Start Date: May 15, 2009
End Date: Jan 31, 2013
A subset of 50 validated Single Nucleotide Polymorphism (SNP) loci derived from Expressed Sequence Tag (EST) sequence data will be used to screen the available western corn rootworm (WCR) Bacterial Artificial Chromosome (BAC) library prepared previously by the Cooperator to determine which clones carry each locus. Screening will be done by PCR at CICGRU. 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" that contains 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." Thus, a PCR amplicon can be associated with an individual BAC using 100 PCR assays (24 to identify the superpool plus 76 to determine the plate, row, and column within the superpool). A genetic linkage map will be constructed with approximately 600 SNP markers developed by CICGRU, using backcross pedigrees of WCR that were collected by the Cooperator and phenotyped for organophosphate resistance. The set of SNP loci to be used in screening will be selected to give an even distribution across the linkage map, but may also target loci for which EST annotations or other information indicates a gene relevant to adaptations to pest management or potential biotechnology applications. PCR primers will be designed from the EST sequences from which the SNP was derived. Optimal PCR conditions for each primer pair will be determined using WCR genomic DNA as a template. In cases where optimal conditions cannot be determined, new primer pairs will be designed and tested. For each SNP locus an initial round of 24 PCR assays followed by agarose gel electrophoresis of the reaction products will determine the superpools that contain the amplicon. Second-round PCR assays and agarose gel electrophoresis on the relevant pool plates will identify the BAC clones containing the amplicon. Because the BAC library is estimated to give 4.6-fold coverage of the WCR genome, we expect, on average, to perform five second-round assays per SNP locus. BAC clones that have been associated with a SNP locus will be digested with HindIII, BamHI, or EcoRI restriction enzymes and separated by agarose gel electrophoresis. The resulting restriction-site fingerprints will be used to arrange the BAC clones associated with each SNP locus into a contiguous region. DNA will be extracted from 1 or 2 BAC clones from each contig associated with a SNP locus for future high-throughput 454 (Roche) pyrosequencing. The clones to be sequenced will be selected to maximize the length of sequence obtained on each side of the SNP.