Location: Corn Insects and Crop Genetics Research2012 Annual Report
1a. Objectives (from AD-416):
1) Improve knowledge of the ecology, genetics, and behavior of corn pests such as corn borer, corn rootworm, and western bean cutworm in relation to pest abundance and insect resistance to transgenic corn; 2) Determine the nature and degree of potential impacts of transgenic corn (e.g., Bt corn) on non-target organisms; and 3) Develop genetic and molecular methods to investigate corn insect biology and plant-insect interactions.
1b. Approach (from AD-416):
Laboratory trials will examine how resistance alleles from European Corn Borer (ECB) colonies impact dispersal and survival of neonates after exposure to various Bacillus thuringiensis (Bt) toxins. Movement and avoidance behaviors will be evaluated using video-tracking and bioassays with lyophilized leaf tissue incorporated into artificial diet. Results of the laboratory trials will guide selection of appropriate treatments for subsequent semi-field experiments. The marker development and screening strategies used for identifying Bt resistance linkage groups in ECB will be adapted for developing molecular markers associated with behavioral adaptations in ECB. We will characterize gene flow and genetic structuring among ECB populations in Pennsylvania (PA). We will use a panel of 74 Single-Nucleotide Polymorphism (SNP) markers, previously identified and verified from Expressed Sequence Tags (EST), in population genetics analyses to determine the effects of potential geographic barriers on ECB gene flow separately for the pheromone races, and determine if genetic structuring is associated with host plant association in the E race. The development of laboratory tests to evaluate possible effects of stressors from GE crops involves two phases. The first phase is development of a standardized protocol for testing an orally administered test substance on a specific insect and life stage. The second phase is using these protocols to parallel test (a.k.a. ring test) the selected insect at three or more laboratories. We will take a mass-screening approach to validate EST-derived SNPs for Western Corn Rootworm (WCR) using the Illumina Golden Gate SNP assay platform. We anticipate that about 75 percent of the 4,111 candidate SNPs we have identified will produce a designable assay, which would translate into nearly 3,100 assays. These candidate SNPs, for which an assay can be designed, will be tested for polymorphism against samples of rotation-resistant WCR populations from Illinois and wild-type populations from Iowa. Information from the Illumina GenCall and GTS Reports software will be used to remove bad SNPs and bad samples. Complementing the genomics and bioinformatics groundwork, we will investigate Bt resistance traits by integrating structural and functional genome information. SNP markers will be used for mapping Quantitative trait loci (QTL) studies of ECB, and population studies of ECB and WBC. Our experiments will investigate separate Native Resistance (NR) sources leading to differential feeding or survival of WCR larvae on roots, of WCR adults on silks, and of Corn Earworm (CEW) larvae on silks. For both lepidopteran and coleopteran pests, we will narrowly define chromosomal segments of the corn genome that harbor NR alleles. We will also create large sets of inbred and hybrid isolines to enable future mechanistic analysis of the effects of the NR alleles on both target and non-target insects.
3. Progress Report:
New Single Nucleotide Polymorphism (SNP) markers were used to genotype Z and E pheromone races of the European Corn Borers (ECB) from multiple locations in Pennsylvania and New York, where topography may reduce gene flow between populations. Preliminary analyses indicate that gene flow in the E race is less than within the Z race, and that topography may isolate populations under certain conditions. Populations of Western Corn Rootworm (WCR) were sampled from their original home range in Colorado and eastern Kansas. Preliminary population genetics analyses with new SNP markers indicate that population structuring exists, which will allow estimates of long-range gene flow. Related to ECB resistance to Bacillus thuringiensis (Bt) corn, differential expression of all genes in midgut tissue of Cry1Ab resistant and susceptible ECB was examined for one backcross family, and potential linkage to resistance is being investigated using other families. Studies were conducted on the dispersal of older larvae (late instars) of ECB on vegetative corn. The late instar studies confirm last year’s results that under high density conditions larvae will disperse during each of the five instars. Most of the dispersal occurs with first instars and appears to be density independent. High dispersal percentages also were found for older larvae, particularly fourth and fifth instars. Also, ECB were successfully selected for plant establishment and plant abandonment, which was related to whether or not they dispersed off a corn plant by spinning silk. Differences in candidate genes (foraging, shaker, and slowmo) were investigated to better understand the genetic mechanisms underlying host plant abandonment. In another study, two families of 50 each ECB siblings were produced and used to prepare a SIRRL (Sequencing Individuals in Reduced Representation Libraries) marker sample with each bar-coded for Illumina sequencing. This is a new type of marker that will be useful for population genetics studies of species with no prior genetic information, such as Western bean cutworm. This test run was successful, demonstrating that the method for library preparation and paired-end sequencing in fact work as well as conceived. Another study related to the WCR identified a gene linked to cyclodiene resistance. Genome and SNP assays were developed to screen for the mutation in populations. F1 crosses were used to determine that maternal genotype influences the inheritance of an extended diapause trait in Northern corn rootworm. Genetic identification of native resistance sources effective against WCR larvae feeding on roots has revealed that multiple factors contribute. In practical terms, direct utilization of this resistance source is feasible, but pyramiding would be required. Mechanistic investigations are proceeding, and could provide biochemical entry points that could be exploited with transgenic, rather than conventional corn breeding approaches.
Coates, B.S., Hellmich II, R.L., Grant, D.M., Abel, C.A. 2012. Mobilizing the genome of Lepidoptera through novel sequence gains and end creation by non-autonomous Lep1 Helitrons. DNA Research. 19(1):11-21.