Source: AGRICULTURAL RESEARCH SERVICE submitted to NRP
GENOMIC RESOURCES TO ACCELERATE WESTERN CORN ROOTWORM RESEARCH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0216553
Grant No.
2009-35302-05263
Cumulative Award Amt.
$749,597.00
Proposal No.
2008-04030
Multistate No.
(N/A)
Project Start Date
Feb 1, 2009
Project End Date
Jan 31, 2014
Grant Year
2009
Program Code
[51.2C]- Arthropod and Nematode Biology and Management (C): Tools, Resources and Genomics
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
RR #3 BOX 45B
AMES,IA 50011
Performing Department
CORN INSECTS AND CROP GENETICS RESEARCH UNIT
Non Technical Summary
The western corn rootworm is a highly destructive pest affecting the U.S. The fundamental goal of this project is to provide researchers who have interests in the genetics and genomics of western corn rootworm 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 sequence data from expressed genes and development of genetic 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, and we have assembled an experienced, interdisciplinary team of investigators with broad expertise in both the genetics of WCR and cutting-edge genomic technologies to accomplish our goal. All of this knowledge will be made available to scientists worldwide via a web interface that can incorporate new discoveries beyond the lifetime of this project. The results of this project will provide a solid foundation for future investigations of the WCR genome as a whole, assist research into the genes that make WCR an adaptable and invasive pest and facilitate the discovery of WCR genes for new biotechnology. In the longer term, the availability of a dense linkage map linked to existing DNA sequence data will be a crucial resource for future WCR genomics projects to produce physical genetic maps and, ultimately, sequence the entire genome. Such an eventual outcome is highly desirable, especially given that the genome of corn itself is now sequenced. It will allow unprecedented insight into the interactions between our most important crop and its most important insect pest.
Animal Health Component
20%
Research Effort Categories
Basic
20%
Applied
20%
Developmental
60%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2111510104050%
2111510108050%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
1510 - Corn;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;
Goals / Objectives
The western corn rootworm (WCR) is the most destructive pest of corn in the US Corn Belt in terms of direct crop losses and control costs. The economic impact of WCR is likely to increase as it affects corn production not only for food but also for the burgeoning biofuels industry. This species is highly adaptable and has evolved resistance to several classes of synthetic insecticides as well as crop rotation, the primary method of cultural control. Safeguarding current management technologies (notably transgenic cultivars expressing Bt-toxins and neonicotinoid seed treatments) from a similar fate will require sound resistance management strategies founded on a thorough understanding of the genetics and evolution of WCR. Furthermore, WCR is a formidably invasive species. In the second half of the 20th century, it expanded its range out of the Great Plains, through the Corn Belt, eventually reaching as far as the east coast and southern Quebec. More recently, it has succeeded in invading much of Europe making it a pest of international importance. Because it is a specialist herbivore of corn, WCR also has significant potential as a source of novel enzymes that could be used for cellulosic ethanol production for biofuels. Both exploiting this potential and managing the risk of future adaptation to control technologies will demand a thorough understanding of the genomics and genetics of WCR. There is an active, highly collaborative international community of researchers that is making steady progress in these fields. Our proposed project will furnish this community with a set of tools and resources that will facilitate existing and future research programs. This project will address Program Priority 1: "Develop innovative approaches for mapping, identification, sequencing and/or expression of genes to enable future studies on genome organization and lead to hypothesis testing research". Outputs will include a high-density linkage map of the WCR genome, populated by single nucleotide polymorphisms, identified from existing expressed sequence tags, and microsatellites. This map will be connected to additional existing resources by associating mapped loci with clones in a bacterial artificial chromosome (BAC) library of the WCR genome. A set of the map-associated BAC clones will be sequenced in full. All of the data generated by the project will be made available to the wider research community via a user-friendly web interface. These resources will aid research into the genetics of adaptive traits, gene discovery for novel biotechnology and lay the foundations for more comprehensive genome-sequencing efforts. The website will be dynamic and colleagues worldwide will be encouraged to incorporate new data as they emerge.
Project Methods
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 ≥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. The results of this project will provide a solid foundation for future investigations of the WCR genome as a whole, assist research into the genes that make WCR an adaptable and invasive pest and facilitate the discovery of WCR genes for new biotechnology. In the longer term, the availability of a dense linkage map linked to existing ESTs and BAC clones will be a crucial resource for future WCR genomics projects to produce physical maps and, ultimately, sequence the entire genome. We have assembled a team of investigators with broad expertise in both the genetics of WCR and cutting-edge genomic technologies. All the prerequisite resources for the project are already in place and our chances of a fully successful outcome are excellent.

Progress 02/01/09 to 01/31/10

Outputs
OUTPUTS: A GoldenGate assay panel was developed to test and genotype 1,536 western corn rootworm SNPs, distributed over 1,135 EST contigs. Candidate SNPs were identified from aligned EST sequence traces using the polyphred computer program. All putative SNPs with a polyphred score of > 95 and at least 50 bases of flanking sequence on either side were submitted to Illumina's GoldenGate Assay Design Tool software pipeline. 4,360 SNPs for which an assay could be developed were screened by eye for potential problems by examining blast alignments of the original sequence reads to the contig consensus sequences. This allowed us to identify and avoid cases where a second SNP was close to the focal SNP, trialleleic loci, and monomorphic loci where the original SNP detection apparently was erroneous. 2,220 SNPs that passed this filter were ranked within the contig from which they originated, permiting selection of loci for the assay panel that were distributed over as many contigs as possible. Criteria for assigning intra-contig ranks were the "designability" score computed by the Assay Design Tool, the score given to the SNP by polyphred, and the number of reads in which the rarer allele was found. The oligonucleotides required for the assay have been purchased and are now ready for use. DNA has been extracted from all 570 individuals in 5 families to be used for construction of the linkage map. These individuals have been genotyped using a panel of 15 microsatellite loci. Genotyping at SNP loci using the GoldenGate assay will begin soon. Extraction of DNA from samples to evaluate the power of the SNP loci to study fine-scale population structure is ongoing. Western corn rootworm adults were collected from 10 locations in its original home range in Colorado and western Kansas, including the species type-location (Wallace County, Kansas). These will be analyzed for genetic diversity and gene flow analyses with the new SNPs, once fully developed. PARTICIPANTS: Thomas W. Sappington, USDA-ARS, Corn Insects & Crop Genetics Research Unit, Ames, IA. PD. Responsible for coordinating overall activities of the project. Nicholas J. Miller, USDA-ARS, Corn Insects & Crop Genetics Research Unit, Ames, IA. Current address, Dept. of Entomology, University of Nebraska-Lincoln. As postdoc at USDA, and now Assistant Professor at UNL, responsible for identifying candidate SNP markers from EST sequence data and, in consultation with Dr. Whitfield, preparing for GoldenGate assay. Charles Whitfield, Dept. of Entomology, University of Illinois, Champaign-Urbana. Responsible for GoldenGate designability assay, and follow-up methodology of further screening by eye to identify poor candidates that algorithms cannot detect. With Dr. Miller, prepared the oligopanel for the GoldenGate assay. Blair D. Siegfried, Dept. of Entomology, University of Nebraska-Lincoln. Provided DNA from 5 western corn rootworm families phenotyped for methyl-parathion resistance, for linkage mapping with new SNPs. TARGET AUDIENCES: The main target right now is other scientists interested in accelerating genomics studies of western corn rootworm, including especially members of the international Diabrotica Genetics Consortium. The consortium is comprised of over 40 scientists in 7 countries. This project is a critical component of a larger effort to obtain the genomic tools necessary for a genome sequencing project. Results, progress, and future plans were disseminated at several venues: 2nd International Conference on Diabrotica Genetics, in Munich, Germany; NCCC-46 Technical Committee (Development, Optimization and Delivery of Management Strategies for Corn Rootworms), Madison, WI; Entomological Society of America annual meeting, Indianapolis, IN; Information Systems for Insect Pests, 1st International Workshop, Rennes, France. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
This project is generating a number of prerequisites for pursuing a WCR genome sequencing project, the most immediate justification for which is simply economic. It will be cheaper in the long run to sequence the genome than for individual laboratories to continue developing genomic tools and working on problems piecemeal. In recent years, a number of technological developments have caused the cost of DNA sequencing to plummet. Thus, sequencing comparatively large genomes from non-model organisms like the WCR (2.5 Gbp) is becoming a realistic proposition.

Publications

  • Gray, M. E., T. W. Sappington, N. J. Miller, J. Moeser, and M. O. Bohn. 2009. Adaptation and invasiveness of western corn rootworm: Intensifying research on a worsening pest. Annu. Rev. Entomol. 54: 303-321.
  • Miller, N. J., T. Guillemaud, R. Giordano, B. D. Siegfried, M. E. Gray, L. J. Meinke, and T. W. Sappington. 2009. Genes, gene flow and adaptation of Diabrotica virgifera virgifera. Agric. For. Entomol. 11: 47-60.