Reduced free asparagine in wheat grain resulting from a natural deletion of TaASN-B2: investigating and exploiting diversity in the asparagine synthetase gene family to improve wheat quality

Abstract

Background Understanding the determinants of free asparagine concentration in wheat grain is necessary to reduce levels of the processing contaminant acrylamide in baked and toasted wheat products. Although crop management strategies can help reduce asparagine concentrations, breeders have limited options to select for genetic variation underlying this trait. Asparagine synthetase enzymes catalyse a critical step in asparagine biosynthesis in plants and, in wheat, are encoded by five homeologous gene triads that exhibit distinct expression profiles. Within this family, TaASN2 genes are highly expressed during grain development but TaASN-B2 is absent in some varieties. Results Natural genetic diversity in the asparagine synthetase gene family was assessed in different wheat varieties revealing instances of presence/absence variation and other polymorphisms, including some predicted to affect the function of the encoded protein. The presence and absence of TaASN-B2 was determined across a range of UK and global common wheat varieties and related species, showing that the deletion encompassing this gene was already present in some wild emmer wheat genotypes. Expression profiling confirmed that TaASN2 transcripts were only detectable in the grain, while TaASN3.1 genes were highly expressed during the early stages of grain development. TaASN-A2 was the most highly expressed TaASN2 homeologue in most assayed wheat varieties. TaASN-B2 and TaASN-D2 were expressed at similar, lower levels in varieties possessing TaASN-B2. Expression of TaASN-A2 and TaASN-D2 did not increase to compensate for the absence of TaASN-B2, so total TaASN2 expression was lower in varieties lacking TaASN-B2. Consequently, free asparagine concentrations in field-produced grain were, on average, lower in varieties lacking TaASN-B2, although the effect was lost when free asparagine accumulated to very high concentrations as a result of sulphur deficiency. Conclusions Selecting wheat genotypes lacking the TaASN-B2 gene may be a simple and rapid way for breeders to reduce free asparagine concentrations in commercial wheat grain.

Understanding the Relationships between Free Asparagine in Grain and Other Traits to Breed Low-Asparagine Wheat

“…It is possible that the association of some soft wheat varieties with consistently low free asparagine content was due to the deletion of one of the asparagine synthetase 2 homeologues, TaASN-B2, which has been shown to be associated with lower grain asparagine content and was more common in the soft wheats used in the trial [26]. The effect of this deletion is only apparent when the plants have adequate sulphur, though, with the effect being overwhelmed by the huge increase in free asparagine concentration that occurs under sulphur deficiency [26], adding more complexity to the control of grain asparagine content. Although the relationship between free asparagine content and the protein composition of grain is complex, there are two factors that are well known to affect both: nitrogen and sulphur fertilisers.…”

“…Curtis et al [ 39 ] did show that varieties with consistently low free asparagine concentration were often soft wheats, but the difference between hard and soft variety groups was not significant, with high and low-asparagine varieties in both groups. It is possible that the association of some soft wheat varieties with consistently low free asparagine content was due to the deletion of one of the asparagine synthetase 2 homeologues, TaASN-B2 , which has been shown to be associated with lower grain asparagine content and was more common in the soft wheats used in the trial [ 26 ]. The effect of this deletion is only apparent when the plants have adequate sulphur, though, with the effect being overwhelmed by the huge increase in free asparagine concentration that occurs under sulphur deficiency [ 26 ], adding more complexity to the control of grain asparagine content.…”

“…Breeding low-asparagine wheat could potentially be achieved in three main ways: directly, by using either existing or induced variation, or indirectly, through selection for related traits ( Figure 3 ). New wheat varieties are commonly developed using existing variation; however, the only multi-environment quantitative trait locus (QTL) for low-asparagine known at present is the one in which the TaASN-B2 gene is either present or deleted, which has been shown to affect the free asparagine content of grain in two different field trials [ 26 ]. Selection for the TaASN-B2 deletion represents an easy gain for breeders, but further trials testing the effect of the deletion should be performed to confirm the stability of the effect across more environments.…”

“…This has been demonstrated by the use of CRISPR/Cas9 technology to ‘knock out’ the TaASN2 genes, reducing grain asparagine content by up to 90% in glasshouse experiments [ 25 ]. The edited lines still need to undergo trials to confirm the stability of this phenotype in the field, but the stability of the ‘natural’ TaASN-B2 deletion phenotype under field conditions [ 26 ] is encouraging, suggesting that the TaASN2 -edited phenotypes may be similarly stable. However, the interaction between the TaASN-B2 deletion and sulphur deficiency implies that TaASN2 variants may not be sufficient to control grain asparagine content during sulphur deficiency or other stresses, again highlighting the effects of E and G × E. On the other hand, the varieties carrying the TaASN-B2 deletion have intact TaASN-A2 and TaASN-D2 genes, whereas the edited lines lack any functional TaASN2 genes, so the edited lines will be valuable for investigating whether this prevents free asparagine accumulation under conditions of sulphur deficiency or other stresses.…”

“… Timeline of asparagine research in wheat since the discovery of acrylamide in food [ 2 , 5 , 6 , 7 , 10 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 ]. Asn (asparagine), G × E (genotype-by-environment interaction), PHS (pre-harvest sprouting), GWAS (genome-wide association study), QTL (quantitative trait locus/loci), RNAseq (RNA sequencing).…”