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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: 5030-22000-018-02
Project Type: Trust

Start Date: Feb 15, 2013
End Date: Dec 31, 2016

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, DvvBACII, will be generated by ARS in Ames, Iowa, using randomly sheared high molecular weight genomic DNA from a Western corn rootworm non-diapausing strain. 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, DvvBACII, by PCR in order to identify regions of the Western corn rootworm genome that encode these genes of interest. The individual DvvBACII clones that contain these candidate gene coding regions will be sequenced, and resulting DNA fragments will be re-assembled and genes identified by standard bioinformatics methods.. These methods will likely result in the sequencing of entire genes or partial gene fragments that represent 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. Additionally, THE COOPERATOR will take a subset of the three-dimensional pools of BAC clones from the same library and generate Illumina sequence from them in order to generate short sequence tags that can be assigned uniquely to individual BAC clones using bioinformatic approaches and assess the utility of pool sequencing as an approach for improving the CRW genome assembly.

Last Modified: 7/28/2016
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