pubs.acs.org/JAFC Published on Web 02/16/2010 © 2010 American Chemical Society 3600 J. Agric. Food Chem. 2010, 58, 3600–3610 DOI:10.1021/jf903705y Impact of Genetics and Environment on the Metabolite Composition of Maize Grain KIRSTEN SKOGERSON, 4, ) GEORGE G. HARRIGAN,* ,†, ) TRACEY L. REYNOLDS, § STEVEN C. HALLS, # MARTIN RUEBELT, ^ ALBERTO IANDOLINO, ^ ANAND PANDRAVADA, X KEVIN C. GLENN, † AND OLIVER FIEHN 4, ) † Product Safety Center, § Global Regulatory Pipeline, # Biotechnology, Monsanto Company, 800 North Lindbergh Boulevard, St. Louis, Missouri 63167, ^ Calgene Campus, Monsanto Company, 1920 Fifth Street, Davis, California 95616, X Corn Breeding, 12849 Gorman Lane, Woodland, California 95695, and 4 Metabolomics Research Laboratory, 451 Health Sciences Drive, Davis, California 95616. ) Authors contributed equally to the work. This study sought to assess genetic and environmental impacts on the metabolite composition of maize grain. Gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) measured 119 identified metabolites including free amino acids, free fatty acids, sugars, organic acids, and other small molecules in a range of hybrids derived from 48 inbred lines crossed against two different tester lines (from the C103 and Iodent heterotic groups) and grown at three locations in Iowa. It was reasoned that expanded metabolite coverage would contribute to a comprehensive evaluation of the grain metabolome, its degree of variability, and, in principle, its relationship to other compositional and agronomic features. The metabolic profiling results established that the small molecule metabolite pool is highly dependent on genotypic variation and that levels of certain metabolite classes may have an inverse genotypic relationship to each other. Different metabolic phenotypes were clearly associated with the two distinct tester populations. Overall, grain from the C103 lines contained higher levels of free fatty acids and organic acids, whereas grain from the Iodent lines were associated with higher levels of amino acids and carbohydrates. In addition, the fold-range of genotype mean values [composed of six samples each (two tester crosses per inbred  three field sites)] for identified metabolites ranged from ∼1.5- to 93-fold. Interestingly, some grain metabolites showed a non-normal distribution over the entire corn population, which could, at least in part, be attributed to large differences in metabolite values within specific inbred crosses relative to other inbred sets. This study suggests a potential role for metabolic profiling in assisting the process of selecting elite germplasm in biotechnology development, or marker- assisted breeding. KEYWORDS: Maize; metabolic profiling; metabolomics; natural variation; Zea mays INTRODUCTION Maize represents one of the most important crops for the production of food, feed, and biofuel. As a major grower, the United States accounted for 13.1 million bushels or approxi- mately 40% of world maize production from 2007 to 2008 at an aggregate value of over $50 billion ( 1 ). As a safe and wholesome commodity, corn remains a focus of further intensive selective breeding to improve quality traits. Key nutritional components in maize grain include starch, protein, fiber, and oil ( 2 ). How- ever, due to its economic value, numerous projects to enhance the food and feed quality of corn grain have also centered on modulating levels of small molecule metabolites. These include attempts to increase R-tocopherol content ( 3 , 4 ) or decrease levels of the antinutrient phytic acid ( 5 -7 ). The metabolite pool size in grain is of low abundance (∼5%) relative to the accumulated biomass of starch, protein, fiber, and oil ( 8 , 9 ). [For the purposes of this discussion, oil (triglycerides) is arbi- trarily considered separate from the remainder of the small molecule metabolite pool of corn grain.] We reasoned that technical developments in nontargeted metabolic profiling would facilitate a comprehensive evaluation of the grain meta- bolome, its degree of variability, and its relationship to other compositional and agronomic features. This study therefore sought to assess genetic and environmental impacts on the small molecule metabolite composition of maize grain by surveying a range of diverse hybrids grown at three different locations in Iowa. The goal was to (i) assess the range of variation in different metabolites and metabolite classes across the study population at all sites, (ii) identify location and genotypic (tester) effects on relative levels of metabolites, and (iii) deter- mine whether metabolites or metabolite classes were more variable within certain hybrid lines and/or across all samples. *Author to whom correspondence should be addressed.