Defining genetic and chemical diversity in wheat grain by 1H-NMR spectroscopy of polar metabolites

Abstract

Scope: The application of high-throughput 1H nuclear magnetic resonance (1H-NMR) of unpurified extracts to determine genetic diversity and the contents of polar components in grain of wheat.

Methods and results: Milled whole wheat grain was extracted with 80:20 D₂ O:CD₃ OD containing 0.05% d₄ -trimethylsilylpropionate. 1H-NMR spectra were acquired under automation at 300°K using an Avance Spectrometer operating at 600.0528 MHz. Regions for individual metabolites were identified by comparison to a library of known standards run under identical conditions. The individual 1H-NMR peaks or levels of known metabolites were then compared by Principal Component Analysis using SIMCA-P software.

Conclusions: High-throughput 1H-NMR is an excellent tool to compare the extent of genetic diversity within and between wheat species, and to quantify specific components (including glycine betaine, choline, and asparagine) in individual genotypes. It can also be used to monitor changes in composition related to environmental factors and to support comparisons of the substantial equivalence of transgenic lines.

Keywords: Grain Composition; Metabolomics; NMR Spectroscopy; Transgenic; Wheat.

Figure 1

Typical 600 MHz 1H‐NMR spectrum of wholemeal flour extracted with 20:80 CD₃OD:D₂O. 1: sucrose; 2: maltose; 3: raffinose; 4: glucose; 5: fructose; 6: galactose; 7: galactinol; 8: glycine betaine; 9: choline; 10: asparagine; 11: malate; 12: citrate; 13: glutamine; 14: glutamate; 15: acetate; 16: alanine; 17: threonine; 18: GABA; 19: valine; 20: leucine; 21: isoleucine; 22: formate; 23: adenosine; 24: fumarate; 25: tryptophan; 26: tyrosine.

Figure 2

PCA analysis of quantified major metabolites from 1H‐NMR (600 MHz) analysis of CD₃OD:D₂O (1:4) extracts of wholemeal flour of 150 bread wheat lines grown at a single location in 2004–2005.

Figure 3

PCA of quantified major metabolites from 1H‐NMR analysis of CD₃OD:D₂O (1:4) extracts of wholemeal flour of 150 bread wheat lines and 50 other cereal lines grown at a single location in 2004–2005.

Figure 4

Asparagine concentrations (mg/g), determined by 1H‐NMR, of wholemeal flour of 26 bread wheat genotypes grown at four locations in 2006–2007 within the EU HEALTHGRAIN project. Data points are colored according to site (Hungary: blue; France: orange; UK: green; Poland: red (24 lines only)) and numbered according to the individual genotypes. 1: Estica; 2: Disponent; 3: Lynx; 4: Riband; 5: San‐Pastore; 6: Cadenza; 7: Tommi; 8: Maris‐Huntsman; 9: CF99105; 10: Campari; 11: Avalon; 12: Chinese‐Spring; 13: Crousty; 14: Herzog; 15: Spartanka; 16: Malacca; 17: Isengrain; 18: Obriy; 19: Tremie; 20: Tiger; 21: Rialto; 22: Claire; 23: Mv‐Emese; 24: Gloria; 25: Atlas‐66; 26: Valoris.

Figure 5

PCA of whole fingerprint data from 1H‐NMR analysis of CD₃OD:D₂O (1:4) extracts of wholemeal flour of two transgenic lines and one control line of wheat cv Cadenza grown in the field in 2013. Four replicate plots of each transgenic line and eight replicate plots of the control line were analyzed each with three technical replicates.

Figure 6

PCA of whole fingerprint data from 1H‐NMR analysis of CD₃OD:D₂O (1:4) extracts of wholemeal flour of two transgenic lines and one control line of wheat cv Cadenza grown in the field in in the United Kingdom in 2013 (data as used in Fig. 5) combined with analyses of 130 winter and 20 spring wheat lines grown in the field in Hungary in 2007 (data as used in Fig. 2).

Figure 7

PCA of whole fingerprint data from 1H‐NMR analysis of CD₃OD:D₂O (1:4) extracts of wholemeal flour of two transgenic lines and one control line of wheat cv Cadenza grown in the field in the United Kingdom in 2012–2013 (data as used in Fig. 5; shown in orange as RRes 2013) combined with analyses of wheat lines grown in the field in Hungary in 2004–2005 (23 lines) and 2005–2006 (26 lines) and in Poland (24 lines), Hungary, France, and the United Kingdom (26 lines) in 2006–2007 (data as used in Fig. 4).