Plant and inflorescence architecture determine the yield potential of crops. Breeders have harnessed natural diversity for inflorescence architecture to improve yields, and induced genetic variation could provide further gains. Wheat is a vital source of protein and calories; however, little is known about the genes that regulate the development of its inflorescence. Here, we report the identification of semidominant alleles for a class III homeodomain-leucine zipper transcription factor, HOMEOBOX DOMAIN-2 ( HB-2 ), on wheat A and D subgenomes, which generate more flower-bearing spikelets and enhance grain protein content. These alleles increase HB-2 expression by disrupting a microRNA 165/166 complementary site with conserved roles in plants; higher HB-2 expression is associated with modified leaf and vascular development and increased amino acid supply to the inflorescence during grain development. These findings enhance our understanding of genes that control wheat inflorescence development and introduce an approach to improve the nutritional quality of grain.

Key message: Yield penalty and increased grain protein content traits associated with Aegilops ventricosa 7D introgression have been mapped for the first time, and they are physically distinct from the eyespot resistance locus Pch1. Wheat wild relatives represent an important source of genetic variation, but introgression of agronomically relevant genes, such as for disease resistance, may lead to the simultaneous introduction of genetically linked deleterious traits. Pch1 is a dominant gene, conferring resistance to eyespot and was introgressed to wheat from Aegilops ventricosa as part of a large segment of the 7D^V chromosome. This introgression has been associated with a significant yield reduction and a concomitant increase in grain protein content. In this study, we evaluated both traits and their relationship to the location of the Pch1 gene. We found that both QTLs were clearly distinct from the Pch1 gene, being located on a different linkage group to Pch1. In addition, we found that the QTL for increased grain protein content was strong and consistent across field trials, whereas the yield penalty QTL was unstable and environmentally dependent. The yield and grain protein content QTLs were genetically linked and located in the same linkage group. This finding is due in part to the small size of the population, and to the restricted recombination between wheat 7D and Ae. ventricosa 7D^v chromosomes. Although recombination in this interval is rare, it does occur. A recombinant line containing Pch1 and 7D_KASP6, the marker associated with increase in grain protein content, but not Xwmc221, the marker associated with the yield penalty effect, was identified.

Cereal inflorescences are complex, highly ordered structures composed of grain-producing florets that form within specialised branches called spikelets. The spikelets of wheat are arranged in two alternating rows along a central rachis, in a pattern determined during early reproductive development. While several genes that control spikelet development have been identified, the molecular processes that regulate their morphology and the formation of supporting structures, such as meristems and the rachis, remain poorly understood. Here, we used spatial transcriptomics to investigate the dynamic transcriptional landscape of a wheat inflorescence during spikelet development. We identified two spatially distinct regions that regulate spikelet architecture, including a primordium region characterised by RAMOSA2 activity, and a boundary region that expresses ALOG1 and known regulators of bract suppression. Developmental assays indicate that spikelets differentiate from meristematic regions, which is accompanied by formation of central vascular regions of the rachis and inflorescence base that express genes controlling spikelet number. The combined spatial transcriptome and genetic data reveal key regulators of spikelet development, including target genes for improving spikelet number and yield.

Breeding for less digestible starch in wheat can improve the health impact of bread and other wheat foods. The application of forward genetic approaches has lately opened opportunities for the discovery of new genes that influence the digestibility of starch, without the burden of detrimental effects on yield or on pasta and bread-making quality. In this study we developed a high-throughput in vitro starch digestibility assay (HTA) for use in forward genetic approaches to screen wheat germplasm. The HTA was validated using standard maize and wheat starches. Using the HTA we measured starch digestibility in hydrothermally processed flour samples and found wide variation among 118 wheat landraces from the A. E. Watkins collection and among eight elite UK varieties (23.5 to 39.9% and 31.2 to 43.5% starch digested after 90 min, respectively). We further investigated starch digestibility in fractions of sieved wholemeal flour and purified starch in a subset of the Watkins lines and elite varieties and found that the matrix properties of flour rather than the intrinsic properties of starch granules conferred lower starch digestibility.

MicroRNA-resistant alleles of HOMEOBOX DOMAIN-2 ( HB-2 ) were recently reported to improve grain protein content (GPC) in wheat by modifying vascular development in stems to increase amino acid distribution to grain. However, it is not known if these alleles increase GPC at the expense of other physiologically important traits, such as senescence and yield-component traits. Here, we evaluated the effects of mutations in HB-2 on grain yield and nitrogen (N)-use efficiency as well as senescence and plant architecture in multi-environment glasshouse and field experiments. In glasshouse experiments, the mutations in HB-2 moderately improved GPC irrespective of N supply and without accelerating senescence or decreasing grain number or weight compared to the wildtype spring wheat cv. Cadenza. The trait analysis indicated mutant plants were more tolerant of N limitation in terms of above-ground N reduction compared to the wild type. The expression of all HB-2 homeologues increased in spikes at the lemma primordium stage in response to N limitation, indicating the later GPC increase could be a result of enhanced N sink strength determined during early development, rather than processes caused by direct changes in HB-2 expression in the grain. In field experiments, the introgression of HB-2 alleles into Australian elite spring wheat cultivars significantly improved GPC without compromising grain yield. The outcomes of this study support the introduction of miRNA-resistant HB-2 alleles, with a preference to HB-A2 , into commercial wheat for improving grain quality without a yield penalty.