Non-lethal genotyping of Tribolium castaneum adults using genomic DNA extracted from wing tissue

The red flour beetle Tribolium castaneum has become the second most important insect model organism and is frequently used in developmental biology, genetics and pest-associated research. Consequently, the methodological
The red flour beetle Tribolium castaneum has become the second most important insect model organism and is frequently used in developmental biology, genetics and pest-associated research. Consequently, the methodological arsenal increases continuously, but many routinely applied techniques for Drosophila melanogaster and other insect species are still unavailable. For example, a protocol for non-lethal genotyping has not yet been adapted but is particularly useful when individuals with known genotypes are required for downstream experiments. In this study, we present a workflow for non-lethal genotyping of T. castaneum adults based on extracting genomic DNA from wing tissue. In detail, we describe a convenient procedure for wing dissection and a custom method for wing digestion that allows PCR-based genotyping of up to fifty adults in less than an afternoon with a success rate of about 86%. The amount of template is sufficient for up to ten reactions while viability and fertility of the beetles are preserved. We prove the applicability of our protocol by genotyping the white / scarlet gene pair alleles from the black-eyed San Bernadino wild-type and white-eyed Pearl recessive mutant strains spanning four generations. Non-lethal genotyping has the potential to improve and accelerate many workflows: Firstly, during the establishment process of homozygous cultures or during stock keeping of cultures that carry recessively lethal alleles, laborious test crossing is replaced by non-lethal genotyping. Secondly, in genome engineering assays, non-lethal genotyping allows the identification of appropriate founders before they are crossed against wild-types, narrowing the efforts down to only the relevant individuals. Thirdly, non-lethal genotyping simplifies experimental strategies, in which genotype and behavior should be correlated, since the genetic configuration of potential individuals can be determined before the actual behavior assays is performed.
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Metadaten
Author:Frederic Strobl, J. Alexander Ross, Ernst H. K. Stelzer
URN:urn:nbn:de:hebis:30:3-439745
DOI:http://dx.doi.org/10.1371/journal.pone.0182564
ISSN:1932-6203
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=28800588
Parent Title (English):PLoS one
Publisher:PLoS
Place of publication:Lawrence, Kan.
Contributor(s):Nikos T. Papadopoulos
Document Type:Article
Language:English
Date of Publication (online):2017/08/14
Date of first Publication:2017/08/11
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2017/08/14
Volume:12
Issue:(8): e0182564
Pagenumber:15
First Page:1
Last Page:15
Note:
Copyright: © 2017 Strobl et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
HeBIS PPN:416310176
Institutes:Biowissenschaften
Exzellenzcluster Makromolekulare Komplexe
Dewey Decimal Classification:570 Biowissenschaften; Biologie
Sammlungen:Universitätspublikationen
Open-Access-Publikationsfonds:Biowissenschaften
Licence (German):License LogoCreative Commons - Namensnennung 4.0

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