Shortening the time taken to release a new crop variety could improve the responsiveness of the plant breeding sector. To gain Plant Breeders’ Rights, and for certain marketing requirements, candidate varieties must be distinct from all known varieties in common knowledge (the variety collection) through assessment against Distinctness, Uniformity and Stability (DUS) criteria. The assessment uses field comparisons of morphological DUS characteristics between the candidate and its most similar varieties from the variety collection. Similar varieties cannot be identified immediately as candidate varieties are received without characteristic data. Here, we investigated if genomic prediction could facilitate most similar variety selection. A barley training population was assembled with 1,171 genotypes, covering ~70% of the UK barley variety collection at the time of study. Genotyping was undertaken to obtain 36,736 genetic markers and phenotypes were compiled for 28 DUS characteristics. Genomic prediction was completed using Random Forest (RF) for classification and Ridge Regression Best Linear Unbiased Prediction (rr-BLUP). RF typically showed the highest proportion of correctly classified calls and marginally higher prediction accuracies for characteristics with high heritability and fewer characteristic notes. Prediction accuracies varied more between methods for characteristics with lower heritability and more notes, where rr-BLUP performed best. Weighting mapped genetic loci in the RF prediction improved prediction accuracies. Comparisons made with historic data showed strong overlap between the most similar varieties identified via predicted or observed characteristics, indicating distinctness comparisons could begin a year earlier. Therefore, the combination of biomolecular technology and genomic prediction could enable faster distinctness assessment.

Using a positional candidate-gene approach we show that semi-sterile desynaptic8 mutants are associated with deleterious variants of the barley homolog of XRCC2 (X-Ray Repair Cross Complementing 2). In barley XRCC2 mutants, the initial meiotic progression is normal, albeit with a small delay in initiation, with completion of synapsis. However, the absence of HvXRCC2 subsequently leads to a dramatic reduction in the number of crossovers, chromosome mis-segregation, and infertility, suggesting that HvXRCC2 plays a major role in recombination. This mutant phenotype is congruent with that reported in mammalian studies but contrasts with the XRCC2 mutant in Arabidopsis which is fertile, exhibits normal chromosome pairing and correct chromosome segregation, and is associated with an increased rate of crossovers. This indicates that the XRCC2 mutant phenotype in Arabidopsis is not representative of all plants and that XRCC2 is not a good candidate for the modulation of recombination in barley.

Land plants secrete a protective outer cuticular layer with diverse functions. Barley (Hordeum vulgare L.) develops two cuticular specialisations: the β-diketone rich wax bloom on vegetative tissues and an adherent grain surface which sticks to the hulls, leading to barley's distinctive 'covered' grain phenotype. Two barley SHINE transcription factors, HvWIN1 and NUD, promote the wax bloom and covered grain phenotypes, respectively, yet we know little about other genes involved. We investigated a barley mutant showing defects in both cuticular specialisations to better understand the networks and processes underlying these traits. We identified the barley BODYGUARD1 (HvBDG1) gene encoding an α/β hydrolase as crucial for leaf cuticular integrity and wax-bloom deposition. Mining tetraploid wheat (Triticum turgidum ssp. durum) mutant populations demonstrated that BDG1 and WIN1 orthologues also control the wax bloom in wheat. We further reveal that NUD and HvWIN1 retain functional, independent overlap in leaf cuticle integrity and hull adhesion in barley, functions which involve upregulation of HvBDG1 and other direct or indirect targets, shared and distinct, depending on the stage and tissue. Our work greatly expands our knowledge of genetic and developmental mechanisms underlying shared and distinctive cuticular features in plants.

A dense genome-wide meta-analysis provides new QTLs, reveals breeding history trends and identifies new candidate genes for yield, plant height, grain weight, and heading time of spring barley. This study contributes new knowledge on quantitative trait loci (QTLs) and candidate genes for adaptive traits and yield in two-rowed spring barley. A meta-analysis of a network of field trials, varying in latitude and sowing date, with 151 cultivars across several European countries, increased QTL detection power compared to single-trial analyses. The traits analysed were heading date (HD), plant height (PH), thousand-grain weight (TGW), and grain yield (GY). Breaking down the analysis by the main genotype-by-environment trends revealed QTLs and candidate genes specific to conditions like sowing date and latitude. A historical look on the evolution of QTL frequencies revealed that early selection focused on PH and TGW, likely due to their high heritability. GY selection occurred later, facilitated by reduced variance in other traits. The study observed that favourable alleles for plant height were often fixed before those for grain yield and TGW. Some regions showed linkage in repulsion, suggesting targets for future breeding. Several candidate genes were identified, including known genes and new candidates based on orthology with rice. Remarkably, the deficiens allele of gene Vrs1 appears associated with higher GY. These findings provide valuable insights for barley breeders aiming to improve yield and other agronomic traits.

This study characterizes the function of a grass-specific E3 ubiquitin ligase, HvST1, in regulating synapsis and crossover (CO) formation during meiosis in barley (Hordeum vulgare). In large-genome cereals, COs are predominantly restricted to distal chromosomal regions, limiting genetic recombination and breeding flexibility. We aimed to identify genetic components regulating CO frequency and distribution. A frameshift mutation in HvST1 was identified in the semi-sterile barley near-isogenic line BW233 through fine mapping of the des12.w locus. The causal role of this mutation was validated via CRISPR/Cas9 gene editing. HvST1 function was investigated using in vitro autoubiquitination and substrate ubiquitination assays, while meiotic progression was assessed using structured illumination microscopy and immunolocalization of key axis and synaptonemal complex (SC) proteins. Loss of HvST1 function led to disrupted SC formation, persistent ASY1 signal, and aberrant ZYP1 polycomplex formation. Despite impaired synapsis, Hvst1 mutants showed a significant increase in distal CO frequency. HvST1 was shown to ubiquitinate ASY1 in vitro, linking its activity to protein turnover at the chromosome axis. HvST1 is critical for normal synapsis and CO regulation during meiosis in barley. Its loss disrupts SC progression but enhances distal CO formation, revealing a previously uncharacterized ubiquitination-based mechanism modulating recombination in grasses.

The outer epidermis of land plants secretes a cuticular layer, a hydrophobic diffusion barrier which minimises water loss into the atmosphere and protects from pests, ultraviolet light and organ fusion. Cuticles typically comprise a polyester cutin matrix embedded and overlaid with cuticular waxes, but their exact chemical make-up, structure and functions can vary widely depending on the tissue and species. Barley shows two such cuticular specialisations: (1) deposition of a thick β-diketone-rich wax bloom on multiple organs at reproductive stage, common in other Poeceae species and linked to yield; and, (2) secretion of a sticky layer on the grain fruit (caryopsis) pericarp cuticle which adheres to inner floral hulls, leading to barley’s distinctive ‘covered’ grain used in animal feed and malting. Two SHINE/WAX-INDUCER transcription factors in barley, HvWIN1 and NUD, promote the wax bloom and hull to caryopsis adhesion, respectively, yet little is understood about other genes involved. Leveraging near-isogenic lines of wax-deficient mutants, we identify the barley BODYGUARD1 ( HvBDG1 ) gene encoding an α/β-hydrolase essential for leaf cuticular integrity and wax bloom deposition. Modelling of functional and defective alleles suggests that HvBDG1 N-terminal region control of protein flexibility is important for HvBDG1 function. In addition to their role in controlling barley epicuticular wax deposition, we show that both HvBDG1 and HvWIN1 are essential for strong hull to caryopsis adhesion. Along with NUD, these gene products differentially contribute to ultrastructural changes on the pericarp associated with a cuticular building programme driven by NUD and HvWIN1 regulation of cuticle metabolism and transport and cell wall-related genes, and correlate with shifts in pericarp surface chemistry. We also show that the previously asserted ‘grain-specific’ role of NUD should be revised, as our findings reveal that it is essential for maintaining leaf cuticle integrity. Our analyses in barley suggest that NUD and HvWIN1 control cuticular specialisations and cuticle integrity in part via promotion of HvBDG1 expression, while HvWIN1 and NUD likely act independently from each other. Lastly, mining tetraploid wheat mutant populations followed by crossing to combine mutated homoeologues demonstrated that BDG1 and WIN1 orthologues also control wax bloom in wheat. Taken together, our work greatly expands the genetic networks and molecular activities important for cuticle development in cereals and the underlying mechanisms for both shared and species-specific cuticular specialisations. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/R010315/1, BB/R010315/1, BB/X018725/1, BB/S019669/1, BB/X019683/1, BBS/E/JI/23NB0001 European Research Council, https://ror.org/0472cxd90, 101000997 Rural and Environment Science and Analytical Services Division, Scottish Government Carnegie UK Trust Council for At-Risk Academics, https://ror.org/04fgrzc24

This study contributes new knowledge on quantitative trait loci (QTLs) and candidate genes for adaptive traits and yield in two-rowed spring barley. A meta-analysis of a network of field trials, varying in latitude and sowing date, with 151 cultivars across several European countries, increased QTL detection power compared to single-trial analyses. The traits analysed were heading date (HD), plant height (PH), thousand-grain weight (TGW), and grain yield (GY). Breaking down the analysis by the main genotype-by-environment trends revealed QTLs and candidate genes specific to conditions like sowing date and latitude. A historical look on the evolution of QTL frequencies revealed that early selection focused on PH and TGW, likely due to their high heritability. GY selection occurred later, facilitated by reduced variance in other traits. The study observed that favourable alleles for plant height were often fixed before those for grain yield and TGW. Some regions showed linkage in repulsion, suggesting targets for future breeding. Several candidate genes were identified, including known genes and new candidates based on orthology with rice. Remarkably, the deficiens allele of gene Vrs1 , appears associated to higher GY. These findings provide valuable insights for barley breeders aiming to improve yield, and other agronomic traits. Highlight A dense genome-wide meta-analysis provides new QTLs, reveals breeding history trends and identifies new candidate genes for yield, plant height, grain weight and heading time of spring barley. ### Competing Interest Statement The authors have declared no competing interest.

Pangenomes are collections of annotated genome sequences of multiple individuals of a species. The structural variants uncovered by these datasets are a major asset to genetic analysis in crop plants. Here, we report a pangenome of barley comprising long-read sequence assemblies of 76 wild and domesticated genomes and short-read sequence data of 1,315 genotypes. An expanded catalogue of sequence variation in the crop includes structurally complex loci that have become hot spots of gene copy number variation in evolutionarily recent times. To demonstrate the utility of the pangenome, we focus on four loci involved in disease resistance, plant architecture, nutrient release, and trichome development. Novel allelic variation at a powdery mildew resistance locus and population-specific copy number gains in a regulator of vegetative branching were found. Expansion of a family of starch-cleaving enzymes in elite malting barleys was linked to shifts in enzymatic activity in micro-malting trials. Deletion of an enhancer motif is likely to change the developmental trajectory of the hairy appendages on barley grains. Our findings indicate that rapid evolution at structurally complex loci may have helped crop plants adapt to new selective regimes in agricultural ecosystems.

In the last century, breeding programs have traditionally favoured yield-related traits, grown under high-input conditions, resulting in a loss of genetic diversity and an increased susceptibility to stresses in crops. Thus, exploiting understudied genetic resources, that potentially harbour tolerance genes, is vital for sustainable agriculture. Northern European barley germplasm has been relatively understudied despite its key role within the malting industry. The European Heritage Barley collection (ExHIBiT) was assembled to explore the genetic diversity in European barley focusing on Northern European accessions and further address environmental pressures. ExHIBiT consists of 363 spring-barley accessions, focusing on two-row type. The collection consists of landraces (~14%), old cultivars (~18%), elite cultivars (~67%) and accessions with unknown breeding history (~1%), with 70% of the collection from Northern Europe. The population structure of the ExHIBiT collection was subdivided into three main clusters primarily based on the accession’s year of release using 26,585 informative SNPs based on 50k iSelect single nucleotide polymorphism (SNP) array data. Power analysis established a representative core collection of 230 genotypically and phenotypically diverse accessions. The effectiveness of this core collection for conducting statistical and association analysis was explored by undertaking genome-wide association studies (GWAS) using 24,876 SNPs for nine phenotypic traits, four of which were associated with SNPs. Genomic regions overlapping with previously characterised flowering genes (HvZTLb) were identified, demonstrating the utility of the ExHIBiT core collection for locating genetic regions that determine important traits. Overall, the ExHIBiT core collection represents the high level of untapped diversity within Northern European barley, providing a powerful resource for researchers and breeders to address future climate scenarios.

The cuticle covering aerial organs of land plants is well known to protect against desiccation. Cuticles also play diverse and specialized functions, including organ separation, depending on plant and tissue. Barley shows a distinctive cuticular wax bloom enriched in beta-diketones on leaf sheaths, stem nodes and internodes and inflorescences. Barley also develops a sticky surface on the outer pericarp layer of its grain fruit leading to strongly adhered hulls, 'covered grain', important for embryo protection and seed dispersal. While the transcription factor-encoding gene HvNUDUM (HvNUD) appears essential for adherent hulls, little is understood about how the pericarp cuticle changes during adhesion or whether changes in pericarp cuticles contribute to another phenotype where hulls partially shed, called 'skinning'. To that end, we screened barley lines for hull adhesion defects, focussing on the Eceriferum (= waxless, cer) mutants. Here, we show that the cer-xd allele causes defective wax blooms and compromised hull adhesion, and results from a mutation removing the last 10 amino acids of the GDS(L) [Gly, Asp, Ser, (Leu)]-motif esterase/lipase HvGDSL1. We used severe and moderate HvGDSL1 alleles to show that complete HvGDSL1 function is essential for leaf blade cuticular integrity, wax bloom deposition over inflorescences and leaf sheaths and pericarp cuticular ridge formation. Expression data suggest that HvGDSL1 may regulate hull adhesion independently of HvNUD. We found high conservation of HvGDSL1 among barley germplasm, so variation in HvGDSL1 unlikely leads to grain skinning in cultivated barley. Taken together, we reveal a single locus which controls adaptive cuticular properties across different organs in barley.

Pangenomes are collections of annotated genome sequences of multiple individuals of a species1. The structural variants uncovered by these datasets are a major asset to genetic analysis in crop plants2. Here we report a pangenome of barley comprising long-read sequence assemblies of 76 wild and domesticated genomes and short-read sequence data of 1,315 genotypes. An expanded catalogue of sequence variation in the crop includes structurally complex loci that are rich in gene copy number variation. To demonstrate the utility of the pangenome, we focus on four loci involved in disease resistance, plant architecture, nutrient release and trichome development. Novel allelic variation at a powdery mildew resistance locus and population-specific copy number gains in a regulator of vegetative branching were found. Expansion of a family of starch-cleaving enzymes in elite malting barleys was linked to shifts in enzymatic activity in micro-malting trials. Deletion of an enhancer motif is likely to change the developmental trajectory of the hairy appendages on barley grains. Our findings indicate that allelic diversity at structurally complex loci may have helped crop plants to adapt to new selective regimes in agricultural ecosystems.

AbstractDuring meiosis, genetic recombination occurs via repair of DNA double-strand breaks (DSBs) as crossovers (COs) resulting in the exchange of parental genetic material (1). Crossovers are important for chromosome segregation and shuffling genetic variation, but their number and distribution are tightly regulated (2). In barley and other large genome cereals, recombination events are limited in number and mainly restricted to the ends of chromosomes (3), constraining progress in plant breeding. Recent studies have highlighted subtle differences in meiotic progression (4, 5) and the distribution of recombination events in barley compared to other plants (6-8), indicating possible evolutionary divergence of the meiotic program in large genome crops. Here we identify a spontaneous loss of function mutation in the grass specific E3 ubiquitin ligaseHvST1(Sticky Telomeres 1) which results in semi-sterility in barley. We show that abnormal synapsis in the absence of HvST1 function increases overall recombination by up to 2.5-fold and that HvST1 is capable of ubiquitinating ASY1, a key component of the lateral elements of the synaptonemal complex. Our findings shed light on a novel—and evolutionarily divergent—pathway regulating synapsis and recombination in cereals. This natural loss of function variant presents new opportunities for the modulation of recombination in large genome cereals.Significance StatementClimate change places significant strain on crop production. Crop secondary gene pools offer an excellent resource for crop improvement. However, linkage drag driven by restrictions to meiotic recombination can impose severe yield or quality penalties from introgression of traits from secondary gene pools to elite varieties. Here, we characterize a spontaneous mutation in the barley E3 ubiquitinHvST1that leads to a significant increase in recombination. Through biochemical analysis of the wild type protein we identified a putative role for this ligase in regulating synapsis. This furthers our understanding of the control of synapsis in large genome cereals and may be of direct use in traditional barley breeding.

Using a positional candidate-gene approach we show that semi-sterile desynaptic8 mutants are associated with deletions in or complete knockout of the barley homolog of XRCC2 ( X-Ray Repair Cross Complementing 2 ). In barley XRCC2 mutants, the initial meiotic progression is normal, albeit with a small delay in initiation, with completion of synapsis. However, the absence of HvXRCC2 subsequently leads to a dramatic reduction in the number of crossovers, chromosome mis-segregation, and infertility, suggesting that HvXRCC2 plays a major role in recombination. This mutant phenotype is congruent with that reported in mammalian studies but contrasts with the XRCC2 mutant in Arabidopsis which is fertile, exhibits normal chromosome pairing and correct chromosome segregation, and is associated with an increased rate of crossovers. This indicates that the XRCC2 mutant phenotype in Arabidopsis is not representative of all plants and that XRCC2 is not a good candidate for the modulation of recombination in barley. Highlight The mutants of the barley homolog of XRCC2 exhibit delays in replication leading to defective meiosis, altered RAD51 orthologue behaviour, and significant reduction in the number of crossovers as in canonical mammalian XRCC2 mutants but unlike those in Arabidopsis.

We report a novel approach for establishing the number and position of CO events in individual homozygous inbred plants by combining low level EMS mutagenesis, speed breeding, whole genome shotgun sequencing and sliding window analysis of the induced molecular variant data. We demonstrate the approach by exploring CO frequency and distribution in self-fertilised progeny of the inbred barley cultivar Bowman and compare these observations to similar data obtained from a Bowman nearly isogenic line (BW230 Hvmlh3) containing a mutation in the DNA mismatch repair gene HvMLH3. We have previously shown that Hvmlh3 decreases both plant fertility and recombination by ~50%. We compare our results to those from previously published traditional genetic analysis of F3 families derived from multiple F2 lines containing WT or mutant alleles of HvMLH3, revealing a high level of correspondence between analyses. We discuss possible applications of the approach in streamlining the assessment of recombination in plant meiosis research.

Faced with terrestrial threats, land plants seal their aerial surfaces with a lipid-rich cuticle. To breathe, plants interrupt their cuticles with adjustable epidermal pores, called stomata, that regulate gas exchange, and develop other specialised epidermal cells such as defensive hairs. Mechanisms coordinating epidermal features remain poorly understood. Addressing this, we studied two loci whose allelic variation causes both cuticular wax-deficiency and misarranged stomata in barley, identifying the underlying genes, Cer-g / HvYDA1 , encoding a YODA-like (YDA) MAPKKK, and Cer-s / HvBRX-Solo , encoding a single BREVIS-RADIX (BRX) domain protein. Both genes control cuticular integrity, the spacing and identity of epidermal cells, and barley’s distinctive epicuticular wax blooms, as well as stomatal patterning in elevated CO 2 conditions. Genetic analyses revealed epistatic and modifying relationships between HvYDA1 and HvBRX-Solo , intimating that their products participate in interacting pathway(s) linking epidermal patterning with cuticular properties in barley. This may represent a mechanism for coordinating multiple adaptive features of the land plant epidermis in a cultivated cereal.

Flowering plants reproduce sexually by combining a haploid male and female gametophyte during fertilization. Male gametophytes are localized in the anthers, each containing reproductive (meiocyte) and non-reproductive tissue necessary for anther development and maturation. Meiosis, where chromosomes pair and exchange their genetic material during a process called recombination, is one of the most important and sensitive stages in breeding, ensuring genetic diversity. Most anther development studies have focused on transcript variation, but very few have been correlated with protein abundance. Taking advantage of a recently published barley anther transcriptomic (BAnTr) dataset and a newly developed sensitive mass spectrometry-based approach to analyse the barley anther proteome, we conducted high-resolution mass spectrometry analysis of barley anthers, collected at six time points and representing their development from pre-meiosis to metaphase. Each time point was carefully staged using immunocytology, providing a robust and accurate staging mirroring our previous BAnTr dataset. We identified >6100 non-redundant proteins including 82 known and putative meiotic proteins. Although the protein abundance was relatively stable throughout prophase I, we were able to quantify the dynamic variation of 336 proteins. We present the first quantitative comparative proteomics study of barley anther development during meiotic prophase I when the important process of homologous recombination is taking place.

Better understanding of the mechanistic basis of plant plasticity will enhance efforts to breed crops resilient to predicted climate change. However, complexity in plasticity's conceptualisation and measurement may hinder fruitful crossover of concepts between disciplines that would enable such advances. We argue active adaptive plasticity is particularly important in shaping the fitness of wild plants, representing the first line of a plant's defence to environmental change. Here, we define how this concept may be applied to crop breeding, suggest appropriate approaches to measure it in crops, and propose a refocussing on active adaptive plasticity to enhance crop resilience. We also discuss how the same concept may have wider utility, such as in ex situ plant conservation and reintroductions.

Plasticity is a widely used concept in plant sciences, but there is inconsistency over its interpretation and measurement. One aspect of plasticity – adaptive plasticity – may be particularly important in shaping plant fitness and reproductive success and represents the first line of a plants defence to environmental change. Here, we define adaptive plasticity, highlight its importance to plant growth and survival, and suggest appropriate approaches for its measurement. We argue that a focus on adaptive plasticity could help address some fundamental challenges in plant ecology and evolutionary biology, including developing insight into climate-change resilience of natural populations and crops.