Genome-Wide Association Study and Pathway-Level Analysis of Tocochromanol Levels in Maize Grain

Abstract

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.

Novel Loci Underlie Natural Variation in Vitamin E Levels in Maize Grain

“…This gene was an eQTL with particularly strong correlations with aT allelic effects. These effects spanned a range of 11.58 mg/g, suggesting that vte4 is key for increasing vitamin E levels, a finding concordant with the large PVEs and clear GWAS results obtained for vte4 in this and previous studies Lipka et al, 2013). The final gene, vte3, encodes a methyltransferase at the branchpoint for d-and g-tocochromanols and was the highest-PVE a priori QTL for dT at 8.2%, with smaller PVEs for four other traits.…”

“…We resolved 14 QTL to the gene level using an approach integrating JL-QTL effect estimates, localization of GWAS signals, and RNAseq data from six stages of developing kernels for the NAM parental genotypes. Only two of the 14 genes identified in this study had been previously associated with natural variation for tocochromanols in maize grain Lipka et al, 2013). These 14 genes included seven of the nine intervals with largest PVEs (Figure 3; Supplemental Table 2) and in an additive model explained 56 to 93% of phenotypic variation attributed to QTL for the traits analyzed in this study (Supplemental Figure 8).…”

“…Condensation of HGA with phytyl-diphosphate (phytyl-DP), geranylgeranyl-diphosphate (GGDP), or solanesyl-diphosphate (solanesyl-DP) yields committed intermediates that are cyclized and methylated to produce the a, b, g, and d isoforms of tocopherols and tocotrienols, and plastochromanol-8 (PC-8), respectively. Tocochromanol biosynthesis is fully elucidated in Arabidopsis thaliana and involves 36 enzymatic activities (encoded by 53 genes) for the biosynthesis of HGA, prenyl-diphosphates, and the core tocochromanol pathway itself (vitamin E [VTE] loci 1 through 6; DellaPenna and Mène-Saffrané, 2011; Lipka et al, 2013). Genes encoding these enzymatic reactions are considered a priori candidates in the Arabidopsis genome that may influence natural variation for tocochromanol traits.…”

“…Recent genome-wide association studies (GWAS) in maize and rice (Oryza sativa) grain Lipka et al, 2013;Wang et al, 2015) showed strong associations of g-tocopherol methyltransferase (vte4) with a-tocopherol concentrations and much weaker associations of tocopherol cyclase (vte1), hggt1, and an arogenate/prephenate dehydratase with tocotrienol traits in maize grain . The panel sizes and density of singlenucleotide polymorphisms (SNPs) in these studies limited both the identification of controlling loci and gene-level resolution of causal variants.…”

Network-Guided GWAS Improves Identification of Genes Affecting Free Amino Acids

“…As such, GWAS has been employed successfully to better resolve the genetic basis of many primary and secondary metabolites (e.g. carotenoids, tocochromanols, glucosinolates, and organic acids) in several model systems, including rice (Oryza sativa), maize (Zea mays), and Arabidopsis (Arabidopsis thaliana; Chan et al, 2011;Riedelsheimer et al, 2012;Gonzalez-Jorge et al, 2013Lipka et al, 2013;Chen et al, 2014;Owens et al, 2014;Verslues et al, 2014). In several cases, GWAS has confirmed genes and their orthologs identified via mutant screens, metabolic QTLs, and other methods (Clarke et al, 1995;Wong et al, 2004;Vallabhaneni and Wurtzel, 2009;Yan et al, 2010;Wurtzel et al, 2012).…”