trap) and volumetric (Hirst-type trap) samplers. Protocols were tested across an increasing complexity of samples, from pure single-species pollen to environmental multi-species samples. A short fragment (about 150 base pairs) of the chloroplast trnL gene was amplified using universal primers for plants. After PCR amplification, amplicons were Sanger-sequenced and taxonomic assignment was accomplished by comparison with a custom-made reference database including chloroplast DNA sequences from most of the anemophilous taxa occurring in the study area (Trentino, northern Italy), representing 46 plant families. Using the classical morphological pollen analysis as a benchmark, we show that DNA metabarcoding is efficient and applicable even in complex samples, provided that protocols for sample preparation, DNA extraction, and metabarcoding analysis are carefully optimized.
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Acknowledgements
This project is supported by FIRS >T (FEM International Research School). We thank Maria Cristina Viola for microscopic analyses and for helping with the taxonomic identification of plants. We thank Heidi Christine Hauffe for comments that greatly improved the manuscript and other members of the Conservation Genetics group of FEM Research and Innovation Centre (Matteo Girardi, Barbara Crestanello, Andrea Gandolfi, Alice Fietta) for providing support in the molecular techniques. Finally, we thank members of the Zooplant Lab group of Milano Bicocca University (Maurizio Casiraghi, Antonella Bruno, and Anna Sandionigi) and other colleagues (Camilla Capelli, Athanasios Charalampopoulos, and Duccio Rocchini) for their useful suggestions.
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Leontidou, K., Vernesi, C., De Groeve, J. et al. DNA metabarcoding of airborne pollen: new protocols for improved taxonomic identification of environmental samples. Aerobiologia 34, 63–74 (2018). https://doi.org/10.1007/s10453-017-9497-z
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DOI: https://doi.org/10.1007/s10453-017-9497-z