2013 Annual Report
1a.Objectives (from AD-416):
The objective of this project is to identify and characterize new inbred lines displaying high and stable resistance to A. flavus and aflatoxin accumulation and to identify and validate new genes and gene-based markers linked to these traits. Germplasm and genes will be ready for use in practical breeding programs to develop resistant varieties for developing world farmers within three years. Inbred lines will be characterized for aflatoxin and A. flavus resistance in growing conditions typical of African and Latin American farmers. Specific and general combining ability will be tested, and information on performance of the lines in hybrid combinations in different environments will be made publically available to maize breeders and small seed companies. Genes for resistance will be identified via association analysis or QTL mapping, and all identified sequences will be run in an independent mapping (QTL or association) population for validation.
1b.Approach (from AD-416):
Considerable work has already been done in the mapping of aflatoxin and A. flavus resistance in maize, and five fully phenotyped and genotyped QTL mapping populations and two association mapping panels are currently available for this project. These resources will allow many gene sequences identified as associated with aflatoxin or A. flavus resistance in one panel to be validated in the other panel as well. Any candidate gene of large phenotypic effect identified via association mapping will be used to develop user-friendly SNP, SSR, or InDel markers from the sequence of the gene. These will allow for the mapping of these genes in any of the QTL mapping populations that segregate for these sequences. We hope that this will identify new QTLs, or allow the immediate fine mapping of previously identified QTLs, and will be further independent validation of the gene(s) as associated with fungus or toxin resistance. QTL mapping will also verify the utility of these markers, which should allow more rapid progress in the ultimate development of resistant hybrids and varieties. In the process of phenotyping the two association mapping panels (one at CHPRRU, one at CIMMYT), most known sources of resistance, and many new potential resistant lines, will be well characterized in a comprehensive manner. New inbred lines with high levels of stable resistance should be identified in the course of this work. These new resistant lines (the best 20 – 30 from each panel), will be field tested in various hybrid combinations and tested for enhanced resistance to aflatoxin accumulation in new hybrids. These hybrids can be used as cultivars for farmers in parts of the world where aflatoxin poisoning has been an ongoing and serious problem. In the first year, aflatoxin resistant inbred lines from CHPRRU will be test crossed onto CIMMYT elite breeding lines and seed will be increased of the inbred lines. The association mapping panel from CIMMYT will be phenotyped (for a second year, as it has been phenotyped in one year already). Association mapping will begin. In the second year, the test crosses made in year one will be phenotyped for yield and disease resistance under local growing conditions, and association mapping will continue, particularly to verify genes identified in the CHPRRU panel. In the third year, the testcrosses will be phentypted again.
The primary objective of this project is to transfer corn germplasm with resistance to Aspergillus flavus infection and aflatoxin accumulation to plant breeders in developing countries and to facilitate integration of resistance into their breeding projects. New sources include new inbred lines displaying high and stable resistance to Aspergillus flavus and aflatoxin accumulation and also new genes and gene-based markers linked to these traits for the faster incorporation of resistance into ongoing breeding programs. Inbred lines displaying high and stable resistance to A. flavus and aflatoxin accumulation in the field tests conducted by ARS scientists at Mississippi State, Mississippi, will be evaluated for adaptation to African growing conditions and disease pressures. Identification of new resistant inbred lines was accomplished during the phenotypic characterization of an association mapping panel of 300 inbred lines of corn. The association mapping panel was created by ARS scientists at Mississippi State, Mississippi, and included 300 diverse inbred lines. Some lines were selected because of their known resistance to aflatoxin contamination; some were selected from environments where Aspergillus flavus is often epidemic; and others were selected to add genetic diversity to the panel. The lines were crossed with a susceptible inbred line that is adapted to the southern United States. The resulting testcrosses were grown in replicated field trials for two years. Grain samples collected from the field trials were analyzed to determine concentration of aflatoxin. The 30 most consistently resistant lines of the panel were identified; seed were increased in the ARS 2012-2013 winter nursery and sent to colleagues at the International Maize and Wheat Improvement Center (CIMMYT) in Mexico. Inbred lines have been planted to increase seed in Mexico and testcrosses have already been generated using two white and two yellow testers of different heterotic groups adapted to Mexican tropical and subtropical growing conditions. The testcrosses will be characterized for resistance to Aspergillus flavus infection and aflatoxin accumulation under growing conditions typical of Latin American farmers. General and specific combining ability will be evaluated, and information on performance of the lines in hybrid combinations in different environments will be made publicly available to corn breeders and small seed companies. A searchable database was created from the genotypic data (created by re-sequencing the genomes of the 300 inbred lines) that was completed prior to initiation of the project. The searchable database of these genomic sequences was created so that all collaborators can examine differences in genes of interest and run association analyses to confirm their effects on resistance to aflatoxin contamination. Candidate gene association analysis has been completed for 30 candidate genes to date: six genes were positively associated. Whole genome scanning has been completed, and 22 genes were positively associated with resistance. Sequences of the 22 genes found to be associated with resistance by whole genome scanning and the 6 genes found to be associated by candidate gene analysis are being used to create user friendly molecular markers. These gene sequences have been passed on to cooperators at CIMMYT who developed an independent association mapping panel. Phenotyping and genotyping of this second panel is nearly complete. The 22 plus 6 genes identified by ARS at Mississippi State, MS, will be added to the genotyping to ensure these genes are analyzed for association in the independent CIMMYT panel to validate their role in resistance to aflatoxin contamination.