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.

High expression of the MADS-box gene VRT2 increases the number of rudimentary basal spikelets in wheat.

Spikelets are the fundamental building blocks of Poaceae inflorescences, and their development and branching patterns determine the various inflorescence architectures and grain yield of grasses. In wheat (Triticum aestivum), the central spikelets produce the most and largest grains, while spikelet size gradually decreases acropetally and basipetally, giving rise to the characteristic lanceolate shape of wheat spikes. The acropetal gradient corresponds with the developmental age of spikelets; however, the basal spikelets are developed first, and the cause of their small size and rudimentary development is unclear. Here, we adapted G&T-seq, a low-input transcriptomics approach, to characterize gene expression profiles within spatial sections of individual spikes before and after the establishment of the lanceolate shape. We observed larger differences in gene expression profiles between the apical, central, and basal sections of a single spike than between any section belonging to consecutive developmental time points. We found that SHORT VEGETATIVE PHASE MADS-box transcription factors, including VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT-A2), are expressed highest in the basal section of the wheat spike and display the opposite expression gradient to flowering E-class SEPALLATA1 genes. Based on multi-year field trials and transgenic lines, we show that higher expression of VRT-A2 in the basal sections of the spike is associated with increased numbers of rudimentary basal spikelets. Our results, supported by computational modeling, suggest that the delayed transition of basal spikelets from vegetative to floral developmental programs results in the lanceolate shape of wheat spikes. This study highlights the value of spatially resolved transcriptomics to gain insights into developmental genetics pathways of grass inflorescences.

A robust KASP marker for selection of four pairs of linked leaf rust and stripe rust resistance genes introgressed on chromosome arm 5DS from different wheat genomes.

Stripe rust and leaf rust are among the most devastating diseases of wheat, limiting its production globally. Wheat wild relatives harbour genetic diversity for new genes and alleles for all major wheat diseases. However, the use of this genetic variation from wild progenitor and non-progenitor species has been limited in the breeding programs. Reasons include limited recombination of donor and recipient genomes and the lack of tertiary gene pool markers. Here, we describe the development of a SNP based marker from the flow-sorted and sequenced Aegilops umbellulata chromosome 5U which can be used for marker assisted selection of four pair of alien leaf rust and stripe rust resistance genes. Lr57-Yr40_CAPS16 marker was reported earlier to be linked with alien leaf and stripe rust resistance genes introgressed on wheat chromosome 5DS. Due to its dominant nature and laborious to work with, a new SNP-based KASP marker, XTa5DS-2754099_kasp23, was developed from the same CAPS marker contig. XTa5DS-2754099_kasp23 was tested in Aegilops umbellulata, Ae. geniculata, Ae. peregrina and Ae. caudata derived alien introgression lines, which harbour four pairs of linked leaf and stripe rust genes; Lr76-Yr70, Lr57-Yr40, LrP- YrP, LrAc-YrAc, respectively. This KASP marker was found to be effective for the selection of the aforesaid four pairs of leaf rust and stripe rust resistance genes. Further, we tested and validated XTa5DS-2754099_kasp23 on commercial varieties and advanced breeding lines from four countries (India, Egypt, Australia and UK) including hexaploid and durum wheat. Our results provide evidence that KASP marker, XTa5DS-2754099_kasp23 can be used in marker-assisted selection of the four pairs of rust resistance alien genes in wheat breeding programmes.

Ectopic expression of Triticum polonicum VRT-A2 underlies elongated glumes and grains in hexaploid wheat in a dosage-dependent manner.

Flower development is an important determinant of grain yield in crops. In wheat (Triticum spp.), natural variation for the size of spikelet and floral organs is particularly evident in Triticum turgidum ssp. polonicum (also termed Triticum polonicum), a tetraploid subspecies of wheat with long glumes, lemmas, and grains. Using map-based cloning, we identified VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT2), which encodes a MADS-box transcription factor belonging to the SHORT VEGETATIVE PHASE family, as the gene underlying the T. polonicum long-glume (P1) locus. The causal P1 mutation is a sequence rearrangement in intron-1 that results in ectopic expression of the T. polonicum VRT-A2 allele. Based on allelic variation studies, we propose that the intron-1 mutation in VRT-A2 is the unique T. polonicum subspecies-defining polymorphism, which was later introduced into hexaploid wheat via natural hybridizations. Near-isogenic lines differing for the P1 locus revealed a gradient effect of P1 across spikelets and within florets. Transgenic lines of hexaploid wheat carrying the T. polonicum VRT-A2 allele show that expression levels of VRT-A2 are highly correlated with spike, glume, grain, and floral organ length. These results highlight how changes in expression profiles, through variation in cis-regulation, can affect agronomic traits in a dosage-dependent manner in polyploid crops.

A roadmap for gene functional characterisation in crops with large genomes: Lessons from polyploid wheat.

Understanding the function of genes within staple crops will accelerate crop improvement by allowing targeted breeding approaches. Despite their importance, a lack of genomic information and resources has hindered the functional characterisation of genes in major crops. The recent release of high-quality reference sequences for these crops underpins a suite of genetic and genomic resources that support basic research and breeding. For wheat, these include gene model annotations, expression atlases and gene networks that provide information about putative function. Sequenced mutant populations, improved transformation protocols and structured natural populations provide rapid methods to study gene function directly. We highlight a case study exemplifying how to integrate these resources. This review provides a helpful guide for plant scientists, especially those expanding into crop research, to capitalise on the discoveries made in Arabidopsis and other plants. This will accelerate the improvement of crops of vital importance for food and nutrition security.

Aegilops umbellulata introgression carrying leaf rust and stripe rust resistance genes Lr76 and Yr70 located to 9.47-Mb region on 5DS telomeric end through a combination of chromosome sorting and sequencing.

KEY MESSAGE: Lr76 and Yr70 have been fine mapped using the sequence of flow-sorted recombinant 5D chromosome from wheat-Ae. umbellulata introgression line. The alien introgression has been delineated to 9.47-Mb region on short arm of wheat chromosome 5D. Leaf rust and stripe rust are among the most damaging diseases of wheat worldwide. Wheat cultivation based on limited number of rust resistance genes deployed over vast areas expedites the emergence of new pathotypes warranting a continuous deployment of new resistance genes. In this paper, fine mapping of Aegilops umbellulata-derived leaf rust and stripe rust resistance genes Lr76 and Yr70 is being reported. We flow sorted and paired-end sequenced 5U chromosome of Ae. umbellulata, recombinant chromosome 5D (5DIL) from wheat-Ae. umbellulata introgression line pau16057 and 5DRP of recurrent parent WL711. Chromosome 5U reads were mapped against the reference Chinese Spring chromosome 5D sequence, and alien-specific SNPs were identified. Chromosome 5DIL and 5DRP sequences were de novo assembled, and alien introgression-specific markers were designed by selecting 5U- and 5D-specific SNPs. Overall, 27 KASP markers were mapped in high-resolution population consisting of 1404 F5 RILs. The mapping population segregated for single gene each for leaf rust and stripe rust resistance. The physical order of the SNPs in pau16057 was defined by projecting the 27 SNPs against the IWGSC RefSeq v1.0 sequence. Based on this physical map, the size of Ae. umbellulata introgression was determined to be 9.47 Mb on the distal most end of the short arm of chromosome 5D. This non-recombining alien segment carries six NB-LRR encoding genes based on NLR annotation of assembled chromosome 5DIL sequence and IWGSC RefSeq v1.1 gene models. The presence of SNPs and other sequence variations in these genes between pau16057 and WL711 suggested that they are candidates for Lr76 and Yr70.

Dominant inhibition of awn development by a putative zinc-finger transcriptional repressor expressed at the B1 locus in wheat.

Awns, bristle-like structures extending from grass lemmas, provide protection against predators, contribute to photosynthesis and aid in grain dispersal. In wheat, selection of awns with minimal extension, termed awnletted, has occurred during domestication by way of loci that dominantly inhibit awn development, such as Tipped1 (B1), Tipped2 (B2), and Hooded (Hd). Here we identify and characterize the B1 gene. B1 was identified using bulked segregant RNA-sequencing of an F2 durum wheat population and through deletion mapping of awned bread wheat mutants. Functional characterization was accomplished by gene overexpression while haplotype analyses assessed B1 polymorphisms and genetic variation. Located on chromosome 5A, B1 is a C2H2 zinc finger encoding gene with ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs. Constitutive overexpression of B1 in awned wheat produced an awnletted phenotype with pleiotropic effects on plant height and fertility. Transcriptome analysis of B1 overexpression plants suggests a role as transcriptional repressor, putatively targeting pathways involved in cell proliferation. Haplotype analysis revealed a conserved B1 coding region with proximal polymorphisms and supported the contention that B1 is mainly responsible for awnletted wheats globally. B1, predominantly responsible for awn inhibition in wheat, encodes a C2H2 zinc finger protein with EAR motifs which putatively functions as a transcriptional repressor.

VERNALIZATION1 controls developmental responses of winter wheat under high ambient temperatures.

Low temperatures are required to regulate the transition from vegetative to reproductive growth via a pathway called vernalization. In wheat, vernalization predominantly involves the cold upregulation of the floral activator VERNALIZATION1 (VRN1). Here, we have used an extreme vernalization response, identified through studying ambient temperature responses, to reveal the complexity of temperature inputs into VRN-A1, with allelic inter-copy variation at a gene expansion of VRN-A1 modulating these effects. We find that the repressors of the reproductive transition, VERNALIZATION2 (VRN2) and ODDSOC2, are re-activated when plants experience high temperatures during and after vernalization. In addition, this re-activation is regulated by photoperiod for VRN2 but was independent of photoperiod for ODDSOC2 We also find this warm temperature interruption affects flowering time and floret number and is stage specific. This research highlights the important balance between floral activators and repressors in coordinating the response of a plant to temperature, and that the absence of warmth is essential for the completion of vernalization. This knowledge can be used to develop agricultural germplasm with more predictable vernalization responses that will be more resilient to variable growth temperatures.

BED-domain-containing immune receptors confer diverse resistance spectra to yellow rust.

Crop diseases reduce wheat yields by ~25% globally and thus pose a major threat to global food security1. Genetic resistance can reduce crop losses in the field and can be selected through the use of molecular markers. However, genetic resistance often breaks down following changes in pathogen virulence, as experienced with the wheat yellow (stripe) rust fungus Puccinia striiformis f. sp. tritici (Pst)2. This highlights the need to (1) identify genes that, alone or in combination, provide broad-spectrum resistance, and (2) increase our understanding of the underlying molecular modes of action. Here we report the isolation and characterization of three major yellow rust resistance genes (Yr7, Yr5 and YrSP) from hexaploid wheat (Triticum aestivum), each having a distinct recognition specificity. We show that Yr5, which remains effective to a broad range of Pst isolates worldwide, is closely related yet distinct from Yr7, whereas YrSP is a truncated version of Yr5 with 99.8% sequence identity. All three Yr genes belong to a complex resistance gene cluster on chromosome 2B encoding nucleotide-binding and leucine-rich repeat proteins (NLRs) with a non-canonical N-terminal zinc-finger BED domain3 that is distinct from those found in non-NLR wheat proteins. We developed diagnostic markers to accelerate haplotype analysis and for marker-assisted selection to expedite the stacking of the non-allelic Yr genes. Our results provide evidence that the BED-NLR gene architecture can provide effective field-based resistance to important fungal diseases such as wheat yellow rust.

Speed breeding is a powerful tool to accelerate crop research and breeding.

The growing human population and a changing environment have raised significant concern for global food security, with the current improvement rate of several important crops inadequate to meet future demand 1 . This slow improvement rate is attributed partly to the long generation times of crop plants. Here, we present a method called 'speed breeding', which greatly shortens generation time and accelerates breeding and research programmes. Speed breeding can be used to achieve up to 6 generations per year for spring wheat (Triticum aestivum), durum wheat (T. durum), barley (Hordeum vulgare), chickpea (Cicer arietinum) and pea (Pisum sativum), and 4 generations for canola (Brassica napus), instead of 2-3 under normal glasshouse conditions. We demonstrate that speed breeding in fully enclosed, controlled-environment growth chambers can accelerate plant development for research purposes, including phenotyping of adult plant traits, mutant studies and transformation. The use of supplemental lighting in a glasshouse environment allows rapid generation cycling through single seed descent (SSD) and potential for adaptation to larger-scale crop improvement programs. Cost saving through light-emitting diode (LED) supplemental lighting is also outlined. We envisage great potential for integrating speed breeding with other modern crop breeding technologies, including high-throughput genotyping, genome editing and genomic selection, accelerating the rate of crop improvement.

Transfer of a starch phenotype from wild wheat to bread wheat by deletion of a locus controlling B-type starch granule content.

Our previous genetic analysis of a tetraploid wild wheat species, Aegilops peregrina, predicted that a single gene per haploid genome, Bgc-1, controls B-type starch granule content in the grain. To test whether bread wheat (Triticum aestivum L.) has orthologous Bgc-1 loci, we screened a population of γ-irradiated bread wheat cv. Paragon for deletions of the group 4 chromosomes spanning Bgc-1. Suitable deletions, each encompassing ~600-700 genes, were discovered for chromosomes 4A and 4D. These two deletions are predicted to have 240 homoeologous genes in common. In contrast to single deletion mutant plants, double deletion mutants were found to lack B-type starch granules. The B-less grains had normal A-type starch granule morphology, normal overall starch content, and normal grain weight. In addition to variation in starch granule size distribution, the B-less wheat grains differed from controls in grain hardness, starch swelling power, and amylose content. We believe that these B-less wheat plants are the only Triticeae cereals available that combine substantial alterations in starch granule size distribution with minimal impact on starch content.

A Metabolic Gene Cluster in the Wheat W1 and the Barley Cer-cqu Loci Determines ß-Diketone Biosynthesis and Glaucousness.

The glaucous appearance of wheat (Triticum aestivum) and barley (Hordeum vulgare) plants, that is the light bluish-gray look of flag leaf, stem, and spike surfaces, results from deposition of cuticular ß-diketone wax on their surfaces; this phenotype is associated with high yield, especially under drought conditions. Despite extensive genetic and biochemical characterization, the molecular genetic basis underlying the biosynthesis of ß-diketones remains unclear. Here, we discovered that the wheat W1 locus contains a metabolic gene cluster mediating ß-diketone biosynthesis. The cluster comprises genes encoding proteins of several families including type-III polyketide synthases, hydrolases, and cytochrome P450s related to known fatty acid hydroxylases. The cluster region was identified in both genetic and physical maps of glaucous and glossy tetraploid wheat, demonstrating entirely different haplotypes in these accessions. Complementary evidence obtained through gene silencing in planta and heterologous expression in bacteria supports a model for a ß-diketone biosynthesis pathway involving members of these three protein families. Mutations in homologous genes were identified in the barley eceriferum mutants defective in ß-diketone biosynthesis, demonstrating a gene cluster also in the ß-diketone biosynthesis Cer-cqu locus in barley. Hence, our findings open new opportunities to breed major cereal crops for surface features that impact yield and stress response.

The Cer-cqu gene cluster determines three key players in a ß-diketone synthase polyketide pathway synthesizing aliphatics in epicuticular waxes.

Aliphatic compounds on plant surfaces, called epicuticular waxes, are the first line of defense against pathogens and pests, contribute to reducing water loss and determine other important phenotypes. Aliphatics can form crystals affecting light refraction, resulting in a color change and allowing identification of mutants in their synthesis or transport. The present study discloses three such Eceriferum (cer) genes in barley - Cer-c, Cer-q and Cer-u - known to be tightly linked and functioning in a biochemical pathway forming dominating amounts of ß-diketone and hydroxy-ß-diketones plus some esterified alkan-2-ols. These aliphatics are present in many Triticeae as well as dicotyledons such as Eucalyptus and Dianthus. Recently developed genomic resources and mapping populations in barley defined these genes to a small region on chromosome arm 2HS. Exploiting Cer-c and -u potential functions pinpointed five candidates, of which three were missing in apparent cer-cqu triple mutants. Sequencing more than 50 independent mutants for each gene confirmed their identification. Cer-c is a chalcone synthase-like polyketide synthase, designated diketone synthase (DKS), Cer-q is a lipase/carboxyl transferase and Cer-u is a P450 enzyme. All were highly expressed in pertinent leaf sheath tissue of wild type. A physical map revealed the order Cer-c, Cer-u, Cer-q with the flanking genes 101kb apart, confirming they are a gene cluster, Cer-cqu. Homology-based modeling suggests that many of the mutant alleles affect overall protein structure or specific active site residues. The rich diversity of identified mutations will facilitate future studies of three key enzymes involved in synthesis of plant apoplast waxes.

Accession-specific modifiers act with ZWILLE/ARGONAUTE10 to maintain shoot meristem stem cells during embryogenesis in Arabidopsis.

BACKGROUND: Stem cells located in the centre of the shoot apical meristem are required for the repetitive formation of new organs such as leaves, branches and flowers. In Arabidopsis thaliana, the ZWILLE/PINHEAD/AGO10 (ZLL) gene encodes a member of the ARGONAUTE (AGO) protein family and is required to maintain shoot meristem stem cells during embryogenesis. In the Landsberg erecta (Ler) acession, ZLL is essential for stem cell maintenance, whereas in the Columbia (Col) accession its requirement appears masked by genetic modifiers. The genetic basis for this variation has remained elusive. RESULTS: To understand the impact of natural variation on shoot stem cell maintenance, we analysed 28 wild-type Arabidopsis accessions from around the world and show that ZLL function is essential for stem cell maintenance in accessions mainly originating from Germany, but is dispensable for accessions from other regions. Quantitative Trait Loci (QTL) mapping using Ler/Col recombinant inbred lines indicated that at least five genomic regions, referred to as FLETSCHE (FHE) 1-5, modify ZLL function in stem cell maintenance. Characterisation of Col zll near isogenic lines confirmed that the major QTL, FHE2, is preferentially maintained as a Ler allele in seedlings lacking stem cells, suggesting that this region harbours an important modifier of ZLL function. Comparison of torpedo-stage embryo expression profiles to QTL map data revealed candidate FHE genes, including the Arabidopsis Cyclophilin-40 homologue SQUINT (SQN), and functional studies revealed a previously uncharacterised role for SQN in stem cell regulation. CONCLUSIONS: Multiple genetic modifiers from different Arabidopsis accessions influence the role of ZLL in embryonic stem cell maintenance. Of the five FHE loci modifying stem cell maintenance in Ler-0 and Col-0, FHE2 was the most prominent and was tightly linked to the SQN gene, which encodes a cofactor that supports AGO1 activity. SQN shows variable embryonic expression levels between accessions and altered ZLL-dependency in transgenic assays, confirming a key role in stem cell maintenance. Reduced SQN expression levels in Col-0 correlate with transposon insertions adjoining the transcriptional start site, which may contribute to stem cell maintenance in other ZLL-independent accessions.

The inhibitor of wax 1 locus (Iw1) prevents formation of ß- and OH-ß-diketones in wheat cuticular waxes and maps to a sub-cM interval on chromosome arm 2BS.

Glaucousness is described as the scattering effect of visible light from wax deposited on the cuticle of plant aerial organs. In wheat, two dominant genes lead to non-glaucous phenotypes: Inhibitor of wax 1 (Iw1) and Iw2. The molecular mechanisms and the exact extent (beyond visual assessment) by which these genes affect the composition and quantity of cuticular wax is unclear. To describe the Iw1 locus we used a genetic approach with detailed biochemical characterization of wax compounds. Using synteny and a large number of F2 gametes, Iw1 was fine-mapped to a sub-cM genetic interval on wheat chromosome arm 2BS, which includes a single collinear gene from the corresponding Brachypodium and rice physical maps. The major components of flag leaf and peduncle cuticular waxes included primary alcohols, ß-diketones and n-alkanes. Small amounts of C19-C27 alkyl and methylalkylresorcinols that have not previously been described in wheat waxes were identified. Using six pairs of BC2 F3 near-isogenic lines, we show that Iw1 inhibits the formation of ß- and hydroxy-ß-diketones in the peduncle and flag leaf blade cuticles. This inhibitory effect is independent of genetic background or tissue, and is accompanied by minor but consistent increases in n-alkanes and C24 primary alcohols. No differences were found in cuticle thickness and carbon isotope discrimination in near-isogenic lines differing at Iw1.

Local maternal control of seed size by KLUH/CYP78A5-dependent growth signaling.

Seed development in plants involves the coordinated growth of the embryo, endosperm, and maternal tissue. Several genes have been identified that influence seed size by acting maternally, such as AUXIN RESPONSE FACTOR2, APETALA2, and DA1. However, given the lack of gain-of-function effects of these genes on seed size, it is unclear whether their activity levels are limiting in WT plants and whether they could thus be used to regulate seed size in development or evolution. Also, whether the altered seed sizes reflect local gene activity or global physiological changes is unknown. Here, we demonstrate that the cytochrome P450 KLUH (KLU) regulates seed size. KLU acts locally in developing flowers to promote seed growth, and its activity level is limiting for seed growth in WT. KLU is expressed in the inner integument of developing ovules, where it non-cell autonomously stimulates cell proliferation, thus determining the growth potential of the seed coat and seed. A KLU-induced increase in seed size leads to larger seedlings and higher relative oil content of the seeds. Genetic analyses indicate that KLU acts independently of other tested maternal factors that influence integument cell proliferation. Thus, the level of KLU-dependent growth factor signaling determines size in ovules and seeds, suggesting this pathway as a target for crop improvement.