Crop Wild Relatives (CWRs), as potential sources of new genetic variants, are being extensively studied to identify genotypes that will be able to confer resistance to biotic stresses. In this study, a collection of barley wild relatives was assessed in the field, and their phenotypic variability was evaluated using a Barley Description List, reflecting the identified ecosites. Overall, the CWRs showed significant field resistance to various fungal diseases. To further investigate their resistance, greenhouse tests were performed, revealing that several CWRs exhibited resistance against Fusarium culmorum, Pyrenophora teres, and Puccinia hordei G.H. Otth. Additionally, to characterize the genetic diversity within the collection, DNA polymorphisms at 21 loci were examined. We successfully employed barley-specific SSR markers, confirming their suitability for identifying H. spontaneum and even H. marinum, i.e., perennial species. The SSR markers efficiently clustered the investigated collection according to species and ecotypes, similarly to the phenotypic assessment. Moreover, SSR markers associated with disease resistance revealed different alleles in comparison to those found in resistant barley cultivars. Overall, our findings highlight that this evaluated collection of CWRs represents a valuable reservoir of genetic variability and resistance genes that can be effectively utilized in breeding programs.

In nature, beneficial bacteria triggering induced systemic resistance (ISR) may protect plants from potential diseases, reducing yield losses caused by diverse pathogens. However, little is known about how the host plant initially responds to different beneficial bacteria. To reveal the impact of different bacteria on barley (Hordeum vulgare), bacterial colonization patterns, gene expression, and composition of seed endophytes were explored.

Response to a high salinity treatment of 300 mM NaCl was studied in a cultivated barley Hordeum vulgare Syrian cultivar Tadmor and in a halophytic wild barley H. marinum. Differential salinity tolerance of H. marinum and H. vulgare is underlied by qualitative and quantitative differences in proteins involved in a variety of biological processes. The major aim was to identify proteins underlying differential salinity tolerance between the two barley species. Analyses of plant water content, osmotic potential and accumulation of proline and dehydrin proteins under high salinity revealed a relatively higher water saturation deficit in H. marinum than in H. vulgare while H. vulgare had lower osmotic potential corresponding with high levels of proline and dehydrins. Analysis of proteins soluble upon boiling isolated from control and salt-treated crown tissues revealed similarities as well as differences between H. marinum and H. vulgare. The similar salinity responses of both barley species lie in enhanced levels of stress-protective proteins such as defense-related proteins from late-embryogenesis abundant family, several chaperones from heat shock protein family, and others such as GrpE. However, there have also been found significant differences between H. marinum and H. vulgare salinity response indicating an active stress acclimation in H. marinum while stress damage in H. vulgare. An active acclimation to high salinity in H. marinum is underlined by enhanced levels of several stress-responsive transcription factors from basic leucine zipper and nascent polypeptide-associated complex families. In salt-treated H. marinum, enhanced levels of proteins involved in energy metabolism such as glycolysis, ATP metabolism, and photosynthesis-related proteins indicate an active acclimation to enhanced energy requirements during an establishment of novel plant homeostasis. In contrast, changes at proteome level in salt-treated H. vulgare indicate plant tissue damage as revealed by enhanced levels of proteins involved in proteasome-dependent protein degradation and proteins related to apoptosis. The results of proteomic analysis clearly indicate differential responses to high salinity and provide more profound insight into biological mechanisms underlying salinity response between two barley species with contrasting salinity tolerance.

Grain development, as a vital process in the crop's life cycle, is crucial for determining crop quality and yield. However, the molecular basis and regulatory network of barley grain development is not well understood at present. Here, we investigated the transcriptional dynamics of barley grain development through RNA sequencing at four developmental phases, including early prestorage phase (3 days post anthesis (DPA)), late prestorage or transition phase (8 DPA), early storage phase (13 DPA), and levels off stages (18 DPA). Transcriptome profiling found that pronounced shifts occurred in the abundance of transcripts involved in both primary and secondary metabolism during grain development. The transcripts' activity was decreased during maturation while the largest divergence was observed between the transitions from prestorage phase to storage phase, which coincided with the physiological changes. Furthermore, the transcription factors, hormone signal transduction-related as well as sugar-metabolism-related genes, were found to play a crucial role in barley grain development. Finally, 4771 RNA editing events were identified in these four development stages, and most of the RNA editing genes were preferentially expressed at the prestore stage rather than in the store stage, which was significantly enriched in "essential" genes and plant hormone signal transduction pathway. These results suggested that RNA editing might act as a 'regulator' to control grain development. This study systematically dissected the gene expression atlas of barley grain development through transcriptome analysis, which not only provided the potential targets for further functional studies, but also provided insights into the dynamics of gene regulation underlying grain development in barley and beyond.

Endogenous phytase plays a crucial role in phytate degradation and is thus closely related to nutrient efficiency in barley products. The understanding of genetic information of phytase in barley can provide a useful tool for breeding new barley varieties with high phytase activity.

Cadmium is an environmental pollutant with high toxicity that negatively affects plant growth and development. To understand the molecular mechanisms of plant response to cadmium stress, we have performed a genome-wide transcriptome analysis on barley plants treated with an increased concentration of cadmium. Differential gene expression analysis revealed 10,282 deregulated transcripts present in the roots and 7,104 in the shoots. Among them, we identified genes related to reactive oxygen species metabolism, cell wall formation and maintenance, ion membrane transport and stress response. One of the most upregulated genes was PLANT CADMIUM RESISTACE 2 (HvPCR2) known to be responsible for heavy metal detoxification in plants. Surprisingly, in the transcriptomic data we identified four different copies of the HvPCR2 gene with a specific pattern of upregulation in individual tissues. Heterologous expression of all five barley copies in a Cd-sensitive yeast mutant restored cadmium resistance. In addition, four HvPCR2 were located in tandem arrangement in a single genomic region of the barley 5H chromosome. To our knowledge, this is the first example showing multiplication of the PCR2 gene in plants.

Sequencing analysis of mitochondrial genomes is important for understanding the evolution and genome structures of various plant species. Barley is a self-pollinated diploid plant with seven chromosomes comprising a large haploid genome of 5.1 Gbp. Wild barley (Hordeum vulgare ssp. spontaneum) and cultivated barley (H. vulgare ssp. vulgare) have cross compatibility and closely related genomes, although a significant number of nucleotide polymorphisms have been reported between their genomes.

Cool season grasses often form reciprocal symbiotic relationships with endophytic fungal species in genus Epichloë. In this study, we characterized three fungal endophytes isolated from the grass Hordeum bogdanii native to northwest China. Based on morphological characteristics and phylogenetic analyses of tefA, tubB, and actG sequences, we identified them as Epichloë sp. HboTG-2 (H. bogdanii Taxonomic Group 2: E. bromicola × E. typhina). Alkaloid synthesis related genes analysis showed that Epichloë sp. HboTG-2 may have the ability only to produce peramine which is toxic to insects but not to animals. In the process of this study, we did not observe sexual structures or epiphyllous growth on leaves of infected plants.

Aquaporins are multifunctional membrane channels which belong to the family of major intrinsic proteins (MIPs) and are best known for their ability to facilitate the movement of water. In the present study, earlier results from microarray experiments were followed up. These experiments had suggested that, in barley (Hordeum vulgare L.), aquaporin family members are expressed in distinct patterns during leaf development. Real-time PCR and in situ hybridization were used to analyse the level and tissue-distribution of expression of candidate aquaporins, focusing on plasma membrane and tonoplast intrinsic proteins (PIPs, TIPs). Water channel function of seven aquaporins, whose transcripts were the most abundant and the most variable, was tested through expression in yeast and, in part, through expression in oocytes. All PIP1 and PIP2 subfamily members changed in expression during leaf development, with expression being much higher or lower in growing compared with mature tissue. The same applied to those TIPs which were expressed at detectable levels. Specific roles during leaf development are proposed for particular aquaporins.

In barley (Hordeum vulgare L.), lateral branches called tillers contribute to grain yield and define shoot architecture, but genetic control of tiller number and developmental rate are not well characterized. The primary objectives of this work were to examine relationships between tiller number and other agronomic and morphological traits and identify natural genetic variation associated with tiller number and rate, and related traits. We grew 768 lines from the USDA National Small Grain Collection in the field and collected data over two years for tiller number and rate, and agronomic and morphological traits. Our results confirmed that spike row-type and days to heading are correlated with tiller number, and as much as 28% of tiller number variance was associated with these traits. In addition, negative correlations between tiller number and leaf width and stem diameter were observed, indicating trade-offs between tiller development and other vegetative growth. Thirty-three quantitative trait loci (QTL) were associated with tiller number or rate. Of these, 40% overlapped QTL associated with days to heading and 22% overlapped QTL associated with spike row-type, further supporting that tiller development is associated with these traits. Some QTL associated with tiller number or rate, including the major QTL on chromosome 3H, were not associated with other traits, suggesting that some QTL may be directly related to rate of tiller development or axillary bud number. These results enhance our knowledge of the genetic control of tiller development in barley, which is important for optimizing tiller number and rate for yield improvement.

Plant disease resistance genes (R-genes) play a critical role in the defense response to pathogens. Barley is one of the most important cereal crops, having a genome recently made available, for which the diversity and evolution of R-genes are not well understood. The main objectives of this research were to conduct a genome-wide identification of barley Coiled-coil, Nucleotide-binding site, Leucine-rich repeat (CNL) genes and elucidate their evolutionary history. We employed a Hidden Markov Model using 52 Arabidopsis thaliana CNL reference sequences and analyzed for phylogenetic relationships, structural variation, and gene clustering. We identified 175 barley CNL genes nested into three clades, showing (a) evidence of an expansion of the CNL-C clade, primarily due to tandem duplications; (b) very few members of clade CNL-A and CNL-B; and (c) a complete absence of clade CNL-D. Our results also showed that several of the previously identified mildew locus A (MLA) genes may be allelic variants of two barley CNL genes, MLOC_66581 and MLOC_10425, which respond to powdery mildew. Approximately 23% of the barley CNL genes formed 15 gene clusters located in the extra-pericentromeric regions on six of the seven chromosomes; more than half of the clustered genes were located on chromosomes 1H and 7H. Higher average numbers of exons and multiple splice variants in barley relative to those in Arabidopsis and rice may have contributed to a diversification of the CNL-C members. These results will help us understand the evolution of R-genes with potential implications for developing durable resistance in barley cultivars.

Aluminium (Al) toxicity is the main factor limiting the crop production in acid soils and barley (Hordeum vulgare L.) is one of the most Al-sensitive of the small-grained cereals. The major gene for Al tolerance in barley is HvAACT1 (HvMATE) on chromosome 4H which encodes a multidrug and toxic compound extrusion (MATE) protein. The HvAACT1 protein facilitates the Al-activated release of citrate from root apices which protects the growing cells and enables root elongation to continue. A 1 kb transposable element-like insert in the 5' untranslated region (UTR) of HvAACT1 is associated with increased gene expression and tolerance and a PCR-based marker is available to score for this insertion.

Land plants are sessile organisms that cannot escape the adverse climatic conditions of a given environment. Hence, adaptation is one of the solutions to surviving in a challenging environment. This study was aimed at detecting adaptive loci in barley landraces that are affected by selection. To that end, a diverse population of barley landraces was analyzed using the genotyping by sequencing approach. Climatic data for altitude, rainfall and temperature were collected from 61 weather sites near the origin of selected landraces across Ethiopia. Population structure analysis revealed three groups whereas spatial analysis accounted significant similarities at shorter geographic distances (< 40 Km) among barley landraces. Partitioning the variance between climate variables and geographic distances indicated that climate variables accounted for most of the explainable genetic variation. Markers by climatic variables association analysis resulted in altogether 18 and 62 putative adaptive loci using Bayenv and latent factor mixed model (LFMM), respectively. Subsequent analysis of the associated SNPs revealed putative candidate genes for plant adaptation. This study highlights the presence of putative adaptive loci among barley landraces representing original gene pool of the farming communities.

Photosynthetic acclimation to cold conditions is an important factor influencing freezing tolerance of plants. Photosynthetic enzyme activities increase as part of a photochemical mechanism underlying photosynthetic acclimation to low temperatures. Additionally, a non-photochemical mechanism may be activated to minimize photooxidative damage. The aim of this study was to test the hypothesis that differences in stomatal conductance in Hordeum vulgare plants with contrasting freezing tolerances induce various strategies for photosynthetic acclimation to cold stress. Different stomatal behaviors during the prehardening step resulted in diverse plant reactions to low-temperature stress. Plants with a relatively low freezing tolerance exhibited decreased stomatal conductance, resulting in decreased photochemical activity, faster induction of the non-photochemical mechanism, and downregulated expression of two Rubisco activase (RcaA) splicing variants. In contrast, plants with a relatively high freezing tolerance that underwent a prehardening step maintained the stomatal conductance at control level and exhibited delayed photochemical activity and RcaA expression decrease, and increased Rubisco activity, which increased net photosynthetic rate. Thus, in barley, the induction of photoinhibition avoidance (i.e., non-photochemical photoacclimation mechanism) is insufficient for an effective cold acclimation. An increase in cold-induced net photosynthetic rate due to open stomata is also necessary.

Temperate cereals, such as wheat (Triticum spp.) and barley (Hordeum vulgare), respond to prolonged cold by becoming more tolerant of freezing (cold acclimation) and by becoming competent to flower (vernalization). These responses occur concomitantly during winter, but vernalization continues to influence development during spring. Previous studies identified VERNALIZATION1 (VRN1) as a master regulator of the vernalization response in cereals. The extent to which other genes contribute to this process is unclear. In this study the Barley1 Affymetrix chip was used to assay gene expression in barley seedlings during short or prolonged cold treatment. Gene expression was also assayed in the leaves of plants after prolonged cold treatment, in order to identify genes that show lasting responses to prolonged cold, which might contribute to vernalization-induced flowering. Many genes showed altered expression in response to short or prolonged cold treatment, but these responses differed markedly. A limited number of genes showed lasting responses to prolonged cold treatment. These include genes known to be regulated by vernalization, such as VRN1 and ODDSOC2, and also contigs encoding a calcium binding protein, 23-KD jasmonate induced proteins, an RNase S-like protein, a PR17d secretory protein and a serine acetyltransferase. Some contigs that were up-regulated by short term cold also showed lasting changes in expression after prolonged cold treatment. These include COLD REGULATED 14B (COR14B) and the barley homologue of WHEAT COLD SPECIFIC 19 (WSC19), which were expressed at elevated levels after prolonged cold. Conversely, two C-REPEAT BINDING FACTOR (CBF) genes showed reduced expression after prolonged cold. Overall, these data show that a limited number of barley genes exhibit lasting changes in expression after prolonged cold treatment, highlighting the central role of VRN1 in the vernalization response in cereals.

Boron (B) is an important micronutrient for plant growth, but is toxic when levels are too high. This commonly occurs in environments with alkaline soils and relatively low rainfall, including many of the cereal growing regions of southern Australia. Four major genetic loci controlling tolerance to high soil B have been identified in the landrace barley, Sahara 3771. Genes underlying two of the loci encode the B transporters HvBot1 and HvNIP2;1.

Real-time quantitative PCR has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. Wild barley (Hordeum brevisubulatum (Trin.) Link) is a wild relative species in Triticeae that has strong tolerance to abiotic stresses and extremely wide adaptation. However, suitable references gene have not been documented for standardization of gene expression in wild barley under abiotic stress.

Barley (Hordeum vulgare L.) represents one of the most important cereals cultivated worldwide. Investigating genetic variability and structure of barley is important for enhancing the crop productivity. This study aimed to investigate the diversity and structure of 40 barley genotypes originated from three European countries (France, the Netherlands, Poland) using amplified fragment length polymorphisms (AFLPs). It also aimed to study 5-aminolevulinic acid (ALA) effect on salinity tolerance of six barley genotypes. The expected heterozygosity (He) diverged from 0.126 to 0.501, with a mean of 0.348. Polymorphic information content (PIC) diverged from 0.103 to 0.482 across barley genotypes, with a mean of 0.316, indicating that barley genotypes are rich in a considerable level of genetic diversity. The 40 barley genotypes were further studied based on their geographical origin (Western Europe and Eastern Europe). The Eastern European region (Poland) has a higher barley variability than the Western European region (France and the Netherlands). Nei's distance-based cluster tree divided the 40 barley accessions into two major clusters; one cluster comprised all the varieties originated from the Eastern European region, while the other major cluster included all accessions originated from the Western European region. Structure analysis results were in a complete concordance with our cluster analysis results. Slaski 2, Damseaux and Urbanowicki genotypes have the highest diversity level, whereas Carmen, Bigo and Cambrinus genotypes have the lowest level. The response of these six varieties to NaCl stress was also investigated. Salt stress (100 mM NaCl) slightly decreased levels of chlorophyll, carotenoid and osmolytes (proteins, soluble sugars, phenolics and flavonoids) in the leaves of Slaski 2, Damseaux and Urbanowicki genotypes at non-significant level, as compared to control samples. However, pigment contents and osmolytes in leaves of Carmen, Bigo and Cambrinus genotypes were significantly decreased by salt stress. Antioxidant enzyme activities were significantly increased in Slaski 2 genotype, but non-significantly increased in Carmen by salt stress. Priming Slaski 2 and Carmen cultivars with ALA under salt stress significantly induced pigment contents, antioxidants enzymes activity and stress-responsive genes expression, relative to NaCl-stressed plants. In conclusion, this study suggested a correlation between variability percentage and degree of salinity resistance. ALA improved salt tolerance in barley.

Potassium retention under saline conditions has emerged as an important determinant for salt tolerance in plants. Halophytic Hordeum brevisubulatum evolves better strategies to retain K+ to improve high-salt tolerance. Hence, uncovering K+-efficient uptake under salt stress is vital for understanding K+ homeostasis. HAK/KUP/KT transporters play important roles in promoting K+ uptake during multiple stresses. Here, we obtained nine salt-induced HAK/KUP/KT members in H. brevisubulatum with different expression patterns compared with H. vulgare through transcriptomic analysis. One member HbHAK1 showed high-affinity K+ transporter activity in athak5 to cope with low-K+ or salt stresses. The expression of HbHAK1 in yeast Cy162 strains exhibited strong activities in K+ uptake under extremely low external K+ conditions and reducing Na+ toxicity to maintain the survival of yeast cells under high-salt-stress. Comparing with the sequence of barley HvHAK1, we found that C170 and R342 in a conserved domain played pivotal roles in K+ selectivity under extremely low-K+ conditions (10 μM) and that A13 was responsible for the salt tolerance. Our findings revealed the mechanism of HbHAK1 for K+ accumulation and the significant natural adaptive sites for HAK1 activity, highlighting the potential value for crops to promote K+-uptake under stresses.

Frost tolerance is a key trait with economic and agronomic importance in barley because it is a major component of winter hardiness, and therefore limits the geographical distribution of the crop and the effective transfer of quality traits between spring and winter crop types. Three main frost tolerance QTL (Fr-H1, Fr-H2 and Fr-H3) have been identified from bi-parental genetic mapping but it can be argued that those mapping populations only capture a portion of the genetic diversity of the species. A genetically broad dataset consisting of 184 genotypes, representative of the barley gene pool cultivated in the Mediterranean basin over an extended time period, was genotyped with 1536 SNP markers. Frost tolerance phenotype scores were collected from two trial sites, Foradada (Spain) and Fiorenzuola (Italy) and combined with the genotypic data in genome wide association analyses (GWAS) using Eigenstrat and kinship approaches to account for population structure.