Improving wheat grain composition for human health by constructing a QTL atlas for essential minerals.

Wheat is an important source of minerals for human nutrition and increasing grain mineral content can contribute to reducing mineral deficiencies. Here, we identify QTLs for mineral micronutrients in grain of wheat by determining the contents of six minerals in a total of eleven sample sets of three biparental populations from crosses between A.E. Watkins landraces and cv. Paragon. Twenty-three of the QTLs are mapped in two or more sample sets, with LOD scores above five in at least one set with the increasing alleles for sixteen of the QTLs being present in the landraces and seven in Paragon. Of these QTLs, the number for each mineral varies between three and five and they are located on 14 of the 21 chromosomes, with clusters on chromosomes 5A (four), 6A (three), and 7A (three). The gene content within 5 megabases of DNA on either side of the marker for the QTL with the highest LOD score is determined and the gene responsible for the strongest QTL (chromosome 5A for Ca) identified as an ATPase transporter gene (TraesCS5A02G543300) using mutagenesis. The identification of these QTLs, together with associated SNP markers and candidate genes, will facilitate the improvement of grain nutritional quality.

Wheat MYOSIN-RESEMBLING CHLOROPLAST PROTEIN controls B-type starch granule initiation timing during endosperm development.

Molecular factors that contribute to the diverse spatial and temporal patterns of starch granule initiation between species and organs are poorly understood. Wheat (Triticum sp.) endosperm contains both large A-type granules initiated during early grain development and small B-type granules that initiate about 10-15 days later. Here we identify that the MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC) is required for the correct timing of B-type granule initiation in wheat endosperm during grain development. MRC is expressed in the endosperm exclusively in early grain development, before B-type granule initiation. We isolated three independent TILLING mutants of tetraploid wheat (Triticum turgidum cv. Kronos) with premature stop or missense mutations in the A-genome homoeolog, which we showed to be the only active homoeolog in tetraploid wheat due to a disruption of the B-genome homoeolog. The mrc mutants had significantly smaller A-type granules and a higher relative volume of B-type granules in the endosperm than the wild type. Whereas B-type granules initiated 15 - 20 days post anthesis (dpa) in the wild type, they appeared as early as 10 dpa in the mrc-1 mutant. These results suggest a temporal role for MRC in repressing B-type granule initiation, providing insight into how the distinct biochemical mechanisms that control A- and B-type granule initiation are regulated. This role of MRC in the wheat endosperm is distinct from the previously described role of Arabidopsis (Arabidopsis thaliana) MRC in promoting granule initiation in leaves, providing an example of functional diversification among granule initiation proteins.

The wheat powdery mildew resistance gene Pm4 also confers resistance to wheat blast.

Wheat blast, caused by the fungus Magnaporthe oryzae, threatens global cereal production since its emergence in Brazil in 1985 and recently spread to Bangladesh and Zambia. Here we demonstrate that the AVR-Rmg8 effector, common in wheat-infecting isolates, is recognized by the gene Pm4, previously shown to confer resistance to specific races of Blumeria graminis f. sp. tritici, the cause of powdery mildew of wheat. We show that Pm4 alleles differ in their recognition of different AVR-Rmg8 alleles, and some confer resistance only in seedling leaves but not spikes, making it important to select for those alleles that function in both tissues. This study has identified a gene recognizing an important virulence factor present in wheat blast isolates in Bangladesh and Zambia and represents an important first step towards developing durably resistant wheat cultivars for these regions.

FIGL1 prevents aberrant chromosome associations and fragmentation and limits crossovers in polyploid wheat meiosis.

Meiotic crossovers (COs) generate genetic diversity and are crucial for viable gamete production. Plant COs are typically limited to 1-3 per chromosome pair, constraining the development of improved varieties, which in wheat is exacerbated by an extreme distal localisation bias. Advances in wheat genomics and related technologies provide new opportunities to investigate, and possibly modify, recombination in this important crop species. Here, we investigate the disruption of FIGL1 in tetraploid and hexaploid wheat as a potential strategy for modifying CO frequency/position. We analysed figl1 mutants and virus-induced gene silencing lines cytogenetically. Genetic mapping was performed in the hexaploid. FIGL1 prevents abnormal meiotic chromosome associations/fragmentation in both ploidies. It suppresses class II COs in the tetraploid such that CO/chiasma frequency increased 2.1-fold in a figl1 msh5 quadruple mutant compared with a msh5 double mutant. It does not appear to affect class I COs based on HEI10 foci counts in a hexaploid figl1 triple mutant. Genetic mapping in the triple mutant suggested no significant overall increase in total recombination across examined intervals but revealed large increases in specific individual intervals. Notably, the tetraploid figl1 double mutant was sterile but the hexaploid triple mutant was moderately fertile, indicating potential utility for wheat breeding.

Systematic identification of wheat spike developmental regulators by integrated multi-omics, transcriptional network, GWAS, and genetic analyses.

The spike architecture of wheat plays a crucial role in determining grain number, making it a key trait for optimization in wheat breeding programs. In this study, we used a multi-omic approach to analyze the transcriptome and epigenome profiles of the young spike at eight developmental stages, revealing coordinated changes in chromatin accessibility and H3K27me3 abundance during the flowering transition. We constructed a core transcriptional regulatory network (TRN) that drives wheat spike formation and experimentally validated a multi-layer regulatory module involving TaSPL15, TaAGLG1, and TaFUL2. By integrating the TRN with genome-wide association studies, we identified 227 transcription factors, including 42 with known functions and 185 with unknown functions. Further investigation of 61 novel transcription factors using multiple homozygous mutant lines revealed 36 transcription factors that regulate spike architecture or flowering time, such as TaMYC2-A1, TaMYB30-A1, and TaWRKY37-A1. Of particular interest, TaMYB30-A1, downstream of and repressed by WFZP, was found to regulate fertile spikelet number. Notably, the excellent haplotype of TaMYB30-A1, which contains a C allele at the WFZP binding site, was enriched during wheat breeding improvement in China, leading to improved agronomic traits. Finally, we constructed a free and open access Wheat Spike Multi-Omic Database (http://39.98.48.156:8800/#/). Our study identifies novel and high-confidence regulators and offers an effective strategy for dissecting the genetic basis of wheat spike development, with practical value for wheat breeding.

A wheat chromosome segment substitution line series supports characterization and use of progenitor genetic variation.

Genome-wide introgression and substitution lines have been developed in many plant species, enhancing mapping precision, gene discovery, and the identification and exploitation of variation from wild relatives. Created over multiple generations of crossing and/or backcrossing accompanied by marker-assisted selection, the resulting introgression lines are a fixed genetic resource. In this study we report the development of spring wheat (Triticum aestivum L.) chromosome segment substitution lines (CSSLs) generated to systematically capture genetic variation from tetraploid (T. turgidum ssp. dicoccoides) and diploid (Aegilops tauschii) progenitor species. Generated in a common genetic background over four generations of backcrossing, this is a base resource for the mapping and characterization of wheat progenitor variation. To facilitate further exploitation the final population was genetically characterized using a high-density genotyping array and a range of agronomic and grain traits assessed to demonstrate the potential use of the populations for trait localization in wheat.

Identification of a novel SNP in the miR172 binding site of Q homoeolog AP2L-D5 is associated with spike compactness and agronomic traits in wheat (Triticum aestivum L.).

KEY MESSAGE: This study found that the compact spike locus of ANK-15 is on chromosome 5D instead of 2B. We have identified a new allele of AP2L-D5 as the candidate causal polymorphism. Spike architecture is a key determinant of wheat yield, a crop which supports much of the human diet but whose yield gains are stagnating. Spike architecture mutants offer opportunities to identify genetic factors contributing to inflorescence development. Here, we investigate the locus underlying the compact spike phenotype of mutant line ANK-15 by conducting mRNA-sequencing and genetic mapping using ANK-15 and its non-compact spike near-isogenic line Novosibirskaya 67 (N67). Previous literature has placed the compact spike locus of ANK-15 to chromosome 2B. However, based on the single nucleotide polymorphisms (SNPs) identified using mRNA-seq data, we were unable to detect polymorphisms between N67 and ANK-15 in the putative chromosome 2B region. We performed differential expression analysis of developing rachis and found that AP2L-D5, the D homoeolog of the domestication Q gene, is upregulated in ANK-15 in comparison to N67. ANK-15 carries a SNP in the microRNA172 binding site of AP2L-D5, which is predicted to lead to higher expression of AP2L-D5 due to decreased miRNA172-mediated degradation. Furthermore, we performed genetic mapping using an ANK-15 × N67 F2 population and found a single quantitative trait locus on chromosome 5D coinciding with the position of AP2L-D5. This result suggests that AP2L-D5 is likely the underlying causal gene for the compact spike phenotype in ANK-15. We performed a field trial to investigate the effect of the AP2L-D5 allele on agronomic traits and found that the AP2L-D5 allele from ANK-15 is associated with a significant reduction in height, increased thousand grain weight (TGW), and increased grain width.

An online database for einkorn wheat to aid in gene discovery and functional genomics studies.

Diploid A-genome wheat (einkorn wheat) presents a nutrition-rich option as an ancient grain crop and a resource for the improvement of bread wheat against abiotic and biotic stresses. Realizing the importance of this wheat species, reference-level assemblies of two einkorn wheat accessions were generated (wild and domesticated). This work reports an einkorn genome database that provides an interface to the cereals research community to perform comparative genomics, applied genetics and breeding research. It features queries for annotated genes, the use of a recent genome browser release, and the ability to search for sequence alignments using a modern BLAST interface. Other features include a comparison of reference einkorn assemblies with other wheat cultivars through genomic synteny visualization and an alignment visualization tool for BLAST results. Altogether, this resource will help wheat research and breeding. Database URL  https://wheat.pw.usda.gov/GG3/pangenome.

Genome wide association in Spanish bread wheat landraces identifies six key genomic regions that constitute potential targets for improving grain yield related traits.

KEY MESSAGE: Association mapping conducted in 189 Spanish bread wheat landraces revealed six key genomic regions that constitute stable QTLs for yield and include 15 candidate genes. Genetically diverse landraces provide an ideal population to conduct association analysis. In this study, association mapping was conducted in a collection of 189 Spanish bread wheat landraces whose genomic diversity had been previously assessed. These genomic data were combined with characterization for yield-related traits, including grain size and shape, and phenological traits screened across five seasons. The association analysis revealed a total of 881 significant marker trait associations, involving 434 markers across the genome, that could be grouped in 366 QTLs based on linkage disequilibrium. After accounting for days to heading, we defined 33 high density QTL genomic regions associated to at least four traits. Considering the importance of detecting stable QTLs, 6 regions associated to several grain traits and thousand kernel weight in at least three environments were selected as the most promising ones to harbour targets for breeding. To dissect the genetic cause of the observed associations, we studied the function and in silico expression of the 413 genes located inside these six regions. This identified 15 candidate genes that provide a starting point for future analysis aimed at the identification and validation of wheat yield related genes.

Einkorn genomics sheds light on history of the oldest domesticated wheat.

Einkorn (Triticum monococcum) was the first domesticated wheat species, and was central to the birth of agriculture and the Neolithic Revolution in the Fertile Crescent around 10,000 years ago1,2. Here we generate and analyse 5.2-Gb genome assemblies for wild and domesticated einkorn, including completely assembled centromeres. Einkorn centromeres are highly dynamic, showing evidence of ancient and recent centromere shifts caused by structural rearrangements. Whole-genome sequencing analysis of a diversity panel uncovered the population structure and evolutionary history of einkorn, revealing complex patterns of hybridizations and introgressions after the dispersal of domesticated einkorn from the Fertile Crescent. We also show that around 1% of the modern bread wheat (Triticum aestivum) A subgenome originates from einkorn. These resources and findings highlight the history of einkorn evolution and provide a basis to accelerate the genomics-assisted improvement of einkorn and bread wheat.

Delayed development of basal spikelets in wheat explains their increased floret abortion and rudimentary nature.

Large differences exist in the number of grains per spikelet across an individual wheat (Triticum aestivum L.) spike. The central spikelets produce the highest number of grains, while apical and basal spikelets are less productive, and the most basal spikelets are commonly only developed in rudimentary form. Basal spikelets are delayed in initiation, yet they continue to develop and produce florets. The precise timing or the cause of their abortion remains largely unknown. Here, we investigated the underlying causes of basal spikelet abortion using shading applications in the field. We found that basal spikelet abortion is likely to be the consequence of complete floret abortion, as both occur concurrently and have the same response to shading treatments. We detected no differences in assimilate availability across the spike. Instead, we show that the reduced developmental age of basal florets pre-anthesis is strongly associated with their increased abortion. Using the developmental age pre-abortion, we were able to predict final grain set per spikelet across the spike, alongside the characteristic gradient in the number of grains from basal to central spikelets. Future efforts to improve spikelet homogeneity across the spike could thus focus on improving basal spikelet establishment and increasing floret development rates pre-abortion.

A two-gene strategy increases iron and zinc concentrations in wheat flour, improving mineral bioaccessibility.

Dietary deficiencies of iron and zinc cause human malnutrition that can be mitigated by biofortified staple crops. Conventional breeding approaches to increase grain mineral concentrations in wheat (Triticum aestivum L.) have had only limited success, and our understanding of the genetic and physiological barriers to altering this trait is incomplete. Here we demonstrate that a transgenic approach combining endosperm-specific expression of the wheat VACUOLAR IRON TRANSPORTER gene TaVIT2-D with constitutive expression of the rice (Oryza sativa) NICOTIANAMINE SYNTHASE gene OsNAS2 significantly increases the total concentration of zinc and relocates iron to white-flour fractions. In two distinct bread wheat cultivars, we show that the so called VIT-NAS construct led to a two-fold increase in zinc in wholemeal flour, to ∼50 µg g-1. Total iron was not significantly increased, but redistribution within the grain resulted in a three-fold increase in iron in highly pure, roller-milled white flour, to ∼25 µg g-1. Interestingly, expression of OsNAS2 partially restored iron translocation to the aleurone, which is iron depleted in grain overexpressing TaVIT2 alone. A greater than three-fold increase in the level of the natural plant metal chelator nicotianamine in the grain of VIT-NAS lines corresponded with improved iron and zinc bioaccessibility in white flour. The growth of VIT-NAS plants in the greenhouse was indistinguishable from untransformed controls. Our results provide insights into mineral translocation and distribution in wheat grain and demonstrate that the individual and combined effects of the two transgenes can enhance the nutritional quality of wheat beyond what is possible by conventional breeding.

Whole genome resequencing and comparative genome analysis of three Puccinia striiformis f. sp. tritici pathotypes prevalent in India.

Stripe rust disease of wheat, caused by Puccinia striiformis f. sp. tritici, (Pst) is one of the most serious diseases of wheat worldwide. In India, virulent stripe rust races have been constantly evolving in the North-Western Plains Zone leading to the failure of some of the most widely grown resistant varieties in the region. With the goal of studying the recent evolution of virulent races in this region, we conducted whole-genome re-sequencing of three prevalent Indian Pst pathotypes Pst46S119, Pst78S84 and Pst110S119. We assembled 58.62, 58.33 and 55.78 Mb of Pst110S119, Pst46S119 and Pst78S84 genome, respectively and found that pathotypes were highly heterozygous. Comparative phylogenetic analysis indicated the recent evolution of pathotypes Pst110S119 and Pst78S84 from Pst46S119. Pathogenicity-related genes classes (CAZyme, proteases, effectors, and secretome proteins) were identified and found to be under positive selection. Higher rate of gene families expansion were also observed in the three pathotypes. A strong association between the effector genes and transposable elements may be the source of the rapid evolution of these strains. Phylogenetic analysis differentiated the Indian races in this study from other known United States, European, African, and Asian races. Diagnostic markers developed for the identification of three Pst pathotypes will help tracking of yellow rust at farmers field and strategizing resistance gene deployment.

A scalable phenotyping approach for female floral organ development and senescence in the absence of pollination in wheat.

In the absence of pollination, female reproductive organs senesce, leading to an irrevocable loss in the reproductive potential of the flower, which directly affects seed set. In self-pollinating crops like wheat (Triticum aestivum), the post-anthesis viability of unpollinated carpels has been overlooked, despite its importance for hybrid seed production systems. To advance our knowledge of carpel development in the absence of pollination, we created a high-throughput phenotyping approach to quantify stigma and ovary morphology. We demonstrate the suitability of the approach, which uses light-microscopy imaging and machine learning, for the analysis of floral organ traits in field-grown plants using fresh and fixed samples. We show that the unpollinated carpel undergoes a well-defined initial growth phase, followed by a peak phase in which stigma area reaches its maximum and the radial expansion of the ovary slows, and a final deterioration phase. These developmental dynamics were consistent across years and could be used to classify male-sterile cultivars. This phenotyping approach provides a new tool for examining carpel development, which we hope will advance research into female fertility of wheat.

Root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

Root angle in crops represents a key trait for efficient capture of soil resources. Root angle is determined by competing gravitropic versus antigravitropic offset (AGO) mechanisms. Here we report a root angle regulatory gene termed ENHANCED GRAVITROPISM1 (EGT1) that encodes a putative AGO component, whose loss-of-function enhances root gravitropism. Mutations in barley and wheat EGT1 genes confer a striking root phenotype, where every root class adopts a steeper growth angle. EGT1 encodes an F-box and Tubby domain-containing protein that is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barley EGT1 locus impacts root angle. Gravitropic assays indicated that Hvegt1 roots bend more rapidly than wild-type. Transcript profiling revealed Hvegt1 roots deregulate reactive oxygen species (ROS) homeostasis and cell wall-loosening enzymes and cofactors. ROS imaging shows that Hvegt1 root basal meristem and elongation zone tissues have reduced levels. Atomic force microscopy measurements detected elongating Hvegt1 root cortical cell walls are significantly less stiff than wild-type. In situ analysis identified HvEGT1 is expressed in elongating cortical and stele tissues, which are distinct from known root gravitropic perception and response tissues in the columella and epidermis, respectively. We propose that EGT1 controls root angle by regulating cell wall stiffness in elongating root cortical tissue, counteracting the gravitropic machinery's known ability to bend the root via its outermost tissues. We conclude that root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

FANCM promotes class I interfering crossovers and suppresses class II non-interfering crossovers in wheat meiosis.

FANCM suppresses crossovers in plants by unwinding recombination intermediates. In wheat, crossovers are skewed toward the chromosome ends, thus limiting generation of novel allelic combinations. Here, we observe that FANCM maintains the obligate crossover in tetraploid and hexaploid wheat, thus ensuring that every chromosome pair exhibits at least one crossover, by localizing class I crossover protein HEI10 at pachytene. FANCM also suppresses class II crossovers that increased 2.6-fold in fancm msh5 quadruple mutants. These data are consistent with a role for FANCM in second-end capture of class I designated crossover sites, whilst FANCM is also required to promote formation of non-crossovers. In hexaploid wheat, genetic mapping reveals that crossovers increase by 31% in fancm compared to wild type, indicating that fancm could be an effective tool to accelerate breeding. Crossover rate differences in fancm correlate with wild type crossover distributions, suggesting that chromatin may influence the recombination landscape in similar ways in both wild type and fancm.

MicroRNA-resistant alleles of HOMEOBOX DOMAIN-2 modify inflorescence branching and increase grain protein content of wheat.

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.

The Triticum ispahanicum elongated glume locus P2 maps to chromosome 6A and is associated with the ectopic expression of SVP-A1.

KEY MESSAGE: We propose the MADS-box transcription factor SVP-A1 as a promising candidate gene for the elongated glume locus P2, which maps to chromosome 6A instead of the previously proposed chromosome 7B. In rice and wheat, glume and floral organ length are positively correlated with grain size, making them an important target to increase grain size and potentially yield. The wheat subspecies Triticum ispahanicum is known to develop elongated glumes and floral organs as well as long grains. These multiple phenotypic effects are controlled by the P2 locus, which was previously mapped to wheat chromosome 7B. Using three mapping populations, we show that the long glume locus P2 does not map to chromosome 7B, but instead maps to a 1.68 Mbp interval on chromosome 6A. Within this interval, we identified SVP-A1, a MADS box transcription factor which is the direct ortholog of the maize gene underlying the 'pod corn' Tunicate locus and is a paralog to the T. polonicum elongated glume P1 gene. In T. ispahanicum, we identified a unique allele which has a 482-bp deletion in the SVP-A1 promoter and is associated with ectopic and higher expression of SVP-A1 in the elongated glumes and floral organs. We used near-isogenic lines (NILs) to show that P2 has a consistent positive effect on the length of glume, lemma, palea, spike and grain. Based on the mapping data, natural variation, biological function of SVP genes in cereals and expression analyses, we propose the MADS-box transcription factor SVP-A1 as a promising candidate for P2.

Pathogen-induced biosynthetic pathways encode defense-related molecules in bread wheat.

Wheat is a widely grown food crop that suffers major yield losses due to attack by pests and pathogens. A better understanding of biotic stress responses in wheat is thus of major importance. The recently assembled bread wheat genome coupled with extensive transcriptomic resources provides unprecedented new opportunities to investigate responses to pathogen challenge. Here, we analyze gene coexpression networks to identify modules showing consistent induction in response to pathogen exposure. Within the top pathogen-induced modules, we identify multiple clusters of physically adjacent genes that correspond to six pathogen-induced biosynthetic pathways that share a common regulatory network. Functional analysis reveals that these pathways, all of which are encoded by biosynthetic gene clusters, produce various different classes of compounds­namely, flavonoids, diterpenes, and triterpenes, including the defense-related compound ellarinacin. Through comparative genomics, we also identify associations with the known rice phytoalexins momilactones, as well as with a defense-related gene cluster in the grass model plant Brachypodium distachyon. Our results significantly advance the understanding of chemical defenses in wheat and open up avenues for enhancing disease resistance in this agriculturally important crop. They also exemplify the power of transcriptional networks to discover the biosynthesis of chemical defenses in plants with large, complex genomes.