Metabolite characterization of durum wheat grain for association studies: exploring the natural variation of free asparagine content.

Acrylamide, a neurotoxic molecule that is also suspected to be carcinogenic, has been detected in many foods after high temperature processing. In wheat derivatives, free asparagine is a precursor of acrylamide formation. Increased levels may drive acrylamide quantity1. Therefore, the control of free asparagine levels in wheat seeds is of considerable interest in contemporary crop and food sciences. Currently, few information is available about the genetic control of this trait2. Our study aims to explore the natural variation of free asparagine levels in durum wheat (Triticum turgidum L. ssp. durum (Desf.)) grains to identify candidate genes controlling this trait. To this purpose, 201 durum wheat genotypes were selected from an international genotyped collection3. These genotypes were sown in an experimental field located in Italy for three years. Wholemeal flour was obtained from harvested grains and free asparagine content was measured using an enzymatic method. A good variability of this trait was shown. Both single-locus and multi-locus genome-wide association studies were performed to identify genomic regions associated with free asparagine content. Six Quantitative Trait Nucleotides on chromosomes 6A, 7A, 2B, 4B, 7B associated with this trait were identified. To deeply characterize metabolite variations associated to asparagine content, the whole grain metabolome of the 201 durum wheat genotypes was investigated by Ultrahigh Pressure Liquid Chromatography coupled to High Resolution Mass Spectrometry (UPLC-HRMS)4. Moreover, 100 selected samples were analysed through High Resolution Proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy. Both spectroscopic determinations showed a good correlation with the enzymatic method for free asparagine content quantification, confirming the good variability observed in our experimental set. These analyses will allow to identify metabolic pathways associated to free asparagine accumulation and to characterize the metabolite composition of the whole grain of the 201 durum wheat genotypes. This work is based upon the work/cooperation in COST Action 21149 - ACRYRED.