2011 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.
In this study, we used individuals from a colony of European corn borers that show an approximate 1400-fold increase in Cry1Ab toxin tolerance to model the inheritance of Bacillus thuringiensis (Bt) resistance traits. Comparative real-time polymerase chain reaction (RT-PCR) indicated that transcripts from the apn1 locus are suppressed approximately 146.2-fold within the midgut of Cry1Ab resistant compared to susceptible larvae. Despite a perfect correlation between apn1 transcript level and inheritance of Cry1Ab resistance, the apn1 locus segregated independently of corresponding apn1 transcription levels and suggests that modification of one or more trans-regulatory factors results in suppression of European corn borer apn1 gene transcription. This project aims to develop a pipeline for the evaluation of complex genetic traits through gene pathway analysis using "next-generation" sequencing (NGS) technologies to evaluate global gene expression changes, specifically using a method called RNA-seq. To date we have generated F1 progeny from a single-pair cross between a Cry1Ab resistant female and a susceptible male moth of the European corn borer. The resulting F1 progeny were used to establish 8 reciprocal backcrosses to the Cry1Ab resistant line, with the anticipation that an approximate 1:1 ratio of resistant to susceptible phenotypes will be recovered from each backcross family. These backcross progeny have been exposed to Cry1Ab toxin concentrations (500 ng cm-2) for 5 days, which allowed for differentiation of larval phenotypes (susceptible larvae remained as neonates and resistant larvae grew to early 3rd instar). Unfortunately, at the 500 ng cm-2 dose, approximately 90% of susceptible larvae did not survive past neonate when transferred to non-Bt toxin-containing diet. Repetition of the biphasic backcross pedigree setup and use of a 100 ng cm-2 exposure for 7 days allowed for both the differentiation of larval phenotypes. Additionally, this modified bioassay protocol allows for the recovery of susceptible 4th instar larvae following transfer to non-Bt toxin-containing diet. Midgut tissue has been dissected in both Cry1Ab resistant (n greater than or equal to 48) and Cry1Ab susceptible larvae (n greater than or equal to 37) from all 8 backcross pedigrees, and are currently stored at -80 degrees C prior to RNA extraction and mass sequencing of cDNA on the Illumina Genome Analyzer 2 (GA2). 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). Progress was monitored with phone calls, emails, and a meeting in Ames, IA.