The coordinated regulation of early meiotic stages is dominated by non-coding RNAs and stage-specific transcription in wheat.

Reproductive success hinges on precisely coordinated meiosis, yet our understanding of how structural rearrangements of chromatin and phase transitions during meiosis are transcriptionally regulated is limited. In crop plants, detailed analysis of the meiotic transcriptome could identify regulatory genes and epigenetic regulators that can be targeted to increase recombination rates and broaden genetic variation, as well as provide a resource for comparison among eukaryotes of different taxa to answer outstanding questions about meiosis. We conducted a meiotic stage-specific analysis of messenger RNA (mRNA), small non-coding RNA (sncRNA), and long intervening/intergenic non-coding RNA (lincRNA) in wheat (Triticum aestivum L.) and revealed novel mechanisms of meiotic transcriptional regulation and meiosis-specific transcripts. Amidst general repression of mRNA expression, significant enrichment of ncRNAs was identified during prophase I relative to vegetative cells. The core meiotic transcriptome was comprised of 9309 meiosis-specific transcripts, 48 134 previously unannotated meiotic transcripts, and many known and novel ncRNAs differentially expressed at specific stages. The abundant meiotic sncRNAs controlled the reprogramming of central metabolic pathways by targeting genes involved in photosynthesis, glycolysis, hormone biosynthesis, and cellular homeostasis, and lincRNAs enhanced the expression of nearby genes. Alternative splicing was not evident in this polyploid species, but isoforms were switched at phase transitions. The novel, stage-specific regulatory controls uncovered here challenge the conventional understanding of this crucial biological process and provide a new resource of requisite knowledge for those aiming to directly modulate meiosis to improve crop plants. The wheat meiosis transcriptome dataset can be queried for genes of interest using an eFP browser located at https://bar.utoronto.ca/efp_wheat/cgi-bin/efpWeb.cgi?dataSource=Wheat_Meiosis.

Long-read sequence assembly: a technical evaluation in barley.

Sequence assembly of large and repeat-rich plant genomes has been challenging, requiring substantial computational resources and often several complementary sequence assembly and genome mapping approaches. The recent development of fast and accurate long-read sequencing by circular consensus sequencing (CCS) on the PacBio platform may greatly increase the scope of plant pan-genome projects. Here, we compare current long-read sequencing platforms regarding their ability to rapidly generate contiguous sequence assemblies in pan-genome studies of barley (Hordeum vulgare). Most long-read assemblies are clearly superior to the current barley reference sequence based on short-reads. Assemblies derived from accurate long reads excel in most metrics, but the CCS approach was the most cost-effective strategy for assembling tens of barley genomes. A downsampling analysis indicated that 20-fold CCS coverage can yield very good sequence assemblies, while even five-fold CCS data may capture the complete sequence of most genes. We present an updated reference genome assembly for barley with near-complete representation of the repeat-rich intergenic space. Long-read assembly can underpin the construction of accurate and complete sequences of multiple genomes of a species to build pan-genome infrastructures in Triticeae crops and their wild relatives.

Topical desiccating agent (DEBRICHEM): an accessible debridement option for removing biofilm in hard-to-heal wounds.

It is now assumed that all hard-to-heal wounds contain biofilm. Debridement plays a key role in wound-bed preparation, as it can remove biofilm along with the devitalised tissue, potentially leaving a clean wound bed that is more likely to progress towards healing. The gold standard methods of debridement (surgical and sharp) are the least used, as they require specialist training and are often not readily available at the point of need. Most other methods can be used by generalists but are slower. They all need regular applications. The topical desiccating agent DEBRICHEM is an innovative alternative, as it is fast, effective and can be used in all clinical settings, as well as typically requiring only a single use. This article describes best practice for achieving optimal outcomes with its use.

Sequencing of Camelina neglecta, a diploid progenitor of the hexaploid oilseed Camelina sativa.

Camelina neglecta is a diploid species from the genus Camelina, which includes the versatile oilseed Camelina sativa. These species are closely related to Arabidopsis thaliana and the economically important Brassica crop species, making this genus a useful platform to dissect traits of agronomic importance while providing a tool to study the evolution of polyploids. A highly contiguous chromosome-level genome sequence of C. neglecta with an N50 size of 29.1 Mb was generated utilizing Pacific Biosciences (PacBio, Menlo Park, CA) long-read sequencing followed by chromosome conformation phasing. Comparison of the genome with that of C. sativa shows remarkable coincidence with subgenome 1 of the hexaploid, with only one major chromosomal rearrangement separating the two. Synonymous substitution rate analysis of the predicted 34 061 genes suggested subgenome 1 of C. sativa directly descended from C. neglecta around 1.2 mya. Higher functional divergence of genes in the hexaploid as evidenced by the greater number of unique orthogroups, and differential composition of resistant gene analogs, might suggest an immediate adaptation strategy after genome merger. The absence of genome bias in gene fractionation among the subgenomes of C. sativa in comparison with C. neglecta, and the complete lack of fractionation of meiosis-specific genes attests to the neopolyploid status of C. sativa. The assembled genome will provide a tool to further study genome evolution processes in the Camelina genus and potentially allow for the identification and exploitation of novel variation for Camelina crop improvement.

Diagnosis and treatment of infected wounds: A multi-centre audit of current clinical practice across the UK, Ireland and Scandinavia.

AIMS AND OBJECTIVES: Surveillance of wound infection including signs of infection alongside antimicrobial usage (types, duration, frequency) can highlight knowledge gaps and inconsistencies. This manuscript aims to highlight these, identify and inform opportunities for practice improvement and to show impact of infection management practice may be having on the issue of antimicrobial resistance. BACKGROUND: Infected wounds pose challenges to healthcare professionals. Balancing risk of wound deterioration and progression to systemic infection with appropriate use of antimicrobials is necessary to minimise development of resistance. METHODS: Analysis consisted of a practice survey of 9661 wounds across 70 community sites over a period of one week. Data were collected from projects between 2017 and 2020. The form was available to providers within the UK, Ireland, Norway, Denmark, Sweden and Finland. EQUATOR research guidelines were followed; STROBE checklist for observational research reporting was completed. RESULTS: Infection rates of 8.9% were reported from wounds assessed. These data indicate inconsistencies with diagnosis across practice with non-specialists more likely to be unsure of wound infection. Greater confidence in infection identification was observed as number of signs increased. Inconsistencies were also observed in appropriate treatment; antimicrobials were used in 35% of wounds considered not infected and not used in 41% of wounds that were identified as infected. CONCLUSIONS: This investigation of infection management practice of over 9000 wounds provides an insight into diagnosis and treatment of infection. Inconsistencies in diagnosis and treatment of wound infections reported highlight the need for increased education, awareness of diagnosis and treatment of infection. RELEVANCE TO CLINICAL PRACTICE: Variability in management of infected wounds highlights opportunities to aid more effective diagnosis and treatment of infected wounds. Incorporation of support tools or evidence-based pathways into practice may enhance confidence in management of local infection, balanced with appropriate use, potentially minimising resistance and improving outcomes.

The development of a core outcome set for clinical effectiveness studies of bordered foam dressings in the treatment of complex wounds.

AIM: The aim of this project was to develop a core outcome set (COS) for clinical effectiveness studies of bordered foam dressings in the treatment of complex wounds. METHODS: The research project followed the Core Outcome Measures in Effectiveness Trials (COMET) initiative and consisted of two phases. The first phase prepared the background and process, while the second phase had three steps: outcome list generation via systematic review and qualitative study, Delphi consensus study, and consensus meeting. The study has been registered in the Core Outcome Measures in Effectiveness Trials database. RESULTS: The systematic review resulted in 82 outcomes and 20 additional outcomes were obtained during the interviews. After refinement, 111 panellists from 23 countries rated a list of 51 outcomes. In the following consensus meeting, six outcomes were prioritized to be included in the core outcome set. After the consensus meeting, a patient-reported outcome was added to the core outcome set. CONCLUSION: The COS for evaluating the effectiveness of bordered foam dressings in treating complex wounds includes 7 outcomes: "ability to stay in place", "leakage", "pain", "dressing related periwound skin changes", "change in wound size over time", and "overall satisfaction". These identified outcomes are correlated with contemporary bioengineering testing and evaluation methods for dressing performance, which underpins the need for a close multidisciplinary collaboration to advance the field of wound dressings. The outcome 'overall satisfaction' reflects the impact of complex wounds and their treatment on a patient's daily life. The use of these outcomes is recommended to improve data synthesis and promote evidence-based practice. Future developments in COS development involve creating measurement instruments and relevant endpoints for these outcomes.

Clinical performance characteristics for bordered foam dressings in the treatment of complex wounds: An international wound dressing technology expert panel review.

The aim of this article is to identify and describe clinical practice performance characteristics for bordered foam dressings in the treatment of complex wounds. Our recently published systematic review of outcomes and applied measurement instruments for the use of bordered foam dressings in complex wounds has led to us identifying a range of important clinical and patient-centred issues related to this dressing class. Specifically, here, we focus on an overview of performance criteria in the areas of application, adhesion, exudate management and debridement functions of bordered foam dressings. Our hope is that by highlighting the clinical performance criteria, future testing standards for wound dressings will more closely match our clinical expectations and, thereby, assist clinicians to make better wound treatment choices based on meaningful and clinically relevant dressing product performance standards. complex wounds, complex wound care, treatment, bordered foam dressings, dressing performance.

Saving the foot: Simple orthopaedic surgical intervention demonstrates improved outcomes and reduced costs.

BACKGROUND: Forefoot ulceration in diabetes requires significant resources, with high cost and low rates of success. The authors present the results of tendon procedures (percutaneous toe tenotomy and percutaneous tendo-achilles lengthening) under local anaesthetic to adjust mechanics in patients with diabetic neuropathic forefoot ulceration. METHODS: Retrospective review of electronic patient record of 19 patients (22 feet) undergoing local anaesthetic tendon procedures between April 2019 and April 2021 with a 12 month follow up period. Size of ulcer, rate of ulcer healing, complication rates and ulcer recurrence were recorded and compared to a population of conservatively-managed patients (14 patients, 15 feet) treated prior to the introduction of tendon procedures. All clinical information obtained from electronic patient records. RESULTS: All patients undergoing tendon procedures achieved complete ulcer healing at a mean time of 3.3 weeks for toe tip ulcers (after toe tenotomy) and 4.5 weeks for metatarsal head ulcers (after Achilles lengthening). There were no admissions for diabetic foot sepsis, reduced recurrence, reduced amputation rates and no mortality. Of the conservatively managed cohort, only 3 of the 15 achieved ulcer resolution without recurrence within the 12 month study period. The cohort managed conservatively had an average cost of £ 9902 per patient, per annum. The intervention cost was £ 1211 per patient, saving an average of £ 8691 per patient, per annum with ulcer resolution (88 % reduction in costs). CONCLUSION: Significant patient benefit, reduction in resource use and cost saving was seen with this simple intervention, which merits full evaluation in a clinical trial. LEVEL OF EVIDENCE: Level-IV.

Genomic sequencing of Thinopyrum elongatum chromosome arm 7EL, carrying fusarium head blight resistance, and characterization of its impact on the transcriptome of the introgressed line CS-7EL.

BACKGROUND: The tall wheatgrass species Thinopyrum elongatum carries a strong fusarium head blight (FHB) resistance locus located on the long arm of chromosome 7 (7EL) as well as resistance to leaf and stem rusts, all diseases with a significant impact on wheat production. Towards understanding the contribution of Th. elongatum 7EL to improvement of disease resistance in wheat, the genomic sequence of the 7EL fragment present in the wheat Chinese Spring (CS) telosomic addition line CS-7EL was determined and the contribution and impact of 7EL on the rachis transcriptome during FHB infection was compared between CS and CS-7EL. RESULTS: We assembled the Th. elongatum 7EL chromosome arm using a reference-guided approach. Combining this assembly with the available reference sequence for CS hexaploid wheat provided a reliable reference for interrogating the transcriptomic differences in response to infection conferred by the 7EL fragment. Comparison of the transcriptomes of rachis tissues from CS and CS-7EL showed expression of Th. elongatum transcripts as well as modulation of wheat transcript expression profiles in the CS-7EL line. Expression profiles at 4 days after infection with Fusarium graminearum, the causal agent of FHB, showed an increased in expression of genes associated with an effective defense response, in particular glucan endo-1,3-beta-glucosidases and chitinases, in the FHB-resistant line CS-7EL while there was a larger increase in differential expression for genes associated with the level of fungal infection in the FHB-susceptible line CS. One hundred and seven 7EL transcripts were expressed in the smallest 7EL region defined to carry FHB resistance. CONCLUSION: 7EL contributed to CS-7EL transcriptome by direct expression and through alteration of wheat transcript profiles. FHB resistance in CS-7EL was associated with transcriptome changes suggesting a more effective defense response. A list of candidate genes for the FHB resistance locus on 7EL has been established.

The Transcriptional Landscape of Polyploid Wheats and Their Diploid Ancestors during Embryogenesis and Grain Development.

Modern wheat production comes from two polyploid species, Triticum aestivum and Triticum turgidum (var durum), which putatively arose from diploid ancestors Triticum urartu, Aegilops speltoides, and Aegilops tauschii How gene expression during embryogenesis and grain development in wheats has been shaped by the differing contributions of diploid genomes through hybridization, polyploidization, and breeding selection is not well understood. This study describes the global landscape of gene activities during wheat embryogenesis and grain development. Using comprehensive transcriptomic analyses of two wheat cultivars and three diploid grasses, we investigated gene expression at seven stages of embryo development, two endosperm stages, and one pericarp stage. We identified transcriptional signatures and developmental similarities and differences among the five species, revealing the evolutionary divergence of gene expression programs and the contributions of A, B, and D subgenomes to grain development in polyploid wheats. The characterization of embryonic transcriptional programming in hexaploid wheat, tetraploid wheat, and diploid grass species provides insight into the landscape of gene expression in modern wheat and its ancestral species. This study presents a framework for understanding the evolution of domesticated wheat and the selective pressures placed on grain production, with important implications for future performance and yield improvements.plantcell;31/12/2888/FX1F1fx1.

Clinical research on the use of bordered foam dressings in the treatment of complex wounds: A systematic review of reported outcomes and applied measurement instruments.

OBJECTIVE: The aim of this review article was to identify reported outcomes and measurement instruments used in clinical research on bordered foam dressings in the treatment of complex wounds. METHODS: MEDLINE (PubMed interface), Cumulative Index to Nursing and Allied Health Literature (CINAHL), EMBASE, and The Cochrane Library were systematically searched using a combination of key terms including; wounds, bordered foam dressing, and treatment. Studies were included if they (1) targeted an adult population, (2) addressed the treatment of complex wounds with a bordered foam dressing as the primary wound dressing, (3) were retrieved from original research, and (4) were published between 2000 and 2022. There were no restrictions on language or study design. Studies that focused primarily on the prevention of complex wounds were excluded. Data extraction included outcome domains, outcomes, instruments, time points, and outcome measures. The OMERACT Filter 2.0 was used as a conceptual framework for the extraction of outcomes. RESULTS: A total of 24 outcome domains and 82 outcomes were identified. The outcomes were categorised into five core areas: (1) impact on life, (2) dressing performance, (3) pathophysiological manifestations, (4) resource use, and (5) adverse events. Thirtynine outcomes (47.0%) were measured at more than one time point. The most frequently reported time point was 'at the end of treatment' (62.7%). Outcomes were measured using self-report instruments, clinical observations, and bio-physiological instruments. CONCLUSION: This systematic review identified reported outcomes and measurement instruments in research on bordered foam dressings in the treatment of complex wounds. The variety and lack of consistency in terms of instruments, time points and outcome measurements made it difficult to compare data directly across different reported studies. A solution to the variety in outcome reporting across studies in complex wound care, and moreover for the treatment with bordered foam dressings, is the development of a Core Outcome Set (COS). The outcomes in this review article will inform the next steps of developing a COS, where patients, clinicians and researchers will be involved to decide on the final outcomes included in a COS for the treatment of complex wounds with bordered foam dressings.

Alternative splicing dynamics and evolutionary divergence during embryogenesis in wheat species.

Among polyploid species with complex genomic architecture, variations in the regulation of alternative splicing (AS) provide opportunities for transcriptional and proteomic plasticity and the potential for generating trait diversities. However, the evolution of AS and its influence on grain development in diploid grass and valuable polyploid wheat crops are poorly understood. To address this knowledge gap, we developed a pipeline for the analysis of alternatively spliced transcript isoforms, which takes the high sequence similarity among polyploid wheat subgenomes into account. Through analysis of synteny and detection of collinearity of homoeologous subgenomes, conserved and specific AS events across five wheat and grass species were identified. A global analysis of the regulation of AS in diploid grass and polyploid wheat grains revealed diversity in AS events not only between the endosperm, pericarp and embryo overdevelopment, but also between subgenomes. Analysis of AS in homoeologous triads of polyploid wheats revealed evolutionary divergence between gene-level and transcript-level regulation of embryogenesis. Evolutionary age analysis indicated that the generation of novel transcript isoforms has occurred in young genes at a more rapid rate than in ancient genes. These findings, together with the development of comprehensive AS resources for wheat and grass species, advance understanding of the evolution of regulatory features of AS during embryogenesis and grain development in wheat.

Structural features of two major nucleolar organizer regions (NORs), Nor-B1 and Nor-B2, and chromosome-specific rRNA gene expression in wheat.

The reference genome sequence of wheat 'Chinese Spring' (CS) is now available (IWGSC RefSeq v1.0), but the core sequences defining the nucleolar organizer regions (NORs) have not been characterized. We estimated that the total copy number of the rDNA units in the wheat genome is 11 160, of which 30.5%, 60.9% and 8.6% are located on Nor-B1 (1B), Nor-B2 (6B) and other NORs, respectively. The total length of the NORs is estimated to be 100 Mb, corresponding to approximately 10% of the unassembled portion of the genome not represented in RefSeq v1.0. Four subtypes (S1-S4) of the rDNA units were identified based on differences within the 3' external transcribed spacer regions in Nor-B1 and Nor-B2, and quantitative PCR indicated locus-specific variation in rDNA subtype contents. Expression analyses of rDNA subtypes revealed that S1 was predominantly expressed and S2 weakly expressed, in contrast to the relative abundance of rDNA subtypes in the wheat genome. These results suggest a regulation mechanism of differential rDNA expression based on sequence differences. S3 expression increased in the ditelosomic lines Dt1BL and Dt6BL, suggesting that S3 is subjected to chromosome-mediated silencing. Structural differences were detected in the regions surrounding the NOR among homoeologous chromosomes of groups 1 and 6. The adjacent regions distal to the major NORs were expanded compared with their homoeologous counterparts, and the gene density of these expanded regions was relatively low. We provide evidence that these regions are likely to be important for autoregulation of the associated major NORs as well as silencing of minor NORs.

WheatCRISPR: a web-based guide RNA design tool for CRISPR/Cas9-mediated genome editing in wheat.

BACKGROUND: CRISPR/Cas9 gene editing has become a revolutionary technique for crop improvement as it can facilitate fast and efficient genetic changes without the retention of transgene components in the final plant line. Lack of robust bioinformatics tools to facilitate the design of highly specific functional guide RNAs (gRNAs) and prediction of off-target sites in wheat is currently an obstacle to effective application of CRISPR technology to wheat improvement. DESCRIPTION: We have developed a web-based bioinformatics tool to design specific gRNAs for genome editing and transcriptional regulation of gene expression in wheat. A collaborative study between the Broad Institute and Microsoft Research used large-scale empirical evidence to devise algorithms (Doech et al., 2016, Nature Biotechnology 34, 184-191) for predicting the on-target activity and off-target potential of CRISPR/SpCas9 (Streptococcus pyogenes Cas9). We applied these prediction models to determine on-target specificity and potential off-target activity for individual gRNAs targeting specific loci in the wheat genome. The genome-wide gRNA mappings and the corresponding Doench scores predictive of the on-target and off-target activities were used to create a gRNA database which was used as a data source for the web application termed WheatCRISPR. CONCLUSION: The WheatCRISPR tool allows researchers to browse all possible gRNAs targeting a gene or sequence of interest and select effective gRNAs based on their predicted high on-target and low off-target activity scores, as well as other characteristics such as position within the targeted gene. It is publicly available at https://crispr.bioinfo.nrc.ca/WheatCrispr/ .

Correction to: MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat.

Following publication of the original article [1], a reader spotted an incorrect citation of the reference 14 [2] in the 'Background'. The male meiocyte isolation work described in this article [2] was carried out in rice and not in Brassica as originally stated in the 'Background' [1]. Thus, the following amendment to the Background section should be noted.

Mapping quantitative trait loci associated with common bunt resistance in a spring wheat (Triticum aestivum L.) variety Lillian.

KEY MESSAGE: Based on their consistency over environments, two QTL identified in Lillian on chromosomes 5A and 7A could be useful targets for marker assisted breeding of common bunt resistance. Common bunt of wheat (Triticum aestivum L.) caused by Tilletia tritici and T. laevis is an economically important disease because of losses in grain yield and reduced grain quality. Resistance can be quantitative, under the control of multiple small effect genes. The Canada Western Red Spring wheat variety Lillian is moderately resistant to common bunt races found on the Canadian prairies. This study was conducted to identify and map quantitative trait loci (QTL) conferring resistance against common bunt in Lillian. A doubled haploid population comprising 280 lines was developed from F1 plants of the cross of Lillian by Vesper. The lines were inoculated at seeding with the two races L16 (T. laevis) and T19 (T. tritici), grown in field near Swift Current, SK, in 2014, 2015 and 2016 and assessed for disease incidence. The lines were genotyped with the 90 K iSelect SNP genotyping assay, and a high-density genetic map was constructed. Quantitative trait locus analysis was performed with MapQTL.6® software. Two relatively stable common bunt resistance QTL, detected in two of the 3 years, were identified on chromosomes 5A and 7A from Lillian. In addition, three less stable QTL, appearing in one out of 3 years, were identified: one was contributed by Lillian on chromosome 3D and two were contributed by Vesper on chromosomes 1D and 2A. Epistatic interaction was identified for the bunt incidence between 3D and 7A resulting in greater bunt resistance. Future bunt resistance breeding will benefit from combining these QTL through gene pyramiding.

A quantum access network.

The theoretically proven security of quantum key distribution (QKD) could revolutionize the way in which information exchange is protected in the future. Several field tests of QKD have proven it to be a reliable technology for cryptographic key exchange and have demonstrated nodal networks of point-to-point links. However, until now no convincing answer has been given to the question of how to extend the scope of QKD beyond niche applications in dedicated high security networks. Here we introduce and experimentally demonstrate the concept of a 'quantum access network': based on simple and cost-effective telecommunication technologies, the scheme can greatly expand the number of users in quantum networks and therefore vastly broaden their appeal. We show that a high-speed single-photon detector positioned at a network node can be shared between up to 64 users for exchanging secret keys with the node, thereby significantly reducing the hardware requirements for each user added to the network. This point-to-multipoint architecture removes one of the main obstacles restricting the widespread application of QKD. It presents a viable method for realizing multi-user QKD networks with efficient use of resources, and brings QKD closer to becoming a widespread technology.

Genome re-sequencing and simple sequence repeat markers reveal the existence of divergent lineages in the Canadian Puccinia striiformis f. sp. tritici population with extensive DNA methylation.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is an important disease in Canada. The worldwide genetic structure of Pst populations have been characterized, excluding Canada. Here, we elucidated the genetic structure of the western Canadian Pst population using molecular markers, revealing the presence of four divergent lineages with predominantly clonal structure. In the worldwide context, two previously reported lineages were identified: PstS0 (22%), representing an old Northwestern-European and PstS1 (35%), an invasive warm-temperature adapted. Additionally, two new, unreported lineages, PstPr (9%) and PstS1-related (35%), were detected, which produced more telia than other lineages and had double the number of unique recombination events. The PstPr was a recent invasion, and likely evolved in a diverse, recombinant population as it was closely related to the PstS5, PstS7/Warrior, PstS8/Kranich, and PstS9 lineages originating from sexually recombining populations in the centre of diversity. The DNA methylation analysis revealed DNA-methyltransferase1-homologs, providing compelling evidence for epigenetic regulation and as a first report, an average of ∼5%, 5hmC in the Puccinia epigenome merits further investigation. The divergent lineages in the Canadian Pst population with the potential for genetic recombination, as well as epigenetic regulation needs consideration in the context of pathogen adaptation and management.

MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat.

BACKGROUND: Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks. RESULTS: We describe an optimized method termed MeioCapture for simultaneous isolation of uncontaminated male meiocytes from wheat (Triticum spp.), specifically from the pre-meiotic G2 and the five sub-stages of meiotic prophase I. The MeioCapture protocol builds on the traditional anther squash technique and the capillary collection method, and involves extrusion of intact sporogenous archesporial columns (SACs) containing meiocytes. This improved method exploits the natural meiotic synchrony between anthers of the same floret, the correlation between the length of anthers and meiotic stage, and the occurrence of meiocytes in intact SACs largely free of somatic cells. The main advantage of MeioCapture, compared to previous methods, is that it allows simultaneous collection of meiocytes from different sub-stages of prophase I at a very high level of purity, through correlation of stages with anther sizes. A detailed description is provided for all steps, including the collection of tissue, isolation and size sorting of anthers, extrusion of intact SACs, and staging of meiocytes. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. The proof-of-concept was successfully established in wheat, and a light microscopic atlas of meiosis, encompassing all stages from pre-meiosis to telophase II, was developed. CONCLUSION: The MeioCapture method provides an essential technique to study the molecular basis of chromosome pairing and exchange of genetic information in wheat, leading to strategies for manipulating meiotic recombination frequencies. The method also provides a foundation for similar studies in other crop species.

The biochemical composition and transcriptome of cotyledons from Brassica napus lines expressing the AtGL3 transcription factor and exhibiting reduced flea beetle feeding.

BACKGROUND: Previously, transgenic trichome-bearing (hairy leaf) Brassica napus lines expressing either the Arabidopsis thaliana GL3 gene (line AtGL3+) [1] or the AtGL3 gene in combination with an RNAi construct to down-regulate TTG1 (line K-5-8) [2] were developed. The leaves of these lines exhibited altered insect feeding (flea beetle) and oviposition (diamondback moth) behaviour compared to the non-transgenic semi-glabrous leaves of B. napus cv. Westar. Interestingly, the cotyledons of these lines remained glabrous, but also showed reduced feeding by flea beetles. Here we examine the composition and global transcriptome of the glabrous cotyledons from these transgenic lines to ascertain the mechanism(s) underlying this unexpected phenomenon. RESULTS: Approximately, 7500 genes were up-regulated in cotyledons of each hairy line, compared with < 30 that were down-regulated. The up-regulated genes included those involved in cell wall synthesis, secondary metabolite production, redox, stress and hormone-related responses that have the potential to impact host plant cues required to elicit defense responses toward insect pests. In particular, the expression of glucosinolate biosynthetic and degradation genes were substantially altered in the glabrous cotyledons of the two hairy leaf lines. The transcriptomic data was supported by glucosinolate and cell wall composition profiles of the cotyledons. Changes in gene expression were much more extreme in the AtGL3+ line compared with the K-5-8 line in terms of diversity and intensity. CONCLUSIONS: The study provides a roadmap for the isolation and identification of insect resistance compounds and proteins in the glabrous cotyledons of these hairy leaf lines. It also confirms the impact of mis-expression of GL3 and TTG1 on types of metabolism other than those associated with trichomes. Finally, the large number of up-regulated genes encoding heat shock proteins, PR proteins, protease inhibitors, glucosinolate synthesis/breakdown factors, abiotic stress factors, redox proteins, transcription factors, and proteins required for auxin metabolism also suggest that these cotyledons are now primed for resistance to other forms of biotic and abiotic stress.