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, 8 (1), 8542

New Universal ITS2 Primers for High-Resolution Herbivory Analyses Using DNA Metabarcoding in Both Tropical and Temperate Zones

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New Universal ITS2 Primers for High-Resolution Herbivory Analyses Using DNA Metabarcoding in Both Tropical and Temperate Zones

Rosemary J Moorhouse-Gann et al. Sci Rep.

Abstract

DNA metabarcoding is a rapidly growing technique for obtaining detailed dietary information. Current metabarcoding methods for herbivory, using a single locus, can lack taxonomic resolution for some applications. We present novel primers for the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) designed for dietary studies in Mauritius and the UK, which have the potential to give unrivalled taxonomic coverage and resolution from a short-amplicon barcode. In silico testing used three databases of plant ITS2 sequences from UK and Mauritian floras (native and introduced) totalling 6561 sequences from 1790 species across 174 families. Our primers were well-matched in silico to 88% of species, providing taxonomic resolution of 86.1%, 99.4% and 99.9% at the species, genus and family levels, respectively. In vitro, the primers amplified 99% of Mauritian (n = 169) and 100% of UK (n = 33) species, and co-amplified multiple plant species from degraded faecal DNA from reptiles and birds in two case studies. For the ITS2 region, we advocate taxonomic assignment based on best sequence match instead of a clustering approach. With short amplicons of 187-387 bp, these primers are suitable for metabarcoding plant DNA from faecal samples, across a broad geographic range, whilst delivering unparalleled taxonomic resolution.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Comparison of amplicon length distribution from available species and NGS datasets for (a) UK dove and pigeon diet, (b) Telfair’s skink diet pool 1 and (c) Telfair’s skink diet pool 2.
Figure 2
Figure 2
Order-level summary of clustering thresholds for the ITS2 region only between 95 and 100% for (a) Mauritius, n = 165 species and (b) UK databases, n = 1116 species. Order names are listed on the y-axis and clustering threshold forms the x-axis. The colour of the cells represents the percentage of species within an order that can be identified to species level at a given clustering threshold; numbers within cells show the number of species that can be resolved at each threshold. Colour gradient from green through to red signifies high species-level resolution moving towards poor species-level resolution.
Figure 3
Figure 3
Schematic diagram of priming sites within the second internal transcribed spacer (ITS2) and flanking regions (5.8S and 26S). The location of S2F and S3R priming sites are shown alongside UniPlantF and UniplantR from this study. The distances of the priming sites from the ITS2 region are shown (bp). Distances are based on a representative Asparagus setaceus sequence (NCBI Accession number KY700230). S2F and UniPlantF overlap by 7 bp. UniPlantR begins on the last 1 bp of ITS2 and continues into 26S. The amplicon size range, across all sequences assessed in this study, of the UniPlant primers is shown. Schematic not to scale.

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