Modulation of physio-biochemical and photosynthesis parameters by overexpressing SbPIP2 gene improved abiotic stress tolerance of transgenic tobacco.

The present study aims to explore the potential of a plasma-membrane localized PIP2-type aquaporin protein sourced from the halophyte Salicornia brachiata to alleviate salinity and water deficit stress tolerance in a model plant through transgenic intervention. Transgenic plants overexpressing SbPIP2 gene showed improved physio-biochemical parameters like increased osmolytes (proline, total sugar, and amino acids), antioxidants (polyphenols), pigments and membrane stability under salinity and drought stresses compared to control plants [wild type (WT) and vector control (VC) plants]. Multivariate statistical analysis showed that, under water and salinity stresses, osmolytes, antioxidants and pigments were correlated with SbPIP2-overexpressing (SbPIP2-OE) plants treated with salinity and water deficit stress, suggesting their involvement in stress tolerance. As aquaporins are also involved in CO2 transport, SbPIP2-OE plants showed enhanced photosynthesis performance than wild type upon salinity and drought stresses. Photosynthetic gas exchange (net CO2 assimilation rate, PSII efficiency, ETR, and non-photochemical quenching) were significantly higher in SbPIP2-OE plants compared to control plants (wild type and vector control plants) under both unstressed and stressed conditions. The higher quantum yield for reduction of end electron acceptors at the PSI acceptor side [Φ( R0 )] in SbPIP2-OE plants compared to control plants under abiotic stresses indicates a continued PSI functioning, leading to retained electron transport rate, higher carbon assimilation, and less ROS-mediated injuries. In conclusion, the SbPIP2 gene functionally validated in the present study could be a potential candidate for engineering abiotic stress resilience in important crops.

Natural and artificial sources of genetic variation used in crop breeding: A baseline comparator for genome editing.

Traditional breeding has successfully selected beneficial traits for food, feed, and fibre crops over the last several thousand years. The last century has seen significant technological advancements particularly in marker assisted selection and the generation of induced genetic variation, including over the last few decades, through mutation breeding, genetic modification, and genome editing. While regulatory frameworks for traditional varietal development and for genetic modification with transgenes are broadly established, those for genome editing are lacking or are still evolving in many regions. In particular, the lack of "foreign" recombinant DNA in genome edited plants and that the resulting SNPs or INDELs are indistinguishable from those seen in traditional breeding has challenged development of new legislation. Where products of genome editing and other novel breeding technologies possess no transgenes and could have been generated via traditional methods, we argue that it is logical and proportionate to apply equivalent legislative oversight that already exists for traditional breeding and novel foods. This review analyses the types and the scale of spontaneous and induced genetic variation that can be selected during traditional plant breeding activities. It provides a base line from which to judge whether genetic changes brought about by techniques of genome editing or other reverse genetic methods are indeed comparable to those routinely found using traditional methods of plant breeding.

Meiosis in allopolyploid Arabidopsis suecica.

Polyploidy is a major force shaping eukaryote evolution but poses challenges for meiotic chromosome segregation. As a result, first-generation polyploids often suffer from more meiotic errors and lower fertility than established wild polyploid populations. How established polyploids adapt their meiotic behaviour to ensure genome stability and accurate chromosome segregation remains an active research question. We present here a cytological description of meiosis in the model allopolyploid species Arabidopsis suecica (2n = 4x = 26). In large part meiosis in A. suecica is diploid-like, with normal synaptic progression and no evidence of synaptic partner exchanges. Some abnormalities were seen at low frequency, including univalents at metaphase I, anaphase bridges and aneuploidy at metaphase II; however, we saw no evidence of crossover formation occurring between non-homologous chromosomes. The crossover number in A. suecica is similar to the combined number reported from its diploid parents Arabidopsis thaliana (2n = 2x = 10) and Arabidopsis arenosa (2n = 2x = 16), with an average of approximately 1.75 crossovers per chromosome pair. This contrasts with naturally evolved autotetraploid A. arenosa, where accurate chromosome segregation is achieved by restricting crossovers to approximately 1 per chromosome pair. Although an autotetraploid donor is hypothesized to have contributed the A. arenosa subgenome to A. suecica, A. suecica harbours diploid A. arenosa variants of key meiotic genes. These multiple lines of evidence suggest that meiosis in the recently evolved allopolyploid A. suecica is essentially diploid like, with meiotic adaptation following a very different trajectory to that described for autotetraploid A. arenosa.

Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production.

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni's α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp_089290's female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.

Schistosoma mansoni venom allergen-like protein 6 (SmVAL6) maintains tegumental barrier function.

The Schistosoma mansoni venom allergen-like protein (SmVAL) superfamily is a collection of at least 29 molecules that have been classified into two distinctive groups (Group 1 and Group 2 SmVALs). The fundamental basis for SmVAL segregation relates to signal peptide and conserved cysteine retention (present in all Group 1 SmVALs, but absent in all Group 2 SmVALs). These structural differences have led to the hypothesis that most Group 1 SmVALs, found as components of schistosome excretory/secretory (E/S) products, predominantly interact with their environment (intermediate or definitive hosts) whereas the Group 2 SmVALs are retained within the schistosome to fulfil parasite-related functions. While experimental evidence to support Group 1 SmVAL/host interactions is growing, similar support for identification of parasite-related Group 2 SmVAL functions is currently lacking. By applying a combination of approaches to the study of SmVAL6, we provide the first known evidence for an essential function of a Group 2 SmVAL in schistosome biology. After whole mount in situ hybridisation (WISH) localised Smval6 to the anterior region of the oesophageal gland (AOG) and cells scattered through the mesenchyme in adult schistosomes, short interfering RNA (siRNA)-mediated silencing of Smval6 was employed to assess loss of function phenotypes. Here, siSmval6-mediated knockdown of transcript and protein levels led to an increase in tegumental permeability as assessed by the quantification of TAMRA-labelled dextran throughout sub-tegumental cells/tissues. Yeast two hybrid screening using SmVAL6 as a bait revealed Sm14 (a fatty acid binding protein) and a dynein light chain (DLC) as directly interacting partners. Interrogation of single-cell RNA-seq (scRNA-seq) data supported these protein interactions by demonstrating the spatial co-expression of Smval6/dlc/Sm14 in a small proportion of adult cell types (e.g. neurons, tegumental cells and neoblasts). In silico modelling of SmVAL6 with Sm14 and DLC provided evidence that opposing faces of SmVAL6 were likely responsible for these protein/protein interactions. Our results suggest that SmVAL6 participates in oesophageal biology, formation of higher order protein complexes and maintenance of tegumental barrier function. Further studies of other Group 2 SmVALs may reveal additional functions of this enigmatic superfamily.

Development and Cytomolecular Identification of Monosomic Alien Addition and Substitution Lines of Triticale (×Triticosecale Wittmack) With 2Sk Chromosome Conferring Leaf Rust Resistance Derived From Aegilops kotschyi Boiss.

Alien chromosome introgression has become a valuable tool to broaden the genetic variability of crop plants via chromosome engineering. This study details the procedure to obtain monosomic addition and monosomic substitution lines of the triticale carrying 2Sk chromosome from Aegilops kotchyi Boiss., which harbors Lr54 + Yr37 leaf and stripe rust-resistant gene loci, respectively. Initially, A. kotschyi × Secale cereale artificial amphiploids (2n = 6x = 42 chromosomes, UUSSRR) were crossed with triticale cv. "Sekundo" (2n = 6x = 42, AABBRR) in order to obtain fertile offspring. Cyto-molecular analyses of five subsequent backcrossing generations revealed that 2Sk chromosome was preferentially transmitted. This allowed for the selection of monosomic 2Sk addition (MA2Sk) lines of triticale. Finally, the 2Sk(2R) substitution plants were obtained by crossing MA2Sk with the nullisomic (N2R) plants of triticale. The presence of 2Sk chromosome in subsequent generations of plants was evaluated using SSR markers linked to Lr54 + Yr37 loci. Disease evaluation of the monosomic 2Sk(2R) substitution plants for the reaction to leaf and stripe rust infection were carried out under controlled conditions in a growth chamber. The results showed significant improvement of leaf rust resistance severity of monosomic substitution plants compared with control ("Sekundo"). In contrast, the introgression of the Lr54 + Yr37 loci did not lead to improvement of stripe rust resistance. In summary, the creation of monosomic addition and monosomic substitution lines of triticale is the starting point for the precise and guided transfer of Lr54 + Yr37 loci. The results showed that the developed materials could be exploited for the development of triticale varieties with resistance to leaf rust.

Cupid, a cell permeable peptide derived from amoeba, capable of delivering GFP into a diverse range of species.

Cell permeating peptides (CPPs) are attracting great interest for use as molecular delivery vehicles for the transport of biologically active cargo across the cell membrane. The sequence of a novel CPP sequence, termed 'Cupid', was identified from the genome of Dictyostelium discoideum. A Cupid-Green Fluorescent Protein (Cupid-GFP) fusion protein was tested on mammalian, whole plant cells, plant leaf protoplast and fungal cell cultures and observed using confocal microscopy. GFP fluorescence builds up within the cell cytosol in 60 min, demonstrating Cupid-GFP has permeated them and folded correctly into its fluorescent form. Our combined data suggest Cupid can act as a molecular vehicle capable of delivering proteins, such as GFP, into the cytosol of a variety of cells.

CDKG1 Is Required for Meiotic and Somatic Recombination Intermediate Processing in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) cyclin-dependent kinase G1 (CDKG1) is necessary for recombination and synapsis during male meiosis at high ambient temperature. In the cdkg1-1 mutant, synapsis is impaired and there is a dramatic reduction in the number of class I crossovers, resulting in univalents at metaphase I and pollen sterility. Here, we demonstrate that CDKG1 is necessary for the processing of recombination intermediates in the canonical ZMM recombination pathway and that loss of CDKG1 results in increased class II crossovers. While synapsis and events associated with class I crossovers are severely compromised in a cdkg1-1 mutant, they can be restored by increasing the number of recombination intermediates in the double cdkg1-1 fancm-1 mutant. Despite this, recombination intermediates are not correctly resolved, leading to the formation of chromosome aggregates at metaphase I. Our results show that CDKG1 acts early in the recombination process and is necessary to stabilize recombination intermediates. Finally, we show that the effect on recombination is not restricted to meiosis and that CDKG1 is also required for normal levels of DNA damage-induced homologous recombination in somatic tissues.

Genetic Transformation of Protoplasts Isolated from Leaves of Lolium temulentum and Lolium perenne.

Transient expression of inserted recombinant DNA in plant protoplasts is a widely used tool for functional genomics research. Recently it has been utilized to screen potential sgRNA guides for CRISPR-mediated genome editing. However, little research has been conducted into the use of transient expression of protoplasts in Lolium perenne (a globally important pasture, hay, and amenity grass), and no studies have been conducted into Lolium temulentum (a weed in cereal crops but a potentially useful model species for Lolium research). In this chapter, we describe a methodology of protoplast extraction and transformation from 14-day-old leaf mesophyll cells from L. perenne and L. temulentum. We believe this is the first report of a procedure for obtaining high density, viable protoplasts from L. temulentum. The method of polyethylene glycol (PEG)-mediated transformation is also described to achieve genetic transformation of protoplasts.

Quantification of Synapsis Using Immunolocalization in Embedded Nuclei of Lolium.

The establishment, formation and disassembly of the synaptonemal complex (SC) is intimately associated with other essential processes that occur during prophase I of meiosis, including recombination. Labeling the SC using primary antibodies raised against key proteins, detected using secondary antibodies conjugated to fluorescent dyes, differentiate between synapsed and unsynapsed regions, revealing the dynamics of the process. Embedding meiotic nuclei in acrylamide pads preserves the three-dimensional (3D) organization of the chromosomes, which can be optically sectioned using confocal laser scanning microscopy to produce a faithful representation of the SC at the point of fixation. Deconvolution, and processing using Imaris allows the axes to be isolated from the nucleus and their features measured. Here, I describe a robust protocol to quantify the SC using immunofluorescence in Lolium perenne and L. temulentum.

Nuclear Disposition of Alien Chromosome Introgressions into Wheat and Rye Using 3D-FISH.

During interphase, the chromosomes of eukaryotes decondense and they occupy distinct regions of the nucleus, called chromosome domains or chromosome territories (CTs). In plants, the Rabl's configuration, with telomeres at one pole of nucleus and centromeres at the other, appears to be common, at least in plants with large genomes. It is unclear whether individual chromosomes of plants adopt defined, genetically determined addresses within the nucleus, as is the case in mammals. In this study, the nuclear disposition of alien rye and barley chromosomes and chromosome arm introgressions into wheat while using 3D-FISH in various somatic tissues was analyzed. All of the introgressed chromosomes showed Rabl's orientation, but their relative positions in the nuclei were less clear. While in most cases pairs of introgressed chromosomes occupied discrete positions, their association (proximity) along their entire lengths was rare, and partial association only marginally more frequent. This arrangement is relatively stable in various tissues and during various stages of the cell cycle. On the other hand, the length of a chromosome arm appears to play a role in its positioning in a nucleus: shorter chromosomes or chromosome arms tend to be located closer to the centre of the nucleus, while longer arms are more often positioned at the nuclear periphery.

Instability of Alien Chromosome Introgressions in Wheat Associated with Improper Positioning in the Nucleus.

Alien introgressions introduce beneficial alleles into existing crops and hence, are widely used in plant breeding. Generally, introgressed alien chromosomes show reduced meiotic pairing relative to the host genome, and may be eliminated over generations. Reduced pairing appears to result from a failure of some telomeres of alien chromosomes to incorporate into the leptotene bouquet at the onset of meiosis, thereby preventing chiasmate pairing. In this study, we analysed somatic nuclei of rye introgressions in wheat using 3D-FISH and found that while introgressed rye chromosomes or chromosome arms occupied discrete positions in the Rabl's orientation similar to chromosomes of the wheat host, their telomeres frequently occupied positions away from the nuclear periphery. The frequencies of such abnormal telomere positioning were similar to the frequencies of out-of-bouquet telomere positioning at leptotene, and of pairing failure at metaphase I. This study indicates that improper positioning of alien chromosomes that leads to reduced pairing is not a strictly meiotic event but rather a consequence of a more systemic problem. Improper positioning in the nuclei probably impacts the ability of introgressed chromosomes to migrate into the telomere bouquet at the onset of meiosis, preventing synapsis and chiasma establishment, and leading to their gradual elimination over generations.

Anthropogenic Impacts on Meiosis in Plants.

As the human population grows and continues to encroach on the natural environment, organisms that form part of such ecosystems are becoming increasingly exposed to exogenous anthropogenic factors capable of changing their meiotic landscape. Meiotic recombination generates much of the genetic variation in sexually reproducing species and is known to be a highly conserved pathway. Environmental stresses, such as variations in temperature, have long been known to change the pattern of recombination in both model and crop plants, but there are other factors capable of causing genome damage, infertility and meiotic abnormalities. Our agrarian expansion and our increasing usage of agrochemicals unintentionally affect plants via groundwater contamination or spray drift; our industrial developments release heavy metals into the environment; pathogens are spread by climate change and a globally mobile population; imperfect waste treatment plants are unable to remove chemical and pharmaceutical residues from sewage leading to the release of xenobiotics, all with potentially deleterious meiotic effects. In this review, we discuss the major classes of exogenous anthropogenic factors known to affect meiosis in plants, namely environmental stresses, agricultural inputs, heavy metals, pharmaceuticals and pathogens. The possible evolutionary fate of plants thrust into their new anthropogenically imposed environments are also considered.

Methyl-CpG-binding (SmMBD2/3) and chromobox (SmCBX) proteins are required for neoblast proliferation and oviposition in the parasitic blood fluke Schistosoma mansoni.

While schistosomiasis remains a significant health problem in low to middle income countries, it also represents a recently recognised threat to more economically-developed regions. Until a vaccine is developed, this neglected infectious disease is primarily controlled by praziquantel, a drug with a currently unknown mechanism of action. By further elucidating how Schistosoma molecular components cooperate to regulate parasite developmental processes, next generation targets will be identified. Here, we continue our studies on schistosome epigenetic participants and characterise the function of a DNA methylation reader, the Schistosoma mansoni methyl-CpG-binding domain protein (SmMBD2/3). Firstly, we demonstrate that SmMBD2/3 contains amino acid features essential for 5-methyl cytosine (5mC) binding and illustrate that adult schistosome nuclear extracts (females > males) contain this activity. We subsequently show that SmMBD2/3 translocates into nuclear compartments of transfected murine NIH-3T3 fibroblasts and recombinant SmMBD2/3 exhibits 5mC binding activity. Secondly, using a yeast-two hybrid (Y2H) screen, we show that SmMBD2/3 interacts with the chromo shadow domain (CSD) of an epigenetic adaptor, S. mansoni chromobox protein (SmCBX). Moreover, fluorescent in situ hybridisation (FISH) mediated co-localisation of Smmbd2/3 and Smcbx to mesenchymal cells as well as somatic- and reproductive- stem cells confirms the Y2H results and demonstrates that these interacting partners are ubiquitously expressed and found within both differentiated as well as proliferating cells. Finally, using RNA interference, we reveal that depletion of Smmbd2/3 or Smcbx in adult females leads to significant reductions (46-58%) in the number of proliferating somatic stem cells (PSCs or neoblasts) as well as in the quantity of in vitro laid eggs. Collectively, these results further expand upon the schistosome components involved in epigenetic processes and suggest that pharmacological inhibition of SmMBD2/3 and/or SmCBX biology could prove useful in the development of future schistosomiasis control strategies.

B chromosomes are associated with redistribution of genetic recombination towards lower recombination chromosomal regions in perennial ryegrass.

Supernumerary 'B' chromosomes are non-essential components of the genome present in a range of plant and animal species-including many grasses. Within diploid and polyploid ryegrass and fescue species, including the forage grass perennial ryegrass (Lolium perenne L.), the presence of B chromosomes has been reported as influencing both chromosome pairing and chiasma frequencies. In this study, the effects of the presence/absence of B chromosomes on genetic recombination has been investigated through generating DArT (Diversity Arrays Technology) marker genetic maps for six perennial ryegrass diploid populations, the pollen parents of which contained either two B or zero B chromosomes. Through genetic and cytological analyses of these progeny and their parents, we have identified that, while overall cytological estimates of chiasma frequencies were significantly lower in pollen mother cells with two B chromosomes as compared with zero B chromosomes, the recombination frequencies within some marker intervals were actually increased, particularly for marker intervals in lower recombination regions of chromosomes, namely pericentromeric regions. Thus, in perennial ryegrass, the presence of two B chromosomes redistributed patterns of meiotic recombination in pollen mother cells in ways which could increase the range of allelic variation available to plant breeders.

A cytological approach to studying meiotic recombination and chromosome dynamics in Arabidopsis thaliana male meiocytes in three dimensions.

During meiotic prophase I chromosomes undergo dramatic conformational changes that accompany chromosome condensation, pairing and recombination between homologs. These changes include the anchoring of telomeres to the nuclear envelope and their clustering to form a bouquet. In plants, these events have been studied and illustrated in intact meiocytes of species with large genomes. Arabidopsis thaliana is an excellent genetic model in which major molecular pathways that control synapsis and recombination between homologs have been uncovered. Yet the study of chromosome dynamics is hampered by current cytological methods that disrupt the three-dimensional (3D) architecture of the nucleus. Here we set up a protocol to preserve the 3D configuration of A. thaliana meiocytes. We showed that this technique is compatible with the use of a variety of antibodies that label structural and recombination proteins and were able to highlight the presence of clustered synapsis initiation centers at the nuclear periphery. By using fluorescence in situ hybridization we also studied the behavior of chromosomes during pre-meiotic G2 and prophase I, revealing the existence of a telomere bouquet during A. thaliana male meiosis. In addition we showed that the number of telomeres in a bouquet and its volume vary greatly, thus revealing the complexity of telomere behavior during meiotic prophase I. Finally, by using probes that label subtelomeric regions of individual chromosomes, we revealed differential localization behaviors of chromosome ends. Our protocol opens new areas of research for investigating chromosome dynamics in A. thaliana meiocytes.

Ndfip1 restricts Th17 cell potency by limiting lineage stability and proinflammatory cytokine production.

While Th17 cells can protect against colonization by pathogenic organisms, they also have the potential to become pathogenic and promote autoimmune and inflammatory diseases. Mechanisms that control their pathogenic potential remain poorly understood. Here we show that Ndfip1, a co-activator of the E3 ubiquitin ligase Itch, restricts the frequency and pathogenicity of Th17 cells. Mice lacking Ndfip1 have increased numbers of Th17 cells, and this increase is cell intrinsic. We found that Ndfip1 restricts production of the proinflammatory cytokines in Th17 cells. Increased cytokine production correlated with reduced degradation and accumulation of RORγT. When transferred in vivo, Th17 cells lacking Ndfip1 were more likely to maintain their ability to make IL-17, were more potent proinflammatory cytokine producers, and were powerful inducers of colitis. Together our data support an essential role for Ndfip1 in degrading RORγT and suppressing Th17 lineage stability, proinflammatory cytokine production, and pathogenicity.

Potential impact of genome editing in world agriculture.

Changeable biotic and abiotic stress factors that affect crop growth and productivity, alongside a drive to reduce the unintended consequences of plant protection products, will demand highly adaptive farm management practices as well as access to continually improved seed varieties. The former is limited mainly by cost and, in theory, could be implemented in relatively short time frames. The latter is fundamentally a longer-term activity where genome editing can play a major role. The first targets for genome editing will inevitably be loss-of-function alleles, because these are straightforward to generate. In addition, they are likely to focus on traits under simple genetic control and where the results of modification are already well understood from null alleles in existing gene pools or other knockout or silencing approaches such as induced mutations or RNA interference. In the longer term, genome editing will underpin more fundamental changes in agricultural performance and food quality, and ultimately will merge with the tools and philosophies of synthetic biology to underpin and enable new cellular systems, processes and organisms completely. The genetic changes required for simple allele edits or knockout phenotypes are synonymous with those found naturally in conventional breeding material and should be regulated as such. The more radical possibilities in the longer term will need societal engagement along with appropriate safety and ethical oversight.

A spontaneous mutation in MutL-Homolog 3 (HvMLH3) affects synapsis and crossover resolution in the barley desynaptic mutant des10.

Although meiosis is evolutionarily conserved, many of the underlying mechanisms show species-specific differences. These are poorly understood in large genome plant species such as barley (Hordeum vulgare) where meiotic recombination is very heavily skewed to the ends of chromosomes. The characterization of mutant lines can help elucidate how recombination is controlled. We used a combination of genetic segregation analysis, cytogenetics, immunocytology and 3D imaging to genetically map and characterize the barley meiotic mutant DESYNAPTIC 10 (des10). We identified a spontaneous exonic deletion in the orthologue of MutL-Homolog 3 (HvMlh3) as the causal lesion. Compared with wild-type, des10 mutants exhibit reduced recombination and fewer chiasmata, resulting in the loss of obligate crossovers and leading to chromosome mis-segregation. Using 3D structured illumination microscopy (3D-SIM), we observed that normal synapsis progression was also disrupted in des10, a phenotype that was not evident with standard confocal microscopy and that has not been reported with Mlh3 knockout mutants in Arabidopsis. Our data provide new insights on the interplay between synapsis and recombination in barley and highlight the need for detailed studies of meiosis in nonmodel species. This study also confirms the importance of early stages of prophase I for the control of recombination in large genome cereals.