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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.

Single-cross hybrids have been critical to the improvement of maize (Zea mays L.), but the characterization of their genetic architectures remains challenging. Previous studies of hybrid maize have shown the contribution of within-locus complementation effects (dominance) and their differential importance across functional classes of loci. However, they have generally considered panels of limited genetic diversity, and have shown little benefit from genomic prediction based on dominance or functional enrichments. This study investigates the relevance of dominance and functional classes of variants in genomic models for agronomic traits in diverse populations of hybrid maize. We based our analyses on a diverse panel of inbred lines crossed with two testers representative of the major heterotic groups in the U.S. (1106 hybrids), as well as a collection of 24 biparental populations crossed with a single tester (1640 hybrids). We investigated three agronomic traits: days to silking (DTS), plant height (PH), and grain yield (GY). Our results point to the presence of dominance for all traits, but also among-locus complementation (epistasis) for DTS and genotype-by-environment interactions for GY. Consistently, dominance improved genomic prediction for PH only. In addition, we assessed enrichment of genetic effects in classes defined by genic regions (gene annotation), structural features (recombination rate and chromatin openness), and evolutionary features (minor allele frequency and evolutionary constraint). We found support for enrichment in genic regions and subsequent improvement of genomic prediction for all traits. Our results suggest that dominance and gene annotations improve genomic prediction across diverse populations in hybrid maize.

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.

Genetically improved maize varieties with high carotenoid levels and dark orange color have been developed to increase dietary consumption of macular carotenoids. However, postharvest and food processing conditions can cause isomerization and oxidation of carotenoids, reducing their potential impact on consumers' health. The purpose of this study was to assess the effectiveness of high barrier pouches in reducing carotenoid losses during the storage of dry-milled corn products. Orange corn grits were packaged in paper pouch bags, and three types of low-oxygen and low-moisture permeable (LOMP) pouches. Grits were packaged in each type of LOMP pouch with (LOMP-oxy) and without (LOMP-noxy) an oxygen scavenger. For six months, all pouches were stored at semi-controlled environmental conditions (22.5 ± 1.3°C, 32 ± 18% RH). After the storage period, orange corn grits stored in paper pouch bags lost 55% of total xanthophylls, whereas grits packaged in LOMP pouches only lost 8% of total xanthophylls. Orange Corn grits packaged in LOMP-oxy pouches had slightly higher carotenoid content than in LOMP-noxy pouches. Relative humidity fluctuation in the storeroom could have caused fluctuation in moisture content in the orange corn grits packaged in paper pouches, which may affect the rate of carotenoid degradation in the orange corn grits. Therefore, an effective control of the moisture content of the packaged dry-milled product and effective control of the temperature of pouches during storage conditions is essential to maximize carotenoid retention during the storage of dry-milled high carotenoid orange corn grits.

Rapid development and adoption of biofortified, provitamin A-dense orange maize (Zea maysL.) varieties could be facilitated by a greater understanding of the natural variation underlying kernel color, including as 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 withy1, encoding the first committed step in carotenoid biosynthesis, and withdxs2, 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. ThelcyEandzep1genes, which can affect carotenoid composition, were also found to be associated with colorimeter values. A pathway-level analysis, focused on genes witha priorievidence of involvement in carotenoid biosynthesis and retention, revealed associations fordxs3anddmes1, involved in isoprenoid biosynthesis;ps1andvp5, within the core carotenoid pathway; andvp14, involved in cleavage of carotenoids. Collectively, these identified genes appear relevant to the accumulation of kernel color.

Increasing adoption of biofortified orange maize in developing countries requires economical storage methods to manage product quality and carotenoid retention. This study assessed the utility of the Purdue Improved Crop Storage (PICS) bags with a specific focus on retention of provitamin A and other carotenoids in two biofortified maize genotypes (OPVI and OPVII). Grain was stored at ambient conditions for eight months in PICS bags with and without an O2 scavenger, (PICS-oxy) and (PICS-noxy), respectively, or in common polypropylene woven bags. After 4 months of storage carotenoid content was significantly higher (p

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.

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.

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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 days. Each diet had five replicates of six 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 50-75% orange corn inclusion could be sufficient to achieve yolk colors similar to diets that involve supplementation with a carotenoid additive.

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.

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.

Vitamin A deficiency remains prevalent in parts of Asia, Latin America and sub-Saharan Africa where maize is a food staple. Extensive natural variation exists for carotenoids in maize grain; to understand its genetic basis, we conducted a joint linkage and genome-wide association study in the U.S. maize nested association mapping panel. Eleven of the 44 detected quantitative trait loci (QTL) were resolved to individual genes. Six of these were expression QTL (eQTL), showing strong correlations between RNA-seq expression abundances and QTL allelic effect estimates across six stages of grain development. These six eQTL also had the largest percent 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 eQTL had highly correlated QTL allelic effect estimates across multiple traits, suggesting that pleiotropy within this pathway is largely regulated at the expression level. Significant pairwise epistatic interactions were also detected. These findings provide the most comprehensive genome-level understanding of the genetic and molecular control of carotenoids in any plant system, and a roadmap to accelerate breeding for provitamin A and other priority carotenoid traits in maize grain that should be readily extensible to other cereals.

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.

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.

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., Breeding has increased crop productivity, but whether it has also changed phenotypic plasticity is unclear. Here, the authors find maize genomic regions selected for high productivity show reduced contribution to genotype by environment variation and provide evidence for regulatory control of phenotypic stability.

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.

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.

Biofortified orange maize open-pollinated varieties and hybrids with higher provitamin A carotenoids (pVACs) have been released in sub-Saharan Africa and will be introduced throughout the local food systems. This study assessed the impact of steeping, a traditional processing method, on retention of carotenoids and starch pasting properties of porridges made from select biofortified maize genotypes. Steeping had a modest effect (&lt, 9% loss) on total carotenoid stability during relatively shorter steeping periods (&lt, 72 h). However, more extended steeping periods (up to 120 h) had a detrimental effect on total carotenoid recovery (61% loss). Xanthophylls showed greater stability (82% retention) compared to carotenes (30% retention) during subsequent wet cooking of fermented flours. Interestingly, steeping of maize did modify pasting properties, with peak viscosities increasing from 24&ndash, 72 h of steeping potentially impacting cooking stability. These results suggest that steeping can impact carotenoid retention and potentially optimal steeping times would be 24&ndash, 72 h for acceptable carotenoid retention.

Heterosis has been key to the development of maize breeding but describing its genetic basis has been challenging. Previous studies of heterosis have shown the contribution of within-locus complementation effects (dominance) and their differential importance across genomic regions. However, they have generally considered panels of limited genetic diversity and have shown little benefit to including dominance effects for predicting genotypic value in breeding populations. This study examined within-locus complementation and enrichment of genetic effects by functional classes in maize. We based our analyses on a diverse panel of inbred lines crossed with two testers representative of the major heterotic groups in the United States (1,106 hybrids), as well as a collection of 24 biparental populations crossed with a single tester (1,640 hybrids). We assayed three agronomic traits: days to silking (DTS), plant height (PH) and grain yield (GY). Our results point to the presence of dominance for all traits, but also among-locus complementation (epistasis) for DTS and genotype-by-environment interactions for GY. Consistently, dominance improved genomic prediction for PH only. In addition, we assessed enrichment of genetic effects in classes defined by genic regions (gene annotation), structural features (recombination rate and chromatin openness), and evolutionary features (minor allele frequency and evolutionary constraint). We found support for enrichment in genic regions and subsequent improvement of genomic prediction for all traits. Our results point to mechanisms by which heterosis arises through local complementation in proximal gene regions and suggest the relevance of dominance and gene annotations for genomic prediction in maize.

Assessing rheological properties of biofortified maize through post-harvest storage is critical for its successful translation to consumer foods. Changes in flour rheology were assessed following post-harvest storage in Purdue Improved Crop Storage (PICS) of a white and two biofortified orange maize genotypes (OPVI and OPVII). Whole grains were stored in PICS bags with (PICS-oxy) or without (PICS-noxy) oxygen scavenger and compared to storage in traditional polypropylene woven bags. Flour pasting profiles were assessed over 8 months of storage. Initial porridge viscosities from biofortified maize were lower than from white maize. After 8 months, higher viscosities (p

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.

Vitamin E refers to eight distinct compounds collectively known as tocochromanols and can be further divided into two classes, tocotrienols and tocopherols. Tocochromanols are the major lipid-soluble antioxidants in maize (Zea mays L.) grain. Enhancing vitamin E content of maize through plant breeding has important implications for human and animal nutrition. Four inbred lines exhibiting unique variation for tocochromanol compounds were chosen from the Goodman maize diversity panel to construct two biparental mapping populations (N6xNC296 and E2558xCo125). The N6xNC296 population was developed to analyze segregation for α-tocopherol and α-tocotrienol content. The E2558WxCo125 population was developed to analyze segregation for the ratio of total tocotrienols to tocopherols. The tocochromanol variation in two replicates of each population was quantified using liquid chromatography-diode array detection. Using high-density linkage mapping, novel quantitative trait loci (QTL) in the N6xNC296 population were mapped using tocopherol ratio traits. These QTL contain the candidate gene homogentisate phytyltransferase (ZmVTE2) within the respective support intervals. This locus was not mapped in a previous genome-wide association study that analyzed tocochromanols in the Goodman diversity panel. Transgressive segregation was observed for γ- and α-tocochromanols in these populations, which facilitated QTL identification. These QTL and transgressive segregant families can be used in selection programs for vitamin E enhancement in maize. This work illustrates the complementary nature of biparental mapping populations and genome-wide association studies to further characterize genetic variation of tocochromanol content in maize grain.

The nutritional value and health benefits derived from carotenoids have prompted increased investment in breeding crop varieties with elevated carotenoid levels for areas where crops like maize with low nutrient density are consumed in large quantities. Twelve exotic donor lines of high β-carotene were crossed to seven elite tropical yellow or orange lines and the F1s were backcrossed to the same or different elite line. Ninety-eight lines derived from these backcrosses (BC), seven recurrent parents, and 24 adapted lines derived from other source populations were included in a trial grown at Ibadan in Nigeria for three years. Carotenoid analyses of samples harvested from this trial found significant differences in accumulating provitamin-A and other carotenoids among lines, which were consistently expressed across years. The lines also displayed distinct carotenoid profiles. Among the best 25 inbred lines containing 5.0–16.6 μg g−1 β-carotene and 8.0–17.4 μg g−1 pro-vitamin A, 24 were BC-derived lines and one was derived from a bi-parental cross of tropical yellow elite lines. The best BC-derived lines accumulated 23–313 % more β-carotene and 32–190 % more provitamin-A than the recurrent parents. These BC-derived lines may be valuable resources for favorable alleles to develop maize varieties with high provitamin-A and for genetic studies to understand the underlying mechanisms regulating carotenoid biosynthesis. These results illustrate the effectiveness of exploiting natural allelic diversity existing in exotic lines through backcrossing combined with visual selection for bright yellow to orange kernel color with semi-flint to flint kernel texture in boosting provitamin-A to a new level in tropical maize.

Provitamin A (proVA) carotenoids are converted into retinol (vitamin A) in the human body, are the subject of human nutrition studies, and are targets for biofortification of staple crops. β-Carotene has been the principal target for enhancing levels of proVA. There is recent interest in enhancing the proVA carotenoid β-cryptoxanthin since it has excellent bioavailability, and in maize may be nearly as effective as β-carotene in providing retinol to humans. This study was designed to enhance our understanding of the genetic control of: levels of β-cryptoxanthin, conversion of β-carotene into β-cryptoxanthin and zeaxanthin, conversion of β-cryptoxanthin into zeaxanthin, and flux into and within the β-branch of carotenoid pathway. A biparental population derived from two inbreds with relatively high levels of β-cryptoxanthin and different ratios of β-carotene to β-cryptoxanthin and β-cryptoxanthin to zeaxanthin was studied. Three field replications of this F2:3 population were grown, grain analyzed by liquid chromatography (LC), and composite interval mapping (CIM) performed to identify 90 quantitative trait loci (QTL) for carotenoids. We detected QTL for β-carotene/(β-cryptoxanthin + zeaxanthin) and (β-carotene + β-cryptoxanthin)/zeaxanthin ratios that contain candidate gene hydroxylase 4 (hyd4), which has not been previously associated with QTL for carotenoids in maize grain. Two color assessment methods, visual score and chromameter reading, were used to phenotype one replicate of the population for initial assessment as simple alternative measuring procedures. A common finding for LC and chromameter analysis included QTL on chromosome 5 that contain candidate gene lycopene β cyclase (lcyβ).

Maize has been targeted as one of the major crops for provitamin enrichment and delivery because it is an inexpensive and easily available source of food for millions of people in sub-Saharan Africa. Although tropical-adapted yellow maize contains provitamin-A carotenoids that can be converted into vitamin A in the human body, they represent less than 25% of the total carotenoids in most widely grown and consumed maize cultivars in Africa. Novel genes conditioning high concentration of β-carotene and other carotenoids were then continually introduced from the temperate zone and tropics to boost provitamin A in tropical-adapted maize. Several promising inbred lines developed from backcrosses involving diverse exotic donor lines displayed provitamin A concentrations that match or surpass the current breeding target of 15 μg g−1. Some of these lines attained high provitamin A content by accumulating mainly high β-carotene while others contained high provitamin A by promoting accumulation of high levels of both carotenes and xanthophylls. Several inbred lines with intermediate to high levels of provitamin A have already been used to develop hybrids and synthetics without compromising grain yield and other adaptive traits that are required to profitably cultivate maize by farmers in West and Central Africa.

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.

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. Abstract 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. Electronic supplementary material The online version of this article (doi:10.1007/s00122-013-2179-5) contains supplementary material, which is available to authorized users.

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.

Increasing levels of provitamin A carotenoids in maize (Zea mays L.) grain through plant breed - ing has potential to help humans suffering from vitamin A deficiency. In parts of Africa where this deficiency is prevalent, there is a consumer pref - erence for white maize grain and an avoidance of yellow maize grain. White grain has minimal lev - els of carotenoids whereas yellow grain can have appreciable levels of carotenoids. There is a new effort to introduce orange maize that contains high levels of provitamin A, which appears to be a more acceptable color than yellow to consum - ers in Africa. The implementation of this program requires backcross selection to convert African- adapted germplasm with white grain to orange. We conducted a study to assess the heritability of visual scores for relative intensity of orange kernel color and identify genetic markers asso - ciated with orange color across and within 10 families of the maize nested association map - ping population. We found visually scored ker nel color to have a moderately high heritability and identified five common quantitative trait loci (QTL) and six rare QTL for intensity of orange color. Notably, half of them coincided with carot - enoid biosynthetic genes. our results indicate that breeders in Africa, Asia, and throughout the world would have flexibility to select for orange kernel color visually and/or with gene-specific markers. Such selection can be combined with marker-assisted selection efforts to increase provitamin A levels in maize grain.

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 (

US maize yield has increased eight-fold in the past 80 years, with half of the gain attributed to selection by breeders. During this time, changes in maize leaf angle and size have altered plant architecture, allowing more efficient light capture as planting density has increased. Through a genome-wide association study (GWAS) of the maize nested association mapping panel, we determined the genetic basis of important leaf architecture traits and identified some of the key genes. Overall, we demonstrate that the genetic architecture of the leaf traits is dominated by small effects, with little epistasis, environmental interaction or pleiotropy. In particular, GWAS results show that variations at the liguleless genes have contributed to more upright leaves. These results demonstrate that the use of GWAS with specially designed mapping populations is effective in uncovering the basis of key agronomic traits.

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.

The emergence of high-throughput, high-density genotyping methods combined with increasingly powerful computing systems has created opportunities to further discover and exploit the genes controlling agronomic performance in elite maize breeding populations. Understanding the genetic basis of population structure in an elite set of materials is an essential step in this genetic discovery process. This paper presents a genotype-based population analysis of all maize inbreds whose Plant Variety Protection certificates had expired as of the end of 2013 (283 inbreds) as well as 66 public founder inbreds. The results provide accurate population structure information and allow for important inferences in context of the historical development of North American elite commercial maize germplasm. Genotypic data was obtained via genotyping-by-sequencing on 349 inbreds. After filtering for missing data, 77,314 high-quality markers remained. The remaining missing data (average per individual was 6.22 percent) was fully imputed at an accuracy of 83 percent. Calculation of linkage disequilibrium revealed that the average r2 of 0.20 occurs at approximately 1.1 Kb. Results of population genetics analyses agree with previously published studies that divide North American maize germplasm into three heterotic groups: Stiff Stalk, Non-Stiff Stalk, and Iodent. Principal component analysis shows that population differentiation is indeed very complex and present at many levels, yet confirms that division into three main sub-groups is optimal for population description. Clustering based on Nei’s genetic distance provides an additional empirical representation of the three main heterotic groups. Overall fixation index (FST), indicating the degree of genetic divergence between the three main heterotic groups, was 0.1361. Understanding the genetic relationships and population differentiation of elite germplasm may help breeders to maintain and potentially increase the rate of genetic gain, resulting in higher overall agronomic performance.

*† ‡ SUMMARY 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 · 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 F1 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.