Evaluation of metabolomics profiles of grain from maize hybrids derived from near-isogenic GM positive and negative segregant inbreds demonstrates that observed differences cannot be attributed unequivocally to the GM trait.

INTRODUCTION: Past studies on plant metabolomes have highlighted the influence of growing environments and varietal differences in variation of levels of metabolites yet there remains continued interest in evaluating the effect of genetic modification (GM). OBJECTIVES: Here we test the hypothesis that metabolomics differences in grain from maize hybrids derived from a series of GM (NK603, herbicide tolerance) inbreds and corresponding negative segregants can arise from residual genetic variation associated with backcrossing and that the effect of insertion of the GM trait is negligible. METHODS: Four NK603-positive and negative segregant inbred males were crossed with two different females (testers). The resultant hybrids, as well as conventional comparator hybrids, were then grown at three replicated field sites in Illinois, Minnesota, and Nebraska during the 2013 season. Metabolomics data acquisition using gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) allowed the measurement of 367 unique metabolite features in harvested grain, of which 153 were identified with small molecule standards. Multivariate analyses of these data included multi-block principal component analysis and ANOVA-simultaneous component analysis. Univariate analyses of all 153 identified metabolites was conducted based on significance testing (α = 0.05), effect size evaluation (assessing magnitudes of differences), and variance component analysis. RESULTS: Results demonstrated that the largest effects on metabolomic variation were associated with different growing locations and the female tester. They further demonstrated that differences observed between GM and non-GM comparators, even in stringent tests utilizing near-isogenic positive and negative segregants, can simply reflect minor genomic differences associated with conventional back-crossing practices. CONCLUSION: The effect of GM on metabolomics variation was determined to be negligible and supports that there is no scientific rationale for prioritizing GM as a source of variation.

Metabolomic Assessment of Key Maize Resources: GC-MS and NMR Profiling of Grain from B73 Hybrids of the Nested Association Mapping (NAM) Founders and of Geographically Diverse Landraces.

The present study expands metabolomic assessments of maize beyond commercial lines to include two sets of hybrids used extensively in the scientific community. One set included hybrids derived from the nested association mapping (NAM) founder lines, a collection of 25 inbreds selected on the basis of genetic diversity and used to investigate the genetic basis of complex plant traits. A second set included 24 hybrids derived from a collection of landraces representative of native diversity from North and South America that may serve as a source of new alleles for improving modern maize hybrids. Metabolomic analysis of grain harvested from these hybrids utilized gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) and (1)H nuclear magnetic resonance spectroscopy ((1)H NMR) techniques. Results highlighted extensive metabolomic variation in grain from both hybrid sets, but also demonstrated that, within each hybrid set, subpopulations could be differentiated in a pattern consistent with the known genetic and compositional variation of these lines. Correlation analysis did not indicate a strong association of the metabolomic data with grain nutrient composition, although some metabolites did show moderately strong correlations with agronomic features such as plant and ear height. Overall, this study provides insights into the extensive metabolomic diversity associated with conventional maize germplasm.

Maize hybrids derived from GM positive and negative segregant inbreds are compositionally equivalent: any observed differences are associated with conventional backcrossing practices.

In this study, we show that compositional differences in grain harvested from genetically modified (GM) maize hybrids derived from near-isogenic trait-positive and trait-negative segregant inbreds are more likely related to backcrossing practices than to the GM trait. To demonstrate this, four paired GM trait-positive (NK603: herbicide tolerance) and trait-negative near-isogenic inbred male lines were generated. These were crossed with two different females (testers) to create a series of trait-positive and trait-negative hybrid variants. The hypothesis was, that compositional variation within the hybrid variants would reflect differences associated with backcrossing practices and provide context to any observed differences between GM and non-GM hybrids. The F1 hybrids, as well as corresponding conventional comparator hybrids, were grown concurrently at four field sites across the United States during the 2013 season. Grain was harvested for compositional analysis; proximates (protein, starch, and oil), amino acids, fatty acids, minerals, tocopherols (α-, δ-, γ-), ß-carotene, phytic acid, and raffinose were measured. Statistical analysis showed that within each hybrid tester set, there were very few significant (p < 0.05) differences between the paired trait-positive and trait-negative hybrids or between the conventional comparators and the trait-positive or trait-negative hybrids. Assessments of the magnitudes of differences and variance component analysis highlighted that growing location, and the tester used in hybrid formation, had a markedly greater effect on composition than did the GM trait. Significantly, for each tester set, compositional differences within the trait-positive and trait-negative hybrid variants were greater than differences between the GM and non-GM comparators. Overall, GM trait insertion is not intrinsically a meaningful contributor to compositional variation, and observed differences between GM and non-GM comparators typically reflect incidental changes associated with conventional breeding practices. These results contribute to ongoing discussions on the relevance of negative segregants as comparators in GM assessments.

Compositional assessments of key maize populations: B73 hybrids of the Nested Association Mapping founder lines and diverse landrace inbred lines.

The present study provides an assessment of the compositional diversity in maize B73 hybrids derived both from the Nested Association Mapping (NAM) founder lines and from a diverse collection of landrace accessions from North and South America. The NAM founders represent a key population of publicly available lines that are used extensively in the maize community to investigate the genetic basis of complex traits. Landraces are also of interest to the maize community as they offer the potential to discover new alleles that could be incorporated into modern maize lines. The compositional analysis of B73 hybrids from the 25 NAM founders and 24 inbred lines derived from landraces included measurements of proximates (protein, fat, ash, and starch), fibers, minerals, amino acids, fatty acids, tocopherols (α-, γ-, and δ-), ß-carotene, phytic acid, and raffinose. Grain was harvested from a replicated trial in New York, USA. For each data set (NAM and landrace) canonical discriminant analysis allowed separation of distinct breeding groups (tropical, temperate, flint, mixed/intermediate) within each data set. Overall, results highlighted extensive variation in all composition components assessed for both sets of hybrids. The variation observed for some components within the landraces may therefore be of value for increasing their levels in modern maize lines. The study described here provided significant information on contributions of conventional breeding to crop compositional variation, as well as valuable information on key genetic resources for the maize community in the development of new improved lines.

Compositional differences between near-isogenic GM and conventional maize hybrids are associated with backcrossing practices in conventional breeding.

Here, we show that differences between genetically modified (GM) and non-GM comparators cannot be attributed unequivocally to the GM trait, but arise because of minor genomic differences in near-isogenic lines. Specifically, this study contrasted the effect of three GM traits (drought tolerance, MON 87460; herbicide resistance, NK603; insect protection, MON 89034) on maize grain composition relative to the effects of residual genetic variation from backcrossing. Important features of the study included (i) marker-assisted backcrossing to generate genetically similar inbred variants for each GM line, (ii) high-resolution genotyping to evaluate the genetic similarity of GM lines to the corresponding recurrent parents and (iii) introgression of the different GM traits separately into a wide range of genetically distinct conventional inbred lines. The F1 hybrids of all lines were grown concurrently at three replicated field sites in the United States during the 2012 growing season, and harvested grain was subjected to compositional analysis. Proximates (protein, starch and oil), amino acids, fatty acids, tocopherols and minerals were measured. The number of statistically significant differences (α = 0.05), as well as magnitudes of difference, in mean levels of these components between corresponding GM variants was essentially identical to that between GM and non-GM controls. The largest sources of compositional variation were the genetic background of the different conventional inbred lines (males and females) used to generate the maize hybrids and location. The lack of any compositional effect attributable to GM suggests the development of modern agricultural biotechnology has been accompanied by a lack of any safety or nutritional concerns.

Compositional analysis of grain and forage from MON 87427, an inducible male sterile and tissue selective glyphosate-tolerant maize product for hybrid seed production.

Conventional maize hybrid seed production has historically relied upon detasseling using either manual methods or semiautomated processes to ensure the purity of the hybrid cross. Monsanto Co. has developed biotechnology-derived MON 87427 maize with tissue-selective glyphosate tolerance to facilitate the production of hybrid maize seed. MON 87427 utilizes a specific promoter and intron combination to drive expression of CP4 EPSPS protein in vegetative and female reproductive tissues, conferring tolerance to glyphosate. This specific combination of regulatory elements also results in limited or no production of CP4 EPSPS protein in two key male reproductive tissues: pollen microspores, which develop into pollen grains, and tapetum cells that supply nutrients to the pollen. Thus, MON 87427 induces a male sterile phenotype after appropriately timed glyphosate applications. To confer additional benefits of herbicide tolerance and/or insect resistance, MON 87427 was combined with MON 89034 and NK603 by conventional breeding to develop MON 87427 × MON 89034 × NK603. The work described here is an assessment of the nutrient, antinutrient, and secondary metabolite levels in grain and forage tissues of MON 87427 and MON 87427 × MON 89034 × NK603. Results demonstrated that MON 87427 is compositionally equivalent to a near-isogenic conventional comparator. Results from this analysis established that the compositional equivalence observed for the single-event product MON 87427 is extendable to the combined-trait product, MON 87427 × MON 89034 × NK603. With increasing global demand for food production, the development of more efficient seed production strategies is important to sustainable agriculture. The study reported here demonstrated that biotechnology can be applied to simplify hybrid maize seed production without affecting crop composition.

Identification and characterization of an Arabidopsis homogentisate phytyltransferase paralog.

Tocochromanols (tocopherols and tocotrienols) are micronutrients with antioxidant properties synthesized by photosynthetic bacteria and plants that play important roles in animal and human nutrition. There is considerable interest in identifying the genes involved in tocochromanol biosynthesis to allow transgenic modification of both tocochromanol levels and tocochromanol composition in agricultural crops. The first committed reaction in tocopherol biosynthesis is the condensation of homogentisic acid (HGA) with phytyldiphosphate or geranylgeranyldiphosphate, catalyzed by the homogentisate phytyltransferase (VTE2) or by the homogentisate geranylgeranyl transferase (HGGT). In this study, we describe the identification of conserved amino acid sequences within VTE2 and HGGT and the application of these conserved sequences for a motif analysis resulting in the discovery of a VTE2-paralog in the Arabidopsis genome. We designated this new gene VTE2-2 and renamed the old VTE2 to VTE2-1. Seed-specific expression of VTE2-2 in Arabidopsis resulted in increased seed-tocopherol levels, similar to the transgenic expression of VTE2-1. Bioinformatics analysis revealed that VTE2-2 is conserved in both monocotyledonous and dicotyledonous plants and is distinct from VTE2-1 and HGGT.

The Arabidopsis vitamin E pathway gene5-1 mutant reveals a critical role for phytol kinase in seed tocopherol biosynthesis.

We report the identification and characterization of a low tocopherol Arabidopsis thaliana mutant, vitamin E pathway gene5-1 (vte5-1), with seed tocopherol levels reduced to 20% of the wild type. Map-based identification of the responsible mutation identified a G-->A transition, resulting in the introduction of a stop codon in At5g04490, a previously unannotated gene, which we named VTE5. Complementation of the mutation with the wild-type transgene largely restored the wild-type tocopherol phenotype. A knockout mutation of the Synechocystis sp PCC 6803 VTE5 homolog slr1652 reduced Synechocystis tocopherol levels by 50% or more. Bioinformatic analysis of VTE5 and slr1652 indicated modest similarity to dolichol kinase. Analysis of extracts from Arabidopsis and Synechocystis mutants revealed increased accumulation of free phytol. Heterologous expression of these genes in Escherichia coli supplemented with free phytol and in vitro assays of recombinant protein produced phytylmonophosphate, suggesting that VTE5 and slr1652 encode phytol kinases. The phenotype of the vte5-1 mutant is consistent with the hypothesis that chlorophyll degradation-derived phytol serves as an important intermediate in seed tocopherol synthesis and forces reevaluation of the role of geranylgeranyl diphosphate reductase in tocopherol biosynthesis.

AmphiNk2-tin, an amphioxus homeobox gene expressed in myocardial progenitors: insights into evolution of the vertebrate heart.

We isolated a full-length cDNA clone of amphioxus AmphiNk2-tin, an NK2 gene similar in sequence to vertebrate NK2 cardiac genes, suggesting a potentially similar function to Drosophila tinman and to vertebrate NK2 cardiac genes during heart development. During the neurula stage of amphioxus, AmphiNk2-tin is expressed first within the foregut endoderm, then transiently in muscle precursor cells in the somites, and finally in some mesoderm cells of the visceral peritoneum arranged in an approximately midventral row running beneath the midgut and hindgut. The peritoneal cells that express AmphiNk2-tin are evidently precursors of the myocardium of the heart, which subsequently becomes morphologically detectable ventral to the gut. The amphioxus heart is a rostrocaudally extended tube consisting entirely of myocardial cells (at both the larval and adult stages); there are no chambers, valves, endocardium, epicardium, or other differentiated features of vertebrate hearts. Phylogenetic analysis of the AmphiNk2-tin sequence documents its close relationship to vertebrate NK2 class cardiac genes, and ancillary evidence suggests a relationship with the Drosophila NK2 gene tinman. Apparently, an amphioxus-like heart, and the developmental program directing its development, was the foundation upon which the vertebrate heart evolved by progressive modular innovations at the genetic and morphological levels of organization.