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United States Department of Agriculture

Agricultural Research Service

Research Project: Effect of Resistance on Insect Pest Management in Transgenic Cotton

Location: Southern Insect Management Research Unit

2012 Annual Report

1a.Objectives (from AD-416):
Objective 1: Determine the impact of a changing cropping landscape on host plant ecology and insect resistance management practices for bollworm using analytical techniques.

Objective 2: Determine gene flow and migration patterns by analyzing tobacco budworm and bollworm populations in temporal and spatial scales using genetic and/or empirical/mathematical approaches.

Objective 3: Identify possible mechanisms of resistance to Bt toxins by profiling gene expression patterns and develop a marker based genetic linkage map.

1b.Approach (from AD-416):
More than 95% of the second generation bollworm within each growing season utilizes field corn as a host. Impact of corn plants expressing multiple Bt toxins on the bollworm populations will be studied by comparing historical pheromone trap data with current and future population estimates influenced by increased acreages of Bt corn expressing multiple Bt toxins. Stable carbon isotope analysis will be used to identify bollworms using corn as a host plant. Influence of local cropping landscape on bollworm populations will be studied using sentential plots of conventional and Bt corn and cotton and early maturing soybeans. Large field cages will be used to evaluate the impact of pyramided-gene Bt corn hybrid/refuge system on resistance management strategies. Expressed genes of tobacco budworm and bollworm will be identified by transcriptome sequencing, and genetic markers developed from polymorphic nucleotide regions will be used in ecological genetic studies of tobacco budworm and bollworm populations. Gene expression profiles will be used to identify biological processes involved in physiological response to ingestion of Bt toxins. Markers developed for candidate loci associated with resistance to Bt toxins will be used to estimate allele frequencies in natural populations. Genetic loci under selection will be identified using statistical methods. A genetic linkage map of the bollworm developed using polymorphic markers will be used to study inheritance of loci of interest to Bt resistance.

3.Progress Report:
Results from the initial year of this project indicated that field corn was still the primary host of H. zea during the growing season even though greater than 50% of the corn planted in the southern U.S. produces one or multiple Bt proteins. Bt corn hybrids that produce multiple Bt proteins suppress H. zea larval numbers and kernel damage, but yields do not necessarily reflect this. Laboratory colonies of H. zea were established from light trap samples taken in concentrated areas of corn, cotton and soybean production in the Mississippi Delta and assayed for susceptibility to Cry1Ac. Variable responses were obtained but no clear association with the different crop areas was evident.

Corn fields were surveyed after harvest to assess the density and maturity of volunteer corn in the fall as well as the abundance of corn earworm in non-Bt corn at various growth stages. Greenhouse and field trials examining the efficacy of various crosses of Bt and non-Bt corn varieties on bollworm were conducted during last year and are being repeated during 2012. Helicoverpa zea colonies with increased tolerance to Cry2Ab and Cry 1Ac were established from insects collected from field locations in the Mississippi Delta. Back-cross genetic mapping populations were developed by mating these colonies with insects from a susceptible laboratory colony maintained at SIMRU and DNA extractions were completed from 96 insects for use in a marker based mapping study.

Nucleotide sequences from 14 DNA pools containing 36,500 clones of a bacterial artificial chromosome (BAC) library were obtained using high-throughput sequencing technology. The sequence reads were assembled to identify candidate Bt resistance genes and genetic markers associated with them. Nucleotide sequences from bollworm BAC clones and transcriptome are also being used to complete assembly and annotation of the bollworm genome by CSIRO, Australia.

Laboratory colonies of Bt resistant and susceptible Old World bollworm, Helicoverpa armigera, exposed to Cry1Ac were used to study the time course of midgut gene expression. RNA extracted from midguts of treated insects was used to obtain short nucleotide reads for RNA-Seq profiling. Validation of differential expression patterns observed in RNA-Seq experiments is in progress.

Computer model simulations examining the risk of corn earworm resistance development on corn expressing two Bt genes versus those expressing a single gene were carried out. At least 350 replicate simulations with randomly drawn parameters were completed for each of four risk assessments. When dual-gene Bt-cotton, planted with a natural refuge and single-gene corn planted with a 50% refuge was simulated, resistance to both toxins simultaneously never occurred within 30 years, but in 38.5% of simulations, resistance evolved to toxin present in single-gene Bt-corn (Cry1A). When both corn and cotton were simulated as dual-gene products, cotton with a natural refuge and corn with a 20% refuge, 3% of simulations evolved resistance to both toxins simultaneously within 30 years, while 10.4% of simulations evolved resistance to the Cry1A toxin.

1. Novel candidate genes that may play a role in resistance to Bacillus thuringiensis toxins in Heliothis virescens were identified. Southern Insect Management Research Unit scientists in Stoneville, MS, used gene expression studies to identify these genes. Understanding the roles of these genes in developing resistance to Bacillus thuringiensis toxins will help develop appropriate insect resistance management strategies to minimize damage to transgenic crops.

Review Publications
Luttrell, R.G., Jackson, R.E. 2012. Helicoverpa zea and Bt Cotton in the United States. GM Crops and Food Biotechnology in Agriculture and the Food Chain. 3(3):213-227.

Perera, O.P., Blanco, C. 2011. Microsatellite variation in Helicoverpa zea (Boddie) populations in the southern United States. Southwest Entomology. 36(3):271-286.

Perera, O.P., Blanco, C.A., Ballard, L., Silva-Brandao, K.L., Domingues, F.A., Abel, C.A. 2011. Evaluation of anonymous and expressed sequence tag derived polymorphic microsatellite markers in the tobacco budworm Heliothis virescens (Lepidoptera: noctuidae). Southwestern Entomologist. 36(3):287-294.

Last Modified: 4/20/2014
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