Pre-harvest contamination of maize grain with aflatoxin is a chronic problem worldwide and particularly in the southeastern U.S. Aflatoxin is a mycotoxin produced by the fungus Aspergillus flavus, an opportunistic ear-rot pathogen of maize (Zea mays). Resistance to aflatoxin accumulation is heritable, and resistant germplasm-lines are available. These lines are derived from “exotic” genetic backgrounds and were released as sources of resistance, not parental inbreds. However, all current sources of resistance are quantitative, which complicates conventional efforts to introgress resistance alleles from unadapted but resistant donor lines to adapted but susceptible recipient lines. Mapping quantitative trait loci (QTL) and their linked markers enables targeted introgression of the desired alleles via marker-assisted selection. Quantitative trait loci were identified in two F2:3 mapping populations, derived from crossing resistant inbreds Mp715 and Mp717 to a common susceptible parent (Va35). The Mp715 x Va35 population was phenotyped for aflatoxin accumulation under artificial inoculation in replicated field trials at Mississippi State (MSU) in 2015 and 2016. The Mp717 x Va35 population was phenotyped at MSU and Lubbock, TX in 2016. Populations were genotyped using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers and linkage maps created in JoinMap4. To locate QTL, linkage maps, genotypes, and phenotypes were analyzed jointly in QTL Cartographer 2.5 using composite interval mapping (CIM) and multiple interval mapping (MIM) procedures. Five QTL with the beneficial allele contributed by Mp715 were identified during CIM in bins 5.01, 6.06, 7.03 10.04 and 10.05. Three QTL with the beneficial allele contributed by Mp717 were identified during CIM in bins 3.07/3.08, 7.02/7.03, and 10.05. In both populations, QTL were identified with the beneficial allele contributed by Va35. Those QTL did not co-locate across populations but four of the six were on chromosome 1. Significant QTL effects from CIM were used as the initial model terms in MIM, where all QTL effects were fit simultaneously and their gene-action and epistatic interactions estimated.

Maize (Zea mays L.) is one of the world’s largest food crops and thus any pathogens of maize are of great importance. Aspergillus flavus is one of these pathogens and it produces a carcinogenic metabolite called aflatoxin. Efforts to reduce infection by A. flavus and subsequent aflatoxin accumulation include the development of maize lines resistant to aflatoxin accumulation. However, resistant lines that have been developed contain agronomically unfavorable traits. Gene-based markers would allow for easier transfer of resistance from resistant inbred lines into maize lines with good agronomic traits. The focus of this research was the development of gene-based markers for resistance to aflatoxin accumulation. To this end, two genes were characterized for their association with reduced aflatoxin accumulation in maize. A gene coding for a photosytem II3 protein shown to be differentially regulated between maize lines Mp313E (resistant) and Va35 (susceptible) was used to develop the marker MpM1. This marker was shown to be associated with resistance to aflatoxin accumulation in three F2:3 mapping populations derived from Mp313E x B73, Mp313E x Va35, and Mp715 x T173 and identified a new quantitative trait locus (QTL) on chromosome 4. The second gene chosen was the chitinase A gene (chiA), which has been shown to inhibit fungal growth and is differentially regulated between resistant and susceptible lines of maize. ChiA also had an association with reduced aflatoxin accumulation in the three F2:3 mapping populations and identified a new QTL in the Mp313E x Va35 population. Together, MpM1 and chiA were associated with 27% of the phenotypic variation in one environment of the Mp313E x B73 population. These markers represent the first two gene-based markers developed for resistance to aflatoxin accumulation, and the methodology developed in this study can be used to screen other candidate genes for potential use as gene-based makers.

Screening for resistance to Aspergillus flavus and aflatoxin contamination in maize is an ongoing effort by universities, state and federal agencies. We evaluated two techniques to screen for resistance; quantitative polymerase reaction (QPCR) and solid-phase microextraction (SPME). Methods were adapted to accurately detect and quantify the fungus in culture and in the vegetative stage of plant tissues. These assays can eliminate microbiological techniques. The primary objectives of the study were to utilize 1) QPCR to detect and quantify fungal biomass in maize stem tissues to evaluate resistance in maize genotypes to A. flavus colonization in situ and in vivo and 2) SPME to identify key MVOC’s to differentiate aflatoxigenic and non-aflatoxigenic strains of A. flavus in situ. A novel QPCR TaqMan probe (OMG3) was designed to detect a region in the aflP gene. The OMG3 probe detected 98.3% of the aflatoxigenic strains. The predominant MVOC’s extracted from both aflatoxigenic and non-aflatoxigenic strains were alcohols, ketones and hydrocarbons. The aflatoxigenic strain produced 39 compounds and the non-aflatoxigenic strain produced 41 compounds. Dimethylsulfide and 2-heptanol were key MVOC biomarkers produced only by the aflatoxigenic strain of A. flavus. Accuracy of the QPCR OMG3 probe, in vivo and in situ procedures were developed. A toothpick inoculation method was used to artificially inoculate maize stems in the vegetative stage five (V5). Plants were harvested at V7 and sampled at predetermined sites. This method was 91% consistent for infecting maize plants. The OMG3 probe was evaluated in in vivo and in situ studies conducted in the greenhouse, growth chamber, and field. Lesion length was greater in susceptible lines in 4 of 7 greenhouse trials. Based on inoculation data, subsequent research should focus on refining tissue-sampling methods and increasing length of plant growth time for tissue sampling post-inoculation.

This study aimed to understand the mechanism of maize inbreds resistance to A. flavus by exploring the proteins that are differentially regulated in presence of pathogen. Silk has been hypothesized as one of the entry routes of fungal growth and so the proteome of silks was investigated by 1) performing a comparative proteomic study to identify silk proteins that are abundant in resistant maize inbreds and down-regulated or absent in susceptible inbreds, 2) identifying the up-regulated proteins in maize resistant and susceptible inbreds when challenged by A. flavus 3) by mapping the proteome of silk proteins in a A. flavus resistant inbred and 4) performing an antiungal assay to test antiungal activity of silk proteins extracted from resistant and susceptible maize inbreds. Using comparative proteomics, proteins that are contributing to the resistance phenotype and could be used for marker-assisted selection in breeding programs were identified from silks collected from resistant (Mp313E, Mp420) and susceptible (SC212m, Mp339) maize inbred 21 and 25 days after silk emergence (DAS) and also, from the silks of ears inoculated at 15 DAS and collected 6 days after inoculation (DAI). Silk proteins were extracted and analyzed by 2-dimensional gel electrophoresis (2-DE). Gel images were analyzed by PD Quest software (Bio-Rad) and proteins that were consistently different were identified using MALDI-TOF-TOF. Two candidate genes that were up-regulated in 21 and 25 DAS in resistant tissues were investigated for polymorphisms and their RNA expression was also studied. Nine proteins from all the differentially regulated proteins were mapped to chromosomes 1, 2, 4 and 6 which are known to have aflatoxin resistance QTLs. Proteome map of Mp313E silks was developed using 2-DE and multi dimensional identification technology (MudPIT) and approximately 971 identified proteins were functionally annotated from the sequences available at AgBase website. The reference map of Mp313E silks could also be used to link proteomics with trancriptomics, metabolic mechanisms and genomics. Antifungal assays using GFP-tagged A.flavus and chitinase assay on silk proteins from resistant and susceptible corn inbreds showed significant activity in the resistant line compared to the susceptible line (p<0.01). A model describing the role of silk proteins in fungal resistance is proposed.

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Public agricultural research has been conducted in Africa for decades and has generated numerous crop technologies, while little is understood on how agricultural research affects the poor and vulnerable groups such as children, and how farmers' perceptions affect their adoption decisions. This dissertation helps fill this gap with three essays on adoption and impacts of improved maize varieties in rural Ethiopia. The first essay estimates poverty impacts. Field-level treatment effects on yield and cost changes with adoption are estimated using instrumental variable techniques, with treatment effect heterogeneity fully accounted for in marginal treatment effect estimation. A backward derivation procedure is then developed within an economic surplus framework to identify the counterfactual income distribution without improved maize varieties. Poverty impacts are estimated by exploiting the differences between the observed and counterfactual income distributions. Improved maize varieties have led to 0.8-1.3 percentage drop in poverty headcount ratio and relative reductions in poverty depth and severity. However, poor producers benefit the least from adoption due to their small land holdings. The second paper assesses the impacts on child nutrition outcomes. The conceptual linkage between maize adoption and child nutrition is first established using an agricultural household model. Instrumental variable (IV) estimation suggests the overall impacts to be positive and significant. Quantile IV regressions further reveal that such impacts are largest among the most severely malnourished. By combining a decomposition procedure with estimates from a system of equations, it is found that the increase in own-produced maize consumption is the major channel such impacts occur. The third paper explores how farmers' perceptions of crop traits affects their willingness to adopt improved maize varieties. Under a random utility framework, a mixed logit procedure is implemented to model farmer's adoption intention, where perceptions of key varietal traits are first identified, and then instrumented using a control function approach to account for potential endogeneity. Perceived yield is found to be the most important trait affecting farmers' adoption intention. Further, yield perceptions among previous adopters appear to be affected by within-village peer effects rather than the real crop performance. / Ph. D.

Doctor of Education / Department of Educational Leadership / Mary Devin / David C. Thompson / School districts throughout the United States have continuous concerns about how to meet the needs of high school students who are at-risk of dropping out of school. Despite multiple resources available for addressing this concern through alternative education schools and programs, there continues to be an unacceptably high number of students who do not graduate from high school. While knowledge about what is working in alternative schools and the students they serve is progressing, additional research is still needed. In Maize USD 266, Complete High School Maize is an award-winning and nationally recognized dropout prevention program that has, over a fifteen year span (1999-2014), helped reduce the dropout rate in Maize. The intent of this qualitative historical analysis was to serve two purposes. First, it delineated the historical evolution of Complete High School Maize as a model for school districts to emulate in an effort to reduce the number of high school dropouts. Secondly, this study provided historical documentation to help preserve and share in the history of the program for future generations of students. This study examined the factors and circumstances present in Maize USD 266 that resulted in the creation of an alternative school for its students. This study also narrated the structural evolution of Complete High School Maize from 1999 to 2014 and examined the conditions and factors that resulted in the progressions. The findings of the study showed that Complete High School Maize had successfully helped reduce the number of dropouts in Maize USD 266.

An in depth review is given of Host Plant Resistance research on borers in general, and on B. fusca in particular. The general biology and economic importance of B. fusca are also discussed. Several aspects of the general methodology of breeding for resistance to B. fusca are discussed. These topics include collection of overwintering larvae, termination of diapause, field infestation methodology and damage assessment. Approximately 20 first instar larvae were applied to the plant whorl when plants were about 30 - 40 cm tall. The primary method of damage assessment was to evaluate leaf damage on a 1 to 5 scale. Further criteria for selection of resistant germplasm are assessment of stunting due to stem boring and yield at harvest. The effect of plant resistance on B. fusca was investigated. There were clearly defined differences in leaf damage evident between different maize genotypes. Heritability of this resistance was demonstrated, and presumed to be an additive mechanism that reduced insect feeding, indicating antibiosis. There were significant differences between the number of larvae recovered from whorl tissue of different cultivars. This was ascribed to two resistance mechanisms exerting their effects within the first few days' feeding by larvae. One mechanism was short lived, but effective, antibiosis resulting in larval death, while the other, also short lived, was repellence, resulting in larval migration. Both mechanisms resulted in fewer larva feeding in the plants. Another longer lasting resistant mechanism affected larval growth and mass gain, resulting in smaller larvae. These mechanisms were found to be heritable traits. Differences in levels of resistance affecting larval mass gain were also determined for various parts of the tassel. For all inbreds, the peduncles were more susceptible than the tassel Cultivars also differed in the levels of resistance in the stern tissue. Resistance in leaf tissue did not necessarily mean that resistance occurred in the stern of that genotype. Some cultivars had resistance mechanisms present in both leaves and sterns, some had only one resistance mechanism in either part, and some were totally susceptible. The effect of the borer on the plant was investigated. Leaf damage was found to not be of any consequence, but severe stern damage caused extensive yield losses. There was good correlation between leaf damage and stern damage. Yield loss was most pronounced in longer season hybrids than in quick maturing hybrids. Methodologies utilized in the development of inbreds, populations and hybrids are discussed. It was concluded that borer resistant hybrids do have a place in the commercial market. However their performance under conditions of low or no infestation must be similar to that of other susceptible hybrids because control measures for B. fusca are not excessively expensive. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1989.

Quality protein maize (QPM), high protein corn (HPC) and food grade maize (FGM) were processed into tortillas and direct expanded extruded snacks. QPM had similar test weight, density and kernel size with 45% more lysine and 38% more tryptophan compared to FGM. HPC had the largest kernel with density and test weight similar to FGM. During alkaline cooking, HPC absorbed water faster than QPM and FGM. White QPM required shorter cooking time and had less dry matter losses compared to FGM. All corn varieties had excellent pericarp removal at the optimum cooking time. Tortillas from QPM had better pliability and rollability after storage compared to FGM and HPC. HPC tortillas had lower rupture force after storage. The use of QPM for tortilla production may reduce energy and sewage cost, and could produce a tortilla with longer shelf stability with improved nutritional value. Decorticated and non-decorticated QPM, FGM and HPC grain were processed into corn meal and direct expanded snacks. A modified short scale dry milling system was used to produce the corn meal. QPM produced more coarse meal with greater fat content compared to FGM. Decortication decreased fiber content and coarse meal yield. Non-decorticated meal had greater protein, fiber and fat content compared to decorticated meal. The modified short flow milling system provides reduced lost fractions for extrusion into nutritionally improved products. Extrusion was performed in a low cost friction extruder. QPM extruded faster than FGM and HPC. FGM required greater specific mechanical energy than QPM. Extrudates from FGM were the most expanded followed by QPM and HPC. Extrudates from the three corn varieties were acceptable to the panelists and decortication did not affect acceptability. The improved nutritional value of QPM, was retained during dry milling and extrusion. Current QPM varieties can be processed into tortillas with longer shelf stability and meal for extrusion into a wide variety of snacks and other foods. These may have application in specialty health foods and in developing countries where maize is a staple food.

The protein quality of maize can be improved by replacing normal Opaque2 alleles with non-functional recessive alleles opaque2 (o2). The allele o2 produces a severe phenotype with soft endosperm enhancing its protein quality but decreasing its agronomical value. Plant breeders have restored a desirable ratio of hard to soft endosperm in o2 germplasm known as Quality Protein Maize (QPM). Neither the mechanism nor the genetic components by which the modification of the endosperm in QPM lines occurs are well understood. To increase the understanding of the genetics of endosperm modification, a population of 146 recombinant inbred lines derived from a cross between the o2 inbred line B73o2 and the QPM inbred line CML161 was evaluated in two Texas locations from 2004 to 2006. Four traits related to endosperm texture were measured and showed significant effect of the inbred lines, high heritability estimates and high genetic correlations. Relative content of the essential amino acids lysine, tryptophan and methionine were measured and showed significant effects of the lines and considerable high genetic correlations and heritabilities. Negative correlation was observed between endosperm texture traits and amino acid content. Quantitative trait loci (QTL) were mapped for traits related to the modification of endosperm texture and the content of lysine, tryptophan and methionine. QTLs clusters for endosperm texture traits were detected on chromosomes 3, 5, 6 and 8 explaining 62-68% of the variation. QTLs clusters for amino acid contents were located on chromosomes 7 and 8 that explained up to 39% of the observed variation. The product of the O2 gene is a transcription factor that affects the expression of a number of endosperm genes. A group of 29 endosperm genes associated with the O2 activity were evaluated in developing endosperm of the recombinant inbred lines. Genomic regions controlling gene transcript abundance in developing endosperm were identified by expression QTL mapping. Evidence is presented of QTL hot spots that segregate in association with endosperm texture modification or amino acid contents and are associated with the regulation of the expression of a group of endosperm genes.