2009 Annual Report
1a.Objectives (from AD-416)
The fundamental goal of this project is to provide researchers who have interests in the genetics and genomics of western corn rootworm (WCR) with a set of resources that will accelerate ongoing and future research programs. During the past half-decade, substantial progress has been made in the study of this species, particularly in the development of Expressed Sequence Tag (EST) data and polymorphic molecular markers. However, these resources have not yet been integrated to the extent that is possible and desirable. This proposal seeks to bring together and expand existing resources via the following objectives:
1) Validate a large number of putative Single Nucleotide Polymorphisms (SNPs) already identified in existing EST data.
2) Construct a comprehensive linkage map of the western corn rootworm genome using validated SNPs and existing microsatellite markers.
3) Associate SNP loci on the linkage map with bacterial artificial chromosome (BAC) clones.
4) Fingerprint SNP-associated BAC clones by restriction mapping and assemble them into contigs.
5) Sequence representative BAC clones from the assembled contigs to characterize the genomic regions surrounding SNP sites.
6) Disseminate the data to the wider research community via a user-friendly website that can accommodate new data in the future.
1b.Approach (from AD-416)
We will take a mass-screening approach for the validation of EST-derived SNPs. This will be done using the Illumina Golden Gate assay platform. Up to 3,072 candidate SNPs with a polyphred score greater than or equal to 95 for which an assay can be designed will be tested for polymorphism against samples of WCR populations from Illinois and Iowa. Markers that are polymorphic in these populations will then be used to genotype the backcross pedigrees to verify that they are Mendelian single-locus markers. DNA extractions from the backcrosses will be prepared at the start of the project so that they are available as soon as they are required. Genotyping of the backcrosses with microsatellites will be done simultaneously with the SNP genotyping. Once genotyping of the backcross pedigrees is completed, linkage maps will be constructed for each pedigree and then merged to form a combined map. The combined map will then be used to select a set of EST sequences associated with validated, mapped SNPs that will be used to design PCR primers to screen the BAC library. BAC clones that are identified as harboring SNP loci will be mapped by restriction digestion and assembled into contigs. Representative BAC clones from each contig will then be sequenced using high throughput 454 pyrosequencing. The BAC clones to be sequenced will be selected based on their location within the SNP-associated contig to maximize the amount of sequence data obtained on both sides of the SNP marker. The genetic maps and associated molecular and other data that will be generated by this project are generally similar in both content and scope to those in other genetic/genomic databases, and in particular to SoyBase. This will allow us to develop prototype databases and web displays in advance of the actual data generation. During the initial development, we will identify a small group of external testers who will be asked to provide input on functionality, displays, etc. This strategy of early development will allow us to release "WCRbase" to the research community as soon as a useful body of data is available, and then extend it as additional data are generated.
Progress of University of Illinois and University of Nebraska collaborators is monitored through frequent email correspondence and telephone calls initiated by both parties. We have begun the single nucleotide polymorphism (SNP) verification portion of the project, which will result in a large number of SNP markers for developing a linkage map. Candidate SNPs were identified from an expressed sequence tag (EST) library from the western corn rootworm (WCR), Diabrotica virgifera virgifera, head and midgut developed previously, and submitted to Illumina's oligonucleotide designability screening service. 4,360 candidate SNPs passed the designability assay. Further inspection by eye has resulted in an updated set of 2,222 candidate SNPs from which the oligonucleotide panels will be ordered. Mapping families of WCR were obtained from our collaborator at the University of Nebraska, and DNA has been extracted from the parents and offspring of backcrosses for these pedigrees. A WCR bacterial artificial chromosome (BAC) library was previously constructed, which 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. DNA preparations representing the entire library, organized into pools and "superpools," was purchased, which will allow efficient screening of the library by polymerase chain reaction (PCR) for sequences of interest. Each superpool contains DNA from 4,608 clones corresponding to twelve 384-well plates of library clones, giving a total of 24 superpools for the entire library. For each superpool, there is an associated pool plate containing 12 pools of DNA from each 384-well plate, 16 pools from each row of all the plates, 24 pools from each column of all the plates, and 24 diagonal pools. These are ready for screening with SNP loci once they are validated.