2009 Annual Report
1a.Objectives (from AD-416)
(1) Develop peanut genomic tools and strategies to elucidate the molecular mechanisms that define crop defense pathways and regulation of resistance to diseases such as tomato spotted wilt virus (TSWV), leaf spots and aflatoxin contamination in peanut. (2) Evaluate corn germplasm that harbors resistance genes that reduce aflatoxin contamination and understand the responding genes and pathways in corn.
1b.Approach (from AD-416)
(1) Replicated laboratory and field screening and evaluation of peanut accessions for disease resistance will be conducted in order to identify the “resistant” germplasm for further genomic studies. The resistant germplasm will be utilized in molecular marker development for marker-assisted breeding. (2) ESTs (expressed sequence tags) will be generated from cDNA libraries constructed from seed and leaf tissues of two genotypes, Tifrunner and GT-C20. EST-derived SSR markers will be developed and peanut oligo-microarray will be produced for gene expression study. (3) Genetic mapping populations (RILs, recombinant inbred lines) will be produced from crosses of Tifrunner and GT-C20, and SunOleic 97R x NC94022. QTL mapping will be conducted for resistance to tomato spotted-wilt virus (TSWV) and leaf spots, and aflatoxin contamination. (4) Microarray experiments will be used to identify candidate genes in corn-Aspergillus flavus and drought stress interactions that are turned on or off during corn kernel development. The candidate genes identified from microarray will be verified or confirmed through real time PCR or other well established methods. Another goal is to develop a macroarray tool (membranes) using these candidate genes from microarray to assess resistance or drought tolerance in corn germplasm for their stability of expression in native crops under environmental conditions (e.g., drought) known to be conducive to aflatoxin contamination. The genes identified in corn kernels also will be applied in searching possible ‘orthologs’ in peanut genome and peanut germplasm.
Peanut grown in the southeastern U.S. are seriously afflicted by tomato spotted wilt virus (TSWV) and routinely contaminated by aflatoxin, induced through infection by fungus Aspergillus flavus/parasiticus. This project has exploited both classical plant breeding and molecular genomic techniques to resolve problems related to these diseases and the abiotic stresses, particularly drought stress, which aggravates disease progression and crop contamination. Two dual purpose populations of recombinant inbred lines (RILs) were derived by single seed descent from a cross between the TSWV resistant cultivar Tifrunner and the aflatoxin resistant breeding line GT-C20, along with a cross between SunOlic 97R and NC94022. Progeny derived from these crosses are in use for the development of germplasm and breeding lines with joint resistance to TSWV and aflatoxin. Additionally, the parents and progeny from these crosses have served as the genetic raw materials to build mapping populations for genetic linkage maps and to develop the EST (expressed sequence tags) database crucial to the development of markers and the discovery of genes that confer resistance and regulate disease and stress responses. ARS scientists at Tifton, GA, have been leading in peanut EST development. Overall, we have released 21,777 seed ESTs and 16,931 leaf ESTs and identified several genes associated with disease resistance in peanut.
Preharvest aflatoxin contamination of developing corn kernels is induced by Aspergillus flavus and is a chronic problem in southern corn production. Research is focused primarily on improving resistance and managing the secondary risk factors associated with this carcinogenic toxin production. Both genomic and classical plant breeding techniques are used to accomplish this assignment. In the last few years, two aflatoxin resistant corn inbreds have been released including GT-601 and GT-602, and GT-P50 is scheduled for final assessment and release in 2009-2010. These inbreds are currently included in an aggressive test hybrid development program with both public and proprietary inbreds, and at least one cooperative hybrid is in pilot production. Corn proteomics and microarray technology are also in use to examine gene expression profiles in the developing kernels under infection and water stress. The expressions of several disease resistance-related genes have been compared among diverse corn lines with water stress and without water stress.
Drought-stress responding genes in corn and resistance to aflatoxin contamination. Throughout the world, aflatoxin contamination is considered one of the most serious food safety issues concerning health. Chronic problems with preharvest aflatoxin contamination occur in the southern U.S. Drought stress is a major factor to contribute to preharvest aflatoxin contamination. Recent studies have demonstrated that higher concentration of defense or stress-related proteins was produced in corn kernels of resistant genotypes compared with susceptible genotypes, suggesting that preharvest field condition (drought stress or no stress) influences gene expression differently in different genotypes resulting in different concentrations of the “end products", disease resistance proteins in the mature kernels. Because of the complexity, better understanding of the mechanisms of genetic resistance will be needed using genomics and proteomics techniques for crop improvement. Genetic improvement of crop resistance to drought stress is one component and will provide a good perspective on the efficacy of control strategy. Proteomic comparisons of corn kernel proteins between resistant or susceptible genotypes have identified stress-related proteins along with antifungal proteins associated with corn kernel resistance. Gene expression studies in corn developing kernels are in agreement with the proteomic studies that defense-related genes could be up- or down-regulated by abiotic stresses and could be correlated with postharvest aflatoxin contamination.
Guo, B., Yu, J., Holbrook Jr, C.C., Cleveland, T.E., Nierman, W.C., Scully, B.T. 2009. Strategies in prevention of preharvest aflatoxin contamination in peanuts: Aflatoxin biosynthesis, genetics and genomics. Peanut Science. 36:11-20.
Guo, B., Chen, Z., Lee R.D., Scully B.T. 2008. Drought stress and preharvest aflatoxin contamination in agricultural commodity: Genetics, genomics and proteomics. Journal of Integrative Plant Biology. 50(10):1281-1291.
Guo, B.Z., Liang, X., Chung, S.Y., Maleki, S.J. 2008. Proteomic screening points to the potential importance of Ara h 3 basic subunit in allergenicity of peanut. Inflammation & Allergy - Drug Targets. 7(3):163-166
Guo, B., Chen, X., Hong, Y., Liang, X., Dang, P.M., Brenneman, T., Holbrook Jr, C.C., Culbreath, A. 2009. Analysis of gene expression profiles in leaf tissues of cultivated peanuts and development of EST-SSR markers and gene discovery.International Journal of Plant Genomics 2009:715605, 14 p. DOI:10.1155/2009/715605.
Liang, X., Chen, X., Hong, Y., Liu, H., Zhou, G., Li, S., Guo, B. 2009. Utility of EST-derived SSR in cultivated peanut (Arachis hypogaea L.) and Arachis wild species. BMC Plant Biology. 9:35.