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United States Department of Agriculture

Agricultural Research Service

Research Project: The Targeted Sequencing of Bacterial Artificial Chromosome (Bac) Clones from Western Corn Rootworm

Location: Corn Insects and Crop Genetics Research

Project Number: 3625-22000-017-28
Project Type: Trust

Start Date: Feb 15, 2013
End Date: Dec 31, 2014

Objective:
To identify genome regions of the Western corn rootworm that contain candidate genes for resistance to Bacillus thuringiensis toxins, and generate genomic DNA sequence of these regions using individual and pooled bacterial artificial chromosome sequencing approaches.

Approach:
A bacterial artificial chromosome (BAC) library, DvvBAC1, was previously created by ARS scientists in Ames, Iowa, using Western corn rootworm non-diapausing strain of genomic DNA digested with the restriction endonuclease HindIII and size selected for genome fragments of ~105 kb. Polymerase chain reaction (PCR) assays will be designed, optimized and used to amplify candidate genes that have been implicated in resistance to Bacillus thuringiensis (Bt) toxins within other arthropods (i.e. candidate gene approach). These DNA fragments that represent amplified regions of candidate genes will be used to screen the BAC library, DvvBAC1, by PCR in order to identify regions of the Western corn rootworm genome that encode these genes of interest. The individual DvvBAC1 clones that contain these candidate gene coding regions will be sequenced on the Roche 454 GS-FLX pyrosequencer, and resulting DNA fragments will be re-assembled and genes idenitifed by standard bioinformatic methods. Additionally, pools of BAC clones from the same library will be sequenced in order to generate larger genome scaffolds and identify additional genomic sequence in proximity to these candidate genes. These methods will likely result in the sequencing of entire genes or partial gene fragments that respresent candidates for future testing for involvement (linkage) with Bt toxin resistance traits in WCR, and will develop tools that may lead to an understanding of Bt toxin resistance and recommendations that could delay the onset of field resistance.

Last Modified: 8/30/2014
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