2009 Annual Report
1a.Objectives (from AD-416)
Use GoldenGate assays to validate Single Nucleotide Polymorphism (SNP) markers and to genotype individual western corn rootworms (WCR) at those loci.
1b.Approach (from AD-416)
A panel of individual western corn rootworm (WCR) sampled from populations in Illinois and Iowa will be used for the initial screening of candidate Single Nucleotide Polymorphism (SNP) markers. The panel will consist of 96 individuals from each state. A mass-screening approach will be used for the validation of Expressed Sequence Tag (EST)-derived SNPs. This will be done using the Illumina GoldenGate assay platform. Validation of candidate SNPs as single-locus markers and their assembly onto a linkage map will be done using six backcross pedigrees. Preserved insects will be transferred from the University of Nebraska-Lincoln to USDA-ARS CICGRU where DNA will be extracted using Qiagen DNeasy Tissue kits. To maximize yield, DNA will be eluted into a volume of 400 ul. Samples will then be concentrated using Millipore Microcon spin columns to ensure adequate DNA concentrations for the GoldenGate genotyping assay. The concentration of each DNA sample will be determined by UV-spectrophotometry and aliquots will be transferred to the University of Illinois for GoldenGate analysis. 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. The Illumina GoldenGate SNP assay is an array-based technique that can be used to genotype 96 individuals at 1,536 SNP loci simultaneously. Illumina, Inc. provides an oligonucleotide design service, included in the cost of purchasing panels of oligonucleotides to perform GoldenGate assays. This service makes use of proprietary bioinformatics techniques not only to design the oligonucleotides for each SNP locus but also to evaluate the probability that the assay will be successful. All of the 5,240 candidate SNPs identified previously by ARS from EST data will be submitted to the assay design and evaluation process. It is expected that 2 panels will be developed to test up to 3,072 candidate SNPs. If the number of potential assays exceeds this number, assays will be selected for inclusion in the panels on the basis of the polyphred score for the SNP, the distribution of potential assays among EST contigs, and the annotations associated with the underlying ESTs. GoldenGate assays for SNP genotyping will be conducted by the W.M. Keck Center for Comparative and Functional Genomics, University of Illinois at Urbana-Champaign. The W.M. Keck Center houses an Illumina Beadstation and related equipment needed for GoldenGate assays. The Keck Center also employs technical staff who have been fully trained to perform the GoldenGate assays. Genotyping assays will be provided as a service to the Cooperator at the University of Illinois.
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. Candidate single nucleotide polymorphisms (SNPs) were identified by searching assembled contiguous sequences from expressed sequence tag (EST) libraries from the western corn rootworm (WCR), Diabrotica virgifera virgifera, head and midgut developed previously. These candidate SNP sequences were submitted to Illumina's oligonucleotide designability screening service at the W. M. Keck Center for Comparative and Functional Genomics, University of Illinois at Urbana-Champaign. We have begun SNP verification, which will result in a large number of SNP markers for developing a genetic linkage map for WCR. 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 for multiplex polymerase chain reactions (PCR) will be ordered.