(1)H-NMR screening for the high-throughput determination of genotype and environmental effects on the content of asparagine in wheat grain

Abstract

Free asparagine in cereals is known to be the precursor of acrylamide, a neurotoxic and carcinogenic product formed during cooking processes. Thus, the development of crops with lower asparagine is of considerable interest to growers and the food industry. In this study, we describe the development and application of a rapid (1)H-NMR-based analysis of cereal flour, that is, suitable for quantifying asparagine levels, and hence acrylamide-forming potential, across large numbers of samples. The screen was applied to flour samples from 150 bread wheats grown at a single site in 2005, providing the largest sample set to date. Additionally, screening of 26 selected cultivars grown for two further years in the same location and in three additional European locations in the third year (2007) provided six widely different environments to allow estimation of the environmental (E) and G x E effects on asparagine levels. Asparagine concentrations in the 150 genotypes ranged from 0.32 to 1.56 mg/g dry matter in wholemeal wheat flours. Asparagine levels were correlated with plant height and therefore, due to recent breeding activities to produce semi-dwarf varieties, a negative relationship with the year of registration of the cultivar was also observed. The multisite study indicated that only 13% of the observed variation in asparagine levels was heritable, whilst the environmental contribution was 36% and the GxE component was 43%. Thus, compared to some other phenotypic traits, breeding for low asparagine wheats presents a difficult challenge.

Keywords: 1H-NMR; Asparagine; G × E; heritability; metabolite profiling; wheat.

Figure 1

NMR quantitation of asparagine in cereal flours. (a) NMR spectrum of typical wheat flour extract made in CD ₃ OD:D₂O (1:4). (b) spectrum of pure asparagine made in CD ₃ OD:D₂O (1:4). (c) expansion of the 3‐H₂ signal (top) and illustration of 3‐H_b of asparagine standard (bottom) which was utilized for quantitation.

Figure 2

Frequency distribution of 151 bread wheat genotypes based on their asparagine concentration.

Figure 3

Heatmap of Pearson correlation coefficients of amino acid and soluble carbohydrate levels derived from ¹H‐NMR metabolite profiling of wholemeal samples of 150 bread wheat genotypes grown in Martonvasar, Hungary in 2005.

Figure 4

Average asparagine concentrations (mg/g d.m.) over 6 environments (Hungary (2005–2007), France (2007), UK (2007) and Poland (2007). Error bars represent observed asparagine concentration range across the 6 years/locations.

Figure 5

Comparison of asparagine concentration (mg/g d.m.) in white flour and wholemeal samples grown at 4 locations (France, Hungary, UK and Poland) in 2007. (a) correlation of asparagine concentrations (mg/g d.m.) in white flours against wholemeals; (b) mean levels of measured asparagine concentration in white flour and wholemeal samples from each of 4 growing locations. Error bars represent standard deviation of 3 replicate samples.

Figure 6

Average asparagine concentrations (mg/g d.m.) from wheat lines grown in 2007 at 4 environments (Hungary, France, UK and Poland). Error bars represent observed asparagine concentration range across the 4 locations. (a) data from white flour samples; (b) data from wholemeal samples.

Figure 7

Pie chart showing computed variance components for asparagine concentration in wholemeal samples from 26 bread wheats grown in 6 environments.