1a.Objectives (from AD-416):
Identify genes and proteins that enhance resistance to Aspergillus (A.) flavus infection and aflatoxin accumulation in maize: (1) isolate and determine functions of genes associated with resistance to A. flavus infection and aflatoxin accumulation in maize; (2) compare expression patterns of genes associated with resistance in maize cultivars with varying levels of resistance or susceptibility to A. flavus/aflatoxin; (3) determine abiotic and biotic factors that affect the expression of genes associated with resistance to A. flavus/aflatoxin; and (4) Evaluate mobile Fourier Transform Infrared for (FT-TR) determining presence of toxigenic and atoxigenic strains of Aspergillus flavus.
1b.Approach (from AD-416):
Maize cultivars with varying levels of resistance or susceptibility to A. flavus infection and aflatoxin accumulation will be grown in field plots. Developing ears will be inoculated with toxin-producing and non-toxin-producing strains of A. flavus at predetermined times after pollination, and tissue will be collected from both inoculated and non-inoculated ears at various intervals throughout the growing season. Plants will be monitored for response to drought and heat stress. Maize and fungal proteins, RNA, and DNA will be extracted from tissue samples. Expression profiles of different tissues collected at various times from resistant and susceptible maize genotypes grown under different conditions will be compared using electrophoresis, microarrays, micro-RNA, and other appropriate proteomic or genomic techniques. Genes and proteins identified in these studies will be developed as molecular markers for enhancing resistance to aflatoxin accumulation in maize hybrids.
Resistance to Aspergillus flavus infection and the subsequent accumulation of aflatoxin in corn are affected by many quantitative trait loci (QTLs) and genes with variable effects. To successfully use molecular markers to transfer resistance to aflatoxin accumulation through breeding, genes controlling resistance must be identified and their individual and cumulative effects quantified. A primary objective of the research included screening a bacterial artificial chromosome (BAC) library of the resistant corn inbred line Mp313E and squencing the BAC clones associated with resistance related genes, proteins, and deoxyribonucleic acid (DNA) markers. To characterize host resistance in corn inbred line Mp313E, genes and DNA markers associated with corn host resistance to Aspergillus flavus infection and aflatoxin accumulation were used to design probes for BAC library screening. Single nucleotide polymorphisms (SNPs), which are highly informative DNA markers, were identified. Fine-mapping of major QTLs and SNP analysis of genome-wide association studies (GWSD) were initiated and will be further improved. The goal of this strategy is to capture the majority of polymorphisms that contribute to the resistance of the quantitative trait. The preliminary data obtained from screening the Mp313E BAC library demonstrated that this technique can be used as a means of identifying and sequencing chromosome regions related to resistance to aflatoxin accumulation. Screening of the library with probes designed from the resistance simple sequence repeat (SSR) marker bnlg2291 identified BAC clones carrying the targeted chromosomal regions. The selected BAC clones were evaluated by end-sequencing and interval polymerase chain reaction (PCR) product sequencing. Comparison of targeted regions from Mp313E to the homologous sequence from B73 revealed abundant SNP markers. Transcriptomic and proteomic profiling of genes and proteins from resistant corn inbred line Mp715 and recombinant inbred lines Mp718 and Mp719 was performed under inoculated and non-inoculated conditions in multiple field environments. Corn inbred line Va35 was used as the susceptible check. Mp718 and Mp719 were derived from Mp715 × Va35. Major computational methods were developed to accommodate the requirement of high throughput data analysis of quantitative real-time polymerase chain reaction (qRT-PCR) data. A nuclear pore complex (NPC) protein found to be involved in the host resistance in Mp313E was also found to be highly expressed in Mp718. The function of NPCs is transport of ribonucleic acid (RNA) and other macromolecules from the nucleus to the cytoplasm. Furthermore, 50 genes selected from the RNA transport pathway were investigated by qRT-PCR. Two-dimensional (2D) protein gel electrophoresis revealed differences in proteomic profiles under different treatments. Differentially expressed proteins across multiple samples were identified and quantitatively evaluated by computational analysis of sets of 2D protein gel images. Resistant inbred lines Mp715 and Mp719 and susceptible inbred lines Va35, Mp04:87 were selected for this study. Developing kernels were collected from the primary ears of corn plants at 14 days after inoculation with Aspergillus flavus. Proteins were extracted, and 2-D protein gel electrophoresis was performed using. Proteins were visualized with florescent dye. A working protocol for quantitative proteomic studies of corn proteins by computational and statistical analysis of 2D protein gel images was developed. This analysis provides a novel procedure for profiling and comparing differentially expressed proteins that will facilitate development of DNA markers for maize resistance breeding.