2010 Annual Report
1a.Objectives (from AD-416)
Identify and develop corn germplasm with resistance to Aspergillus flavus infection/aflatoxin contamination and ear-feeding insects and release this germplasm together with information on molecular markers and methodology that will expedite its deployment into commercially available corn hybrids. Specific objectives include the following: (1) determine the effects of indigenous fungal species and ear-feeding insects on A. flavus infection and aflatoxin accumulation in corn grain; (2) identify new sources of corn germplasm with resistance to A. flavus infection and aflatoxin accumulation and/or resistance to damage by southwestern corn borer, fall armyworm, and corn earworm; (3) identify quantitative trait loci, genes, and proteins associated with resistance in corn to A. flavus infection, aflatoxin accumulation, and insect damage; and (4) enhance corn germplasm with resistance to A. flavus infection, aflatoxin accumulation, and insect damage and release germplasm lines as sources of resistance.
1b.Approach (from AD-416)
Objective 1. Determine the effects of indigenous fungal species and ear-feeding insects on A. flavus infection and aflatoxin accumulation in corn grain. Colonization of corn grain is rarely by a single fungal species, but rather a mixture of fungi. Fusarium verticillioides (syn. F. moniliforme) is the most commonly reported fungus infecting corn in the USA, and it is frequently found together with A. flavus. Acremonium zeae is a common contaminant of preharvest corn in the Southeast. It has been reported to suppress growth of both A. flavus and F. verticillioides in laboratory experiments. The interactions of these fungi will be investigated to determine whether F. verticillioides and A. zeae affect A. flavus infection of corn grain and the subsequent accumulation of aflatoxin, and if so, whether these fungi are impediments to the identification of aflatoxin-resistant corn germplasm. The association between insect damage and aflatoxin accumulation in different corn genotypes will be investigated and the extent to which resistance to damage by southwestern corn borer, Diatraea grandiosella; fall armyworm, Spodoptera frugiperda; or corn earworm, Helicoverpa zea, reduces aflatoxin contamination will be determined. Objective 2. Identify new sources of corn germplasm with resistance to A. flavus infection and aflatoxin accumulation and/or resistance to damage by southwestern corn borer, fall armyworm, and corn earworm. Corn germplasm from diverse backgrounds will be screened for resistance to A. flavus/aflatoxin, southwestern corn borer, fall armyworm, and corn earworm. Information on the effects of other fungi or insects on A. flavus/aflatoxin accumulation (Objective.
1)will be used to refine and improve techniques for evaluating germplasm for resistance. Newly identified sources of resistance will be used to pursue Objectives 3 and 4. Objective 3. Identify quantitative trait loci, genes, and proteins associated with resistance in corn to A. flavus infection, aflatoxin accumulation, and insect damage. Populations of F2:3 families and recombinant inbred lines derived from crosses between aflatoxin or insect resistant inbred lines and susceptible lines will be used to identify quantitative trait loci (QTL) associated with resistance. Resistant and susceptible corn inbred lines and recombinant inbred lines will be used in complementary investigations to identify candidate genes and proteins associated with resistance. Molecular markers identified in these investigations will be used in developing improved germplasm lines (Objective 4). Objective 4. Enhance corn germplasm with resistance to A. flavus infection, aflatoxin accumulation, and insect damage and release germplasm lines as sources of resistance. Both breeding methods based on phenotypic performance and those based on molecular markers will be used to enhance germplasm with resistance to aflatoxin contamination and insect damage. The effectiveness of molecular markers based on QTL, genes, and proteins identified in Objective 3 in transferring resistance to A. flavus/aflatoxin and insect damage into germplasm lines with desirable agronomic qualities will be determined.
Germplasm accessions obtained through the Germplasm Enhancement of Maize (GEM) project and from the International Maize and Wheat Improvement Center (CIMMYT) were evaluated for resistance to aflatoxin accumulation in 2009. The lines exhibiting the lowest levels of aflatoxin were retained for inclusion in the breeding program. Hybrids were produced by crossing lines with different levels of resistance to Aspergillus (A.) flavus/aflatoxin to Bacillus thuringiensis (BT) and non-BT versions of proprietary lines. The hybrids were evaluated for aflatoxin accumulation and corn earworm damage in Mississippi, Georgia, and North Carolina. Damage from ear-feeding insects differed significantly among locations. Aflatoxin accumulation was generally lower in BT hybrids than in their non-BT versions; however, the interaction of inbred lines and BT/non-BT testers was not significant. A panel of 300 diverse lines from U.S. breeding programs and the CIMMYT was chosen for an association mapping project to identify genes associated with reduced aflatoxin accumulation. These lines were crossed with Va35, a susceptible line; the testcrosses were evaluated for A. flavus infection, aflatoxin accumulation, and corn earworm damage in field tests conducted in two locations in Mississippi and two locations in Texas (6406-21000-011-07S and 6406-21000-09S) in 2009 and again in 2010. The results should be useful in identifying genes and groups of genes associated with resistance to A. flavus/aflatoxin and in determining which lines possess unique genes for resistance. A diallel cross produced from eight parental inbred lines varying in level of resistance to aflatoxin accumulation was evaluated for Aspergillus ear rot, aflatoxin accumulation, and A. flavus biomass. Biomass was determined by quantitative real-time polymerase chain reaction (q-RT-PCR). A. flavus biomass and aflatoxin concentration were highly correlated (r = 0.90) in corn grain produced in the 2-year field trial. Estimates of general combining ability (GCA) effects for germplasm lines developed and released as sources of resistance to aflatoxin accumulation (Mp313E, Mp715, Mp717) indicated that these lines, when used in hybrids, contribute to both reduced aflatoxin and reduced fungal biomass. Experiments to identify quantitative trait loci (QTL) for resistance to A. flavus/aflatoxin in Mp313E, Mp715, and Mp717 provided valuable information on molecular markers for resistance to aflatoxin accumulation in corn. Additional genetic analyses and fine mapping of the QTL reduced their size and improved the identification of useful molecular markers for resistance to A. flavus/aflatoxin. The refined estimates of QTL associated with resistance are being used to develop near-isogenic lines from backcrosses of Mp313E, Mp715, and Mp717 to B73, T173, and Va35. Several lines selected from crosses between Mp313E × Va35 and Mp715 × Va35 exhibited not only high levels of resistance to aflatoxin accumulation, but also desirable agronomic qualities in several environments. Confirmation of QTLs associated with resistance to aflatoxin accumulation in the lines is underway.
Identification of Quantitative Trait Loci (QTL) for Resistance to Aflatoxin Accumulation in Corn. Aflatoxin contamination of corn grain, especially in the Southeast, is a serious food and feed safety problem that can result in major economic losses. Growing corn hybrids with genetic resistance to Aspergillus flavus infection and the subsequent accumulation of aflatoxin is widely considered the best way to reduce these losses, but sources of resistance are limited. ARS scientists at Mississippi State have identified, developed, and released corn germplasm lines with resistance to aflatoxin accumulation. Nine QTL, or chromosomal regions, associated with resistance were identified in three of the resistant lines: Mp717 (1 QTL on Chromosome 7), Mp715 (4 QTL on Chromosomes 3, 5, 7), Mp313E (4 QTL on Chromosomes 2, 3, 4). Some QTL were initially quite large and included up to half the chromosome, but re-mapping reduced the portion of the genome included in the QTL by almost a half. This will permit the use of these QTL to transfer resistance to aflatoxin accumulation through marker assisted selection into elite germplasm lines and ultimately into commercial corn hybrids while reducing the chances of transferring undesirable characteristics from the resistant donor lines.
Warburton, M.L., Setimela, P., Franco, J., Cordova, H., Pixley, K., Banziger, M., Dreisigacker, S., Bedoya, C., Macrobert, J. 2010. Toward a Cost-Effective Fingerprinting Methodology to Distinguish Maize Open-Pollinated Varieties. Crop Science. 50:467-477.
Shivaji, R., Camas, A., Ankala, A., Engelberth, J., Tumlinson, J.H., Williams, W.P., Wilkinson, J.R., Luthe, D.S. 2010. Plants on Constant Alert: Elevated Levels of Jasmonic Acid and Jasmonate-Induced Transcripts in Caterpillar Resistant Maize. Journal of Chemical Ecology. 36:179-191.
Windham, G.L., Hawkins, L.K., Williams, W.P. 2010. Aflatoxin Accumulation and Kernel Infection of Maize Hybrids Inoculated with Aspergillus flavus and A. parasiticus. World Mycotoxin Journal. 3:89-93.
Ankala, A., Luthe, D.S., Williams, W.P., Wilkinson, J.R. 2009. Integration of Ethylene and Jasmonic Acid Signaling Pathways in the Expression of Novel Maize Defense Protein Mir1-CP. Molecular Plant-Microbe Interactions. 22:1555-1564.
Mideros, S.X., Windham, G.L., Williams, W.P., Nelson, R.J. 2009. Aspergillus flavus Biomass in Maize Estimated by Quantitative Real-Time Polymerase Chain Reaction is Strongly Correlated with Aflatoxin Concentration. Plant Disease. 93:1163-1170.
Yan, J., Yang, X., Shah, T., Sanchez-Viellda, H., Li, J., Warburton, M.L., Zhou, Y., Jonathan, J., Xu, Y. 2010. High-throughput SNP Genotyping with the GoldenGate Assay in Maize. Molecular Breeding. 25:441-451.
Fu, Z.Y., Yan, J.B., Zheng, Y.P., Warburton, M.L., Crouch, J.H., Li, J.S. 2010. Nucelotide Diversity and Molecular Evolution of the PSY1 Gene in Zea mays Compared to some Other Grass Species. Theoretical and Applied Genetics. 120:709-720.
Li, Q., Yang, X., Bai, G., Warburton, M.L., Mahuku, G., Gore, M., Dai, J., Li, J., Yan, J. 2010. Cloning and Characterization of a Putative GS3 Ortholog Involved in Maize Kernel Development. Theoretical and Applied Genetics. 120:753-763.
Williams, W.P., Windham, G.L. 2009. Diallel Analysis of Fumonisin Accumulation in Maize. Field Crops Research. 114:324-326.
Yan, J., Shah, T., Warburton, M.L., Buckler IV, E.S., McMullen, M.D., Crouch, J. 2009. Genetic Characterization and Linkage Disequilibrium Estimation of a Global Maize Collection Using SNP Markers. PLoS One. 4:1-14.
Henry, W.B., Williams, W.P., Windham, G.L., Hawkins, L.K. 2009. Evaluation of Maize Inbred Lines for Resistance to Aspergillus and Fusarium Ear Rot and Mycotoxin Accumulation. Agronomy Journal. 101:1219-1226.
Petthambaran, B., Hawkins, L.K., Windham, G.L., Williams, W.P., Luthe, D.S. 2009. Anti-fungal Activity of Maize Silk Proteins and Role of Chitinases in Aspergillus flavus Resistance. Journal of Toxicology Toxins Reviews. 29:27-39.
Kelley, R.Y., Williams, W.P., Mylroie, J.E., Boykin, D.L., Hawkins, L.K., Windham, G.L., Brooks, T.D., Bridges, S.M., Scheffler, B.E., Wilkinson, J.R. 2009. Genomic Profile of Maize Response to Aspergillus flavus Infection. Toxin Reviews. 28:129-141.
Williams, W.P., Buckley, P.M. 2008. Fall Armyworm (Lepidoptera: Noctuidae) and Southwestern Corn Borer (Lepidoptera: Noctuidae)Leaf Feeding Damage and Its Effect on Larval Growth on Diets Prepared from Lyophilized Corn. Journal of Agricultural and Urban Entomology. 25:1-11.
Matthews Jr., G.A., Williams, W.P., Daves, C.A. 2009. Diallel Analysis of Corn Earworm (Lepidoptera: Noctuidae) Resistance in Maize. Journal of Agricultural and Urban Entomology. 24:59-66.