DNA metabarcoding of airborne pollen: new protocols for improved taxonomic identification of environmental samples

Abstract

Metabarcoding is a promising DNA-based method for identifying airborne pollen from environmental samples with advantages over microscopic methods. This method requires several preparatory steps of the samples, with the extraction protocol being of fundamental importance to obtain an optimal DNA yield. Currently, there is no consensus in sample preparation and DNA extraction, especially for gravimetric pollen samplers. Therefore, the aim of this study was to develop protocols to process environmental samples for pollen DNA extraction and further metabarcoding analysis, and to assess the efficacy of these protocols for the taxonomic assignment of airborne pollen, collected by gravimetric (Tauber trap) and volumetric samplers (Burkard spore trap). Protocols were tested across an increasing complexity of samples, from single-species pure pollen to environmental samples. A short fragment (about 150 base pair) of chloroplast DNA was amplified by universal primers for plants (trnL). After PCR amplification, amplicons were Sanger-sequenced and taxonomic assignment was accomplished by comparison to a custom-made reference database including chloroplast DNA sequences of 46 plant families, including most of the anemophilous taxa occurring in the study area (Trentino, Italy, Eastern Italian Alps). Using as a benchmark the classical morphological pollen analysis, it emerged that DNA metabarcoding is applicable efficiently across a complexity of samples, provided that sample preparation, DNA extraction and amplification protocols are specifically optimized.

Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring

“…This is important because commercial column-based extraction kits are currently the most commonly used methods in metabarcoding for freshwater bioassessment studies (Andújar et al, 2018;Carew et al, 2013;Deiner et al, 2015;Gibson et al, 2014;Hermans et al, 2018;Linard et al, 2016), thereby requiring further assessment on the potential of magnetic bead technology (e.g. Leontidou et al, 2018;Krehenwinkel et al, 2018), or even other approaches such as the salting-out protocol (Elbrecht & Steinke, 2019;Elbrecht, Vamos, et al, 2017). Fourth, we suggest that the results of preservative ethanol metabarcoding are significantly improved when subsampling 7-14 days after field collection rather than earlier on, though this is less important when using the more efficient BEAD protocol.…”

Advancing DNA Barcoding and Metabarcoding Applications for Plants Requires Systematic Analysis of Herbarium Collections—An Australian Perspective

“…Despite this appetite for genetic plant identification, systematic development of barcoding reference databases that comprise whole (or majority) flora of particular regions are sparse (but see de Vere et al, 2012;Saarela et al, 2013;Kuzmina et al, 2017). Instead, most studies develop partial reference databases and/or utilize genetic repositories such as GenBank to augment their resources which then requires post hoc data quality control (Leontidou et al, 2018;e.g., Kumar et al, 2018). The responsibility for accurate initial specimen identification and sequence data quality lies solely with the data generator for standard submissions to GenBank and there is often very little information upon which veracity can be assessed (e.g., voucher specimen meta-data, raw sequencing data files).…”

Plant assemblages in atmospheric deposition

“…Precipitation (rain and snow) is analyzed for wet deposition of plant-associated materials such as phosphorus (Doskey and Ugoagwu, 1989) and organic carbon compounds from the atmosphere (Noll and Khalili, 1990;Mullaugh et al, 2014). However, pollen identification in the majority of aero-palynological studies is based on micromorphological characteristics (Levetin, 2004), which are subjective and limited in their resolving power. Recent studies have used DNA-based methods for accurate characterization of pollen diversity and assemblages in the atmosphere (Leontidou et al, 2018;Núñez et al, 2017).…”