Abstract Background Barley ( Hordeum vulgare L.) provides a suitable model for studying domestication-driven plant-microbiome interactions. Although wild, landrace, and modern genotypes host distinct rhizosphere communities, the extent to which roots and microbes reciprocally influence each other remains unclear. Here, we applied an integrated multi-omics approach combining long-read metagenomics, root transcriptomics, and plant genomics to understand genotype-specific host-microbiome coordination. Results Oxford Nanopore whole metagenome sequencing (WMS) revealed genotype-associated shifts in rhizosphere communities across seasons. Functional profiling showed a conserved metabolic backbone including amino acid metabolism, energy production, and secondary metabolite biosynthesis, alongside genotype-dependent variation in carbohydrate metabolism and transport-associated pathways. Genome-resolved analysis through metagenome-assembled genomes (MAGs) further detailed the taxonomic and functional architecture of key rhizosphere lineages. Root transcriptome profiling identified extensive differential expression associated with microbial perception, signaling, defense, and metabolic processes. Integration of host and microbiome data revealed coordinated molecular patterns, indicating that barley genotypes are associated with distinct microbial assemblages and corresponding transcriptional responses. Conclusions These findings indicate that domestication has shaped coordinated associations between barley genotypes and their rhizosphere microbiomes, reflected in both microbial community composition and host transcriptional regulation. This work provides new insights into the evolutionary tuning of plant-microbiome relationships and highlights opportunities for microbiome-informed strategies in barley improvement.

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.

Abstract Background Melissa officinalis (balm) is a perennial medicinal. Climate change and high cultivation costs necessitate the breeding of new cultivars with improved stress tolerance and high metabolite content. However, the high costs of phenotyping large collections limit breeding progress. This study characterized the genetic diversity and population structure of 256 genotypes from 215 international accessions using flow cytometry and genotyping by sequencing (GBS). The primary objective was to identify untapped genetic resources and establish core sets to streamline future breeding and research efforts. Results Morphological characterization and flow cytometry revealed a clear division by ploidy and subspecies. The collection comprised 209 diploid and three polyploid genotypes of ssp. officinalis (2C = 1.79 pg) and 44 tetraploid genotypes of ssp. altissima (4C = 3.57 pg). GBS generated 29,307 and 9,909 SNPs for the entire collection and a subset of ssp. officinalis genotypes, respectively. We identified significant genetic differentiation between the subspecies, as confirmed by PCA (PC1 = 69.9%), STRUCTURE, and hierarchical clustering. With 21,770 private alleles and HE = 0.38 subspecies altissima exhibited greater genetic divergence than ssp. officinalis (2,953 private alleles, HE = 0.07). Within ssp. officinalis, several clusters of untapped genetic diversity from Armenia, France, Georgia, and Spain were identified. Furthermore, genomic analysis revealed inconsistencies in varietal purity in three out of four investigated traditional cultivars, where genotypes bearing the same name were assigned to different genetic clusters. Based on a clustering approach, two core sets were constructed. The first core set (Core15, 5.7% of the collection) of 9 officinalis and 6 altissima genotypes aimed to comprise the diversity of the entire collection with minimal redundancy for long-term breeding programs. An extended core set of 30 genotypes (Core30, 11.3%) focused on the subspecies officinalis and included more cultivated material. Conclusions This study provides a genomic characterization of a comprehensive M. officinalis collection. The identification of distinct genetic clusters and the development of optimized core sets significantly reduce the resources required for future evaluations. These core sets serve as a robust foundation for breeding programs aimed at enhancing agronomic traits such as drought tolerance, while increasing pharmaceutical quality.

Caraway (Carum carvi L.) is a major essential oil crop with biennial and annual flowering types. As basic research resource, we developed a draft genome assembly using long-read ONT sequencing and provide a structural and functional gene annotation for an annual caraway inbred line. To elucidate the genetic control of flowering, a genotyping-by-sequencing (GBS) was conducted for an F2 population (N = 187) generating 731 strictly filtered SNPs. A linkage map was constructed spanning 663 cM across 10 linkage groups with in total 634 (full map) or 259 (thinned map) SNPs. Contrary to its dominance in F1, annual flowering occurred in only 36% of F2 plants under late sowing conditions. Furthermore, the annual F2 plants exhibited delayed flowering compared to the annual parent. QTL analysis identified five significant QTLs for (adjusted) flowering time (LG02, LG03, LG05, LG08 and LG10) explaining 6.1% to 10.5% (in total 42.7%) of phenotypic variance. The results support a polygenic predominately additive model for flowering induction in caraway. In addition, two QTLs for flower color (LG01, LG10) were detected explaining 10.9% and 26.0% of phenotypic variance, respectively. This study provides a comprehensive genomic resource for caraway, bridging the gap between traditional breeding and molecular improvement.

Background Barley ( Hordeum vulgare L.) provides a suitable model for studying domestication-driven plant-microbiome interactions. Although wild, landrace, and modern genotypes host distinct rhizosphere communities, the extent to which roots and microbes reciprocally influence each other remains unclear. Here, we applied an integrated multi-omics approach combining long-read metagenomics, root transcriptomics, and plant genomics to understand genotype-specific host-microbiome coordination. Results Oxford Nanopore whole metagenome sequencing (WMS) revealed clear genotype-dependent shifts in rhizosphere communities across seasons. Functional profiling showed a conserved metabolic backbone including amino acid metabolism, energy production, and secondary metabolite biosynthesis accompanied by genotype-specific differences in carbohydrate metabolism and transport-associated pathways. Genome-resolved analysis through metagenome-assembled genomes (MAGs) further detailed the taxonomic and functional architecture of key rhizosphere lineages. Root transcriptomics identified extensive differential expression linked to microbial perception, signaling, defense, and metabolic reprogramming. Integrating host and microbiome data revealed coordinated molecular responses, indicating that barley genotypes not only shape microbial assembly but also program their transcriptional activity in response to microbial cues. Conclusions These findings demonstrate that domestication has shaped a bidirectional interaction network in which barley genotypes and their rhizosphere microbiomes jointly modulate microbial community structure and host transcriptional regulation. The coordinated exchange provides new insights into the evolutionary tuning of plant-microbiome relationships and highlights opportunities for microbiome-informed crop improvement.

A pan-transcriptome describes the transcriptional and post-transcriptional consequences of genome diversity from multiple individuals within a species. We developed a barley pan-transcriptome using 20 inbred genotypes representing domesticated barley diversity by generating and analyzing short- and long-read RNA-sequencing datasets from multiple tissues. To overcome single reference bias in transcript quantification, we constructed genotype-specific reference transcript datasets (RTDs) and integrated these into a linear pan-genome framework to create a pan-RTD, allowing transcript categorization as core, shell or cloud. Focusing on the core (expressed in all genotypes), we observed significant transcript abundance variation among tissues and between genotypes driven partly by RNA processing, gene copy number, structural rearrangements and conservation of promotor motifs. Network analyses revealed conserved co-expression module::tissue correlations and frequent functional diversification. To complement the pan-transcriptome, we constructed a comprehensive cultivar (cv.) Morex gene-expression atlas and illustrate how these combined datasets can be used to guide biological inquiry.

A pan-transcriptome describes the transcriptional and post-transcriptional consequences of genome diversity from multiple individuals within a species, revealing an assortment of functions that drive biological outcomes. We developed a barley pan-transcriptome using twenty inbred genotypes representing domesticated* barley diversity by generating and analysing extensive short- and long-read RNA sequencing datasets from multiple tissues. To overcome single reference bias and facilitate downstream analyses we constructed genotype-specific reference transcript datasets (RTDs) and integrated these into a linear pan-genome framework to create a single pan-RTD. Categorising transcripts based upon presence or absence across genotypes defined them as core (expressed in all), shell (absent in one or more) or cloud (expressed in only one). Focusing on the core we observed significant transcript abundance variation among tissues and between genotypes. We show that drivers of transcript abundance variation in this category include RNA processing, gene copy number, large structural rearrangements and degree of conservation of promotor motifs. We reveal conserved patterns of co-expression module-tissue correlations encompassing distinct biological functions, as well as frequent functional diversification. We complement the pan-transcriptome by integrating extensive and diverse replicated public RNA-seq datasets from the reference cultivar (cv.) Morex into a comprehensive gene-expression atlas

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.

AbstractThe genomes of many plants, animals, and fungi frequently comprise dispensable B chromosomes that rely upon various chromosomal drive mechanisms to counteract the tendency of non-essential genetic elements to be purged over time. The B chromosome of rye – a model system for nearly a century – undergoes targeted nondisjunction during first pollen mitosis, favouring segregation into the generative nucleus, thus increasing their numbers over generations. However, the genetic mechanisms underlying this process are poorly understood. Here, using a newly-assembled, ~430 Mb-long rye B chromosome pseudomolecule, we identify five candidate genes whose role as trans-acting moderators of the chromosomal drive is supported by karyotyping, chromosome drive analysis and comparative RNA-seq. Among them, we identify DCR28, coding a microtubule-associated protein related to cell division, and detect this gene also in the B chromosome of Aegilops speltoides. The DCR28 gene family is neo-functionalised and serially-duplicated with 15 B chromosome-located copies that are uniquely highly expressed in the first pollen mitosis of rye.

The removal of introns by the spliceosome is a key gene regulatory mechanism in eukaryotes, with the U1 snRNP subunit playing a crucial role in the early stages of splicing. Studies in metazoans show that the U1 snRNP also conducts splicing-independent functions, but the lack of genetic tools and knowledge about U1 snRNP-associated proteins have limited the study of such splicing-independent functions in plants. Here we describe an RNA-centric approach that identified more than 200 proteins associated with the Arabidopsis U1 snRNP and revealed a tight link to mRNA cleavage and polyadenylation factors. Interestingly, we found that the U1 snRNP protects mRNAs against premature cleavage and polyadenylation within introns-a mechanism known as telescripting in metazoans-while also influencing alternative polyadenylation site selection in 3 '-UTRs. Overall, our work provides a comprehensive view of U1 snRNP interactors and reveals novel functions in regulating mRNA 3 '-end processing in Arabidopsis, laying the groundwork for understanding non-canonical functions of plant U1 snRNPs.

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.

The removal of introns by the spliceosome is a key gene regulatory mechanism in eukaryotes, with the U1 snRNP subunit of the spliceosome playing a crucial role in the early stages of splicing. Studies in metazoans show that the U1 snRNP also conducts splicing-independent functions, but the lack of genetic tools and knowledge about U1 snRNP-associated proteins have limited the study of such splicing-independent functions in plants. Here, we describe an RNA-centric approach that identified more than 200 proteins associated with the Arabidopsis U1 snRNP, among them mRNA cleavage and polyadenylation factors. The loss of U1 core components is linked to premature cleavage and polyadenylation within gene bodies and alternative polyadenylation site selection in 3’-UTRs. Overall, our work provides a comprehensive view of U1 snRNP interactors and reveals novel functions in regulating mRNA 3’-end processing in Arabidopsis, thus establishing the groundwork for a better understanding of non-canonical functions of plant U1 snRNPs.

In the context of a continuously increasing human population that needs to be fed, with environmental pro-tection in mind, nitrogen use efficiency (NUE) improvement is becoming very important. To understand the natural variation of traits linked to nitrogen uptake efficiency (UPE), one component of NUE, the multiparent advanced generation intercross (MAGIC) winter wheat population WM-800 was phenotyped under two con-trasting nitrogen (N) levels in a high-throughput phenotyping facility for six weeks. Three biomass-related, three root-related, and two reflectance-related traits were measured weekly under each treatment. Subsequently, the population was genetically analysed using a total of 13,060 polymorphic haplotypes and singular SNPs for a genome-wide association study (GWAS). In total, we detected 543 quantitative trait loci (QTL) across all time points and traits, which were pooled into 42 stable QTL (sQTL; present in at least three of the six weeks). Besides Rht-B1 and Rht-D1, candidate genes playing a role in gibberellic acid-regulated growth and nitrate transporter genes from the NPF gene family, like NRT 1.1, were linked to sQTL. Two novel sQTL on chromosomes 5 A and 6D showed pleiotropic effects on several traits. The high number of N-specific sQTL indicates that selection for UPE is useful specifically under N-limited conditions.

EARLY FLOWERING 3 (ELF3) is an important regulator of various physiological and developmental processes and hence may serve to improve plant adaptation which will be essential for future plant breeding. To expand the limited knowledge on barley ELF3 in determining agronomic traits, we conducted field studies with heterogeneous inbred families (HIFs) derived from selected lines of the wild barley nested association mapping population HEB-25. During two growing seasons, phenotypes of nearly isogenic HIF sister lines, segregating for exotic and cultivated alleles at the ELF3 locus, were compared for 10 developmental and yield-related traits. We determine novel exotic ELF3 alleles and show that HIF lines, carrying the exotic ELF3 allele, accelerated plant development compared with the cultivated ELF3 allele, depending on the genetic background. Remarkably, the most extreme effects on phenology could be attributed to one exotic ELF3 allele differing from the cultivated Barke ELF3 allele in only one single nucleotide polymorphism (SNP). This SNP causes an amino acid substitution (W669G), which as predicted has an impact on the protein structure of ELF3. Consequently, it may affect phase separation behaviour and nano-compartment formation of ELF3 and, potentially, also its local cellular interactions causing significant trait differences between HIF sister lines.

The use of wild plant species or their halophytic relatives has been considered in plant breeding programs to improve salt and drought tolerance in crop plants. Aeluropus littoralis serves as halophyte model for identification and isolation of novel stress adaptation genes. A. littoralis, a perennial monocot grass, grows in damp or arid areas, often salt-impregnated places and wasteland in cultivated areas, can survive periodically high water salinity, and tolerate high salt concentrations in the soil up to 1,100 mM sodium chloride. Therefore, it serves as valuable genetic resource to understand molecular mechanisms of stress-responses in monocots. The knowledge can potentially be used for improving tolerance to abiotic stresses in economically important crops. Several morphological, anatomical, ecological, and physiological traits of A. littoralis have been investigated so far. After watering with salt water the grass is able to excrete salt via its salt glands. Meanwhile, a number of ESTs (expressed sequence tag), genes and promoters induced by the salt and drought stresses were isolated, sequenced and annotated at a molecular level. Transfer of stress related genes to other species resulted in enhanced stress resistance. Here we describe the genome sequence and structure of A. littoralis analyzed by whole genome sequencing and histological analysis. The chromosome number was determined to be 20 (2n = 2x = 20). The genome size was calculated to be 354 Mb. This genomic information provided here, will support the functional investigation and application of novel genes improving salt stress resistance in crop plants. The utility of the sequence information is exemplified by the analysis of the DREB-transcription factor family.

Increased salinity is one of the major consequences of climatic change affecting global crop production. The early stages in the barley (Hordeum vulgare L.) life cycle are considered the most critical phases due to their contributions to final crop yield. Particularly, the germination and seedling development are sensitive to numerous environmental stresses, especially soil salinity. In this study, we aimed to identify SNP markers linked with germination and seedling development at 150 mM NaCl as a salinity treatment. We performed a genome-wide association study (GWAS) using a panel of 208 intermedium-spike barley (H. vulgare convar. intermedium (Körn.) Mansf.) accessions and their genotype data (i.e., 10,323 SNPs) using the genome reference sequence of "Morex". The phenotypic results showed that the 150 mM NaCl salinity treatment significantly reduced all recorded germination and seedling-related traits compared to the control treatment. Furthermore, six accessions (HOR 11747, HOR 11718, HOR 11640, HOR 11256, HOR 11275 and HOR 11291) were identified as the most salinity tolerant from the intermedium-spike barley collection. GWAS analysis indicated that a total of 38 highly significantly associated SNP markers under control and/or salinity traits were identified. Of these, two SNP markers on chromosome (chr) 1H, two on chr 3H, and one on chr 4H were significantly linked to seedling fresh and dry weight under salinity stress treatment. In addition, two SNP markers on chr 7H were also significantly associated with seedling fresh and dry weight but under control condition. Under salinity stress, one SNP marker on chr 1H, 5H and 7H were detected for more than one phenotypic trait. We found that in most of the accessions exhibiting the highest salinity tolerance, most of the salinity-related QTLs were presented. These results form the basis for detailed studies, leading to improved salt tolerance breeding programs in barley.

19 Adaptation of crops to an increasing range of environmental conditions will be substantial for 20 future plant breeding. EARLY FLOWERING 3 ( ELF3 ) is an important regulator of various 21 physiological and developmental processes and hence may serve to improve plant adaptation. 22 To expand the limited knowledge on barley ELF3 in determining yield formation, we 23 conducted field studies with heterogeneous inbred families (HIFs) derived from selected lines 24

The multi-parent-advanced-generation-intercross (MAGIC) population WM-800 was developed by intercrossing eight modern winter wheat cultivars to enhance the genetic diversity present in breeding populations. We cultivated WM-800 during two seasons in seven environments under two contrasting nitrogen fertilization treatments. WM-800 lines exhibited highly significant differences between treatments, as well as high heritabilities among the seven agronomic traits studied. The highest-yielding WM-line achieved an average yield increase of 4.40 dt/ha (5.2%) compared to the best founder cultivar Tobak. The subsequent genome-wide-association-study (GWAS), which was based on haplotypes, located QTL for seven agronomic traits including grain yield. In total, 40, 51, and 46 QTL were detected under low, high, and across nitrogen treatments, respectively. For example, the effect of QYLD_3A could be associated with the haplotype allele of cultivar Julius increasing yield by an average of 4.47 dt/ha (5.2%). A novel QTL on chromosome 2B exhibited pleiotropic effects, acting simultaneously on three-grain yield components (ears-per-square-meter, grains-per-ear, and thousand-grain-weight) and plant-height. These effects may be explained by a member of the nitrate-transporter-1 (NRT1)/peptide-family, TaNPF5.34, located 1.05 Mb apart. The WM-800 lines and favorable QTL haplotypes, associated with yield improvements, are currently implemented in wheat breeding programs to develop advanced nitrogen-use efficient wheat cultivars.

Metabolites play a key role in plants as they are routing plant developmental processes and are involved in biotic and abiotic stress responses. Their analysis can offer important information on the underlying processes. Regarding plant breeding, metabolite concentrations can be used as biomarkers instead of or in addition to genetic markers to predict important phenotypic traits (metabolic prediction). In this study, we applied a genome-wide association study (GWAS) in a wild barley nested association mapping (NAM) population to identify metabolic quantitative trait loci (mQTL). A set of approximately 130 metabolites, measured at early and late sampling dates, was analysed. For four metabolites from the early and six metabolites from the late sampling date significant mQTL (grouped as 19 mQTL for the early and 25 mQTL for the late sampling date) were found. Interestingly, all of those metabolites could be classified as sugars. Sugars are known to be involved in signalling, plant growth and plant development. Sugar-related genes, encoding mainly sugar transporters, have been identified as candidate genes for most of the mQTL. Moreover, several of them co-localized with known flowering time genes like Ppd-H1 , HvELF3 , Vrn-H1 , Vrn-H2 and Vrn-H3 , hinting on the known role of sugars in flowering. Furthermore, numerous disease resistance-related genes were detected, pointing to the signalling function of sugars in plant resistance. An mQTL on chromosome 1H in the region of 13 Mbp to 20 Mbp stood out, that alone explained up to 65% of the phenotypic variation of a single metabolite. Analysis of family-specific effects within the diverse NAM population showed the available natural genetic variation regarding sugar metabolites due to different wild alleles. The study represents a step towards a better understanding of the genetic components of metabolite accumulation, especially sugars, thereby linking them to biological functions in barley.

Straw biomass and stability are crucial for stable yields. Moreover, straw harbors the potential to serve as a valuable raw material for bio-economic processes. The peduncle is the top part of the last shoot internode and carries the spike. This study investigates the genetic control of barley peduncle morphology. Therefore, 1411 BC1S3 lines of the nested association mapping (NAM) population "Halle Exotic Barley 25" (HEB-25), generated by crossing the spring barley elite cultivar Barke with an assortment of 25 exotic barley accessions, were used. Applying 50k Illumina Infinium iSelect SNP genotyping yielded new insights and a better understanding of the quantitative trait loci (QTL) involved in controlling the peduncle diameter traits, we found the total thickness of peduncle tissues and the area of the peduncle cross-section. We identified three major QTL regions on chromosomes 2H and 3H mainly impacting the traits. Remarkably, the exotic allele at the QTL on chromosome 3H improved all three traits investigated in this work. Introgressing this QTL in elite cultivars might facilitate to adjust peduncle morphology for improved plant stability or enlarged straw biomass production independent of flowering time and without detrimental effects on grain yield.