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Abstract 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.

Abstract 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.

The FANCM helicase functions in limiting crossovers (COs) by unwinding inter-homolog repair intermediates. Here, the authors generate null mutants of fancm in tetraploid and hexaploid wheat and show that FANCM promotes class I interfering COs and suppresses class II noninterfering COs in wheat meiosis.

Abstract Background Polyploidy, especially allopolyploidy, which entails merging divergent genomes via hybridization and whole-genome duplication (WGD), is a major route to speciation in plants. The duplication among the parental genomes (subgenomes) often leads to one subgenome becoming dominant over the other(s), resulting in subgenome asymmetry in gene content and expression. Polyploid wheats are allopolyploids with most genes present in two (tetraploid) or three (hexaploid) functional copies, which commonly show subgenome expression asymmetry. It is unknown whether a similar subgenome asymmetry exists during translation. We aim to address this key biological question and explore the major contributing factors to subgenome translation asymmetry. Results Here, we obtain the first tetraploid wheat translatome and reveal that subgenome expression asymmetry exists at the translational level. We further perform in vivo RNA structure profiling to obtain the wheat RNA structure landscape and find that mRNA structure has a strong impact on translation, independent of GC content. We discover a previously uncharacterized contribution of RNA structure in subgenome translation asymmetry. We identify 3564 single-nucleotide variations (SNVs) across the transcriptomes between the two tetraploid wheat subgenomes, which induce large RNA structure disparities. These SNVs are highly conserved within durum wheat cultivars but are divergent in both domesticated and wild emmer wheat. Conclusions We successfully determine both the translatome and in vivo RNA structurome in tetraploid wheat. We reveal that RNA structure serves as an important modulator of translational subgenome expression asymmetry in polyploids. Our work provides a new perspective for molecular breeding of major polyploid crops.

Plants respond to warmth via growth processes termed thermomorphogenesis. Here, via a phosphoproteomics approach, the authors show that the mitogen activated protein kinase TOT3 regulates thermomorphogenesis in both wheat and Arabidopsis and modifies brassinosteroid signaling in Arabidopsis.

Brinton, Uauy and colleagues utilize genomic data from the 10+ Wheat Genome Project to develop a useful tool for studying and generating new wheat cultivars. This framework uses advanced exploitation of wheat haplotypes to bring newfound precision and efficiency to wheat breeding.

Gene regulatory networks are powerful tools which facilitate hypothesis generation and candidate gene discovery. However, the extent to which the network predictions are biologically relevant is often unclear. Recently a GENIE3 network which predicted targets of wheat transcription factors was produced. Here we used an independent RNA-Seq dataset to test the predictions of the wheat GENIE3 network for the senescence-regulating transcription factor NAM-A1 (TraesCS6A02G108300). We re-analyzed the RNA-Seq data against the RefSeqv1.0 genome and identified a set of differentially expressed genes (DEGs) between the wild-type and nam-a1 mutant which recapitulated the known role of NAM-A1 in senescence and nutrient remobilisation. We found that the GENIE3-predicted target genes of NAM-A1 overlap significantly with the DEGs, more than would be expected by chance. Based on high levels of overlap between GENIE3-predicted target genes and the DEGs, we identified candidate senescence regulators. We then explored genome-wide trends in the network related to polyploidy and found that only homeologous transcription factors are likely to share predicted targets in common. However, homeologs which vary in expression levels across tissues are less likely to share predicted targets than those that do not, suggesting that they may be more likely to act in distinct pathways. This work demonstrates that the wheat GENIE3 network can provide biologically-relevant predictions of transcription factor targets, which can be used for candidate gene prediction and for global analyses of transcription factor function. The GENIE3 network has now been integrated into the KnetMiner web application, facilitating its use in future studies.

Abstract Background Functional characterisation of genes using transgenic methods is increasingly common in cereal crops. Yet standard methods of gene over-expression can lead to undesirable developmental phenotypes, or even embryo lethality, due to ectopic gene expression. Inducible expression systems allow the study of such genes by preventing their expression until treatment with the specific inducer. When combined with the Cre-Lox recombination system, inducible promoters can be used to initiate constitutive expression of a gene of interest. Yet while these systems are well established in dicot model plants, like Arabidopsis thaliana, they have not yet been implemented in grasses. Results Here we present an irreversible heat-shock inducible system developed using Golden Gate-compatible components which utilises Cre recombinase to drive constitutive gene expression in barley and wheat. We show that a heat shock treatment of 38 °C is sufficient to activate the construct and drive expression of the gene of interest. Modulating the duration of heat shock controls the density of induced cells. Short durations of heat shock cause activation of the construct in isolated single cells, while longer durations lead to global construct activation. The system can be successfully activated in multiple tissues and at multiple developmental stages and shows no activation at standard growth temperatures (~ 20 °C). Conclusions This system provides an adaptable framework for use in gene functional characterisation in cereal crops. The developed vectors can be easily adapted for specific genes of interest within the Golden Gate cloning system. By using an environmental signal to induce activation of the construct, the system avoids pitfalls associated with consistent and complete application of chemical inducers. As with any inducible system, care must be taken to ensure that the expected construct activation has indeed taken place.

Genebanks hold comprehensive collections of wild species, wild relatives, and landraces that are useful for genetic improvement. Here, the authors report the genotype of nearly 80,000 wheat accessions using DArTseq technology to show the less explored genetic diversity.

Abstract Background Little is known about the initial, symptomless (latent) phase of the devastating wheat disease Septoria tritici blotch. However, speculations as to its impact on fungal success and disease severity in the field have suggested that a long latent phase is beneficial to the host and can reduce inoculum build up in the field over a growing season. The winter wheat cultivar Stigg is derived from a synthetic hexaploid wheat and contains introgressions from wild tetraploid wheat Triticum turgidum subsp. dicoccoides, which contribute to cv. Stigg’s exceptional STB resistance, hallmarked by a long latent phase. We compared the early transcriptomic response to Zymoseptoria tritici of cv. Stigg to a susceptible wheat cultivar, to elucidate the mechanisms of and differences in pathogen recognition and disease response in these two hosts. Results The STB-susceptible cultivar Longbow responds to Z. tritici infection with a stress response, including activation of hormone-responsive transcription factors, post translational modifications, and response to oxidative stress. The activation of key genes associated with these pathways in cv. Longbow was independently observed in a second susceptible wheat cultivar based on an independent gene expression study. By comparison, cv. Stigg is apathetic in response to STB, and appears to fail to activate a range of defence pathways that cv. Longbow employs. Stigg also displays some evidence of sub-genome bias in its response to Z. tritici infection, whereas the susceptible cv. Longbow shows even distribution of Z. tritici responsive genes across the three wheat sub-genomes. Conclusions We identify a suite of disease response genes that are involved in early pathogen response in susceptible wheat cultivars that may ultimately lead to susceptibility. In comparison, we hypothesise that rather than an active defence response to stave off disease progression, cv. Stigg’s defence strategy is molecular lethargy, or a lower-amplitude of pathogen recognition that may stem from cv. Stigg’s wild wheat-derived ancestry. Overall, we present insights into cv. Stigg’s exceptional resistance to STB, and present key biological processes for further characterisation in this pathosystem.

Abstract Background Wheat blast, caused by Magnaporthe oryzae Triticum (MoT) pathotype, is a global threat to wheat (Triticum aestivum L.) production. Few blast resistance (R) genes have been identified to date, therefore assessing potential sources of resistance in wheat is important. The Brazilian wheat cultivar BR 18-Terena is considered one of the best sources of resistance to blast and has been widely used in Brazilian breeding programmes, however the underlying genetics of this resistance are unknown. Results BR 18-Terena was used as the common parent in the development of two recombinant inbred line (RIL) F6 populations with the Brazilian cultivars Anahuac 75 and BRS 179. Populations were phenotyped for resistance at the seedling and heading stage using the sequenced MoT isolate BR32, with transgressive segregation being observed. Genetic maps containing 1779 and 1318 markers, were produced for the Anahuac 75 × BR 18-Terena and BR 18-Terena × BRS 179 populations, respectively. Five quantitative trait loci (QTL) associated with seedling resistance, on chromosomes 2B, 4B (2 QTL), 5A and 6A, were identified, as were four QTL associated with heading stage resistance (1A, 2B, 4A and 5A). Seedling and heading stage QTL did not co-locate, despite a significant positive correlation between these traits, indicating that resistance at these developmental stages is likely to be controlled by different genes. BR 18-Terena provided the resistant allele for six QTL, at both developmental stages, with the largest phenotypic effect conferred by a QTL being 24.8% suggesting that BR 18-Terena possesses quantitative resistance. Haplotype analysis of 100 Brazilian wheat cultivars indicates that 11.0% of cultivars already possess a BR 18-Terena-like haplotype for more than one of the identified heading stage QTL. Conclusions This study suggests that BR 18-Terena possesses quantitative resistance to wheat blast, with nine QTL associated with resistance at either the seedling or heading stage being detected. Wheat blast resistance is also largely tissue-specific. Identification of durable quantitative resistances which can be combined with race-specific R gene-mediated resistance is critical to effectively control wheat blast. Collectively, this work facilitates marker-assisted selection to develop new varieties for cultivation in regions at risk from this emerging disease.

Bread wheat (Triticum aestivum L.) is one of the world’s most important crops. Maintaining wheat yield gains across all of its major production areas is a key target toward underpinning global food security. Brazil is a major wheat producer in South America, generating grain yields of around 6.8 million tons per year. Here, we establish and genotype a wheat association mapping resource relevant to contemporary Brazilian wheat breeding programs. The panel of 558 wheat accessions was genotyped using an Illumina iSelect 90,000 single nucleotide polymorphism array. Following quality control, the final data matrix consisted of 470 accessions and 22,475 polymorphic genetic markers (minor allele frequency ≥5%, missing data

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.

Roots are the main channel for water and nutrient uptake in plants. Optimization of root architecture provides a viable strategy to improve nutrient and water uptake efficiency and maintain crop productivity under water-limiting and nutrient-poor conditions. We know little, however, about the genetic control of root development in wheat, a crop supplying 20% of global calorie and protein intake. To improve our understanding of the genetic control of seminal root development in wheat, we conducted a high-throughput screen for variation in seminal root number using an exome-sequenced mutant population derived from the hexaploid wheat cultivar Cadenza. The screen identified seven independent mutants with homozygous and stably altered seminal root number phenotypes. One mutant, Cadenza0900, displays a recessive extra seminal root number phenotype, while six mutants (Cadenza0062, Cadenza0369, Cadenza0393, Cadenza0465, Cadenza0818 and Cadenza1273) show lower seminal root number phenotypes most likely originating from defects in the formation and activation of seminal root primordia. Segregation analysis in F2 populations suggest that the phenotype of Cadenza0900 is controlled by multiple loci whereas the Cadenza0062 phenotype fits a 3:1 mutant:wild-type segregation ratio characteristic of dominant single gene action. This work highlights the potential to use the sequenced wheat mutant population as a forward genetic resource to uncover novel variation in agronomic traits, such as seminal root architecture.

Abstract Background NAC transcription factors contain five highly conserved subdomains which are required for protein dimerisation and DNA binding. Few residues within these subdomains have been identified as essential for protein function, and fewer still have been shown to be of biological relevance in planta. Here we use a positive regulator of senescence in wheat, NAM-A1, to test the impact of missense mutations at specific, highly conserved residues of the NAC domain on protein function. Results We identified missense mutations in five highly conserved residues of the NAC domain of NAM-A1 in a tetraploid TILLING population. TILLING lines containing these mutations, alongside synonymous and non-conserved mutation controls, were grown under glasshouse conditions and scored for senescence. Four of the five mutations showed a significant and consistent delay in peduncle senescence but had no consistent effects on flag leaf senescence. All four mutant alleles with the delayed senescence phenotype also lost the ability to interact with the homoeolog NAM-B1 in a yeast two-hybrid assay. Two of these residues were previously shown to be involved in NAC domain function in Arabidopsis, suggesting conservation of residue function between species. Three of these four alleles led to an attenuated cell death response compared to wild-type NAM-A1 when transiently over-expressed in Nicotiana benthamiana. One of these mutations was further tested under field conditions, in which there was a significant and consistent delay in both peduncle and leaf senescence. Conclusions We combined field and glasshouse studies of a series of mutant alleles with biochemical analyses to identify four residues of the NAC domain which are required for NAM-A1 function and protein interaction. We show that mutations in these residues lead to a gradient of phenotypes, raising the possibility of developing allelic series of mutations for traits of agronomic importance. We also show that mutations in NAM-A1 more severely impact peduncle senescence, compared to the more commonly studied flag leaf senescence, highlighting this as an area deserving of further study. The results from this integrated approach provide strong evidence that conserved residues within the functional domains of NAC transcription factors have biological significance in planta.

Abstract Bread wheat is an allohexaploid species originating from two successive and recent rounds of hybridization between three diploid species that were very similar in terms of chromosome number, genome size, TE content, gene content and synteny. As a result, it has long been considered that most of the genes were in three pairs of homoeologous copies. However, these so‐called triads represent only one half of wheat genes, while the remaining half belong to homoeologous groups with various number of copies across subgenomes. In this study, we examined and compared the distribution, conservation, function, expression and epigenetic profiles of triads with homoeologous groups having undergone a deletion (dyads) or a duplication (tetrads) in one subgenome. We show that dyads and tetrads are mostly located in distal regions and have lower expression level and breadth than triads. Moreover, they are enriched in functions related to adaptation and more associated with the repressive H3K27me3 modification. Altogether, these results suggest that triads mainly correspond to housekeeping genes and are part of the core genome, while dyads and tetrads belong to the Triticeae dispensable genome. In addition, by comparing the different categories of dyads and tetrads, we hypothesize that, unlike most of the allopolyploid species, subgenome dominance and biased fractionation are absent in hexaploid wheat. Differences observed between the three subgenomes are more likely related to two successive and ongoing waves of post‐polyploid diploidization, that had impacted A and B more significantly than D, as a result of the evolutionary history of hexaploid wheat.

Durum wheat is an important cereal grown in Ethiopia, a country which is also its center for genetic diversity. Yellow (stripe) rust caused by Puccinia striiformis fsp tritici is one of the most devastating diseases threatening Ethiopian wheat production. To identify sources of genetic resistance and combat this pathogen, we conducted a genome wide association study of yellow rust resistance on 300 durum wheat accessions comprising 261 landraces and 39 cultivars. The accessions were evaluated for their field resistance using a modified Cobb scale at Meraro, Kulumsa and Chefe Donsa in the 2015 and 2016 main growing seasons. Analysis of the 35K Axiom Array genotyping data of the panel resulted in a total of 8,797 polymorphic SNPs of which 7,093 were used in subsequent analyses. Population structure analysis suggested two groups in which the cultivars clearly stood out separately from the landraces. Eleven SNPs significantly associated with yellow rust resistance were identified on four chromosomes (1A, 1B, 2B, and 5A) which defined at least five genomic loci. Six of the SNPs were consistently identified on chromosome 1B singly at each and combined overall environments which explained 62.6-64.0% of the phenotypic variation (R2). Resistant allele frequency ranged from 14.0-71.0%; Zooming in to the identified resistance loci revealed the presence of disease resistance related genes involved in the plant defense system such as the ABC transporter gene family, disease resistance protein RPM1 (NBS-LRR class), Receptor kinases and Protein kinases. This study has provided SNPs for tracking the loci associated with yellow rust resistance and a diversity panel which can be used for association study of other agriculturally important traits in durum wheat.

Fusarium head blight (FHB) is a disease of wheat (Triticum aestivum L.) that causes major yield losses in South America, as well as many other wheat growing regions around the world. FHB results in low quality, contaminated grain due to the production of mycotoxins such as deoxynivalenol (DON). In Brazil, FHB outbreaks are increasing in frequency and are currently controlled by fungicides which are costly and potentially harmful to the wider environment. To identify the genetic basis of resistance to FHB in Brazilian wheat, two mapping populations (Anahuac 75 × BR 18-Terena and BR 18-Terena × BRS 179) segregating for FHB resistance were phenotyped and quantitative trait loci (QTL) analysis was undertaken to identify genomic regions associated with FHB-related traits. A total of 14 QTL associated with FHB visual symptoms were identified, each of which explained 3.7-17.3% of the phenotypic variance. Two of these QTL were stable across environments. This suggests FHB resistance in Anahuac 75, BR 18-Terena and BRS 179 is controlled by multiple genetic loci that confer relatively minor differences in resistance. A major, novel QTL associated with DON accumulation was also identified on chromosome 4B (17.8% of the phenotypic variance), as well as a major QTL associated with thousand-grain weight on chromosome 6B (16.8% phenotypic variance). These QTL could be useful breeding targets, when pyramided with major sources of resistance such as Fhb1, to improve grain quality and reduce the reliance on fungicides in Brazil and other countries affected by FHB.

In barley (Hordeum vulgare L.), Agrobacterium-mediated transformation efficiency is highly dependent on genotype with very few cultivars being amenable to transformation. Golden Promise is the cultivar most widely used for barley transformation and developing embryos are the most common donor tissue. We tested whether barley mutants with abnormally large embryos were more or less amenable to transformation and discovered that mutant M1460 had a transformation efficiency similar to that of Golden Promise. The large-embryo phenotype of M1460 is due to mutation at the LYS3 locus. There are three other barley lines with independent mutations at the same LYS3 locus, and one of these, Risø1508 has an identical missense mutation to that in M1460. However, none of the lys3 mutants except M1460 were transformable showing that the locus responsible for transformation efficiency, TRA1, was not LYS3 but another locus unique to M1460. To identify TRA1, we generated a segregating population by crossing M1460 to the cultivar Optic, which is recalcitrant to transformation. After four rounds of backcrossing to Optic, plants were genotyped and their progeny were tested for transformability. Some of the progeny lines were transformable at high efficiencies similar to those seen for the parent M1460 and some were not transformable, like Optic. A region on chromosome 2H inherited from M1460 is present in transformable lines only. We propose that one of the 225 genes in this region is TRA1.

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.

The influence of wheat (modern wheat, both bread and pasta, their wild ancestors and synthetic hybrids) on the microbiota of their roots and surrounding soil is characterized. We isolated lines of bread wheat by hybridizing diploid (Aegilops tauschii) with tetraploid Triticum durum and crossed it with a modern cultivar of Triticum aestivum. The newly created, synthetic hybrid wheat, which recapitulate the breeding history of wheat through artificial selection, is found to support a microbiome enriched in beneficial Glomeromycetes fungi, but also in, potentially detrimental, Nematoda. We hypothesize that during wheat domestication this plant-microbe interaction diminished, suggesting an evolutionary tradeoff; sacrificing advantageous nutrient acquisition through fungal interactions to minimize interaction with pathogenic fungi. Increased plant selection for Glomeromycetes and Nematoda is correlated with the D genome derived from A. tauschii. Despite differences in their soil microbiota communities, overall wheat plants consistently show a low ratio of eukaryotes to prokaryotes. We propose that this is a mechanism for protection against soil-borne fungal disease and appears to be deeply rooted in the wheat genome. We suggest that the influence of plants on the composition of their associated microbiota is an integral factor, hitherto overlooked, but intrinsic to selection during wheat domestication.

Abstract Background Transposable elements (TEs) are major components of large plant genomes and main drivers of genome evolution. The most recent assembly of hexaploid bread wheat recovered the highly repetitive TE space in an almost complete chromosomal context and enabled a detailed view into the dynamics of TEs in the A, B, and D subgenomes. Results The overall TE content is very similar between the A, B, and D subgenomes, although we find no evidence for bursts of TE amplification after the polyploidization events. Despite the near-complete turnover of TEs since the subgenome lineages diverged from a common ancestor, 76% of TE families are still present in similar proportions in each subgenome. Moreover, spacing between syntenic genes is also conserved, even though syntenic TEs have been replaced by new insertions over time, suggesting that distances between genes, but not sequences, are under evolutionary constraints. The TE composition of the immediate gene vicinity differs from the core intergenic regions. We find the same TE families to be enriched or depleted near genes in all three subgenomes. Evaluations at the subfamily level of timed long terminal repeat-retrotransposon insertions highlight the independent evolution of the diploid A, B, and D lineages before polyploidization and cases of concerted proliferation in the AB tetraploid. Conclusions Even though the intergenic space is changed by the TE turnover, an unexpected preservation is observed between the A, B, and D subgenomes for features like TE family proportions, gene spacing, and TE enrichment near genes.

Abstract Background There is an urgent need to increase global crop production. Identifying and combining specific genes controlling distinct biological processes holds the potential to enhance crop yields. Transcriptomics is a powerful tool to gain insights into the complex gene regulatory networks that underlie such traits, but relies on the availability of a high-quality reference sequence and accurate gene models. Previously, we identified a grain weight QTL on wheat chromosome 5A (5A QTL) which acts during early grain development to increase grain length through cell expansion in the pericarp. In this study, we performed RNA-sequencing on near isogenic lines (NILs) segregating for the 5A QTL and used the latest gene models to identify differentially regulated genes and pathways that potentially influence pericarp cell size and grain weight in wheat. Results We sampled grains at 4 and 8 days post anthesis and found that genes associated with metabolism, biosynthesis, proteolysis and the defence response are upregulated during this stage of grain development in both NILs. We identified a specific set of 112 transcripts differentially expressed (DE) between 5A NILs at either time point, including eight potential candidates for the causal 5A gene and its downstream targets. The 112 DE transcripts had functional annotations including non-coding RNA, transposon-associated, cell-cycle control, ubiquitin-related, heat-shock, transcription and histone-related. Many of the genes identified belong to families that have been previously associated with seed/grain development in other species. Notably, we identified DE transcripts at almost all steps of the pathway associated with ubiquitin-mediated protein degradation. In the promoters of a subset of DE transcripts we identified enrichment of binding sites associated with C2H2, MYB/SANT, YABBY, AT-HOOK and Trihelix transcription factor families. Conclusions In this study, we identified DE transcripts with a diverse range of predicted biological functions, reflecting the complex nature of the pathways that control early grain development. Few of these are the direct orthologues of grain size genes in other species and none have been previously characterised in wheat. Further functional characterisation of these candidates and how they interact will provide novel insights into the control of grain size in cereals.

Many important genes in agriculture correspond to transcription factors (TFs) that regulate a wide range of pathways from flowering to responses to disease and abiotic stresses. In this study, we identified 5776 TFs in hexaploid wheat (Triticum aestivum) and classified them into gene families. We further investigated the NAC family exploring the phylogeny, C-terminal domain (CTD) conservation, and expression profiles across 308 RNA-seq samples. Phylogenetic trees of NAC domains indicated that wheat NACs divided into eight groups similar to rice (Oryza sativa) and barley (Hordeum vulgare). CTD motifs were frequently conserved between wheat, rice, and barley within phylogenetic groups; however, this conservation was not maintained across phylogenetic groups. Three homeologous copies were present for 58% of NACs, whereas evidence of single homeolog gene loss was found for 33% of NACs. We explored gene expression patterns across a wide range of developmental stages, tissues, and abiotic stresses. We found that more phylogenetically related NACs shared more similar expression patterns compared to more distant NACs. However, within each phylogenetic group there were clades with diverse expression profiles. We carried out a coexpression analysis on all wheat genes and identified 37 modules of coexpressed genes of which 23 contained NACs. Using gene ontology (GO) term enrichment, we obtained putative functions for NACs within coexpressed modules including responses to heat and abiotic stress and responses to water: these NACs may represent targets for breeding or biotechnological applications. This study provides a framework and data for hypothesis generation for future studies on NAC TFs in wheat.

Polyploidization has played an important role in plant evolution. However, upon polyploidization, the process of meiosis must adapt to ensure the proper segregation of increased numbers of chromosomes to produce balanced gametes. It has been suggested that meiotic gene (MG) duplicates return to a single copy following whole genome duplication to stabilize the polyploid genome. Therefore, upon the polyploidization of wheat, a hexaploid species with three related (homeologous) genomes, the stabilization process may have involved rapid changes in content and expression of MGs on homeologous chromosomes (homeologs). To examine this hypothesis, sets of candidate MGs were identified in wheat using co-expression network analysis and orthology informed approaches. In total, 130 RNA-Seq samples from a range of tissues including wheat meiotic anthers were used to define co-expressed modules of genes. Three modules were significantly correlated with meiotic tissue samples but not with other tissue types. These modules were enriched for GO terms related to cell cycle, DNA replication, and chromatin modification and contained orthologs of known MGs. Overall, 74.4% of genes within these meiosis-related modules had three homeologous copies which was similar to other tissue-related modules. Amongst wheat MGs identified by orthology, rather than co-expression, the majority (93.7%) were either retained in hexaploid wheat at the same number of copies (78.4%) or increased in copy number (15.3%) compared to ancestral wheat species. Furthermore, genes within meiosis-related modules showed more balanced expression levels between homeologs than genes in non-meiosis-related modules. Taken together, our results do not support extensive gene loss nor changes in homeolog expression of MGs upon wheat polyploidization. The construction of the MG co-expression network allowed identification of hub genes and provided key targets for future studies.

Durum wheat (Triticum turgidum) derives from a hybridization event approximately 400,000 years ago which led to the creation of an allotetraploid genome. The evolutionary recent origin of durum wheat means that its genome has not yet been fully diploidised. As a result, many of the genes present in the durum genome act in a redundant fashion, where loss-of-function mutations must be present in both gene copies to observe a phenotypic effect. Here, we use a novel set of induced variation within the cv. Kronos TILLING population to identify a locus controlling a dominant, environmentally dependent chlorosis phenotype. We carried out a forward screen of the sequenced cv. Kronos TILLING lines for senescence phenotypes and identified a line with a dominant early senescence and chlorosis phenotype. Mutant plants contained less chlorophyll throughout their development and displayed premature flag leaf senescence. A segregating population was classified into discrete phenotypic groups and subjected to bulked-segregant analysis using exome capture followed by next-generation sequencing. This allowed the identification of a single region on chromosome 3A, Yellow Early Senescence 1 (YES-1), which was associated with the mutant phenotype. While this phenotype was consistent across 4 years of field trials in the United Kingdom, the mutant phenotype was not observed when grown in Davis, CA (United States). To obtain further SNPs for fine-mapping, we isolated chromosome 3A using flow sorting and sequenced the entire chromosome. By mapping these reads against both the cv. Chinese Spring reference sequence and the cv. Kronos assembly, we could identify high-quality, novel EMS-induced SNPs in non-coding regions within YES-1 that were previously missed in the exome capture data. This allowed us to fine-map YES-1 to 4.3 Mb, containing 59 genes. Our study shows that populations containing induced variation can be sources of novel dominant variation in polyploid crop species, highlighting their importance in future genetic screens. We also demonstrate the value of using cultivar-specific genome assemblies alongside the gold-standard reference genomes particularly when working with non-coding regions of the genome. Further fine-mapping of the YES-1 locus will be pursued to identify the causal SNP underpinning this dominant, environmentally dependent phenotype.

Bread wheat is an important and the most consumed cereal worldwide. However, people with predominantly cereal-based diets are increasingly affected by micronutrient deficiencies, suggesting the need for biofortified wheat varieties. The limited genetic diversity in hexaploid wheat warrants exploring the wider variation present in wheat wild relatives, among these Aegilops tauschii, the wild progenitor of the bread wheat D genome. In this study, a panel of 167 Ae. tauschii accessions was phenotyped for grain Fe, Zn, Cu, and Mn concentrations for 3 years and was found to have wide variation for these micronutrients. Comparisons between the two genetic subpopulations of Ae. tauschii revealed that lineage 2 had higher mean values for Fe and Cu concentration than lineage 1. To identify potentially new genetic sources for improving grain micronutrient concentration, we performed a genome-wide association study (GWAS) on 114 non-redundant Ae. tauschii accessions using 5,249 genotyping-by-sequencing (GBS) markers. Best linear unbiased predictor (BLUP) values were calculated for all traits across the three growing seasons. A total of 19 SNP marker trait associations (MTAs) were detected for all traits after applying Bonferroni corrected threshold of -log10(P-value) ≥ 4.68. These MTAs were found on all seven chromosomes. For grain Fe, Zn, Cu, and Mn concentrations, five, four, three, and seven significant associations were detected, respectively. The associations were linked to the genes encoding transcription factor regulators, transporters, and phytosiderophore synthesis. The results demonstrate the utility of GWAS for understanding the genetic architecture of micronutrient accumulation in Ae. tauschii, and further efforts to validate these loci will aid in using them to diversify the D-genome of hexaploid wheat.

Polyploidization is a fundamental process in plant evolution. One of the biggest challenges faced by a new polyploid is meiosis, particularly discriminating between multiple related chromosomes so that only homologous chromosomes synapse and recombine to ensure regular chromosome segregation and balanced gametes. Despite its large genome size, high DNA repetitive content and similarity between homoeologous chromosomes, hexaploid wheat completes meiosis in a shorter period than diploid species with a much smaller genome. Therefore, during wheat meiosis, mechanisms additional to the classical model based on DNA sequence homology, must facilitate more efficient homologous recognition. One such mechanism could involve exploitation of differences in chromosome structure between homologs and homoeologs at the onset of meiosis. In turn, these chromatin changes, can be expected to be linked to transcriptional gene activity. In this study, we present an extensive analysis of a large RNA-seq data derived from six different genotypes: wheat, wheat–rye hybrids and newly synthesized octoploid triticale, both in the presence and absence of the Ph1 locus. Plant material was collected at early prophase, at the transition leptotene-zygotene, when the telomere bouquet is forming and synapsis between homologs is beginning. The six genotypes exhibit different levels of synapsis and chromatin structure at this stage; therefore, recombination and consequently segregation, are also different. Unexpectedly, our study reveals that neither synapsis, whole genome duplication nor the absence of the Ph1 locus are associated with major changes in gene expression levels during early meiotic prophase. Overall wheat transcription at this meiotic stage is therefore highly resilient to such alterations, even in the presence of major chromatin structural changes. Further studies in wheat and other polyploid species will be required to reveal whether these observations are specific to wheat meiosis.

Nucleotide-binding leucine-rich-repeat (LRR) receptors (NLRs) with non-canonical integrated domains (NLR-IDs) are widespread in plant genomes. Zinc-finger BED (named after the Drosophila proteins Boundary Element-Associated Factor and DNA Replication-related Element binding Factor, named BED hereafter) are among the most frequently found IDs. Five BED-NLRs conferring resistance against bacterial and fungal pathogens have been characterized. However, it is unknown whether BED-NLRs function in a manner similar to other NLR-IDs. Here, we used chromosome-level assemblies of wheat to explore the Yr7 and Yr5a genomic regions and show that, unlike known NLR-ID loci, there is no evidence for a NLR-partner in their vicinity. Using neighbor-network analyses, we observed that BED domains from BED-NLRs share more similarities with BED domains from single-BED proteins and from BED-containing proteins harboring domains that are conserved in transposases. We identified a nuclear localization signal (NLS) in Yr7, Yr5, and the other characterized BED-NLRs. We thus propose that this is a feature of BED-NLRs that confer resistance to plant pathogens. We show that the NLS was functional in truncated versions of the Yr7 protein when expressed in N. benthamiana. We did not observe cell-death upon the overexpression of Yr7 full-length, truncated, and ‘MHD’ variants in N. benthamiana. This suggests that either this system is not suitable to study BED-NLR signaling or that BED-NLRs require additional components to trigger cell death. These results define novel future directions to further understand the role of BED domains in BED-NLR mediated resistance.

Wheat breeding nowadays must address producers and consumers' desire. According to the last FAO report, a dramatic decrease in wheat production is expected in the next decades mainly due to the upcoming climate change. The identification of the processes which are triggered by heat stress and how thermotolerance develops in wheat is an active research topic. Genomic approach may help wheat breeding since it allows direct study on the genotype and relationship with the phenotype. Here the isolation and characterization of four members of the chloroplast-localized small heat shock proteins (sHSP) encoded by the Hsp26 gene family is reported. Furthermore, two high throughput TILLING (Targeting Induced Local Lesions In Genomes) approaches in vivo and in silico were used for the identification of new alleles within this family. Small heat shock proteins are known to prevent the irreversible aggregation of misfolded proteins and contribute to the acquisition of thermotolerance. Chloroplast-localized sHSPs protect the photosynthetic machinery during episodes of high temperature stress. The modulation of the newly discovered genes within the sHsp26 family has been analyzed in vivo and by the ExpVIP platform widening the abiotic stress analysis; and their involvement in the heat stress response has been demonstrated. In addition, in this study a total of 50 TILLING mutant lines have been identified. A set of KASP (Kompetitive Allele Specific PCR) markers was also developed to follow the specific mutations in the ongoing backcrosses, applicable to high throughput genotyping approaches and usable in marker assisted selection breeding programs.

Pre-harvest sprouting (PHS) is an important cause of quality loss in many cereal crops and is particularly prevalent and damaging in wheat. Resistance to PHS is therefore a valuable target trait in many breeding programs. The Phs-A1 locus on wheat chromosome arm 4AL has been consistently shown to account for a significant proportion of natural variation to PHS in diverse mapping populations. However, the deployment of sprouting resistance is confounded by the fact that different candidate genes, including the tandem duplicated Plasma Membrane 19 (PM19) genes and the mitogen-activated protein kinase kinase 3 (TaMKK3-A) gene, have been proposed to underlie Phs-A1. To further define the Phs-A1 locus, we constructed a physical map across this interval in hexaploid and tetraploid wheat. We established close proximity of the proposed candidate genes which are located within a 1.2 Mb interval. Genetic characterization of diverse germplasm used in previous genetic mapping studies suggests that TaMKK3-A, and not PM19, is the major gene underlying the Phs-A1 effect in European, North American, Australian and Asian germplasm. We identified the non-dormant TaMKK3-A allele at low frequencies within the A-genome diploid progenitor Triticum urartu genepool, and show an increase in the allele frequency in modern varieties. In United Kingdom varieties, the frequency of the dormant TaMKK3-A allele was significantly higher in bread-making quality varieties compared to feed and biscuit-making cultivars. Analysis of exome capture data from 58 diverse hexaploid wheat accessions identified fourteen haplotypes across the extended Phs-A1 locus and four haplotypes for TaMKK3-A. Analysis of these haplotypes in a collection of United Kingdom and Australian cultivars revealed distinct major dormant and non-dormant Phs-A1 haplotypes in each country, which were either rare or absent in the opposing germplasm set. The diagnostic markers and haplotype information reported in the study will help inform the choice of germplasm and breeding strategies for the deployment of Phs-A1 resistance into breeding germplasm.

Aegilops tauschii, the D-genome progenitor of Triticum aestivum, encompasses huge diversity for various traits of potential economic importance such as yield, biotic and abiotic stress tolerance, quality and nutrition. In the present study, variation for grain size in Ae. tauschii germplasm was studied and its genetic basis dissected using genome-wide association study (GWAS). Grain length, width, and weight evaluated in 177 Ae. tauschii accessions over 3 years showed near normal distribution with 1.74-, 1.75-, and 2.82-fold variation, respectively. These lines were genetically characterized using genotyping-by-sequencing (GBS) protocol that produced 11,489 single nucleotide polymorphic (SNP) markers. Genetic diversity analysis revealed the presence of two distinct subgroups (designated as lineage 1 and 2) in Ae. tauschii. Based on GBS markers, the genetic similarity was calculated between the accessions and GWAS was conducted using 114 non-redundant accessions and 5,249 SNP markers. A total of 17 SNPs associated with grain size traits distributed over all the seven chromosomes were revealed with 6D, 5D, and 2D harboring most significant marker–trait associations. Some of the chromosomal regions such as 6D_66.4–71.1 cM, 1D_143.5–156.7 cM, and 2D_89.9–92.5 cM had associations with multiple traits. Candidate genes associated with cell division and differentiation were identified for some of the associated SNP markers. Further efforts to validate these loci will help to understand their role in determining grain size and allelic diversity in current germplasm and its effect on grain size upon transfer to bread wheat background.