2012 Annual Report
1a.Objectives (from AD-416):
Associate SNP markers with existing AFLP linkage map and sequence midgut transcriptome for scaffolding RNAseq data.
1b.Approach (from AD-416):
Feeding damage to corn by larval European Corn Borer (ECB), Ostrinia nubilalis, is controlled by transgenic hydrids that express the Bacillus thuringiensis (Bt) toxin Cry1F. Field screens have shown the existence of Cry1F resistant ECB phenotypes in midwestern fields, and following mapping of this trait with Amplified Fragment Length Polymorphism (AFLP) markers we have identified a Quantitative Trait Locus (QTL) on Linkage Group (LG) 12. More detailed mapping of this QTL interval will require the generation of a large number of novel genetic markers in order to identify the causal gene(s). To do this, we will predict the presence of Single Nucleotide Polymorphism (SNP) loci from homolgous Expressed Sequence Tag (EST) reads, and develop molecular assays to detect variation using high-throughput genotyping on the SEQUENOM MassARRAY platform. A high number of SNP markers will be used to genotype backcross progeny generated in a biphasic mapping approach that exploits the lack of meiotic recombination in female Lepidoptera, and likely result in a refined QTL interval with a greater number of markers compared to our prior AFLP-based map. Additionally, we will use the Illumina Genome Analyzer II for ultra-deep sequencing to detect changes in quantity and structure (mutational- and splosomal-level) of larval ECB midgut transcripts among full-sib backcross progeny that show a segregating Cry1F resistance trait. This sequencing approach will detect expression changes, termed expression QTL (eQTL), that co-segregate with larval resistance traits. Structural changes in eQTL transcripts will be used to develop additional SNP markers, and applied to genotyping full-sibs within the same backcross pedigrees and refining the original QTL defined by AFLP-mapping. Our approach of using traditional QTL and eQTL analyses will test for both co-segregation of SNP markers (loci) and differentially-expressed transcripts with larval phenotypes. The goal of this research will be to develop a diagnostic molecular assay to detect population-level changes in the frequency of alleles conferring resistance to Cry1F toxin in O. nubilalis, which will be a valuable tool for stakeholders to use in evaluating IRM strategies and recommendations.
In a previous project, a laboratory European corn borer, Ostrinia nubilalis, colony was selected for being 12,000-fold more tolerance to the Bacillus thuringiensis (Bt)-corn toxin Cry1F compared to a susceptible control colony. Progeny (called F1s) were established by mating a female from this Cry1F resistance colony with a male susceptible moth. Backcrosses were carried out for seven F1 females separately to male resistant moths, called Backcross Male (BCM) families, and five F1 males to female resistant moths, called Backcross Female (BCF) families. Bioassays were preformed that identified Cry1F toxin resistant and susceptible traits segregating within BCM and BCF families. Midgut tissues were dissected from nearly 956 susceptible and 808 resistant larvae for RNA extraction (needed for gene expression analyses) and remaining tissues for DNA extraction for Single-Nucleotide Polymorphism (SNP) marker genotyping. A subset of three reciprocal backcross families have been genotyped using 684 SNP markers, and all progeny are currently being genotyped for the four backcrosses that have the highest number of segregating markers. Total RNA was extracted from 24 larval midguts (12 Cry1F resistant and 12 susceptible), quantified and submitted to the Iowa State University DNA Sequencing and Syntheses Facility for RNAseq library construction and single end 50 cycle cluster generation. The project is related to the objective “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” of the in-house project (3625-22000-017-00D).