Genome-wide circadian gating of a cold temperature response in bread wheat.

Circadian rhythms coordinate the responses of organisms with their daily fluctuating environments, by establishing a temporal program of gene expression. This schedules aspects of metabolism, physiology, development and behaviour according to the time of day. Circadian regulation in plants is extremely pervasive, and is important because it underpins both productivity and seasonal reproduction. Circadian regulation extends to the control of environmental responses through a regulatory process known as circadian gating. Circadian gating is the process whereby the circadian clock regulates the response to an environmental cue, such that the magnitude of response to an identical cue varies according to the time of day of the cue. Here, we show that there is genome-wide circadian gating of responses to cold temperatures in plants. By using bread wheat as an experimental model, we establish that circadian gating is crucial to the programs of gene expression that underlie the environmental responses of a crop of major socioeconomic importance. Furthermore, we identify that circadian gating of cold temperature responses are distributed unevenly across the three wheat subgenomes, which might reflect the geographical origins of the ancestors of modern wheat.

Differential effects of anti-CD20 therapy on CD4 and CD8 T cells and implication of CD20-expressing CD8 T cells in MS disease activity.

A small proportion of multiple sclerosis (MS) patients develop new disease activity soon after starting anti-CD20 therapy. This activity does not recur with further dosing, possibly reflecting deeper depletion of CD20-expressing cells with repeat infusions. We assessed cellular immune profiles and their association with transient disease activity following anti-CD20 initiation as a window into relapsing disease biology. Peripheral blood mononuclear cells from independent discovery and validation cohorts of MS patients initiating ocrelizumab were assessed for phenotypic and functional profiles using multiparametric flow cytometry. Pretreatment CD20-expressing T cells, especially CD20dimCD8+ T cells with a highly inflammatory and central nervous system (CNS)-homing phenotype, were significantly inversely correlated with pretreatment MRI gadolinium-lesion counts, and also predictive of early disease activity observed after anti-CD20 initiation. Direct removal of pretreatment proinflammatory CD20dimCD8+ T cells had a greater contribution to treatment-associated changes in the CD8+ T cell pool than was the case for CD4+ T cells. Early disease activity following anti-CD20 initiation was not associated with reconstituting CD20dimCD8+ T cells, which were less proinflammatory compared with pretreatment. Similarly, this disease activity did not correlate with early reconstituting B cells, which were predominantly transitional CD19+CD24highCD38high with a more anti-inflammatory profile. We provide insights into the mode-of-action of anti-CD20 and highlight a potential role for CD20dimCD8+ T cells in MS relapse biology; their strong inverse correlation with both pretreatment and early posttreatment disease activity suggests that CD20-expressing CD8+ T cells leaving the circulation (possibly to the CNS) play a particularly early role in the immune cascades involved in relapse development.

Crossover-active regions of the wheat genome are distinguished by DMC1, the chromosome axis, H3K27me3, and signatures of adaptation.

The hexaploid bread wheat genome comprises over 16 gigabases of sequence across 21 chromosomes. Meiotic crossovers are highly polarized along the chromosomes, with elevation in the gene-dense distal regions and suppression in the Gypsy retrotransposon-dense centromere-proximal regions. We profiled the genomic landscapes of the meiotic recombinase DMC1 and the chromosome axis protein ASY1 in wheat and investigated their relationships with crossovers, chromatin state, and genetic diversity. DMC1 and ASY1 chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed strong co-enrichment in the distal, crossover-active regions of the wheat chromosomes. Distal ChIP-seq enrichment is consistent with spatiotemporally biased cytological immunolocalization of DMC1 and ASY1 close to the telomeres during meiotic prophase I. DMC1 and ASY1 ChIP-seq peaks show significant overlap with genes and transposable elements in the Mariner and Mutator superfamilies. However, DMC1 and ASY1 ChIP-seq peaks were detected along the length of each chromosome, including in low-crossover regions. At the fine scale, crossover elevation at DMC1 and ASY1 peaks and genes correlates with enrichment of the Polycomb histone modification H3K27me3. This indicates a role for facultative heterochromatin, coincident with high DMC1 and ASY1, in promoting crossovers in wheat and is reflected in distalized H3K27me3 enrichment observed via ChIP-seq and immunocytology. Genes with elevated crossover rates and high DMC1 and ASY1 ChIP-seq signals are overrepresented for defense-response and immunity annotations, have higher sequence polymorphism, and exhibit signatures of selection. Our findings are consistent with meiotic recombination promoting genetic diversity, shaping host-pathogen co-evolution, and accelerating adaptation by increasing the efficiency of selection.

Development of a next generation SNP genotyping array for wheat.

High-throughput genotyping arrays have provided a cost-effective, reliable and interoperable system for genotyping hexaploid wheat and its relatives. Existing, highly cited arrays including our 35K Wheat Breeder's array and the Illumina 90K array were designed based on a limited amount of varietal sequence diversity and with imperfect knowledge of SNP positions. Recent progress in wheat sequencing has given us access to a vast pool of SNP diversity, whilst technological improvements have allowed us to fit significantly more probes onto a 384-well format Axiom array than previously possible. Here we describe a novel Axiom genotyping array, the 'Triticum aestivum Next Generation' array (TaNG), largely derived from whole genome skim sequencing of 204 elite wheat lines and 111 wheat landraces taken from the Watkins 'Core Collection'. We used a novel haplotype optimization approach to select SNPs with the highest combined varietal discrimination and a design iteration step to test and replace SNPs which failed to convert to reliable markers. The final design with 43 372 SNPs contains a combination of haplotype-optimized novel SNPs and legacy cross-platform markers. We show that this design has an improved distribution of SNPs compared to previous arrays and can be used to generate genetic maps with a significantly higher number of distinct bins than our previous array. We also demonstrate the improved performance of TaNGv1.1 for Genome-wide association studies (GWAS) and its utility for Copy Number Variation (CNV) analysis. The array is commercially available with supporting marker annotations and initial genotyping results freely available.

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.

Identification, characterization, and rescue of CRISPR/Cas9 generated wheat SPO11-1 mutants.

Increasing crop yields through plant breeding is time consuming and laborious, with the generation of novel combinations of alleles being limited by chromosomal linkage blocks and linkage-drag. Meiotic recombination is essential to create novel genetic variation via the reshuffling of parental alleles. The exchange of genetic information between homologous chromosomes occurs at crossover (CO) sites but CO frequency is often low and unevenly distributed. This bias creates the problem of linkage-drag in recombination 'cold' regions, where undesirable variation remains linked to useful traits. In plants, programmed meiosis-specific DNA double-strand breaks, catalysed by the SPO11 complex, initiate the recombination pathway, although only ~5% result in the formation of COs. To study the role of SPO11-1 in wheat meiosis, and as a prelude to manipulation, we used CRISPR/Cas9 to generate edits in all three SPO11-1 homoeologues of hexaploid wheat. Characterization of progeny lines shows plants deficient in all six SPO11-1 copies fail to undergo chromosome synapsis, lack COs and are sterile. In contrast, lines carrying a single copy of any one of the three wild-type homoeologues are phenotypically indistinguishable from unedited plants both in terms of vegetative growth and fertility. However, cytogenetic analysis of the edited plants suggests that homoeologues differ in their ability to generate COs and in the dynamics of synapsis. In addition, we show that the transformation of wheat mutants carrying six edited copies of SPO11-1 with the TaSPO11-1B gene, restores synapsis, CO formation, and fertility and hence opens a route to modifying recombination in this agronomically important crop.

Bacterial neurotoxic metabolites in multiple sclerosis cerebrospinal fluid and plasma.

The identification of intestinal dysbiosis in patients with neurological and psychiatric disorders has highlighted the importance of gut-brain communication, and yet the question regarding the identity of the components responsible for this cross-talk remains open. We previously reported that relapsing remitting multiple sclerosis patients treated with dimethyl fumarate have a prominent depletion of the gut microbiota, thereby suggesting that studying the composition of plasma and CSF samples from these patients may help to identify microbially derived metabolites. We used a functional xenogeneic assay consisting of cultured rat neurons exposed to CSF samples collected from multiple sclerosis patients before and after dimethyl fumarate treatment to assess neurotoxicity and then conducted a metabolomic analysis of plasma and CSF samples to identify metabolites with differential abundance. A weighted correlation network analysis allowed us to identify groups of metabolites, present in plasma and CSF samples, whose abundance correlated with the neurotoxic potential of the CSF. This analysis identified the presence of phenol and indole group metabolites of bacterial origin (e.g. p-cresol sulphate, indoxyl sulphate and N-phenylacetylglutamine) as potentially neurotoxic and decreased by treatment. Chronic exposure of cultured neurons to these metabolites impaired their firing rate and induced axonal damage, independent from mitochondrial dysfunction and oxidative stress, thereby identifying a novel pathway of neurotoxicity. Clinical, radiological and cognitive test metrics were also collected in treated patients at follow-up visits. Improved MRI metrics, disability and cognition were only detected in dimethyl fumarate-treated relapsing remitting multiple sclerosis patients. The levels of the identified metabolites of bacterial origin (p-cresol sulphate, indoxyl sulphate and N-phenylacetylglutamine) were inversely correlated to MRI measurements of cortical volume and directly correlated to the levels of neurofilament light chain, an established biomarker of neurodegeneration. Our data suggest that phenol and indole derivatives from the catabolism of tryptophan and phenylalanine are microbially derived metabolites, which may mediate gut-brain communication and induce neurotoxicity in multiple sclerosis.

Unravelling mechanisms that govern meiotic crossover formation in wheat.

Wheat is a major cereal crop that possesses a large allopolyploid genome formed through hybridisation of tetraploid and diploid progenitors. During meiosis, crossovers (COs) are constrained in number to 1-3 per chromosome pair that are predominantly located towards the chromosome ends. This reduces the probability of advantageous traits recombining onto the same chromosome, thus limiting breeding. Therefore, understanding the underlying factors controlling meiotic recombination may provide strategies to unlock the genetic potential in wheat. In this mini-review, we will discuss the factors associated with restricted CO formation in wheat, such as timing of meiotic events, chromatin organisation, pre-meiotic DNA replication and dosage of CO genes, as a means to modulate recombination.

Tangible Experiences of Grace: A Qualitative Investigation of Divine Grace in Roman Catholics.

According to Catholic theology, God offers a gift of love, known as divine grace, to all of humanity. This gift of divine grace is the gift of redemption and forgiveness of sins from God that is offered to everyone who decides to acknowledge and accept it. Grace is central to the lived experience of many Christians. This qualitative study examined how Catholics perceive and experience divine grace using interviews that assessed perceptions of divine grace in 29 practicing adult Catholics. A grounded theory analysis resulted in themes indicating that these Catholics view God's divine grace as a tangible gift that is undeserved though continuously offered. The participants' experience of God's grace is not just an abstract theological concept but an embodied aspect of religious life with which believers can interact in many powerful ways. Three characteristics of God's divine grace (i.e., salvific grace, cooperation through free will, primacy of conscience and the afterlife) and three mechanisms to experiencing God's grace (i.e., sacraments, prayer and meditation, saints) are presented.

Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2.

Free asparagine is the precursor for acrylamide, which forms during the baking, toasting and high-temperature processing of foods made from wheat. In this study, CRISPR/Cas9 was used to knock out the asparagine synthetase gene, TaASN2, of wheat (Triticum aestivum) cv. Cadenza. A 4-gRNA polycistronic gene was introduced into wheat embryos by particle bombardment and plants were regenerated. T1 plants derived from 11 of 14 T0 plants were shown to carry edits. Most edits were deletions (up to 173 base pairs), but there were also some single base pair insertions and substitutions. Editing continued beyond the T1 generation. Free asparagine concentrations in the grain of plants carrying edits in all six TaASN2 alleles (both alleles in each genome) were substantially reduced compared with wildtype, with one plant showing a more than 90 % reduction in the T2 seeds. A plant containing edits only in the A genome alleles showed a smaller reduction in free asparagine concentration in the grain, but the concentration was still lower than in wildtype. Free asparagine concentration in the edited plants was also reduced as a proportion of the free amino acid pool. Free asparagine concentration in the T3 seeds remained substantially lower in the edited lines than wildtype, although it was higher than in the T2 seeds, possibly due to stress. In contrast, the concentrations of free glutamine, glutamate and aspartate were all higher in the edited lines than wildtype. Low asparagine seeds showed poor germination but this could be overcome by exogenous application of asparagine.

MutS homologue 4 and MutS homologue 5 Maintain the Obligate Crossover in Wheat Despite Stepwise Gene Loss following Polyploidization.

Crossovers (COs) ensure accurate chromosome segregation during meiosis while creating novel allelic combinations. Here, we show that allotetraploid (AABB) durum wheat (Triticum turgidum ssp. durum) utilizes two pathways of meiotic recombination. The class I pathway requires MSH4 and MSH5 (MutSγ) to maintain the obligate CO/chiasma and accounts for ∼85% of meiotic COs, whereas the residual ∼15% are consistent with the class II CO pathway. Class I and class II chiasmata are skewed toward the chromosome ends, but class II chiasmata are significantly more distal than class I chiasmata. Chiasma distribution does not reflect the abundance of double-strand breaks, detected by proxy as RAD51 foci at leptotene, but only ∼2.3% of these sites mature into chiasmata. MutSγ maintains the obligate chiasma despite a 5.4-kb deletion in MSH5B rendering it nonfunctional, which occurred early in the evolution of tetraploid wheat and was then domesticated into hexaploid (AABBDD) common wheat (Triticum aestivum), as well as an 8-kb deletion in MSH4D in hexaploid wheat, predicted to create a nonfunctional pseudogene. Stepwise loss of MSH5B and MSH4D following hybridization and whole-genome duplication may have occurred due to gene redundancy (as functional copies of MSH5A, MSH4A, and MSH4B are still present in the tetraploid and MSH5A, MSH5D, MSH4A, and MSH4B are present in the hexaploid) or as an adaptation to modulate recombination in allopolyploid wheat.

AutoCloner: automatic homologue-specific primer design for full-gene cloning in polyploids.

BACKGROUND: Polyploid organisms such as wheat complicate even the simplest of procedures in molecular biology. Whilst knowledge of genomic sequences in crops is increasing rapidly, the scientific community is still a long way from producing a full pan-genome for every species. Polymerase chain reaction and Sanger sequencing therefore remain widely used as methods for characterizing gene sequences in many varieties of crops. High sequence similarity between genomes in polyploids means that if primers are not homeologue-specific via the incorporation of a SNP at the 3' tail, sequences other than the target sequence will also be amplified. Current consensus for gene cloning in wheat is to manually perform many steps in a long bioinformatics pipeline. RESULTS: Here we present AutoCloner ( www.autocloner.com ), a fully automated pipeline for crop gene cloning that includes a free-to-use web interface for users. AutoCloner takes a sequence of interest from the user and performs a basic local alignment search tool (BLAST) search against the genome assembly for their particular polyploid crop. Homologous sequences are then compiled with the input sequence into a multiple sequence alignment which is mined for single-nucleotide polymorphisms (SNPs). Various combinations of potential primers that cover the entire gene of interest are then created and evaluated by Primer3; the set of primers with the highest score, as well as all possible primers at every SNP location, are then returned to the user for polymerase chain reaction (PCR). We have successfully used AutoCloner to clone various genes of interest in the Apogee wheat variety, which has no current genome sequence. In addition, we have successfully run the pipeline on ~ 80,000 high-confidence gene models from a wheat genome assembly. CONCLUSION: AutoCloner is the first tool to fully-automate primer design for gene cloning in polyploids, where previously the consensus within the wheat community was to perform this process manually. The web interface for AutoCloner provides a simple and effective polyploid primer-design method for gene cloning, with no need for researchers to download software or input any other details other than their sequence of interest.

Variation in key leaf photosynthetic traits across wheat wild relatives is accession dependent not species dependent.

The wild relatives of modern wheat represent an underutilized source of genetic and phenotypic diversity and are of interest in breeding owing to their wide adaptation to diverse environments. Leaf photosynthetic traits underpin the rate of production of biomass and yield and have not been systematically explored in the wheat relatives. This paper identifies and quantifies the phenotypic variation in photosynthetic, stomatal, and morphological traits in up to 88 wheat wild relative accessions across five genera. Both steady-state measurements and dynamic responses to step changes in light intensity are assessed. A 2.3-fold variation for flag leaf light and CO2 -saturated rates of photosynthesis Amax was observed. Many accessions showing higher and more variable Amax , maximum rates of carboxylation, electron transport, and Rubisco activity when compared with modern genotypes. Variation in dynamic traits was also significant; with distinct genus-specific trends in rates of induction of nonphotochemical quenching and rate of stomatal opening. We conclude that utilization of wild relatives for improvement of photosynthesis is supported by the existence of a high degree of natural variation in key traits and should consider not only genus-level properties but variation between individual accessions.

Prior treatment status: impact on the efficacy and safety of teriflunomide in multiple sclerosis.

BACKGROUND: In this pooled, post hoc analysis of a phase 2 trial and the phase 3 TEMSO, TOWER, and TENERE clinical trials, long-term efficacy and safety of teriflunomide were assessed in subgroups of patients with relapsing multiple sclerosis (MS) defined by prior treatment status. METHODS: Patients were classified according to their prior treatment status in the core and core plus extension periods. In the core period, patients were grouped according to treatment status at the start of the study: treatment naive (no prior disease-modifying therapy [DMT] or DMT > 2 years prior to randomization), previously treated with another DMT (DMT > 6 to ≤24 months prior to randomization), and recently treated with another DMT (DMT ≤6 months prior to randomization). In the core plus extension period, patients were re-baselined to the time of starting teriflunomide 14 mg and grouped according to prior treatment status at that time point. Efficacy endpoints included annualized relapse rate (ARR), probability of confirmed disability worsening (CDW) over 12 weeks, and Expanded Disability Status Scale (EDSS) score. The incidence of adverse events was also assessed. RESULTS: Most frequently received prior DMTs at baseline were glatiramer acetate and interferon beta-1a across treatment groups. Teriflunomide 14 mg significantly reduced ARR versus placebo in the core period, regardless of prior treatment status. In the core and extension periods, adjusted ARRs were low (0.193-0.284) in patients treated with teriflunomide 14 mg across all subgroups. Probability of CDW by Year 4 was similar across subgroups; by Year 5, the percentage of patients with 12-week CDW was similar in treatment-naive patients and patients recently treated with another DMT (33.9 and 33.7%, respectively). EDSS scores were stable over time in all prior-treatment subgroups. There were no new or unexpected safety signals. Limitations include selective bias due to patient attrition, variability in subgroup size, and lack of magnetic resonance imaging outcomes. CONCLUSIONS: The efficacy and safety of teriflunomide 14 mg was similar in all patients with relapsing MS, regardless of prior treatment history. TRIAL REGISTRATION: Phase 2 trial core: NCT01487096 ; Phase 2 trial extension: NCT00228163 ; TEMSO core: NCT00134563 ; TEMSO extension: NCT00803049 ; TOWER: NCT00751881 ; TENERE: NCT00883337 .

Development of an Agrobacterium-delivered CRISPR/Cas9 system for wheat genome editing.

CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with co-transferred plasmid backbone and low edit rate in wheat, a large T0 transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium-delivered CRISPR/Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR/Cas9 system, we have developed 68 edit mutants for four grain-regulatory genes, TaCKX2-1, TaGLW7, TaGW2, and TaGW8, in T0 , T1 , and T2 generation plants at an average edit rate of 10% without detecting off-target mutations in the most Cas9-active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160-bp deletion in TaCKX2-D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium-delivered CRISPR/Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR/Cas9 remains active for novel mutations through generations.

Conversion of array-based single nucleotide polymorphic markers for use in targeted genotyping by sequencing in hexaploid wheat (Triticum aestivum).

Wheat breeders and academics alike use single nucleotide polymorphisms (SNPs) as molecular markers to characterize regions of interest within the hexaploid wheat genome. A number of SNP-based genotyping platforms are available, and their utility depends upon factors such as the available technologies, number of data points required, budgets and the technical expertise required. Unfortunately, markers can rarely be exchanged between existing and newly developed platforms, meaning that previously generated data cannot be compared, or combined, with more recently generated data sets. We predict that genotyping by sequencing will become the predominant genotyping technology within the next 5-10 years. With this in mind, to ensure that data generated from current genotyping platforms continues to be of use, we have designed and utilized SNP-based capture probes from several thousand existing and publicly available probes from Axiom® and KASP™ genotyping platforms. We have validated our capture probes in a targeted genotyping by sequencing protocol using 31 previously genotyped UK elite hexaploid wheat accessions. Data comparisons between targeted genotyping by sequencing, Axiom® array genotyping and KASP™ genotyping assays, identified a set of 3256 probes which reliably bring together targeted genotyping by sequencing data with the previously available marker data set. As such, these probes are likely to be of considerable value to the wheat community. The probe details, full probe sequences and a custom built analysis pipeline may be freely downloaded from the CerealsDB website (http://www.cerealsdb.uk.net/cerealgenomics/CerealsDB/sequence_capture.php).

High-density genotyping of the A.E. Watkins Collection of hexaploid landraces identifies a large molecular diversity compared to elite bread wheat.

The importance of wheat as a food crop makes it a major target for agricultural improvements. As one of the most widely grown cereal grains, together with maize and rice, wheat is the leading provider of calories in the global diet, constituting 29% of global cereal production in 2015. In the last few decades, however, yields have plateaued, suggesting that the green revolution, at least for wheat, might have run its course and that new sources of genetic variation are urgently required. The overall aim of our work was to identify novel variation that may then be used to enable the breeding process. As landraces are a potential source of such diversity, here we have characterized the A.E. Watkins Collection alongside a collection of elite accessions using two complementary high-density and high-throughput genotyping platforms. While our results show the importance of using the appropriate SNP collection to compare diverse accessions, they also show that the Watkins Collection contains a substantial amount of novel genetic diversity which has either not been captured in current breeding programmes or which has been lost through previous selection pressures. As a consequence of our analysis, we have identified a number of accessions which carry an array of novel alleles along with a number of interesting chromosome rearrangements which confirm the variable nature of the wheat genome.

Introgression of Aegilops speltoides segments in Triticum aestivum and the effect of the gametocidal genes.

Background and Aims: Bread wheat (Triticum aestivum) has been through a severe genetic bottleneck as a result of its evolution and domestication. It is therefore essential that new sources of genetic variation are generated and utilized. This study aimed to generate genome-wide introgressed segments from Aegilops speltoides. Introgressions generated from this research will be made available for phenotypic analysis. Methods: Aegilops speltoides was crossed as the male parent to T. aestivum 'Paragon'. The interspecific hybrids were then backcrossed to Paragon. Introgressions were detected and characterized using the Affymetrix Axiom Array and genomic in situ hybridization (GISH). Key Results: Recombination in the gametes of the F1 hybrids was at a level where it was possible to generate a genetic linkage map of Ae. speltoides. This was used to identify 294 wheat/Ae. speltoides introgressions. Introgressions from all seven linkage groups of Ae. speltoides were found, including both large and small segments. Comparative analysis showed that overall macro-synteny is conserved between Ae. speltoides and T. aestivum, but that Ae. speltoides does not contain the 4A/5A/7B translocations present in wheat. Aegilops speltoides has been reported to carry gametocidal genes, i.e. genes that ensure their transmission through the gametes to the next generation. Transmission rates of the seven Ae. speltoides linkage groups introgressed into wheat varied. A 100 % transmission rate of linkage group 2 demonstrates the presence of the gametocidal genes on this chromosome. Conclusions: A high level of recombination occurs between the chromosomes of wheat and Ae. speltoides, leading to the generation of large numbers of introgressions with the potential for exploitation in breeding programmes. Due to the gametocidal genes, all germplasm developed will always contain a segment from Ae. speltoides linkage group 2S, in addition to an introgression from any other linkage group.

Analysis of the bread wheat genome using whole-genome shotgun sequencing.

Bread wheat (Triticum aestivum) is a globally important crop, accounting for 20 per cent of the calories consumed by humans. Major efforts are underway worldwide to increase wheat production by extending genetic diversity and analysing key traits, and genomic resources can accelerate progress. But so far the very large size and polyploid complexity of the bread wheat genome have been substantial barriers to genome analysis. Here we report the sequencing of its large, 17-gigabase-pair, hexaploid genome using 454 pyrosequencing, and comparison of this with the sequences of diploid ancestral and progenitor genomes. We identified between 94,000 and 96,000 genes, and assigned two-thirds to the three component genomes (A, B and D) of hexaploid wheat. High-resolution synteny maps identified many small disruptions to conserved gene order. We show that the hexaploid genome is highly dynamic, with significant loss of gene family members on polyploidization and domestication, and an abundance of gene fragments. Several classes of genes involved in energy harvesting, metabolism and growth are among expanded gene families that could be associated with crop productivity. Our analyses, coupled with the identification of extensive genetic variation, provide a resource for accelerating gene discovery and improving this major crop.