To exploit allelic variation in Hordeum vulgare subsp. spontaneum, the Wild Barley Diversity Collection was subjected to paired-end Illumina sequencing at ∼9 × depth and evaluated for several agronomic traits. We discovered 240.2 million single nucleotide polymorphisms (SNPs) after alignment to the Morex V3 assembly and 24.4 million short (1 to 50 bp) insertions and deletions. A genome-wide association study of lemma color identified one marker-trait association (MTA) on chromosome 1H close to HvBlp, the cloned gene controlling black lemma. Four MTAs were identified for seedling stem rust resistance, including 2 novel loci on chromosomes 1H and 6H and one co-locating to the complex RMRL1-RMRL2 locus on 5H. The whole-genome sequence data described herein will facilitate the identification and utilization of new alleles for barley improvement.

Stripe or yellow rust (YR), caused by Puccinia striiformis Westend. f. sp. tritici (Pst), is a devastating fungal disease of wheat. Here, we report the map-based cloning of Yr26 (synonyms: Yr24, YrCH42, and YrG22), a major stripe rust resistance gene, and its validation by gene silencing, mutation analysis and transgenic complementation. Yr26, which firstly arises in emmer wheat and recently has introgressed into bread wheat, is a newly evolved gene encoding a transmembrane protein. AlphaFold v3.0 predictes a C-terminal four-helical bundle domain that is critical for triggering Ca2+-dependent cell death, a hallmark of the hypersensitive response. The cloning of Yr26 offers not only a genetic resource for pyramiding stripe rust resistance genes in wheat breeding but also an opportunity to study host–pathogen co-evolution. Yellow rust (YR) is a devastating fungal disease of wheat. Here, the authors report the cloning of Yr26 as a transmembrane protein and reveal its evolves within the Triticeae lineage as an orphan gene.

Malawi has diverse local banana germplasms that are preferred by its population. However, the epidemics of banana bunchy top disease (BBTD), caused by the banana bunchy top virus (BBTV) is wiping out the preferred germplasms and limiting their cultivation. A survey was conducted to characterize banana germplasm and evaluate the presence, incidence and prevalence of banana viruses. PCR products from infected germplasm were sequenced and aligned for each detected virus to construct a phylogenetic tree. BBTV, banana mild mosaic virus (BanMMV) and six banana streak virus (BSV) species were detected in Malawi. Malawi's BBTV isolates belonged to the Pacific Indian Ocean group, and BanMMV isolates clustered to three sub-branches. The six BSV species detected in Malawi belonged to clade 1. Among the genetic groups of Musa, the characterized banana germplasms belonged to AA, AAA, AAB, and ABB groups with some germplasms being unique compared to those already genotyped. The ABB group was dominant in Malawi and was significantly more often infected by BSV species (possibly originating from endogenous viral sequences), while BBTV and BanMMV infected the AAA and AAB group more frequently, respectively. The primary source of banana planting materials was banana propagule exchange among relatives which posed a higher risk of spreading virus diseases. The survey underlined the importance of establishing a banana seed industry and implementing policies that promote farmers' access to virus-tested planting materials, ultimately helping to prevent future virus epidemics.

Goatgrasses with U- and M-genomes are important sources of new alleles for wheat breeding to maintain yield and quality under extreme conditions. However, the introgression of beneficial traits from wild Aegilops species into wheat has been limited by poor knowledge of their genomes and scarcity of molecular tools. Here, we present the first linkage map of allotetraploid Aegilops biuncialis Vis., developed using 224 F2 individuals derived from a cross between MvGB382 and MvGB642 accessions. The map comprises 5663 DArTseq markers assigned to 15 linkage groups corresponding to 13 chromosomes. Chromosome 1Mb could not be constructed due to a lack of recombination caused by rearrangements in the MvGB382 accession. The genetic map spans 2518 cM with an average marker density of 2.79 cM. The skeleton map contains 920 segregating markers, divided between the Mb sub-genome (425 markers) and the Ub sub-genome (495 markers). Chromosomes of the Mb sub-genome, originating from Aegilops comosa Sm. in Sibth. et Sm., show well-preserved collinearity with Triticum aestivum L. chromosomes. In contrast, chromosomes of the Ub sub-genome, originating from Aegilops umbellulata Zhuk., exhibit a varying degree of collinearity, with 1Ub, 3Ub, and 5Ub retaining a substantial level of collinearity with Triticum aestivum, while 2Ub, 4Ub, 6Ub, and 7Ub show significant rearrangements. A quantitative trait locus affecting fertility was identified near the centromere on the long arm of chromosome 3Mb, explaining 23.5% of the variance. The genome structure of Aegilops biuncialis, highlighted by the genetic map, provides insights into the speciation within the species and will support alien gene transfer into wheat.

Wheat and its close relatives have large and complex genomes, making gene cloning difficult. Nevertheless, developments in genomics over the past decade have made it more feasible. The large and complex genomes of cereals, especially bread wheat, have always been a challenge for gene mapping and cloning. Nevertheless, recent advances in genomics have led to significant progress in this field. Currently, high-quality reference sequences are available for major wheat species and their relatives. New high-throughput genotyping platforms and next-generation sequencing technologies combined with genome complexity reduction techniques and mutagenesis have opened new avenues for gene cloning. In this review, we provide a comprehensive overview of the genes cloned in wheat so far and discuss the strategies used for cloning these genes. We highlight the advantages and drawbacks of individual approaches and show how particular genomic progress contributed to wheat gene cloning. A wide range of new resources and approaches have led to a significant increase in the number of successful cloning projects over the past decade, demonstrating that it is now feasible to perform rapid gene cloning of agronomically important genes, even in a genome as large and complex as that of wheat.

Bread wheat, a global staple food, has seen a significant reduction in genetic diversity due to domestication and intensive breeding. Diploid einkorn wheat (Triticum monococcum L.) is a valuable resource for enhancing the genetic pool of bread wheat. A linkage map spanning 1033 cM was constructed using 81 recombinant inbred lines (RILs) derived from a cross between cultivated Triticum monococcum 'DV92' and wild Triticum boeoticum 'G3116.' The RILs were phenotyped in up to eight environments for 24 agronomically important traits, including plant height, tiller number, plant architecture, leaf pubescence, ear emergence time, and spike- and grain-related traits. QTL analyses revealed 167 quantitative trait loci (QTLs) distributed across all chromosomes, of which 36 QTLs were mapped in intervals of less than 10 cM, and 77 QTLs explained more than 10% of the phenotypic variance for a trait, with a maximum of 56.2% (the drought resistance-related leaf pubescence). Verification using 407 F2 genotypes confirmed that over 30% of the QTLs were highly reproducible. In addition, we have demonstrated that the identified QTLs can be readily characterized by fine-mapping of a novel QTL for leaf pubescence, QLP.ieb-3A, on chromosome 3Am. This study provides valuable insights into loci influencing key agronomic traits, such as yield and resistance to abiotic stress, and is suitable for bread wheat gene pool enrichment. Additionally, the findings facilitate map-based cloning of the underlying genes, paving the way for functional studies and efficient application of the loci in wheat breeding programs.

The wheat-Parastagonospora nodorum pathosystem has emerged as a model system for plant-necrotrophic fungal pathogen interactions. In this system, fungal necrotrophic effectors are recognized by specific host genes in an inverse gene-for-gene manner to induce programmed cell death and other host responses, which leads to disease. We previously cloned a wheat gene (Snn3-D1) encoding protein kinase and major sperm protein domains that recognizes the P. nodorum necrotrophic effector SnTox3. Here, we identified an Snn3-D1 homoeolog (Snn3-B1) and a paralog (Snn3-B2) that also recognize SnTox3, leading to susceptibility. DNA sequence divergence of Snn3-B1 and Snn3-B2 and differences in transcriptional expression patterns and three-dimensional protein conformation were associated with a more severe programmed cell death response conferred by Snn3-B2 compared with Snn3-B1. Both Snn3 proteins were localized to the nucleus and cytoplasm in wheat protoplasts, suggesting that they may have acquired novel functions compared with previously characterized major sperm protein domain-containing proteins in other species. Snn3-B2 was previously shown to govern osmotic stress and salt tolerance, indicating that protein kinase-major sperm protein genes can act in plant defense responses to both biotic and abiotic stresses. Evaluation of a large collection of wheat lines showed that several alleles of each gene, including absent alleles, exist within the germplasm. Diagnostic markers were developed for the absent alleles of both genes, which will prove useful for marker-assisted selection in wheat to eliminate SnTox3 sensitivity and achieve better disease resistance. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2025.

SHORT SUMMARY:This study cloned the wheat powdery mildew resistance gene Pm6 via map-based cloning, which encodes an NLR protein. Through GWAS, its cognate pathogen effector AvrPm6 was characterized, revealing a direct physical interaction that triggers resistance. The findings provide crucial insights into the molecular mechanism of wheat immunity against powdery mildew.

Wild wheat relative Aegilops biuncialis offers valuable traits for crop improvement through interspecific hybridization. However, gene transfer from Aegilops has been hampered by difficulties in detecting introgressed Ub- and Mb-genome chromatin in the wheat background at high resolution. The present study applied DArTseq technology to genotype two backcrossed populations (BC382, BC642) derived from crosses of wheat line Mv9kr1 with Ae. biuncialis accession, MvGB382 (early flowering and drought-tolerant) and MvGB642 (leaf rust-resistant). A total of 11,952 Aegilops-specific Silico-DArT markers and 8,998 wheat-specific markers were identified. Of these, 7,686 markers were assigned to Ub-genome chromosomes and 4,266 to Mb-genome chromosomes and were ordered using chromosome scale reference assemblies of hexaploid wheat and Ae. umbellulata. Ub-genome chromatin was detected in 5.7

Abstract The challenge of in-situ handling and high-resolution, low-dose imaging of intact, sensitive and moist samples in their native state at the nanometer scale or live samples is met by Advanced Environmental Scanning Electron Microscopy (A-ESEM). This new generation of ESEM uses machine learning-based optimization of thermodynamic conditions with respect to sample specifics to employ a low temperature method and an ionization secondary electron detector with an electrostatic separator in an in-house modified electron microscope equipped with temperature, humidity and gas pressure sensors for in-situ and real-time monitoring of the sample. An invisible thin film of ionic liquid is used to increase the thermal and electrical conductivity of the samples and minimize damage from free radicals. To validate the power of the new method, we analyze condensed mitotic metaphase chromosomes to reveal new structural features of their perichromosomal layer, and the organization of chromatin fibers, not directly observed before by any microscopic technique.

Some species of the genus Aegilops, a wild relative of wheat, carry chromosomes that after introducing to wheat exhibit preferential transmission to progeny. Their selective retention is a result of the abortion of gametes lacking them due to induced chromosomal aberrations. These chromosomes are termed Gametocidal (Gc) and, based on their effects, they are categorized into three types: mild, intense or severe, and very strong. Gc elements within the same homoeologous chromosome groups of Aegilops (II, III, or IV) demonstrate similar Gc action. This review explores the intriguing dynamics of Gc chromosomes and encompasses comprehensive insights into their source species, behavioral aspects, mode of action, interactions, suppressions, and practical applications of the Gc system in wheat breeding. By delving into these areas, this work aims to contribute to the development of novel plant genetic resources for wheat breeding. The insights provided herein shed light on the utilization of Gc chromosomes to produce chromosomal rearrangements in wheat and its wild relatives, thereby facilitating the generation of chromosome deletions, translocations, and telosomic lines. The Gc approach has significantly advanced various aspects of wheat genetics, including the introgression of novel genes and alleles, molecular markers and gene mapping, and the exploration of homoeologous relationships within Triticeae species. The mystery lies in why gametes possessing Gc genes maintain their normality while those lacking Gc genes suffer abnormalities, highlighting an unresolved research gap necessitating deeper investigation.

The challenge of in-situ handling and high-resolution low-dose imaging of intact, sensitive and wet samples in their native state at nanometer scale, including live samples is met by Advanced Environmental Scanning Electron Microscopy (A-ESEM). This new generation of ESEM utilises machine learning-based optimization of thermodynamic conditions with respect to sample specifics to employ a low temperature method and an ionization secondary electron detector with an electrostatic separator. A modified electron microscope was used, equipped with temperature, humidity and gas pressure sensors for in-situ and real-time monitoring of the sample. A transparent ultra-thin film of ionic liquid is used to increase thermal and electrical conductivity of the samples and to minimize sample damage by free radicals. To validate the power of the new method, we analyze condensed mitotic metaphase chromosomes to reveal new structural features of their perichromosomal layer, and the organization of chromatin fibers, not observed before by any microscopic technique. The ability to resolve nano-structural details of chromosomes using A-ESEM is validated by measuring gold nanoparticles with achievable resolution in the lower nanometre units.

DNA damage response (DDR) is an essential mechanism by which living organisms maintain their genomic stability. In plants, DDR is important also for normal growth and yield. Here, we explored the DDR of a temperate model crop barley (Hordeum vulgare) at the phenotypic, physiological, and transcriptomic levels. By a series of in vitro DNA damage assays using the DNA strand break (DNA-SB) inducing agent zeocin, we showed reduced root growth and expansion of the differentiated zone to the root tip. Genome-wide transcriptional profiling of barley wild-type and plants mutated in DDR signaling kinase ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED (hvatr.g) revealed zeocin-dependent, ATR-dependent, and zeocin-dependent/ATR-independent transcriptional responses. Transcriptional changes were scored also using the newly developed catalog of 421 barley DDR genes with the phylogenetically-resolved relationships of barley SUPRESSOR OF GAMMA 1 (SOG1) and SOG1-LIKE (SGL) genes. Zeocin caused up-regulation of specific DDR factors and down-regulation of cell cycle and histone genes, mostly in an ATR-independent manner. The ATR dependency was obvious for some factors associated with DDR during DNA replication and for many genes without an obvious connection to DDR. This provided molecular insight into the response to DNA-SB induction in the large and complex barley genome.

Tiller number is a key component of wheat plant architecture having a direct impact on grain yield. Because of their viability, biotic resistance, and abiotic stress tolerance, wild relative species are a valuable gene source for increasing wheat genetic diversity, including yield potential. Agropyron glael , a perennial hybrid of Thinopyrum intermedium and Th. ponticum , was created in the 1930s. Recent genome analyses identified five evolutionarily distinct subgenomes (J, J st , J vs , J r , and St), making A. glael an important gene source for transferring useful agronomical traits into wheat. During a bread wheat × A. glael crossing program, a genetically stable translocation line, WT153397, was developed. Sequential in situ hybridizations (McGISH) with J-, St-, and D-genomic DNA probes and pSc119.2, Afa family, pTa71, and (GAA) 7 DNA repeats, as well as molecular markers specific for the wheat 6D chromosome, revealed the presence of a 6DS.6J vs Robertsonian translocation in the genetic line. Field trials in low-input and high-input breeding nurseries over four growing seasons demonstrated the Agropyron chromosome arm’s high compensating ability for the missing 6DL, as spike morphology and fertility of WT153397 did not differ significantly from those of wheat parents, Mv9kr1 and ‘Mv Karizma.’ Moreover, the introgressed 6J vs chromosome arm significantly increased the number of productive tillers, resulting in a significantly higher grain yield potential compared to the parental wheat cultivars. The translocated chromosome could be highly purified by flow cytometric sorting due to the intense fluorescent labeling of (GAA) 7 clusters on the Thinopyrum chromosome arm, providing an opportunity to use chromosome genomics to identify Agropyron gene variant(s) responsible for the tillering capacity. The translocation line WT153397 is an important genetic stock for functional genetic studies of tiller formation and useful breeding material for increasing wheat yield potential. The study also discusses the use of the translocation line in wheat breeding.

Aegilops species, wild relatives of wheat, offer valuable traits for wheat improvement through interspecific hybridization. Despite many Ae. biuncialis accessions maintained in gene banks, only a few have been utilized in wheat breeding. Gene transfer from Aegilops has been hampered by difficulties in detecting introgressed Ub- and Mb-genome chromatin at high resolution in the wheat background. The present study applied DArTseq technology, to genotype two backcrossed populations (BC382, BC642) derived from crosses of a wheat line Mv9kr1 with Ae. biuncialis accession, MvGB382 (earlyflowering and drought tolerant) and MvGB642 (leaf rust-resistant). 11,952 Aegilops specific Silico-DArT markers were obtained, while 8998 markers were specific to wheat. Using the results obtained on diploid progenitors and wheat-Aegilops addition lines, 7686 markers were assigned to the Ub-genome chromosomes and 4266 for Mb-genome chromosomes and ordered by the use of chromosome assemblies of hexaploid wheat and Ae. umbellulata. In the BC3 populations, 5.7% of BC382 and 22.7% of BC642 lines contained Ub-genome chromatin, while Mb-genome chromatin was detected in 88.5% of BC382 and 84% of BC642 lines. Notably, chromosomes 4Mb and 5Mb were frequently present in both populations, whereas chromosomes 3Mb and 6Mb were rarer. A Mv9kr1-Ae. biuncialis MvGB382 4Mb and 5Mb disomic addition lines were identified together with a new 5DS.5DL-5MbL recombination. Moreover, previously developed Mv9kr1-MvGB642 introgression lines were identified more precisely by DArTseq. A possible effect of the 5MbL distal region on seed length has been also observed and discussed. The new cytogenetic stocks represent valuable genetic resources for wheat improvement.

Cultivated and wild species of the genus rye (Secale) are important but underexploited gene sources for increasing the genetic diversity of bread wheat. Gene transfer is possible via bridge genetic materials derived from intergeneric hybrids. During this process, it is essential to precisely identify the rye chromatin in the wheat genetic background. In the present study, backcross generation BC2F8 from a cross between Triticum aestivum (Mv9kr1) and S. cereanum ('Kriszta,' a cultivar from the artificial hybrid of S. cereale and S. strictum) was screened using in-situ hybridization (GISH and FISH) and analyzed by DArTseq genotyping in order to select potentially agronomically useful genotypes for prebreeding purposes. Of the 329,267 high-quality short sequence reads generated, 27,822 SilicoDArT and 8,842 SNP markers specific to S. cereanum 1R-7R chromosomes were identified. Heatmaps of the marker densities along the 'Lo7' rye reference pseudomolecules revealed subtle differences between the FISH- and DArTseq-based results. This study demonstrates that the "exotic" rye chromatin of S. cereanum introgressed into wheat can be reliably identified by high-throughput DArTseq genotyping. The Mv9kr1-'Kriszta' addition and translocation lines presented here may serve as valuable prebreeding genetic materials for the development of stress-tolerant or disease-resistant wheat varieties.

Nucleotide-binding leucine-rich repeat (NLR) disease resistance genes typically confer resistance against races of a single pathogen. Here, we report that Yr87/Lr85, an NLR gene from Aegilops sharonensis and Aegilops longissima, confers resistance against both P. striiformis tritici (Pst) and Puccinia triticina (Pt) that cause stripe and leaf rust, respectively. Yr87/Lr85 confers resistance against Pst and Pt in wheat introgression as well as transgenic lines. Comparative analysis of Yr87/Lr85 and the cloned Triticeae NLR disease resistance genes shows that Yr87/Lr85 contains two distinct LRR domains and that the gene is only found in Ae. sharonensis and Ae. longissima. Allele mining and phylogenetic analysis indicate multiple events of Yr87/Lr85 gene flow between the two species and presence/absence variation explaining the majority of resistance to wheat leaf rust in both species. The confinement of Yr87/Lr85 to Ae. sharonensis and Ae. longissima and the resistance in wheat against Pst and Pt highlight the potential of these species as valuable sources of disease resistance genes for wheat improvement. Leaf rust and stripe rust of wheat are two important fungal diseases of cultivated wheat and they are caused by infection of different pathogens. Here, the authors report the nucleotide-binding leucine-rich repeat (NLR) protein encoding gene Yr87/Lr85 confers resistance to both diseases.

Powdery mildew poses a significant threat to wheat crops worldwide, emphasizing the need for durable disease control strategies. The wheat- Dasypyrum villosum T5AL·5 V#4 S and T5DL·5 V#4 S translocation lines carrying powdery mildew resistant gene Pm55 shows developmental-stage and tissue-specific resistance, whereas T5DL·5 V#5 S line carrying Pm5V confers resistance at all stages. Here, we clone Pm55 and Pm5V , and reveal that they are allelic and renamed as Pm55a and Pm55b , respectively. The two Pm55 alleles encode coiled-coil, nucleotide-binding site-leucine-rich repeat (CNL) proteins, conferring broad-spectrum resistance to powdery mildew. However, they interact differently with a linked inhibitor gene, SuPm55 to cause different resistance to wheat powdery mildew. Notably, Pm55 and SuPm55 encode unrelated CNL proteins, and the inactivation of SuPm55 significantly reduces plant fitness. Combining SuPm55 / Pm55a and Pm55b in wheat does not result in allele suppression or yield penalty. Our results provide not only insights into the suppression of resistance in wheat, but also a strategy for breeding durable resistance.

SUMMARY Restoring cytonuclear stoichiometry is necessary after whole‐genome duplication (WGD) and interspecific/intergeneric hybridization in plants. We investigated this phenomenon in auto‐ and allopolyploids of the Festuca‐Lolium complex providing insights into the mechanisms governing cytonuclear interactions in early polyploid and hybrid generations. Our study examined the main processes potentially involved in restoring the cytonuclear balance after WGD comparing diploids and new and well‐established autopolyploids. We uncovered that both the number of chloroplasts and the number of chloroplast genome copies were significantly higher in the newly established autopolyploids and grew further in more established autopolyploids. The increase in the copy number of the chloroplast genome exceeded the rise in the number of chloroplasts and fully compensated for the doubling of the nuclear genome. In addition, changes in nuclear and organelle gene expression were insignificant. Allopolyploid Festuca × Lolium hybrids displayed potential structural conflicts in parental protein variants within the cytonuclear complexes. While biased maternal allele expression has been observed in numerous hybrids, our results suggest that its role in cytonuclear stabilization in the Festuca × Lolium hybrids is limited. This study provides insights into the restoration of the cytonuclear stoichiometry, yet it emphasizes the need for future research to explore post‐transcriptional regulation and its impact on cytonuclear gene expression stoichiometry. Our findings may enhance the understanding of polyploid plant evolution, with broader implications for the study of cytonuclear interactions in diverse biological contexts.

The barley Mla locus contains functionally diversified genes that encode intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) and confer strain-specific immunity to biotrophic and hemibiotrophic fungal pathogens. In this study, we isolated a barley gene Scs6, which is an allelic variant of Mla genes but confers susceptibility to the isolate ND90Pr (BsND90Pr) of the necrotrophic fungus Bipolaris sorokiniana. We generated Scs6 transgenic barley lines and showed that Scs6 is sufficient to confer susceptibility to BsND90Pr in barley genotypes naturally lacking the receptor. The Scs6-encoded NLR (SCS6) is activated by a nonribosomal peptide (NRP) effector produced by BsND90Pr to induce cell death in barley and Nicotiana benthamiana. Domain swaps between MLAs and SCS6 reveal that the SCS6 leucine-rich repeat domain is a specificity determinant for receptor activation by the NRP effector. Scs6 is maintained in both wild and domesticated barley populations. Our phylogenetic analysis suggests that Scs6 is a Hordeum-specific innovation. We infer that SCS6 is a bona fide immune receptor that is likely directly activated by the nonribosomal peptide effector of BsND90Pr for disease susceptibility in barley. Our study provides a stepping stone for the future development of synthetic NLR receptors in crops that are less vulnerable to modification by necrotrophic pathogens.