2010 Annual Report
1a.Objectives (from AD-416)
Our first objective is to use "Next-Generation" Sequencing (NGS) on the Illumina Genome Analyzer II for ultra-deep sequencing of midgut transcripts, and detect changes in quantity and structure (mutational- and splicosomal-level) among transcripts from multiple full-sib Cry1Ab resistant and susceptible Ostrinia nubilalis larvae from backcross pedigrees. Our second objective is to describe expression differences between phenotypes, termed expression quantitative trait loci (eQTL), which co-segregate with phenotypic traits. Our third objective is to use single nucleotide polymorphisms (SNPs) within eQTL transcripts as markers for genotyping full-sibs within the same backcross pedigrees as used to define eQTL.
1b.Approach (from AD-416)
Control measures suppress populations of larval Lepidoptera, but applications present challenges for long-term sustainability. Phenotypic plasticity within populations can form differential response of multiple genes or gene pathways to common environments and control practices. Portions of quantitative phenotypic variation within populations is attributed to differential response at the transcriptional level. As a robust microarray alternative, we will use "Next-Generation" Sequencing (NGS) by the Illumina Genome Analyzer II for ultra-deep sequencing of midgut transcripts to detect changes in quantity and structure (mutational- and splicosomal-level) among transcripts from multiple full-sib Cry1Ab resistant and susceptible Ostrinia nubilalis larvae from backcross pedigrees. Constitutive expression level changes between phenotypes, termed expression quantative trait loci (eQTL), that co-segregate with phenotypic traits will be identified. Transcript levels of genes within a gene regulatory network co-segregate, such that validation of eQTL involvement in trait determination cannot use futher expression assays. Structural changes in eQTL transcripts will be used for single nucleotide polymorphism (SNP) marker development, and markers applied to genotyping full-sibs within the same backcross pedigrees (mentioned above). Subsequent QTL analyses will test for co-segregation of genomic loci for candiate eQTL with the larval phenotype. These procedures for lepidopteran transcriptome analysis by NGS technologies include protocols for contig assembly, gene annotation, and SNP and splice variant predictions. NGS data will have added value due to analysis of different larval phenotypes segregating in pedigrees, such that eQTL will be identified. NGS application also will be achieved through validation of eQTL via QTL mapping of associated SNP loci.
A laboratory colony of European corn borer, Ostrinia nubilalis, was selected for increased tolerance to the Bacillus thuringiensis (Bt) Cry1Ab toxin, at a level approximately 2,500-fold higher than susceptible larvae. A diagnostic dose of 30 ng cm-2 of a Cry1Ab toxin overlaying a standard diet used to rear the insect was shown to be sufficient to distinguish between resistant and susceptible individuals within backcross populations, and correlates 100% with an approximately 150-fold reduction in a specific protein enzyme level [aminopeptidase N 1 (apn1)] as detected by a method that quantifies gene expression over time [quantitative real-time Polymerase Chain Reaction (PCR)]. Corn borer family lines with known pedigrees have been established, and backcrosses to the Cry1Ab resistant line are currently in progress. Progress is monitored with phone calls, emails, and discussions at professional meetings.