Submitted to: Biomed Central (BMC) Genomics
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/5/2009
Publication Date: 11/5/2009
Publication URL: www.biomedcentral.com/1741-7007/7/73
Citation: Trauner, J., Schinko, J.B., Lorenzen, M.D., Shippy, T.D., Wimmer, E.A., Beeman, R.W., Klingler, M., Bucher, G., Brown, S.J. 2009. Large-Scale Insertional Mutagenesis of the Coleopteran Stored Grain Pest, the Red Flour Beetle Tribolium castaneum, Identifies Embryonic Lethal Mutations and Enhancer Traps. Biomed Central (BMC) Genomics. 7:73. Interpretive Summary: No methods exist for high-throughput discovery of essential genes in pest insects. The red flour beetle, Tribolium castaneum, was the first agronomic pest insect to have its genome completely sequenced. This resource, in combination with the sophisticated genetic toolkit available for Tribolium, makes it the best laboratory model for gene discovery in pest insect species. In order to further improve the usefulness of Tribolium for new gene discovery and functional analysis we developed highly efficient methods for randomly labelling genes with fluorescent tags. These tags make it possible to determine whether the tagged gene is necessary for insect survival, and also provide clues about gene function. We used the method to identify almost 500 new genes that are required for insect health and survival, as well as more than 500 additional genes whose expression patterns could be identified, giving clues about their likely functions. The method should be applicable to other pest insect species.
Technical Abstract: Given its sequenced genome and efficient systemic RNA interference response, Tribolium castaneum is a model organism well suited for the application of “reverse genetics” approaches to elucidate the function of candidate genes. However, there is still a continuing need for “forward genetic” analysis to avoid the bias inherent in candidate gene approaches. In order to produce easy-to-maintain insertional mutations and to obtain fluorescent marker lines to facilitate phenotypic analysis, we undertook a large-scale transposon mutagenesis screen. The screen used a highly efficient, in vivo system to yield more than 6,500 new piggyBac insertions. These included 421 recessive lethal, 75 semi-lethal and 18 recessive sterile mutations, while 505 displayed novel enhancer-trap patterns. Insertion junctions were determined for 403 lines, and often proved to be located within transcription units of genes. Insertion sites appeared to be randomly distributed throughout the genome, with the exception of a noticeable preference for reinsertion near the donor site. This collection of enhancer-trap and embryonic lethal lines has been made available to the research community and will facilitate investigations into diverse fields of insect biology, pest control, and evolution. Because the genetic elements used in this screen are species-nonspecific, and because the crossing scheme does not depend on balancer chromosomes, the methods presented herein should be broadly applicable for many insect species.