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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

2013 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:
Stable carbon isotope analysis of Helicoverpa (H.) zea moths collected from pheromone traps in Arkansas, Georgia, Louisiana, Missouri, Mississippi, South Carolina, Texas, Tennesse and Virginia from 2005 through 2012 continues. Once analysis of the complete dataset is obtained, an ecological analysis of host origin (C3 or C4 plant) will be conducted relative to local crop landscape and abundance of Bt crops.

Colonies of H. zea collected from Bacillus thuringiensis (Bt) and non Bt corn have been assayed for susceptibility to Cry1Ac and Cry2Ab2 and Dipel. Conventional and Bollgard cotton exposed to first generation progeny of field collected H. zea exhibited fruit damage. Higher damage was recorded in conventional cotton as expected. Additional studies are being conducted to associate field control with assay response.

Variability in susceptibility of H. zea to Cry1Ac was measured in progeny of females captured in black-light traps in different cropping areas of the Mississippi Delta. Current data do not indicate strong relationships to local crop structure. Field studies are being conducted on commercial farms to measure insecticide use on Bt and conventional cottons. In addition, baseline studies have been initiated to benchmark susceptibility of lepidopteran larvae in soybean to Cry1Ac.

Plant tissue assays with H. zea, Heliothis virescens, and Spodoptera (S.) frugiperda larvae exposed to different conventional and Bt cottons resulted in significant differences in survivorship of the three species on Bt cotton and signficiant differences in the insecticidal activity of different Bt cottons studies.

Microarrays developed using H. virescens transcriptome sequences were used to identify gene expression patterns in larvae exposed to the Bacillus thuringiensis (Bt) toxin Cry1Ac. Gene expression patterns between susceptible and Cry1Ac resistant strains of H. virescens were analyzed to identify statistically significant differences.

Nucleotide sequences obtained by high-throughput sequencing of a bacterial artificial chromosome (BAC) library of H. zea were screened to identify simple sequence repeat (SSR or microsatellite) markers. Two back-cross families of H.zea developed by mating a laboratory colony (established from field collected insects) with high tolerance to the Bt toxin Cry1Ac with a susceptible laboratory colony were analyzed by genotype by sequencing technology (GBS) to identify polymorphic single nucleotide markers suitable for genetic linkage mapping.


4.Accomplishments
1. Transcriptome Heliothis virescens was assembled, annotated, and a reference gene set was developed for use in expression profiling experiments. Arylphorin, a storage protein in insects was identified as a biomarker that was differentially expressed in Cry1Ac resistant insects.

2. More than 29,000 simple secquence repeat (SSR) containing sequences were identified from DNA sequence assemblies representing approximately 80% of the 350 million base-pair Helicoverpa zea genome. Probes representing 3,900 tri-nucleotide repeat containing SSR markers were designed for high-throughput genotyping of filed populations and genetic crosses of H. zea. In addition, Genotyping by sequencing (GBS) analysis identified 2,664 polymorphic single nucleotide markers (SNP) from H. zea. A preliminary genetic linkage map of H. zea consisting of 33 linkage groups was developed. In collaboration with Southern Insect Management Research Unit, Stoneville, MS, H. zea genome sequencing and annotation was completed by the Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia.


Review Publications
Sanchez, J., La Spina, M., Perera, O.P. 2012. Analysis of the population structure of Macrolophus pygmaeus (Rambur) (Heteroptera: miridae) in the palaearctic region using microsatellite markers. Ecology and Evolution. 2(12):3145-3159. DOI: 10.1002/ece3.420.

Domingues, F.A., Silva-Brandao, K.L., Abreu, A.G., Perera, O.P., Blanco, C.A., Consoli, F.L., Omoto, C. 2012. Genetic structure and gene flow among Brazilian populations of Heliothis virescens (F.) (Lepidoptera: Noctuidae). Journal of Economic Entomology. 105(6)2136-46.

Last Modified: 7/28/2014
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