Assessment of ITS1, ITS2, 5′-ETS, and trnL-F DNA Barcodes for Metabarcoding of Poaceae Pollen

Denis O Omelchenko,Elena E Severova,Olga V Chesnokova,Artem S Kasianov,Anna S Speranskaya,Svetlana V Polevova,Anastasia A Krinitsina

doi:10.3390/d14030191

Abstract

Grass pollen is one of the major causes of allergy. Aerobiological monitoring is a necessary element of the complex of anti-allergic measures, but the similar pollen morphology of Poaceae species makes it challenging to discriminate species in airborne pollen mixes, which impairs the quality of aerobiological monitoring. One of the solutions to this problem is the metabarcoding approach employing DNA barcodes for taxonomical identification of species in a mix by high-throughput sequencing of the pollen DNA. A diverse set of 14 grass species of different genera were selected to create a local reference database of nuclear ITS1, ITS2, 5′-ETS, and plastome trnL-F DNA barcodes. Sequences for the database were Sanger sequenced from live field and herbarium specimens and collected from GenBank. New Poaceae-specific primers for 5′-ETS were designed and tested to obtain a 5′-ETS region less than 600 bp long, suitable for high-throughput sequencing. The DNA extraction method for single-species pollen samples and mixes was optimized to increase the yield for amplification and sequencing of pollen DNA. Barcode sequences were analyzed and compared by the barcoding gap and intra- and interspecific distances. Their capability to correctly identify grass pollen was tested on artificial pollen mixes of various complexity. Metabarcoding analysis of the artificial pollen mixes showed that nuclear DNA barcodes ITS1, ITS2, and 5′-ETS proved to be more efficient than the plastome barcode in both amplification from pollen DNA and identification of grass species. Although the metabarcoding results were qualitatively congruent with the actual composition of the pollen mixes in most cases, the quantitative results based on read-counts did not match the actual ratio of pollen grains in the mixes.

Highlights

One of the major disadvantages of pollen light microscopy analysis is that similar pollen morphology of Poaceae species makes it challenging to discriminate species in airborne pollen mixes, which impairs the quality of aerobiological monitoring [6,7]
Designed primers for External transcribed spacer (ETS) amplification for some of the Poaceae genera and species could amplify a fragment of ~500–900 bp [30,45,46], but we aimed to obtain a shorter amplicon for a broad spectrum of Poaceae species of diverse genera that could be sequenced entirely using high-throughput sequencing (HTS) and is suitable for metabarcoding analysis
ITS1 and ITS2 proved to be the most effective qualitatively and quantitatively, and we recommend using them for Poaceae pollen analysis

Summary

Introduction

One of the major disadvantages of pollen light microscopy analysis is that similar pollen morphology of Poaceae species makes it challenging to discriminate species in airborne pollen mixes, which impairs the quality of aerobiological monitoring [6,7]. DNA metabarcoding is an alternative approach that has been actively developing recently, allowing qualitative (to the level of species or genus for some taxa) and quantitative (to some extent) composition analysis of complex biological mixes. It employs high-throughput sequencing (HTS) and comparative analysis of specific DNA sequences called “DNA barcodes” to discriminate species present in the mix. DNA barcoding has been widely used in various areas of botanical research; for example, the phylogeny of wild cherry [8], archaeobotany of grapevine [9], authentication and identification of medicinal [10] and poisonous [11] plants, and plant species composition of honey [12,13]

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