1a.Objectives (from AD-416):
Identify molecular markers associated with resistance to Cry3Bb1 Bt corn in replicated colonies of WCR populations that are reared on isoline or Bt corn.
1b.Approach (from AD-416):
A genome scan using a large number of SNP markers will be used to identify markers associated with the resistant phenotype. The SNP markers (about 1000) will be developed within the next year as part of a NRI grant recently awarded to a scientist at the Corn Insects and Crop Genetics Research Unit. Markers will be genotyped from a total of 960 individual western corn rootworms from replicated lines selected for Bt-corn resistance and their associated unselected control lines.
DNA has been extracted from about 1000 individual western corn rootworms (WCR) from each of three laboratory lines selected for resistance to transgenic Bacillus thuringiensis (Bt) corn in Columbia, Missouri, as well as from their unselected control lines. In addition, 900 of these individuals, from three samples of 100 (resistant line, unselected control line, and an ancestor line) from each of the three replicated colonies of different geographic origins, have been genotyped at microsatellite marker loci so that the effective population size of each colony can be determined. Because of their complicated history, custom simulation software has been written for each colony to determine the amount of genetic divergence expected between the resistant and control lines in the absence of selection and based on the effective population size. The next step will be to genotype each individual at about 800 hundred single nucleotide polymorphism markers that we recently developed and verified as part of another project. This type of genetic marker (single nucleotide polymorphisms) can demonstrate the relative position of genes. Markers that exceed the expected level of divergence (based on the simulations) in all three selected lines in an experiment called a "genome scan" are potentially located so physically near a resistance gene on a chromosome that they are inherited together, or "linked." Thus, such markers will be considered candidate markers linked to resistance genes that have responded to selection. It is easier to detect linked markers in an individual than to detect the resistance gene itself, at least until the resistance gene is identified in the future. Three major goals were accomplished in preparation for the genome scan of the replicated Bt resistant colonies to identify significant outliers associated with resistance. The first was verification of 661 unique single nucleotide polymorphism (SNP) markers from among 1419 candidate SNPs identified from expressed sequence tag (EST) libraries. Secondly, we have created a linkage map of the WCR genome, populated with these SNPs and previously developed microsatellites. The map will be further saturated as the new SNPs come online, and it will help in the future identification of loci associated with resistance. Third, comprehensive simulation studies have been conducted to optimize the strategy for genome scanning. Simulation results showed that for both recessive and dominant resistance alleles, the power to detect selection rises to a peak at six to eight generations of selection. Subsequently, the power to detect selection declines as recombination degrades the linkage disequilibrium between resistance alleles and marker alleles. This information will allow us to focus our efforts on studying only the generations of replicated resistant colonies that give the best opportunity to detect selected markers. Results also showed that additional markers will be needed to supplement the currently available SNPs in order to achieve the desired power to detect selection. Specifically, the number of markers should be increased four- to ten-fold. This information is being used to optimize the genotyping-by-sequencing strategy that will provide the supplemental markers.