Cargando…

Taxonomic Characterization of Honey Bee (Apis mellifera) Pollen Foraging Based on Non-Overlapping Paired-End Sequencing of Nuclear Ribosomal Loci

Identifying plant taxa that honey bees (Apis mellifera) forage upon is of great apicultural interest, but traditional methods are labor intensive and may lack resolution. Here we evaluate a high-throughput genetic barcoding approach to characterize trap-collected pollen from multiple North Dakota ap...

Descripción completa

Detalles Bibliográficos
Autores principales: Cornman, R. Scott, Otto, Clint R. V., Iwanowicz, Deborah, Pettis, Jeffery S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4689544/
https://www.ncbi.nlm.nih.gov/pubmed/26700168
http://dx.doi.org/10.1371/journal.pone.0145365
_version_ 1782406863119187968
author Cornman, R. Scott
Otto, Clint R. V.
Iwanowicz, Deborah
Pettis, Jeffery S.
author_facet Cornman, R. Scott
Otto, Clint R. V.
Iwanowicz, Deborah
Pettis, Jeffery S.
author_sort Cornman, R. Scott
collection PubMed
description Identifying plant taxa that honey bees (Apis mellifera) forage upon is of great apicultural interest, but traditional methods are labor intensive and may lack resolution. Here we evaluate a high-throughput genetic barcoding approach to characterize trap-collected pollen from multiple North Dakota apiaries across multiple years. We used the Illumina MiSeq platform to generate sequence scaffolds from non-overlapping 300-bp paired-end sequencing reads of the ribosomal internal transcribed spacers (ITS). Full-length sequence scaffolds represented ~530 bp of ITS sequence after adapter trimming, drawn from the 5’ of ITS1 and the 3’ of ITS2, while skipping the uninformative 5.8S region. Operational taxonomic units (OTUs) were picked from scaffolds clustered at 97% identity, searched by BLAST against the nt database, and given taxonomic assignments using the paired-read lowest common ancestor approach. Taxonomic assignments and quantitative patterns were consistent with known plant distributions, phenology, and observational reports of pollen foraging, but revealed an unexpected contribution from non-crop graminoids and wetland plants. The mean number of plant species assignments per sample was 23.0 (+/- 5.5) and the mean species diversity (effective number of equally abundant species) was 3.3 (+/- 1.2). Bray-Curtis similarities showed good agreement among samples from the same apiary and sampling date. Rarefaction plots indicated that fewer than 50,000 reads are typically needed to characterize pollen samples of this complexity. Our results show that a pre-compiled, curated reference database is not essential for genus-level assignments, but species-level assignments are hindered by database gaps, reference length variation, and probable errors in the taxonomic assignment, requiring post-hoc evaluation. Although the effective per-sample yield achieved using custom MiSeq amplicon primers was less than the machine maximum, primarily due to lower “read2” quality, further protocol optimization and/or a modest reduction in multiplex scale should offset this difficulty. As small quantities of pollen are sufficient for amplification, our approach might be extendable to other questions or species for which large pollen samples are not available.
format Online
Article
Text
id pubmed-4689544
institution National Center for Biotechnology Information
language English
publishDate 2015
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-46895442015-12-31 Taxonomic Characterization of Honey Bee (Apis mellifera) Pollen Foraging Based on Non-Overlapping Paired-End Sequencing of Nuclear Ribosomal Loci Cornman, R. Scott Otto, Clint R. V. Iwanowicz, Deborah Pettis, Jeffery S. PLoS One Research Article Identifying plant taxa that honey bees (Apis mellifera) forage upon is of great apicultural interest, but traditional methods are labor intensive and may lack resolution. Here we evaluate a high-throughput genetic barcoding approach to characterize trap-collected pollen from multiple North Dakota apiaries across multiple years. We used the Illumina MiSeq platform to generate sequence scaffolds from non-overlapping 300-bp paired-end sequencing reads of the ribosomal internal transcribed spacers (ITS). Full-length sequence scaffolds represented ~530 bp of ITS sequence after adapter trimming, drawn from the 5’ of ITS1 and the 3’ of ITS2, while skipping the uninformative 5.8S region. Operational taxonomic units (OTUs) were picked from scaffolds clustered at 97% identity, searched by BLAST against the nt database, and given taxonomic assignments using the paired-read lowest common ancestor approach. Taxonomic assignments and quantitative patterns were consistent with known plant distributions, phenology, and observational reports of pollen foraging, but revealed an unexpected contribution from non-crop graminoids and wetland plants. The mean number of plant species assignments per sample was 23.0 (+/- 5.5) and the mean species diversity (effective number of equally abundant species) was 3.3 (+/- 1.2). Bray-Curtis similarities showed good agreement among samples from the same apiary and sampling date. Rarefaction plots indicated that fewer than 50,000 reads are typically needed to characterize pollen samples of this complexity. Our results show that a pre-compiled, curated reference database is not essential for genus-level assignments, but species-level assignments are hindered by database gaps, reference length variation, and probable errors in the taxonomic assignment, requiring post-hoc evaluation. Although the effective per-sample yield achieved using custom MiSeq amplicon primers was less than the machine maximum, primarily due to lower “read2” quality, further protocol optimization and/or a modest reduction in multiplex scale should offset this difficulty. As small quantities of pollen are sufficient for amplification, our approach might be extendable to other questions or species for which large pollen samples are not available. Public Library of Science 2015-12-23 /pmc/articles/PMC4689544/ /pubmed/26700168 http://dx.doi.org/10.1371/journal.pone.0145365 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
spellingShingle Research Article
Cornman, R. Scott
Otto, Clint R. V.
Iwanowicz, Deborah
Pettis, Jeffery S.
Taxonomic Characterization of Honey Bee (Apis mellifera) Pollen Foraging Based on Non-Overlapping Paired-End Sequencing of Nuclear Ribosomal Loci
title Taxonomic Characterization of Honey Bee (Apis mellifera) Pollen Foraging Based on Non-Overlapping Paired-End Sequencing of Nuclear Ribosomal Loci
title_full Taxonomic Characterization of Honey Bee (Apis mellifera) Pollen Foraging Based on Non-Overlapping Paired-End Sequencing of Nuclear Ribosomal Loci
title_fullStr Taxonomic Characterization of Honey Bee (Apis mellifera) Pollen Foraging Based on Non-Overlapping Paired-End Sequencing of Nuclear Ribosomal Loci
title_full_unstemmed Taxonomic Characterization of Honey Bee (Apis mellifera) Pollen Foraging Based on Non-Overlapping Paired-End Sequencing of Nuclear Ribosomal Loci
title_short Taxonomic Characterization of Honey Bee (Apis mellifera) Pollen Foraging Based on Non-Overlapping Paired-End Sequencing of Nuclear Ribosomal Loci
title_sort taxonomic characterization of honey bee (apis mellifera) pollen foraging based on non-overlapping paired-end sequencing of nuclear ribosomal loci
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4689544/
https://www.ncbi.nlm.nih.gov/pubmed/26700168
http://dx.doi.org/10.1371/journal.pone.0145365
work_keys_str_mv AT cornmanrscott taxonomiccharacterizationofhoneybeeapismelliferapollenforagingbasedonnonoverlappingpairedendsequencingofnuclearribosomalloci
AT ottoclintrv taxonomiccharacterizationofhoneybeeapismelliferapollenforagingbasedonnonoverlappingpairedendsequencingofnuclearribosomalloci
AT iwanowiczdeborah taxonomiccharacterizationofhoneybeeapismelliferapollenforagingbasedonnonoverlappingpairedendsequencingofnuclearribosomalloci
AT pettisjefferys taxonomiccharacterizationofhoneybeeapismelliferapollenforagingbasedonnonoverlappingpairedendsequencingofnuclearribosomalloci