2013 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 genomic sequencing technology to identify and verify large numbers of molecular genetic markers called Single Nucleotide Polymorphisms (SNPs) from western corn rootworm to create a linkage map and to be used in future population genetics studies. A linkage map constructed with SNPs 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. Over 1,500 candidate markers were chosen for further testing and evaluation in collaboration with the University of Illinois W. M. Keck Center for Comparative and Functional Genomics. SNP genotyping revealed 1710 segregating markers from five families. Of these, initial statistical screening identified 661 unique markers exhibiting Mendelian inheritance, which were used to determine genetic linkage. These resulted in the prediction of about nine Linkage Groups (LGs) per family, with about eight markers per LG. In subsequent analysis we used the LG association data among SNP markers to construct a consensus genetic linkage map that integrated independent information among the five families. This resulted in a total of 353 of the 661 unique markers being integrated into ten consensus LGs, with about 35 markers per LG. A bioinformatic pipeline was designed for identifying polymorphisms (SNPs and small insertion/deletions) from next generation sequencing of genetically variable insect samples, adapting methods from human genetic studies. Genome sequencing work was also conducted. Nineteen Bacterial Artificial Chromosomes (BACs) were sequenced at the High-Throughput Sequencing and Genotyping Unit, W.M. Keck Center. The data were submitted to the High-Throughput Genome Sequencing database, and are being used in aiding the assembly of sequences from a whole genome sequencing effort. This project is now complete.