Location: Corn Host Plant Resistance Research
Project Number: 6064-21000-015-001-I
Project Type: Interagency Reimbursable Agreement
Start Date: Sep 30, 2013
End Date: Sep 30, 2022
Objective:
Identify maize germplasm with resistance to Aspergillus flavus infection and subsequent aflatoxin accumulation and develop and release this germplasm together with information on molecular markers and breeding methodology that will expedite deployment of maize inbred lines, hybrids, and synthetic varieties with resistance to aflatoxin contamination for the USA, Africa, and Mexico. Specific objectives include: (1) validate putative genes for resistance identified in past association and quantitative trait loci (QTL) mapping experiments, (2) use RNA interference (RNAi), synthetic peptides, and transgenic lines to identify and interfere with fungal growth, (3) produce near isogenic lines (NILs), recombinant inbred lines (RILs), and transgenic lines to validate newly identified genes for resistance to aflatoxin contamination, (4) characterize protein products of gene exhibiting expression correlated with resistance, (5) identify germplasm with resistance to Aspergillus flavus infection and aflatoxin accumulation, and (6) develop resistant maize varieties and parental inbred lines.
Approach:
Objective 1: Validate putative genes for resistance identified in past association and QTL mapping experiments. Candidate resistance genes will be validated using expression studies via quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Gene expression in known resistant and susceptible lines, inoculated with Aspergillus flavus or water will be monitored. RNA will be extracted from kernels, and gene-specific primers for candidate resistant and housekeeping genes will be designed for the qRT-PCR reaction. Gene expression will be determined, and the effect of Aspergillus flavus inoculation on patterns of gene expression observed over time among the resistant and susceptible genotypes will be compared. Objective 2: Use RNA interference (RNAi), synthetic peptides, and transgenic lines to identify and interfere with fungal growth. Objective 3: Produce near isogenic lines (NILs), recombinant inbred lines (RILs), and transgenic lines to validate newly identified genes for resistance to aflatoxin contamination. Final validation of candidate genes will be through comparison of aflatoxin accumulation in field trials of a maize line that has the positive allele for the candidate gene and a line that differs only in that it has the negative allele for that gene. Any increase in resistance in the line containing the positive allele will be attributed to the positive allele. Lines will be ready for testing of phenotypic effect in replicated field trials in multiple environments in the second or third year of this study. Objective 4: Characterize protein products of gene exhibiting expression correlated with resistance. For genes with insufficient gene annotation data available in the literature or in public databases, protein functional analysis will be performed by heterologous expression of the gene of interest in an expression vector. Purity of the expressed protein will be determined and protein identity will be confirmed. Structural comparison of the expressed proteins will be compared using predictive tools for 3D-protein modeling and by NMR spectroscopy combined with isotopic labeling. Protein function will be determined. Objective 5: Identify germplasm with resistance to Aspergillus flavus infection and aflatoxin accumulation. Current efforts to screen new germplasm accessions from the Germplasm Enhancement of Maize (GEM) project, International Maize and Wheat Improvement Center (CIMMYT), and International Institute of Tropical Agriculture (IITA), southern USA breeding programs, and other sources will continue. Objective 6: Develop resistant maize varieties and parental inbred lines. Resistant lines from CIMMYT, IITA, and USDA will be shared among partners and crossed to local germplasm lines used as testers to determine heterotic patterns within grain color groups. Experimental hybrids will be generated and evaluated for resistance to aflatoxin contamination, local adaptation, and yield. Synthetic populations will be generated as new varieties and breeding population stocks and improved by reciprocal recurrent and genomic selection using molecular markers previously identified.
