Genetic control of grain amino acid composition in a UK soft wheat mapping population.

Wheat (Triticum aestivum L.) is a major source of nutrients for populations across the globe, but the amino acid composition of wheat grain does not provide optimal nutrition. The nutritional value of wheat grain is limited by low concentrations of lysine (the most limiting essential amino acid) and high concentrations of free asparagine (precursor to the processing contaminant acrylamide). There are currently few available solutions for asparagine reduction and lysine biofortification through breeding. In this study, we investigated the genetic architecture controlling grain free amino acid composition and its relationship to other traits in a Robigus × Claire doubled haploid population. Multivariate analysis of amino acids and other traits showed that the two groups are largely independent of one another, with the largest effect on amino acids being from the environment. Linkage analysis of the population allowed identification of quantitative trait loci (QTL) controlling free amino acids and other traits, and this was compared against genomic prediction methods. Following identification of a QTL controlling free lysine content, wheat pangenome resources facilitated analysis of candidate genes in this region of the genome. These findings can be used to select appropriate strategies for lysine biofortification and free asparagine reduction in wheat breeding programs.

Reducing Dietary Acrylamide Exposure from Wheat Products through Crop Management and Imaging.

The nutritional safety of wheat-based food products is compromised by the presence of the processing contaminant acrylamide. Reduction of the key acrylamide precursor, free (soluble, non-protein) asparagine, in wheat grain can be achieved through crop management strategies, but such strategies have not been fully developed. We ran two field trials with 12 soft (biscuit) wheat varieties and different nitrogen, sulfur, potassium, and phosphorus fertilizer combinations. Our results indicated that a nitrogen-to-sulfur ratio of 10:1 kg/ha was sufficient to prevent large increases in free asparagine, whereas withholding potassium or phosphorus alone did not cause increases in free asparagine when sulfur was applied. Multispectral measurements of plants in the field were able to predict the free asparagine content of grain with an accuracy of 71%, while a combination of multispectral, fluorescence, and morphological measurements of seeds could distinguish high free asparagine grain from low free asparagine grain with an accuracy of 86%. The acrylamide content of biscuits correlated strongly with free asparagine content and with color measurements, indicating that agronomic strategies to decrease free asparagine would be effective and that quality control checks based on product color could eliminate high acrylamide biscuit products.

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.

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.

Deciphering the Theobroma cacao self-incompatibility system: from genomics to diagnostic markers for self-compatibility.

Cocoa self-compatibility is an important yield factor and has been described as being controlled by a late gameto-sporophytic system expressed only at the level of the embryo sac. It results in gametic non-fusion and involves several loci. In this work, we identified two loci, located on chromosomes 1 and 4 (CH1 and CH4), involved in cocoa self-incompatibility by two different processes. Both loci are responsible for gametic selection, but only one (the CH4 locus) is involved in the main fruit drop. The CH1 locus acts prior to the gamete fusion step and independently of the CH4 locus. Using fine-mapping and genome-wide association studies, we focused analyses on restricted regions and identified candidate genes. Some of them showed a differential expression between incompatible and compatible reactions. Immunolocalization experiments provided evidence of CH1 candidate genes expressed in ovule and style tissues. Highly polymorphic simple sequence repeat (SSR) diagnostic markers were designed in the CH4 region that had been identified by fine-mapping. They are characterized by a strong linkage disequilibrium with incompatibility alleles, thus allowing the development of efficient diagnostic markers predicting self-compatibility and fruit setting according to the presence of specific alleles or genotypes. SSR alleles specific to self-compatible Amelonado and Criollo varieties were also identified, thus allowing screening for self-compatible plants in cocoa populations.

High throughput, high resolution selection of polymorphic microsatellite loci for multiplex analysis.

BACKGROUND: Large-scale genetic profiling, mapping and genetic association studies require access to a series of well-characterised and polymorphic microsatellite markers with distinct and broad allele ranges. Selection of complementary microsatellite markers with non-overlapping allele ranges has historically proved to be a bottleneck in the development of multiplex microsatellite assays. The characterisation process for each microsatellite locus can be laborious and costly given the need for numerous, locus-specific fluorescent primers. RESULTS: Here, we describe a simple and inexpensive approach to select useful microsatellite markers. The system is based on the pooling of multiple unlabelled PCR amplicons and their subsequent ligation into a standard cloning vector. A second round of amplification utilising generic labelled primers targeting the vector and unlabelled locus-specific primers targeting the microsatellite flanking region yield allelic profiles that are representative of all individuals contained within the pool. Suitability of various DNA pool sizes was then tested for this purpose. DNA template pools containing between 8 and 96 individuals were assessed for the determination of allele ranges of individual microsatellite markers across a broad population. This helped resolve the balance between using pools that are large enough to allow the detection of many alleles against the risk of including too many individuals in a pool such that rare alleles are over-diluted and so do not appear in the pooled microsatellite profile. Pools of DNA from 12 individuals allowed the reliable detection of all alleles present in the pool. CONCLUSION: The use of generic vector-specific fluorescent primers and unlabelled locus-specific primers provides a high resolution, rapid and inexpensive approach for the selection of highly polymorphic microsatellite loci that possess non-overlapping allele ranges for use in large-scale multiplex assays.