A simple PCR-based approach for rapid detection of Ips typographus and Ips duplicatus in the presence of (associated) sy
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ORIGINAL ARTICLE
A simple PCR‑based approach for rapid detection of Ips typographus and Ips duplicatus in the presence of (associated) symbionts and parasites Matthias Becker1 · Stephan König1 · Björn Hoppe1 Received: 8 September 2020 / Accepted: 22 September 2020 © The Author(s) 2020
Abstract In plant pest diagnosis, Sanger sequencing of marker genes (DNA-barcoding) is the most applied and appropriate method for the identification of insects. Standard PM7/129 of the European and Mediterranean Plant Protection Organization (EPPO) includes a number of primers and PCR protocols for diagnosing insect pests. LCO1490 and HCO2198 primers recommended herein were shown to be excellent tools for amplifying a fragment of the COI gene from a vast range of arthropods. The COI barcoding region is available for thousands of arthropod taxa in public databases and ready-to-use for evolutionary studies. However, we found that LCO1490 and HCO2198 primers are not working for bark beetles of genus Ips. The attempt to amplify this gene fragment from an individual organism using the barcoding primers led to DNA amplification of associated wasps and nematodes, which were apparently vectored by the beetle. Thus, new primers for Ips that bind specifically to another (non-barcoding) region of the COI gene were developed in the past years. These primers were successfully applied in phylogenetic analyses of this genus, resulting in the adverse effect that COI-based Ips phylogenies cannot be expanded to higher systematic categories without sequencing the outgroups (as they are not available in databases yet). Here we provide new primers for Ips that differ significantly from DNA sequences of Ips-associated wasps and nematodes and bind to a COI fragment that largely overlaps with the barcoding region proposed in the EPPO standard. Furthermore, using these primers we developed a quick PCR-based test for detecting Ips duplicatus, a quarantine pest currently emerging in many European countries. Keywords Ips duplicatus · DNA-barcoding · Primer design · Molecular diagnosis · Insect pests
Introduction The suitability of the mitochondrial gene encoding subunit I of Cytochrome oxidase (COI, CO1 or COX1) as a molecular marker for evolutionary studies of a wide range of organisms (particularly animals) was already shown more than Electronic supplementary material The online version of this article (https://doi.org/10.1007/s41348-020-00388-w) contains supplementary material, which is available to authorized users. * Matthias Becker matthias.becker@julius‑kuehn.de * Björn Hoppe bjoern.hoppe@julius‑kuehn.de 1
Julius Kühn-Institut, Institute for National and International Plant Health, Messeweg 11/12, 38104 Braunschweig, Germany
two decades ago (Simon et al. 1994, Lunt et al. 1996). Since then many PCR primers have been designed for more or less conserved regions of the gene in order to enable identification and phylogenetic analyses of diverse species across eukaryotes (often called “DNA barcoding”). The European and Mediterranean Plant Protec
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