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.
Genomic DNA and midgut complementary DNA (cDNA) have been prepared from three backcross families. Each of these resulted from a replication of initial crossing of a Cry1Ab resistant female to a susceptible male, then performing reciprocal backcrosses between F1 progeny to a resistant parent. Preliminary Single-Nucleotide Polymorphism (SNP) genotyping was used to identify one backcross family as having the greatest number of segregating markers. A combined assembly of O. nubilalis midgut Express Sequence Tag (EST) DNA was generated from GenBank accessions. Additionally, the midgut transcriptome of O. nubilalis was sequenced on a 1/2 plate of a Roche 454 Titanium run; and the assembly of these data is ongoing. Midgut cDNA from the same backcross family progeny were used as template in real-time polymerase chain reaction (RT-PCR) reactions and showed approximately 50 percent of individuals with approximately 150-fold reductions in apn1 transcript levels compared to the remainder of individuals. The cDNA from both classes of apn1 transcript level were prepared for sequencing on an Illumina HiSeq, and are currently in the queue for 1 x 50 cycle sequencing at the Iowa State University DNA Sequencing and Synthesis Facility.