A foundation for provitamin A biofortification of maize: genome-wide association and genomic prediction models of carotenoid levels.

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

Characterization of three members of the Arabidopsis carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family

Arabidopsis thaliana has nine genes that constitute a family of putative carotenoid cleavage dioxygenases (CCDs). While five members of the family are believed to be involved in synthesis of the phytohormone abscisic acid, the functions of the other four enzymes are less clear. Recently two of the enzymes, CCD7/MAX3 and CCD8/MAX4, have been implicated in synthesis of a novel apocarotenoid hormone that controls lateral shoot growth. Here, we report on the molecular and genetic interactions between CCD1, CCD7/MAX3 and CCD8/ MAX4. CCD1 distinguishes itself from other reported CCDs as being the only member not targeted to the plastid. Unlike ccd7/max3 and ccd8/max4, both characterized as having highly branched phenotypes, ccd1 loss-of-function mutants are indistinguishable from wild-type plants. Thus, even though CCD1 has similar enzymatic activity to CCD7/MAX3, it does not have a role in synthesis of the lateral shoot growth inhibitor. Rather, it may have a role in synthesis of apocarotenoid flavor and aroma volatiles, especially in maturing seeds where loss of function leads to significantly higher carotenoid levels

Functional Analysis of β- and ɛ-Ring Carotenoid Hydroxylases in Arabidopsis

Lutein and zeaxanthin are dihydroxy xanthophylls that are produced from their corresponding carotene precursors by the action of β- and ɛ-ring carotenoid hydroxylases. Two genes that encode β-ring hydroxylases (β-hydroxylases 1 and 2) have been identified in the Arabidopsis genome and are highly active toward β-rings but only weakly active toward ɛ-rings. A third distinct activity required for ɛ-ring hydroxylation has been defined by mutation of the LUTEIN1 (LUT1) locus, but LUT1 has not yet been cloned. To address the individual and overlapping functions of the three Arabidopsis carotenoid hydroxylase activities in vivo, T-DNA knockout mutants corresponding to β-hydroxylases 1 and 2 (b1 and b2, respectively) were isolated and all possible hydroxylase mutant combinations were generated. β-Hydroxylase single mutants do not exhibit obvious growth defects and have limited impact on carotenoid composition relative to the wild type, suggesting that the encoded proteins have a significant degree of functional redundancy in vivo. Surprisingly, the b1 b2 double mutant, which lacks both known β-hydroxylase enzymes, still contains significant levels of β-carotene–derived xanthophylls, suggesting that additional β-ring hydroxylation activity exists in vivo. The phenotype of double and triple hydroxylase mutants indicates that at least a portion of this activity resides in the LUT1 gene product. Despite the severe reduction of β-carotene–derived xanthophylls (up to 90% in the lut1 b1 b2 triple mutant), the double and triple hydroxylase mutants still contain at least 50% of the wild-type amount of hydroxylated β-rings. This finding suggests that it is the presence of minimal amounts of hydroxylated β-rings, rather than minimal amounts of specific β-carotene–derived xanthophylls, that are essential for light-harvesting complex II assembly and function in vivo. The carotenoid profiles in wild-type seeds and the effect of single and multiple hydroxylase mutations are distinct from those in photosynthetic tissues, indicating that the activities of each gene product differ in the two tissues. Overall, the hydroxylase mutants provide insight into the unexpected overlapping activity of carotenoid hydroxylases in vivo

Genetic basis for natural variation in seed vitamin E levels in Arabidopsis thaliana

Vitamin E is an essential nutrient for humans and is obtained primarily from food, especially oil, derived from the seed of plants. Genes encoding the committed steps in vitamin E synthesis in plants (VTE, loci 1–5) have been isolated and used for tocopherol pathway engineering with various degrees of success. As a complement to such approaches we have used quantitative trait loci analysis with two sets of Arabidopsis thaliana recombinant inbred lines and have identified 14 QVE (quantitative vitamin E) loci affecting tocopherol content and composition in seeds. Five QVE intervals contain VTE loci that are likely QVE gene candidates. Nine QVE intervals do not contain VTE loci and therefore identify novel loci affecting seed tocopherol content and composition. Several near-isogenic lines containing introgressions of the accession with increased vitamin E levels were shown to confer significantly elevated tocopherol levels compared with the recurrent parent. Fine-mapping has narrowed QVE7 (a γ-tocopherol quantitative trait loci) to an 8.5-kb interval encompassing two genes. Understanding the basis of the QVE loci in Arabidopsis promises to provide insight into the regulation and/or metabolism of vitamin E in plants and has clear ramifications for improving the nutritional content of crops through marker-assisted selection and metabolic engineering

Raw_GBS_SweetCorn_384indiv_955K_RefGen_v2.hmp

Raw genotypes from 384 sweet corn inbred lines using genotyping-by-sequencing (GBS) with 955,690 high confidence single-nucleotide polymorphism markers (SNPs) that were called using default parameters in the TASSEL 5 GBSv1 production pipeline with the ZeaGBSv2.7 Production TagsOnPhysicalMapfile in B73 RefGen_v2 coordinates. File in Hapmap format

Genome-Wide Association and Genomic Prediction Models of Tocochromanols in Fresh Sweet Corn Kernels

Sweet corn ( L.), a highly consumed fresh vegetable in the United States, varies for tocochromanol (tocopherol and tocotrienol) levels but makes only a limited contribution to daily intake of vitamin E and antioxidants. We performed a genome-wide association study of six tocochromanol compounds and 14 derivative traits across a sweet corn inbred line association panel to identify genes associated with natural variation for tocochromanols and vitamin E in fresh kernels. Concordant with prior studies in mature maize kernels, an association was detected between γ-tocopherol methyltransferase (vte4) and α-tocopherol content, along with () and () for tocotrienol variation. Additionally, two kernel starch synthesis genes, () and (), were associated with tocotrienols, with the strongest evidence for in combination with fixed, strong and alleles, accounting for the greater amount of tocotrienols in and lines. In prediction models with genome-wide markers, predictive abilities were higher for tocotrienols than tocopherols, and these models were superior to those that used marker sets targeting a priori genes involved in the biosynthesis and/or genetic control of tocochromanols. Through this quantitative genetic analysis, we have established a key step for increasing tocochromanols in fresh kernels of sweet corn for human health and nutrition