2010 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.
The goal of this project is to use next-generation sequencing technology (Illumina GoldenGate assay) to identify and verify large numbers of molecular genetic markers called single nucleotide polymorphisms from western corn rootworm to create a linkage map and to be used in future population genetics studies. A linkage map constructed with this type of genetic marker (single nucleotide polymorphism) will show the relative position of genes on each chromosome. The closer genes are, the more likely they will be inherited together. The linkage map will be useful to many researchers for years to come, as they attempt to locate and identify the genes that cause certain traits, such as resistance to insecticides, specialized behaviors, or anything else important to developing sophisticated tools to manage this pest. In 2010, the verification portion of the project was initiated, in which candidate genetic markers were identified from an expressed sequence tag library, and submitted to Illumina's oligonucleotide designability screening service. Of the 2,222 candidate markers that we determined to be potentially useful, 1,536 were chosen for further testing in collaboration with the University of Illinois W. M. Keck Center for Comparative and Functional Genomics. A western corn rootworm bacterial artificial chromosome library was constructed. A bacterial artificial chromosome is a type of tool, called a vector, that inserts long stretches of DNA from any species into a bacterium. Once the foreign DNA has been cloned into the bacterium, many copies of it can be made and sequenced. This library was screened by us at the Corn Insects and Crop Genetics Research Unit, Ames, Iowa, and collaborators at the University of Nebraska to identify genes coding for important proteins such as receptors and enzymes that are often involved in insecticide resistance. DNA from ten of these clones has been extracted, and is now being purified and prepared for sequencing at the University of Illinois. We anticipate that the sequence data for the single nucleotide polymorphism markers and bacterial artificial chromosome clones will be available soon, and the final phase of sequence analysis and linkage map construction will be completed within the next year. Progress is monitored through frequent email correspondence, telephone calls initiated by both parties, conference calls, and discussions at jointly attended technical meetings and conferences.