Progress 08/25/06 to 06/27/08
Outputs
Progress Report Objectives (from AD-416) The objective of this cooperative research project is to create a BAC (Bacterial Artificial Chromosome) library for Western corn rootworm (WCR) (Diabrotica virgifera virgifera), and begin characterizing it in preparation for proposing a WCR genome sequencing project. Approach (from AD-416) We will create F1 sibling crosses of WCR from a laboratory colony. It is hoped that enough offspring can be obtained from a single pair to obtain adequate quantities of genomic DNA to construct a BAC library. However, such crosses are often less fertile due to inbreeding depression than crosses of unrelated individuals. Alternatively, mass mating of siblings may be necessary. The DNA from the offspring will be used to create a BAC library, a highly technical job that will be contracted out to a commercial enterprise specializing in such work. Given the large genome size of WCR, it is anticipated that genome coverage of 4X or 5X may be achievable. Characterization of the library will be initiated, and will ultimately include more precise estimates of the genome size, BAC-end sequencing, and restriction-mapping of fragments, all in preparation for proposing a WCR genome sequencing project. Significant Activities that Support Special Target Populations Progress is monitored through discussions at various technical meetings and conferences attended by both parties, and by frequent email correspondence and telephone calls initiated by both parties. Controlled family crosses of western corn rootworm (WCR) were successfully made, and genomic DNA was isolated from the parents and offspring. The BAC (Bacterial Artificial Chromosome) library was successfully constructed by Amplicon Express by partially digesting WCR genomic DNA with HindIII and cloning the resulting restriction fragments into pCC1BAC. This was a particularly difficult library to construct, requiring many optimization steps. The library contains 110,592 clones of an average length of 105 kb and thus represents an approximate 4.6-fold coverage of the 2.5-Gbp WCR genome. Some preliminary characterization of the library has been performed by sequencing the ends of 1,122 clones to produce 2,142 sequence reads. A BLASTN search was used to query these sequences against the GenBank non-redundant database. Unsurprisingly, given that these sequences are essentially random genomic sequences, 1,875 (87.5%) did not return a significant match. Of the sequences that did return a significant match, 87 (4.1%) corresponded to 11 different transposon-like elements. One hundred and eighty sequences (8.4%) gave matches to a total of 113 different gene sequences. This BAC library will likely prove to be a powerful resource for gene discovery in the future. A copy is held in secure long-term storage by Amplicon Express, and researchers have the option of purchasing additional copies for their own research projects. DNA preparations representing the entire library, organized into pools and "superpools," can be purchased to efficiently screen the library by Polymerase Chain Reaction (PCR) for sequences of interest. This research addresses National Program 304 Action Plan Component III, B, by characterizing regional differences in genetics of pest arthropods. It addresses Component V, D, by improving monitoring for insect resistance management.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) The objective of this cooperative research project is to create a BAC (Bacterial Artificial Chromosome) library for Western corn rootworm (WCR) (Diabrotica virgifera virgifera), and begin characterizing it in preparation for proposing a WCR genome sequencing project. Approach (from AD-416) We will create F1 sibling crosses of WCR from a laboratory colony. It is hoped that enough offspring can be obtained from a single pair to obtain adequate quantities of genomic DNA to construct a BAC library. However, such crosses are often less fertile due to inbreeding depression than crosses of unrelated individuals. Alternatively, mass mating of siblings may be necessary. The DNA from the offspring will be used to create a BAC library, a highly technical job that will be contracted out to a commercial enterprise specializing in such work. Given the large genome size of WCR, it is anticipated that genome coverage of 4X or 5X may be achievable. Characterization of the library will be initiated, and will ultimately include more precise estimates of the genome size, BAC-end sequencing, and restriction-mapping of fragments, all in preparation for proposing a WCR genome sequencing project. Significant Activities that Support Special Target Populations This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the University of Nebraska. Additional details of research can be found in the report for the parent project #3625-22000-016-00D, Ecologically-Based Management of Insect Pests of Corn. Progress is monitored through discussions at various technical meetings and conferences attended by both parties, and by frequent email correspondence and telephone calls initiated by both parties. Controlled family crosses of western corn rootworm (Diabrotica virgifera virgifera) were successfully made, and genomic DNA was isolated from the parents and offspring. The BAC (Bacterial Artificial Chromosome) library was successfully constructed by a commercial company. The average insert size per clone in the library is 105 kb. 110,592 clones were included in the library for an estimated genome coverage of about 4. 6X. BAC end sequencing has been initiated for a portion of the clones in the library.
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