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

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

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

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

Aerobiological Monitoring and Metabarcoding of Grass Pollen

Grass pollen is one of the leading causes of pollinosis, affecting 10–30% of the world’s population. The allergenicity of pollen from different Poaceae species is not the same and is estimated from moderate to high. Aerobiological monitoring is a standard method that allows one to track and predict the dynamics of allergen concentration in the air. Poaceae is a stenopalynous family, and thus grass pollen can usually be identified only at the family level with optical microscopy. Molecular methods, in particular the DNA barcoding technique, can be used to conduct a more accurate analysis of aerobiological samples containing the DNA of various plant species. This study aimed to test the possibility of using the ITS1 and ITS2 nuclear loci for determining the presence of grass pollen from air samples via metabarcoding and to compare the analysis results with the results of phenological observations. Based on the high-throughput sequencing data, we analyzed the changes in the composition of aerobiological samples taken in the Moscow and Ryazan regions for three years during the period of active flowering of grasses. Ten genera of the Poaceae family were detected in airborne pollen samples. The representation for most of them for ITS1 and ITS2 barcodes was similar. At the same time, in some samples, the presence of specific genera was characterized by only one sequence: either ITS1 or ITS2. Based on the analysis of the abundance of both barcode reads in the samples, the following order could describe the change with time in the dominant species in the air: Poa, Alopecurus, and Arrhenatherum in early mid-June, Lolium, Bromus, Dactylis, and Briza in mid-late June, Phleum, Elymus in late June to early July, and Calamagrostis in early mid-July. In most samples, the number of taxa found via metabarcoding analysis was higher compared to that in the phenological observations. The semi-quantitative analysis of high-throughput sequencing data well reflects the abundance of only major grass species at the flowering stage