Location: Corn Insects and Crop Genetics Research2013 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.
3. Progress Report:
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 shows 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. A SNP genotyping assay 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 western corn rootworm Bacterial Artificial Chromosome (BAC) library was constructed. A BAC 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 USDA Researchers in Ames, IA 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. A total of 38 western corn rootworm BAC library clones were successfully identified as containing specific genes of interest. DNA from these clones was extracted, sequenced in pieces, and re-assembled. In addition to providing direct data about the target genes, information from sequence analysis has been useful in characterizing the overall structure of the western corn rootworm genome which made it possible to begin a project to sequence and re-assemble the whole genome. The data were submitted to the High-Throughput Genome Sequencing database, and are being used in aiding the assembly of sequences from the whole genome sequencing effort. Results indicate that genes occupy only about nine percent of the genome, whereas other regions are composed of repeated and mobile DNA sequences. Mobile DNA sequences were shown to be responsible for the large genome size. DNA from laboratory colonies of insects, and from a number of wild populations, including western corn rootworms resistant to control by corn-soybean planting rotation, has been extracted and is ready for linkage and genome scanning experiments. The genome scan will be used to identify genetic markers linked to rotation resistance genes. The size of the western corn rootworm genome was determined using flow cytometry (a technique for counting microscopic particles) and is very large. 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.