Oligonucleotide Primers for Targeted Amplification of Single-Copy Nuclear Genes in Apocritan Hymenoptera

Gerrit Hartig,Janus Borner,Bernhard Misof,Claudia Etzbauer,Ralph S Peters,Oliver Niehuis

doi:10.1371/journal.pone.0039826

Gerrit Hartig, Janus Borner + Show 4 more

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https://doi.org/10.1371/journal.pone.0039826

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Abstract

BackgroundPublished nucleotide sequence data from the mega-diverse insect order Hymenoptera (sawflies, bees, wasps, and ants) are taxonomically scattered and still inadequate for reconstructing a well-supported phylogenetic tree for the order. The analysis of comprehensive multiple gene data sets obtained via targeted PCR could provide a cost-effective solution to this problem. However, oligonucleotide primers for PCR amplification of nuclear genes across a wide range of hymenopteran species are still scarce.FindingsHere we present a suite of degenerate oligonucleotide primer pairs for PCR amplification of 154 single-copy nuclear protein-coding genes from Hymenoptera. These primers were inferred from genome sequence data from nine Hymenoptera (seven species of ants, the honeybee, and the parasitoid wasp Nasonia vitripennis). We empirically tested a randomly chosen subset of these primer pairs for amplifying target genes from six Hymenoptera, representing the families Chrysididae, Crabronidae, Gasteruptiidae, Leucospidae, Pompilidae, and Stephanidae. Based on our results, we estimate that these primers are suitable for studying a large number of nuclear genes across a wide range of apocritan Hymenoptera (i.e., all hymenopterans with a wasp-waist) and of aculeate Hymenoptera in particular (i.e., apocritan wasps with stingers).ConclusionsThe amplified nucleotide sequences are (a) with high probability from single-copy genes, (b) easily generated at low financial costs, especially when compared to phylogenomic approaches, (c) easily sequenced by means of an additionally provided set of sequencing primers, and (d) suitable to address a wide range of phylogenetic questions and to aid rapid species identification via barcoding, as many amplicons contain both exonic and fast-evolving intronic nucleotides.

Highlights

Targeted amplification of single-copy genes is still a cornerstone of molecular phylogenetics despite the emergence of phylogenomic approaches analyzing transcriptome data and entire genomes
The amplified nucleotide sequences are (a) with high probability from single-copy genes, (b) generated at low financial costs, especially when compared to phylogenomic approaches, (c) sequenced by means of an provided set of sequencing primers, and (d) suitable to address a wide range of phylogenetic questions and to aid rapid species identification via barcoding, as many amplicons contain both exonic and fast-evolving intronic nucleotides
Phylogenomic approaches provide a plethora of nucleotide sequence data and facilitate addressing difficult phylogenetic questions

Summary

Introduction

Targeted amplification of single-copy genes is still a cornerstone of molecular phylogenetics despite the emergence of phylogenomic approaches analyzing transcriptome data and entire genomes. Degenerate oligonucleotide PCR primers designed to amplify a large set of single-copy nuclear genes in species of interest could close the gap between the two approaches and could be a viable alternative to both of them. We present such a suite of PCR primers for amplifying single-copy nuclear genes from Hymenoptera (sawflies, bees, wasps, and ants). Oligonucleotide primers for PCR amplification of nuclear genes across a wide range of hymenopteran species are still scarce

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