2011 Annual Report
1a.Objectives (from AD-416)
1. Develop and implement marker-assisted corn breeding strategy. Identify and characterize novel markers associated with aflatoxin-resistance, e.g., resistance-associated proteins (RAPs), in developing and mature kernels through proteomic and genomic comparisons of resistant and susceptible corn genotypes.
2. Identify new sources of corn germplasm and develop new germplasm resistant to fungal infection and aflatoxin contamination with national and international collaboration, using laboratory and field inoculations of corn kernels with tester fungi designed for rapid resistance screening.
3. Evaluate the contribution of novel RAP genes from corn (see Objective.
1)for resistance to A. flavus growth and aflatoxin production and use these genes or others to develop transgenic cotton with enhanced resistance to aflatoxin contamination under greenhouse and field conditions. Identify and transfer resistant varieties to cooperating plant breeders for development of varieties for commercial application.
4. Develop rapid, non-destructive hyperspectral imaging methodology to: a) measure fungal growth and aflatoxin contamination in corn as a tool for use in enhancement of Homeland Security, and b) measure spectral signatures associated with traits for resistance to fungal infection and aflatoxin contamination in corn kernels.
1b.Approach (from AD-416)
Resistance to aflatoxin contamination will be enhanced in corn and cottonseed through marker assisted breeding and genetic engineering, respectively. In order to accomplish these goals, complex natural resistance mechanisms in corn kernels will be elucidated in resistant corn inbreds through identification of resistance associated proteins using proteomics and other resistance associated compounds through chemical analysis. Understanding the molecular basis of seed based resistances will lead to identification of biochemical factors correlated with resistance for use in marker assisted breeding and/or when pertinent resistance genes are identified and cloned, for use in enhancement of resistance in crops through genetic engineering. This strategy is especially pertinent to cottonseed, which does not possess practical levels of natural resistance to aflatoxin producing fungi in its germplasm base. Another goal is to assess resistance related
biochemical products for their stability of expression in native and transgenic crops under environmental conditions (e.g. drought) known to be conducive to aflatoxin contamination. Also as a part of this project, rapid, non destructive detection methodology based upon hyperspectral imaging will be developed to measure fungal growth and aflatoxin in corn kernels and spectral signatures associated with traits for resistance to fungal infection and aflatoxin contamination in corn kernels, and also to measure physical and biochemical attributes in kernels potentially useful in resistance marker selection.
Progress reported for project 6435-42000-021-00D (initiated in January 2011) represents a continuation of much of the work that was reported in project 6435-42000-019-00D, terminated in December 2010. We are continuing to analyze the role of candidate corn resistance-associated protein (RAP) genes/proteins as antifungal factors using ribonucleic acid interference (RNAi)-based technology in corn. In addition, we are looking at the ability of corn lines with high levels of carotenoids (pigments) to resist Aspergillus flavus growth and aflatoxin production. We continue to transform both cotton and our tobacco model system with candidate antifungal genes such as those encoding lytic peptides, thionins, and corn RAPs to determine their effectiveness at controlling fungal and bacterial pathogens in laboratory and green house assays. We are in the process of cotton seed increase for additional field trials of D4e1-expressing cottons in Arizona or Texas where aflatoxin contamination is endemic. In association with Agricultural Research Service colleagues at the Citrus Research Station, Ft. Pierce, Florida, the D4e1 gene is being evaluated in citrus to control the devastating bacterial “greening” disease. To overcome the problems associated with detection of the small D4e1 peptide, we have transformed tobacco with a hemagglutinin (HA)-tagged version of the peptide to understand localization, quantification, and track expression patterns in planta. We have initiated a collaboration with scientists at Louisiana State University to study the contribution of stress resistance genes (from Spartina alternifolia, a Louisiana marsh grass species) towards aflatoxin contamination in cotton. Studies are being performed to confirm and further characterize the fluorescence peak shift phenomenon that was identified among groups of kernels with different aflatoxin contamination levels.