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ABSTRACT: Carotenoids, which are pigments known to have many health benefits, such as their antioxidant properties, are being researched for their potential as a feed additive for production animals. These pigments are found in varying quantities in different breeds of corn, and their impact on the chicken microbiome requires further investigation. This 35 d laying hen (Novagen White) feeding trial involved varying the levels and composition of carotenoids by changing the corn source: white (0.9 µg total carotinoids/g total diet), yellow (5.7 µg/g), and orange (24.9 µg/g). For each of the three corn diet treatments, 6 replicate cages were randomly assigned. The cecal microbial community composition of the hens was then studied by 16S rRNA gene amplicon sequencing. The composition of the cecal bacterial community, as determined by Bray-Curtis dissimilarity, was different (P < 0.05) in chickens fed the orange corn diet, compared to chickens on the white corn diet, but there was no statistical difference between animals fed yellow corn compared to the white or orange corn groups. There was no change in the alpha diversity between any of the groups. Within Lactobacillus, which is one of the most abundant genera, 2 amplicon sequence variants (ASVs) were decreased and one ASV was increased in the orange corn group compared to both the white and yellow corn groups. While previous studies showed that orange corn did not alter the community composition in broilers, it appears that orange corn based feed may alter the community composition of laying hens.

SUMMARY: Consumers associate nutritional quality and potential health benefits with yolks showing deeper colors produced by healthy layers fed a well-balanced, nutrient-rich diet. However, cereals and oilseed meals generally used in poultry feed do not provide sufficient carotenoids to achieve deeper yolk colors, thus carotenoid additives may be supplemented. High carotenoid corn genotypes have been developed, and when included in poultry diets increase carotenoid concentrations in yolks. Seven different diets were fed to 210 Novogen White laying hens over 21 d. Each diet had 5 replicates of 6 hens housed in conventional cages. The only difference in diet treatments was percent inclusion rate of orange corn or inclusion of a carotenoid additive (Oro Glo). Yolk color was assessed by colorimeter and DSM YolkFan. Yolk carotenoid concentrations were determined by high performance liquid chromatography. Yolks from hens fed diets with a 100% orange corn inclusion rate had the highest YolkFan scores and total xanthophyll levels at the end of the study. Orange corn inclusion rate showed a linear relationship with colorimeter a* values (r = 0.96) and YolkFan scores (r = 0.90) for egg yolks. Orange corn inclusion of 50 and 75% produced yolks with a* axis color value equivalent to that found in yolks from hens fed 100% yellow corn plus 35 g/kg of Oro Glo. Data indicates that orange corn can be a suitable replacement for carotenoid additives. A 50 to 75% orange corn inclusion could be sufficient to achieve yolk colors similar to diets that involve supplementation with a carotenoid additive.

Footpad dermatitis (FPD), damage and inflammation of the plantar surface of the foot, is of concern for poultry because FPD affects the birds' welfare and production value. Footpad dermatitis is painful and causes costly chicken paw downgrades, carcass condemnations, and reduced live weights. However, a universal preventative has not been found. The hypothesis was that diets containing orange corn, when compared with diets containing yellow or white corn, would reduce the severity of footpad dermatitis in broiler chickens on wet litter. When compared with yellow and white corn, orange corn contains higher quantities of carotenoids, antioxidant pigments, believed to play a role in skin and feather health. This experiment was a randomized block, 3 × 2 factorial design: orange, yellow, and white corn diets with birds raised on wet or dry litter (control group). Female Ross 708 broilers (n = 960) were used to create 4 replicates of each diet x litter treatment combination. Footpads were scored at day 19, 27, 35, and 42, following the Global Animal Partnership standard's 0–2 scale of visual increasing severity: 0 indicates minimal damage and 1 and 2 indicate mild to severe lesions and ulceration, dark papillae, and/or bumble foot. At 42 d of age, birds on the wet litter had greater severity of FPD, scores 1 and 2, compared with the control group (88 vs. 13% respectively; P < 0.0001). At 42 d of age, prevalence of more severe footpad scores, 1 or 2, was lowest on the orange corn diet (33%), followed by white corn (56%) and yellow corn (63%). Birds fed the orange corn diet had higher BW throughout the study (P = 0.004) and had fat pads and livers with higher yellow pigment deposition (P < 0.005). Litter moisture content altered microbiome composition but corn type did not. In conclusion, the main determinant of FPD in this study was exposure to wet litter. When compared with yellow and white corn, orange corn was associated with improved bird growth and reduced severity of footpad dermatitis, especially at later time points.

Abstract  High-oil maize as a product of long-term selection provides a unique resource for functional genomics. In this study, the abundant soluble proteins of early developing germs from high-oil and normal lines of maize were compared using two-dimensional gel electrophoresis (2-DGE) in combination with mass spectrometry (MS). More than 1100 protein spots were detected on electrophoresis maps of both high-oil and normal lines by using silver staining method. A total of 83 protein spots showed significant differential expression (>two-fold change; t-test: P  [ABSTRACT FROM AUTHOR]

Plant breeding has developed corn genotypes with grain higher in levels of carotenoids. Dietary consumption of specific carotenoids by humans has been associated with improved eye health, notably with some protection against age-related macular degeneration. Increasing dietary sources of macular carotenoids in the standard American diet might be accomplished by using high carotenoid Orange Corn in poultry diets to increase macular carotenoid concentrations in egg yolks. Three hundred sixty laying hens (Novogen White) were fed three different diets over 31 days. Each diet had six replicates of 20 hens housed in enrichable colony cages. The only difference was the type of corn included - white, yellow, and orange, in order to assess the impact of each type of corn on egg production, yolk pigmentation, and carotenoid deposition. This study assessed yolk color and carotenoid densities using a portable colorimeter and the DSM YolkFan, and by high performance liquid chromatography (HPLC) on eggs from the feeding study and on 43 cartons of 12 eggs commercially available and produced in various production settings: conventional cage, cage-free, cage-free organic, free-range/pasture, and free-range/pasture organic. Yolks from hens fed with the Orange Corn diet produced eggs with higher (P < 0.01) DSM yolk color (6 to 10) and total xanthophylls (23.5 to 35.3 μg/g of egg yolk) compared to the yellow diet (5 to 6 DSM and 12.3 to 17.7 μg/g xanthophylls) and white diet (1 to 2 DSM and 2.5 to 3.0 μg/g xanthophylls). Egg yolks reached a maximum xanthophyll accumulation with the Orange Corn diet (35.3 μg/g of egg yolk) after twelve days of treatment and maintained steady levels at subsequent time points. In general, xanthophyll levels in yolks from the Orange Corn diet were superior (30-61% higher) to any of the commercial egg brands, suggesting that feeding high carotenoid Orange Corn increases xanthophyll density in eggs., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Vitamin A deficiency remains prevalent in parts of Asia, Latin America, and sub-Saharan Africa where maize (Zea mays) is a food staple. Extensive natural variation exists for carotenoids in maize grain. Here, to understand its genetic basis, we conducted a joint linkage and genome-wide association study of the US maize nested association mapping panel. Eleven of the 44 detected quantitative trait loci (QTL) were resolved to individual genes. Six of these were correlated expression and effect QTL (ceeQTL), showing strong correlations between RNA-seq expression abundances and QTL allelic effect estimates across six stages of grain development. These six ceeQTL also had the largest percentage of phenotypic variance explained, and in major part comprised the three to five loci capturing the bulk of genetic variation for each trait. Most of these ceeQTL had strongly correlated QTL allelic effect estimates across multiple traits. These findings provide an in-depth genome-level understanding of the genetic and molecular control of carotenoids in plants. In addition, these findings provide a roadmap to accelerate breeding for provitamin A and other priority carotenoid traits in maize grain that should be readily extendable to other cereals., (� American Society of Plant Biologists 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Despite its importance to plant function and human health, the genetics underpinning element levels in maize grain remain largely unknown. Through a genome-wide association study in the maize Ames panel of nearly 2,000 inbred lines that was imputed with ∼7.7 million SNP markers, we investigated the genetic basis of natural variation for the concentration of 11 elements in grain. Novel associations were detected for the metal transporter genes rte2 (rotten ear2) and irt1 (iron-regulated transporter1) with boron and nickel, respectively. We also further resolved loci that were previously found to be associated with one or more of five elements (copper, iron, manganese, molybdenum, and/or zinc), with two metal chelator and five metal transporter candidate causal genes identified. The nas5 (nicotianamine synthase5) gene involved in the synthesis of nicotianamine, a metal chelator, was found associated with both zinc and iron and suggests a common genetic basis controlling the accumulation of these two metals in the grain. Furthermore, moderate predictive abilities were obtained for the 11 elemental grain phenotypes with two whole-genome prediction models: Bayesian Ridge Regression (0.33-0.51) and BayesB (0.33-0.53). Of the two models, BayesB, with its greater emphasis on large-effect loci, showed ∼4-10% higher predictive abilities for nickel, molybdenum, and copper. Altogether, our findings contribute to an improved genotype-phenotype map for grain element accumulation in maize., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Genomic prediction provides an efficient alternative to conventional phenotypic selection for developing improved cultivars with desirable characteristics. New and improved methods to genomic prediction are continually being developed that attempt to deal with the integration of data types beyond genomic information. Modern automated weather systems offer the opportunity to capture continuous data on a range of environmental parameters at specific field locations. In principle, this information could characterize training and target environments and enhance predictive ability by incorporating weather characteristics as part of the genotype-by-environment (G×E) interaction component in prediction models. We assessed the usefulness of including weather data variables in genomic prediction models using a naïve environmental kinship model across 30 environments comprising the Genomes to Fields (G2F) initiative in 2014 and 2015. Specifically four different prediction scenarios were evaluated (i) tested genotypes in observed environments; (ii) untested genotypes in observed environments; (iii) tested genotypes in unobserved environments; and (iv) untested genotypes in unobserved environments. A set of 1,481 unique hybrids were evaluated for grain yield. Evaluations were conducted using five different models including main effect of environments; general combining ability (GCA) effects of the maternal and paternal parents modeled using the genomic relationship matrix; specific combining ability (SCA) effects between maternal and paternal parents; interactions between genetic (GCA and SCA) effects and environmental effects; and finally interactions between the genetics effects and environmental covariates. Incorporation of the genotype-by-environment interaction term improved predictive ability across all scenarios. However, predictive ability was not improved through inclusion of naive environmental covariates in G×E models. More research should be conducted to link the observed weather conditions with important physiological aspects in plant development to improve predictive ability through the inclusion of weather data., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jarquin, de Leon, Romay, Bohn, Buckler, Ciampitti, Edwards, Ertl, Flint-Garcia, Gore, Graham, Hirsch, Holland, Hooker, Kaeppler, Knoll, Lee, Lawrence-Dill, Lynch, Moose, Murray, Nelson, Rocheford, Schnable, Schnable, Smith, Springer, Thomison, Tuinstra, Wisser, Xu, Yu and Lorenz.)

High-dimensional and high-throughput genomic, field performance, and environmental data are becoming increasingly available to crop breeding programs, and their integration can facilitate genomic prediction within and across environments and provide insights into the genetic architecture of complex traits and the nature of genotype-by-environment interactions. To partition trait variation into additive and dominance (main effect) genetic and corresponding genetic-by-environment variances, and to identify specific environmental factors that influence genotype-by-environment interactions, we curated and analyzed genotypic and phenotypic data on 1918 maize (Zea mays L.) hybrids and environmental data from 65 testing environments. For grain yield, dominance variance was similar in magnitude to additive variance, and genetic-by-environment variances were more important than genetic main effect variances. Models involving both additive and dominance relationships best fit the data and modeling unique genetic covariances among all environments provided the best characterization of the genotype-by-environment interaction patterns. Similarity of relative hybrid performance among environments was modeled as a function of underlying weather variables, permitting identification of weather covariates driving correlations of genetic effects across environments. The resulting models can be used for genomic prediction of mean hybrid performance across populations of environments tested or for environment-specific predictions. These results can also guide efforts to incorporate high-throughput environmental data into genomic prediction models and predict values in new environments characterized with the same environmental characteristics., (Published by Oxford University Press on behalf of Genetics Society of America 2021. This work is written by US Government employees and is in the public domain in the US.)

Objectives: Advanced tools and resources are needed to efficiently and sustainably produce food for an increasing world population in the context of variable environmental conditions. The maize genomes to fields (G2F) initiative is a multi-institutional initiative effort that seeks to approach this challenge by developing a flexible and distributed infrastructure addressing emerging problems. G2F has generated large-scale phenotypic, genotypic, and environmental datasets using publicly available inbred lines and hybrids evaluated through a network of collaborators that are part of the G2F's genotype-by-environment (G × E) project. This report covers the public release of datasets for 2014-2017., Data Description: Datasets include inbred genotypic information; phenotypic, climatic, and soil measurements and metadata information for each testing location across years. For a subset of inbreds in 2014 and 2015, yield component phenotypes were quantified by image analysis. Data released are accompanied by README descriptions. For genotypic and phenotypic data, both raw data and a version without outliers are reported. For climatic data, a version calibrated to the nearest airport weather station and a version without outliers are reported. The 2014 and 2015 datasets are updated versions from the previously released files [1] while 2016 and 2017 datasets are newly available to the public.

Rapid development and adoption of biofortified, provitamin A-dense orange maize ( Zea mays L.) varieties could be facilitated by a greater understanding of the natural variation underlying kernel color, including as it relates to carotenoid biosynthesis and retention in maize grain. Greater abundance of carotenoids in maize kernels is generally accompanied by deeper orange color, useful for distinguishing provitamin A-dense varieties to consumers. While kernel color can be scored and selected with high-throughput, low-cost phenotypic methods within breeding selection programs, it remains to be well established as to what would be the logical genetic loci to target for selection for kernel color. We conducted a genome-wide association study of maize kernel color, as determined by colorimetry, in 1,651 yellow and orange inbreds from the Ames maize inbred panel. Associations were found with y1 , encoding the first committed step in carotenoid biosynthesis, and with dxs2 , which encodes the enzyme responsible for the first committed step in the biosynthesis of the isoprenoid precursors of carotenoids. These genes logically could contribute to overall carotenoid abundance and thus kernel color. The lcyE and zep1 genes, which can affect carotenoid composition, were also found to be associated with colorimeter values. A pathway-level analysis, focused on genes with a priori evidence of involvement in carotenoid biosynthesis and retention, revealed associations for dxs3 and dmes1 , involved in isoprenoid biosynthesis; ps1 and vp5 , within the core carotenoid pathway; and vp14 , involved in cleavage of carotenoids. Collectively, these identified genes appear relevant to the accumulation of kernel color., (Copyright © 2019 Owens et al.)

Objectives: Crop improvement relies on analysis of phenotypic, genotypic, and environmental data. Given large, well-integrated, multi-year datasets, diverse queries can be made: Which lines perform best in hot, dry environments? Which alleles of specific genes are required for optimal performance in each environment? Such datasets also can be leveraged to predict cultivar performance, even in uncharacterized environments. The maize Genomes to Fields (G2F) Initiative is a multi-institutional organization of scientists working to generate and analyze such datasets from existing, publicly available inbred lines and hybrids. G2F's genotype by environment project has released 2014 and 2015 datasets to the public, with 2016 and 2017 collected and soon to be made available., Data Description: Datasets include DNA sequences; traditional phenotype descriptions, as well as detailed ear, cob, and kernel phenotypes quantified by image analysis; weather station measurements; and soil characterizations by site. Data are released as comma separated value spreadsheets accompanied by extensive README text descriptions. For genotypic and phenotypic data, both raw data and a version with outliers removed are reported. For weather data, two versions are reported: a full dataset calibrated against nearby National Weather Service sites and a second calibrated set with outliers and apparent artifacts removed.

Translation of the breeding efforts designed to biofortify maize ( Z. mays) genotypes with higher levels of provitamin A carotenoid (pVAC) content for sub-Saharan Africa is dependent in part on the stability of carotenoids during postharvest through industrial and in-home food processing operations. The purpose of this study was to simulate production of commercial milled products by determining the impact of dry milling and extrusion processing on carotenoid stability in three higher pVAC maize genotypes (C17xDE3, Orange ISO, Hi27xCML328). Pericarp and germ removal of biofortified maize kernels resulted in ∼10% loss of total carotenoids. Separating out the maize flour fraction (<212 μm) resulted in an additional ∼15% loss of total carotenoids. Carotenoid degradation was similar across milled maize fractions. Dry-milled products of Orange ISO and Hi27xCML328 genotypes showed ∼28% pVAC loss after 90-days storage. Genotype C17xDE3, with highest levels of all- trans-β-carotene, showed a 68% pVAC loss after 90-day storage. Extrusion processing conditions were optimal at 35% extrusion moisture, producing fully cooked instant maize flours with high pVAC retention (70-93%). These results support the notion that postharvest losses in maize milled fractions may be dependent, in part, on genotype and that extrusion processing may provide an option for preserving biofortified maize products.

Remarkable productivity has been achieved in crop species through artificial selection and adaptation to modern agronomic practices. Whether intensive selection has changed the ability of improved cultivars to maintain high productivity across variable environments is unknown. Understanding the genetic control of phenotypic plasticity and genotype by environment (G × E) interaction will enhance crop performance predictions across diverse environments. Here we use data generated from the Genomes to Fields (G2F) Maize G × E project to assess the effect of selection on G × E variation and characterize polymorphisms associated with plasticity. Genomic regions putatively selected during modern temperate maize breeding explain less variability for yield G × E than unselected regions, indicating that improvement by breeding may have reduced G × E of modern temperate cultivars. Trends in genomic position of variants associated with stability reveal fewer genic associations and enrichment of variants 0-5000 base pairs upstream of genes, hypothetically due to control of plasticity by short-range regulatory elements.

Biofortification of crops to enhance provitamin A carotenoids is a strategy to increase the intake where vitamin A deficiency presents a widespread problem. Heat, light, and oxygen cause isomerization and oxidation of carotenoids, reducing provitamin A activity. Understanding provitamin A retention is important for assessing efficacy of biofortified foods. Retention of carotenoids in high-xanthophyll and high-β-carotene maize was assessed after a long-term storage at three temperatures. Carotenoid retention in high-β-cryptoxanthin maize was determined in muffins, non-nixtamalized tortillas, porridge, and fried puffs made from whole-grain and sifted flour. Retention in eggs from hens fed high-β-cryptoxanthin maize was assessed after frying, scrambling, boiling, and microwaving. Loss during storage in maize was accelerated with increasing temperature and affected by genotype. Boiling whole-grain maize into porridge resulted in the highest retention of all cooking and sifting methods (112%). Deep-fried maize and scrambled eggs had the lowest carotenoid retention rates of 67-78 and 84-86%, respectively., Competing Interests: The authors declare no competing financial interest.

Tocopherols, tocotrienols, and plastochromanols (collectively termed tocochromanols) are lipid-soluble antioxidants synthesized by all plants. Their dietary intake, primarily from seed oils, provides vitamin E and other health benefits. Tocochromanol biosynthesis has been dissected in the dicot Arabidopsis thaliana , which has green, photosynthetic seeds, but our understanding of tocochromanol accumulation in major crops, whose seeds are nonphotosynthetic, remains limited. To understand the genetic control of tocochromanols in grain, we conducted a joint linkage and genome-wide association study in the 5000-line U.S. maize ( Zea mays ) nested association mapping panel. Fifty-two quantitative trait loci for individual and total tocochromanols were identified, and of the 14 resolved to individual genes, six encode novel activities affecting tocochromanols in plants. These include two chlorophyll biosynthetic enzymes that explain the majority of tocopherol variation, which was not predicted given that, like most major cereal crops, maize grain is nonphotosynthetic. This comprehensive assessment of natural variation in vitamin E levels in maize establishes the foundation for improving tocochromanol and vitamin E content in seeds of maize and other major cereal crops., (© 2017 American Society of Plant Biologists. All rights reserved.)

Maize is a staple crop that has been the subject of biofortification efforts to increase the natural content of provitamin A carotenoids. Although significant progress toward increasing provitamin A carotenoid content in maize varieties has been made, postharvest handling factors that influence carotenoid stability during storage have not been fully established. The objectives of this study were to determine carotenoid profiles of six selected provitamin A biofortified maize genotypes at various developmental stages and assess the stability of carotenoids in maize kernels during controlled storage conditions (12 month period), including elevated temperature and relative humidity. There were no significant changes in the content of individual carotenoids within genotypes during kernel development from 45 days after pollination through the time of harvest. Carotenoid losses through traditional grain drying were also minimal (<9%). However, the stability of carotenoids in maize kernels over storage time after harvest was found to be dependent on both temperature and humidity, with variation observed among genotypes. Different forms of provitamin A carotenoids follow similar degradation rates. The genotype C17xDE3 had a degradation rate 2 times faster than those of the other genotypes evaluated (P < 0.001). These differences in carotenoid stability under controlled storage were attributed, in part, to observed differences in the physical properties of the kernels (surface area and porosity). These results support the notion that effective control of moisture content and temperature of the kernels during storage conditions is essential to reduce the speed of degradative reactions.

Efforts are underway for development of crops with improved levels of provitamin A carotenoids to help combat dietary vitamin A deficiency. As a global staple crop with considerable variation in kernel carotenoid composition, maize (Zea mays L.) could have a widespread impact. We performed a genome-wide association study (GWAS) of quantified seed carotenoids across a panel of maize inbreds ranging from light yellow to dark orange in grain color to identify some of the key genes controlling maize grain carotenoid composition. Significant associations at the genome-wide level were detected within the coding regions of zep1 and lut1, carotenoid biosynthetic genes not previously shown to impact grain carotenoid composition in association studies, as well as within previously associated lcyE and crtRB1 genes. We leveraged existing biochemical and genomic information to identify 58 a priori candidate genes relevant to the biosynthesis and retention of carotenoids in maize to test in a pathway-level analysis. This revealed dxs2 and lut5, genes not previously associated with kernel carotenoids. In genomic prediction models, use of markers that targeted a small set of quantitative trait loci associated with carotenoid levels in prior linkage studies were as effective as genome-wide markers for predicting carotenoid traits. Based on GWAS, pathway-level analysis, and genomic prediction studies, we outline a flexible strategy involving use of a small number of genes that can be selected for rapid conversion of elite white grain germplasm, with minimal amounts of carotenoids, to orange grain versions containing high levels of provitamin A., (Copyright © 2014 by the Genetics Society of America.)

Key Message: Genetic control of maize grain carotenoid profiles is coordinated through several loci distributed throughout three secondary metabolic pathways, most of which exhibit additive, and more importantly, pleiotropic effects. The genetic basis for the variation in maize grain carotenoid concentrations was investigated in two F2:3 populations, DEexp × CI7 and A619 × SC55, derived from high total carotenoid and high β-carotene inbred lines. A comparison of grain carotenoid concentrations from population DEexp × CI7 grown in different environments revealed significantly higher concentrations and greater trait variation in samples harvested from a subtropical environment relative to those from a temperate environment. Genotype by environment interactions was significant for most carotenoid traits. Using phenotypic data in additive, environment-specific genetic models, quantitative trait loci (QTL) were identified for absolute and derived carotenoid traits in each population, including those specific to the isomerization of β-carotene. A multivariate approach for these correlated traits was taken, using carotenoid trait principal components (PCs) that jointly accounted for 97 % or more of trait variation. Component loadings for carotenoid PCs were interpreted in the context of known substrate-product relationships within the carotenoid pathway. Importantly, QTL for univariate and multivariate traits were found to cluster in close proximity to map locations of loci involved in methyl-erythritol, isoprenoid and carotenoid metabolism. Several of these genes, including lycopene epsilon cyclase, carotenoid cleavage dioxygenase1 and beta-carotene hydroxylase, were mapped in the segregating populations. These loci exhibited pleiotropic effects on α-branch carotenoids, total carotenoid profile and β-branch carotenoids, respectively. Our results confirm that several QTL are involved in the modification of carotenoid profiles, and suggest genetic targets that could be used for the improvement of total carotenoid and β-carotene in future breeding populations.

Tocopherols and tocotrienols, collectively known as tocochromanols, are the major lipid-soluble antioxidants in maize (Zea mays L.) grain. Given that individual tocochromanols differ in their degree of vitamin E activity, variation for tocochromanol composition and content in grain from among diverse maize inbred lines has important nutritional and health implications for enhancing the vitamin E and antioxidant contents of maize-derived foods through plant breeding. Toward this end, we conducted a genome-wide association study of six tocochromanol compounds and 14 of their sums, ratios, and proportions with a 281 maize inbred association panel that was genotyped for 591,822 SNP markers. In addition to providing further insight into the association between ZmVTE4 (γ-tocopherol methyltransferase) haplotypes and α-tocopherol content, we also detected a novel association between ZmVTE1 (tocopherol cyclase) and tocotrienol composition. In a pathway-level analysis, we assessed the genetic contribution of 60 a priori candidate genes encoding the core tocochromanol pathway (VTE genes) and reactions for pathways supplying the isoprenoid tail and aromatic head group of tocochromanols. This analysis identified two additional genes, ZmHGGT1 (homogentisate geranylgeranyltransferase) and one prephenate dehydratase parolog (of four in the genome) that also modestly contribute to tocotrienol variation in the panel. Collectively, our results provide the most favorable ZmVTE4 haplotype and suggest three new gene targets for increasing vitamin E and antioxidant levels through marker-assisted selection.

The relationship of dietary vitamin A transfer from mother to fetus is not well understood. The difference in swine offspring liver reserves was investigated between single-dose vitamin A provided to the mother post-conception compared with continuous provitamin A carotenoid dietary intake from biofortified (enhanced provitamin A) orange maize (OM) fed during gestation and lactation. Vitamin A-depleted sows were fed OM (n = 5) or white maize (WM) + 1.05 mmol retinyl palmitate administered at the beginning of gestation (n = 6). Piglets (n = 102) were killed at 0, 10, 20, and 28 d after birth. Piglets from sows fed OM had higher liver retinol reserves (P < 0.0001) and a combined mean concentration from d 10 to 28 of 0.11 ± 0.030 μmol/g. Piglets from sows fed WM had higher serum retinol concentrations (0.56 ± 0.25 μmol/L; P = 0.0098) despite lower liver retinol concentrations of 0.068 ± 0.026 μmol/g from d 10 to 28. Milk was collected at 0, 5, 10, 20, and 28 d. Sows fed OM had a higher milk retinol concentration (1.36 ± 1.30 μmol/L; P = 0.038), than those fed WM (0.93 ±1.03 μmol/L). Sow livers were collected at the end of the study (n = 3/group) and had identical retinol concentrations (0.22 ± 0.05 μmol/g). Consumption of daily provitamin A carotenoids by sows during gestation and lactation increased liver retinol status in weanling piglets, illustrating the potential for provitamin A carotenoid consumption from biofortified staple foods to improve vitamin A reserves. Biofortified OM could have a measurable impact on vitamin A status in deficient populations if widely adopted.

Background: Vitamin A deficiency is associated with poor health outcomes related to reproduction, growth, vision, and immunity. Biofortification of staple crops is a novel strategy for combating vitamin A deficiency in high-risk populations where staple food intakes are high. African populations are proposed beneficiaries of maize (Zea mays) biofortified with provitamin A carotenoids, often called "orange maize" because of its distinctive deep yellow-orange kernels. The color facilitates ready recognition but presents a cultural challenge to maize-consuming populations, including those in much of Africa, who traditionally eat white varieties., Objective: This study explores the intake patterns of, as well as adaptation to, traditional foods made with provitamin A-biofortified maize compared with white maize in rural Zambian children 3 to 5 years of age (n = 189) during a 3-month feeding trial., Methods: The subjects were fed a breakfast of maize porridge (sweet mush), a lunch of maize nshima (stiff mush) with various side dishes, and an afternoon snack based on a 6-day rotating menu. The trial was conducted in 2010. The orange maize used in the trial came from three different sources. O1 maize was from the 2009 harvest and was stored in a freezer until use in 2010. O2 maize was also from the 2009 harvest and was stored in a cold room until 2010. O3 ("fresh") maize was from the 2010 harvest and was fed immediately after harvest in week 9 of the study and then stored in a freezer until milling for the final four weeks., Results: Consumption of menu items, except snacks, was influenced by week (p < .0084). The intakes of porridge and nshima made with orange maize equaled those of porridge and nshima made with white maize from week 2 onward. The intakes of porridge and nshima prepared from O1 and O2 did not differ, but intakes became significantly higher when meals made from O3 were introduced (p < .014 for porridge and p < or = .013 for nshima)., Conclusions: These results demonstrate quick adaptation to orange maize, a preference for recently harvested maize, and an optimistic outlook for similar adaptation patterns in other biofortified-maize target countries.

The laying hen has a natural ability to deposit carotenoids into its egg yolks, especially the xanthophyll carotenoid lutein that is used commercially as an egg colorant. Can this ability to deposit carotenoids be used to enrich egg yolk provitamin A value? After a 10-d carotenoid depletion period in hens (n = 24), the effects of a 20-d intervention with high-β-cryptoxanthin, high-β-carotene, or typical yellow maize on color and carotenoid profile were compared with the effects of a white maize diet (n = 6/treatment). Eggs were collected every other day and yolks were analyzed by using a portable colorimeter to define the color space and by using an HPLC to determine the carotenoid profile. The high-β-cryptoxanthin and yellow maize increased β-cryptoxanthin in the yolk (0.55 ± 0.08 to 4.20 ± 0.56 nmol/g and 0.55 ± 0.08 to 1.06 ± 0.12 nmol/g, respectively; P < 0.001). Provitamin A equivalents increased in eggs from hens fed high-β-cryptoxanthin maize (P < 0.001) but not the high-β-carotene maize. The color (L*, a*, and b*) assessment of the yolks showed an increase in the high-β-cryptoxanthin treatment for the red-green a* scale (P < 0.001) and a decrease for the light-dark L* scale (P < 0.001). No appreciable change was noted in the yellow-blue b* scale for the high-β-cryptoxanthin treatment; but significant changes were noted for the yellow (P = 0.002) and high-β-carotene maize (P = 0.005) treatments, which were most evident at the end of the washout period with white maize. β-Cryptoxanthin-biofortified maize is a potential vehicle to elevate provitamin A equivalents and to enhance the color of yolks. This could lead to a human health benefit if widely adopted.

Background: β-Carotene-biofortified maize is being developed through plant breeding as a sustainable agronomic approach to alleviate vitamin A deficiency., Objective: Our objective was to quantify the vitamin A equivalence of the β-carotene in β-carotene-biofortified maize based on consumption of a single serving of maize porridge., Design: Six healthy women each consumed three 250-g portions of maize porridge as follows: 1) β-carotene-biofortified maize porridge containing 527 μg (0.98 μmol) total β-carotene, 2) white maize porridge with a β-carotene reference dose containing 595 μg (1.11 μmol) added β-carotene, and 3) white maize porridge with a vitamin A reference dose containing 286 μg retinol activity equivelent (1.00 μmol) added retinyl palmitate. Each portion contained 8.0 g added sunflower oil. The porridges were consumed in random order separated by ≥2 wk. Blood samples were collected over 9 h. Retinyl palmitate was analyzed in plasma triacylglycerol-rich lipoprotein (TRL) fractions by HPLC with coulometric array electrochemical detection., Results: Mean (± SD) areas under the curve for retinyl palmitate in the TRL fractions (nmol ⋅ h) were 24.0 ± 9.4, 89.7 ± 34.7, and 80.1 ± 24.8 after ingestion of the β-carotene-biofortified maize porridge, the white maize porridge with the β-carotene reference dose, and the white maize porridge with the vitamin A reference dose, respectively. On average, 6.48 ± 3.51 μg (mean ± SD) of the β-carotene in β-carotene-biofortified maize porridge and 2.34 ± 1.61 μg of the β-carotene in the reference dose were each equivalent to 1 μg retinol., Conclusion: β-Carotene in biofortified maize has good bioavailability as a plant source of vitamin A.

Breeding to increase beta-carotene levels in cereal grains, termed provitamin A biofortification, is an economical approach to address dietary vitamin A deficiency in the developing world. Experimental evidence from association and linkage populations in maize (Zea mays L.) demonstrate that the gene encoding beta-carotene hydroxylase 1 (crtRB1) underlies a principal quantitative trait locus associated with beta-carotene concentration and conversion in maize kernels. crtRB1 alleles associated with reduced transcript expression correlate with higher beta-carotene concentrations. Genetic variation at crtRB1 also affects hydroxylation efficiency among encoded allozymes, as observed by resultant carotenoid profiles in recombinant expression assays. The most favorable crtRB1 alleles, rare in frequency and unique to temperate germplasm, are being introgressed via inexpensive PCR marker-assisted selection into tropical maize germplasm adapted to developing countries, where it is most needed for human health.

High-oil maize is a useful genetic resource for genomic investigation in plants. To determine the genetic basis of oil concentration and composition in maize grain, a recombinant inbred population derived from a cross between normal line B73 and high-oil line By804 was phenotyped using gas chromatography, and genotyped with 228 molecular markers. A total of 42 individual QTL, associated with fatty acid compositions and oil concentration, were detected in 21 genomic regions. Five major QTL were identified for measured traits, one each of which explained 42.0% of phenotypic variance for palmitic acid, 15.0% for stearic acid, 27.7% for oleic acid, 48.3% for linoleic acid, and 15.7% for oil concentration in the RIL population. Thirty-six loci were involved in 24 molecular marker pairs of epistatic interactions across all traits, which explained phenotypic variances ranging from 0.4 to 6.1%. Seven of 18 mapping candidate genes related to lipid metabolism were localized within or were close to identified individual QTL, explaining 0.7-13.2% of the population variance. These results demonstrated that a few major QTL with large additive effects could play an important role in attending fatty acid compositions and increasing oil concentration in used germplasm. A larger number of minor QTL and a certain number of epistatic QTL, both with additive effects, also contributed to fatty acid compositions and oil concentration.

We characterized allelic variation at barren inflorescence2 (bif2), a maize co-ortholog of the Arabidopsis PINOID protein kinase (PID), and tested for trait associations with bif2 in both an association mapping population of 277 diverse maize inbreds and in the inter-mated B73 x Mo17 (IBM) linkage population. Results from the quantitative analyses were compared with previous reports of bif2 phenotypes in mutagenesis studies. All three approaches (association, linkage, and mutagenesis) detect a significant effect of bif2 on tassel architecture. Association mapping implicates bif2 in an unexpectedly wide range of traits including plant height, node number, leaf length, and flowering time. Linkage mapping finds a significant interaction effect for node number between bif2 and other loci, in keeping with previous reports that bif2;spi1 and Bif2;Bif1 double mutants produce fewer phytomers. The Mo17 allele is associated with a reduced tassel branch zone and shows lower expression than the B73 allele in hybrid B73-Mo17 F(1) inflorescences, consistent with the complete absence of tassel branches in the bif2 knockout mutant. Overall, these data suggest that allelic variation at bif2 affects maize architecture by modulating auxin transport during vegetative and inflorescence development.

High-oil maize as a product of long-term selection provides a unique resource for functional genomics. In this study, the abundant soluble proteins of early developing germs from high-oil and normal lines of maize were compared using two-dimensional gel electrophoresis (2-DGE) in combination with mass spectrometry (MS). More than 1100 protein spots were detected on electrophoresis maps of both high-oil and normal lines by using silver staining method. A total of 83 protein spots showed significant differential expression (>two-fold change; t-test: P < 0.05) between high-oil and normal inbred lines. Twenty-seven protein spots including 25 non-redundant proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Functional categorization of these proteins was carbohydrate metabolism, cytoskeleton, protein metabolism, stress response, and lipid metabolism. Three such proteins involved in lipid metabolism, namely putative enoyl-ACP reductase (ENR), putative stearoyl-ACP desaturase (SAD) and putative acetyl-CoA C-acyltransferase (ACA), had more abundant expressions in high-oil lines than in normal. At the mRNA expression level, SAD, ENR and ACA were expressed at significantly higher levels in high-oil lines than in normal. The results demonstrated that high expressions of SAD, ENR and ACA might be associated to increasing oil concentration in high-oil maize. This study represents the first proteomic analysis of high-oil maize and contributes to a better understanding of the molecular basis of oil accumulation in high-oil maize.

Maize with enhanced provitamin A carotenoids (biofortified), accomplished through conventional plant breeding, maintains vitamin A (VA) status in Mongolian gerbils (Meriones unguiculatus). Two studies in gerbils compared the VA value of beta-cryptoxanthin with beta-carotene. Study 1 (n 47)examined oil supplements and study 2 (n 46) used maize with enhanced beta-cryptoxanthin and beta-carotene. After 4 weeks' depletion, seven or six gerbils were killed; remaining gerbils were placed into weight-matched groups of 10. In study 1, daily supplements were cottonseed oil, and 35, 35 or 17.5 nmol VA (retinyl acetate), beta-cryptoxanthin or beta-carotene, respectively, for 3 weeks. In study 2, one group of gerbils was fed a 50% biofortified maize diet which contained 2.9 nmol beta-cryptoxanthin and 3.2 nmol beta-carotene/g feed. Other groups were given equivalent b-carotene or VA supplements based on prior-day intake from the biofortified maize or oil only for 4 weeks. In study 1, liver retinol was higher in the VA (0.74 (SD 0.11) micromol) and beta-cryptoxanthin (0.5 (SD 0.10) micromol) groups than in the beta-carotene (0.49 (SD 0.13) micromol) and control (0.41 (SD 0.16) micromol)groups (P<0.05). In study 2, the VA (1.17 (SD 0.19) micromol) and maize (0.71 (SD 0.18) micromol) groups had higher liver retinol than the control (0.42 (SD 0.16) micromol) group (P<0.05), whereas the beta-carotene (0.57 (SD 0.21) micromol) group did not. Bioconversion factors (i.e. 2.74 microg beta-cryptoxanthin and 2.4 microg beta-carotene equivalents in maize to 1 microg retinol) were lower than the Institute of Medicine values.

Carotenoids are essential for photosynthesis and photoprotection; they also serve as precursors to signaling molecules that influence plant development and biotic/abiotic stress responses. With potential to improve plant yield and nutritional quality, carotenoids are targets for metabolic breeding/engineering, particularly in the Poaceae (grass family), which includes the major food crops. Depending on genetic background, maize (Zea mays) endosperm carotenoid content varies, and therefore breeding-enhanced carotenoid levels have been of ongoing interest. The first committed step in the plastid-localized biosynthetic pathway is mediated by the nuclear-encoded phytoene synthase (PSY). The gene family in maize and other grasses contains three paralogs with specialized roles that are not well understood. Maize endosperm carotenoid accumulation requires PSY1 expression. A maize antibody was used to localize PSY1 to amyloplast envelope membranes and to determine PSY1 accumulation in relation to carotenoid accumulation in developing endosperm. To test when and if PSY transcript levels correlated with carotenoid content, advantage was taken of a maize germplasm diversity collection that exhibits genetic and chemical diversity. Total carotenoid content showed statistically significant correlation with endosperm transcript levels at 20 d after pollination for PSY1 but not PSY2 or PSY3. Timing of PSY1 transcript abundance, previously unknown, provides critical information for choosing breeding alleles or properly controlling introduced transgenes. PSY1 was unexpectedly found to have an additional role in photosynthetic tissue, where it was required for carotenogenesis in the dark and for heat stress tolerance. Leaf carotenogenesis was shown to require phytochrome-dependent and phytochrome-independent photoregulation of PSY2 plus nonphotoregulated PSY1 expression.

High beta-carotene maize, biofortified with beta-carotene through plant breeding, is being developed as a cost-effective, sustainable agronomic approach to alleviating the problem of vitamin A deficiency in Africa. We used high beta-carotene maize (10.49+/-0.16 microg beta-carotene/g) to prepare traditional maize porridges and compared the carotenoid contents in the following: (1) whole kernels; (2) wet milled flour; (3) wet milled flour, fermented; (4) wet milled flour, cooked; (5) wet milled flour, fermented and cooked. The cumulative losses of beta-carotene in the final, cooked products were 24.5% (95% CI 22.8-26.2%) and 24.8% (95% CI 23.1-26.5%), for the fermented and unfermented porridges, respectively. Thus, fermentation, a traditional technology with documented nutritional and other health benefits, does not adversely affect the retention of beta-carotene in porridges prepared with high beta-carotene maize. The relatively good retention of beta-carotene during traditional maize processing provides additional experimental support for the feasibility of maize biofortification as a means to alleviate vitamin A deficiency.

Genetic control of grass inflorescence architecture is critical given that cereal seeds provide most of the world's food. Seeds are borne on axillary branches, which arise from groups of stem cells in axils of leaves and whose branching patterns dictate most of the variation in plant form. Normal maize (Zea mays) ears are unbranched, and tassels have long branches only at their base. The ramosa2 (ra2) mutant of maize has increased branching with short branches replaced by long, indeterminate ones. ra2 was cloned by chromosome walking and shown to encode a LATERAL ORGAN BOUNDARY domain transcription factor. ra2 is transiently expressed in a group of cells that predicts the position of axillary meristem formation in inflorescences. Expression in different mutant backgrounds places ra2 upstream of other genes that regulate branch formation. The early expression of ra2 suggests that it functions in the patterning of stem cells in axillary meristems. Alignment of ra2-like sequences reveals a grass-specific domain in the C terminus that is not found in Arabidopsis thaliana. The ra2-dm allele suggests this domain is required for transcriptional activation of ra1. The ra2 expression pattern is conserved in rice (Oryza sativa), barley (Hordeum vulgare), sorghum (Sorghum bicolor), and maize, suggesting that ra2 is critical for shaping the initial steps of grass inflorescence architecture.

Aflatoxin B(1) formed by Aspergillus flavus Fr:Link has been associated with animal disease and liver cancer in humans. We performed genetic studies in progenies derived from maize inbred Tex6, associated with relatively low levels of aflatoxin production, crossed with the historically important inbred B73. (Tex6 x B73) x B73 BC(1)S(1) and Tex6 x B73 F(2:3) mapping populations were produced and evaluated in 1996 and 1997 in Champaign, Ill. Ears were inoculated 20 to 24 days after midsilk using a pinboard method and a mixture of conidia of A. flavus Link:Fr. isolates. Aflatoxin B(1) levels in harvested ears were determined using an indirect competitive ELISA. Molecular markers were assayed on the populations and used to generate maps. Molecular marker - QTL associations for lower levels of aflatoxin production were determined using multiple regression (MR) and composite interval analysis with multiple regression (CIM MR). MR revealed sets of markers associated with lower aflatoxin production in 1996 and 1997, and CIM MR detected a smaller subset of loci significant in 1997. QTLs for lower aflatoxin were attributed to both Tex6 and B73 parental sources. Environment strongly influenced the detection of QTLs for lower aflatoxin production in different years. There were very few chromosome regions associated with QTLs in more than 1 year or population with MR analysis, and none with CIM MR analysis. In 1997, QTLs for lower aflatoxin were detected with CIM MR in bins 5.01-2 and 5.04-5 in the BC(1)S(1) population, and in bins 3.05-6, 4.07-8 and 10.05-10.07 in the F(2:3) population. These QTL associations appear the most promising for further study.

Comparative mapping of cereals has shown that chromosomes of barley, wheat, and maize can be described in terms of rice "linkage segments." However, little is known about marker order in the junctions between linkage blocks or whether this will impair comparative analysis of major genes that lie in such regions. We used genetic and physical mapping to investigate the relationship between the distal part of rice chromosome 7L, which contains the Hd2 heading date gene, and the region of barley chromosome 2HS containing the Ppd-H1 photoperiod response gene, which lies near the junction between rice 7 and rice 4 linkage segments. RFLP markers were mapped in maize to identify regions that might contain Hd2 or Ppd-H1 orthologs. Rice provided useful markers for the Ppd-H1 region but comparative mapping was complicated by loss of colinearity and sequence duplications that predated the divergence of rice, maize, and barley. The sequences of cDNA markers were used to search for homologs in the Arabidopsis genome. Homologous sequences were found for 13 out of 16 markers but they were dispersed in Arabidopsis and did not identify any candidate equivalent region. The implications of the results for comparative trait mapping in junction regions are discussed.