Microbiome properties in the root nodules of Prosopis cineraria, a leguminous desert tree.

We conducted a comprehensive analysis of the total microbiome and transcriptionally active microbiome communities in the roots and root nodules of Prosopis cineraria, an important leguminous tree in arid regions of many Asian countries. Mature P. cineraria trees growing in the desert did not exhibit any detected root nodules. However, we observed root nodules on the roots of P. cineraria growing on a desert farm and on young plants growing in a growth chamber, when inoculated with rhizosphere soil, including with rhizosphere soil from near desert tree roots that had no nodules. Compared to nearby soil, non-nodulated roots were enriched with Actinobacteria (e.g., Actinophytocola sp.), whereas root nodules sampled from the desert farm and growth chamber had abundant Alphaproteobacteria (e.g., Ensifer sp.). These nodules yielded many microbes in addition to such nitrogen-fixing bacteria as Ensifer and Sinorhizobium species. Significant differences exist in the composition and abundance of microbial isolates between the nodule surface and the nodule endosphere. Shotgun metagenome analysis of nodule endospheres revealed that the root nodules comprised over 90% bacterial DNA, whereas metatranscriptome analysis showed that the plant produces vastly more transcripts than the microbes in these nodules. Control inoculations demonstrated that four out of six Rhizobium, Agrobacterium, or Ensifer isolates purified from P. cineraria nodules produced nodules in the roots of P. cineraria seedlings under greenhouse conditions. The best nodulation was achieved when seedlings were inoculated with a mixture of those bacterial strains. Though root nodulation could be achieved under water stress conditions, nodule number and nodule biomass increased with copious water availability. .IMPORTANCEMicrobial communities were investigated in roots and root nodules of Prosopis cineraria, a leguminous tree species in arid Asian regions that is responsible for exceptionally important contributions to soil fertility in these dramatically dry locations. Soil removed from regions near nodule-free roots on these mature plants contained an abundance of bacteria with the genetic ability to generate nodules and fix nitrogen but did not normally nodulate in their native rhizosphere environment, suggesting a very different co-evolved relationship than that observed for herbaceous legumes. The relative over-expression of the low-gene-density plant DNA compared to the bacterial DNA in the nodules was also unexpected, indicating a very powerful induction of host genetic contributions within the nodule. Finally, the water dependence of nodulation in inoculated seedlings suggested a possible link between early seedling growth (before a deep root system can be developed) and the early development of nitrogen-fixing capability.

Near-chromosomal-level genome of the red palm weevil (Rhynchophorus ferrugineus), a potential resource for genome-based pest control.

The red palm weevil (RPW) is a highly destructive pest that mainly affects palms, particularly date palms (Phoenix dactylifera), in the Arabian Gulf region. In this study, we present a near-chromosomal-level genome assembly of the RPW using a combination of PacBio HiFi and Dovetail Omini-C reads. The final genome assembly is around 779 Mb in size, with an N50 of ~43 Mb, consistent with our previous flow cytometry estimates. The completeness of the genome was confirmed through BUSCO analysis, which indicates the presence of 99.5% of BUSCO single copy orthologous genes. The genome annotation identified a total of 29,666 protein-coding, 1,091 tRNA and 543 rRNA genes. Overall, the proposed genome assembly is significantly superior to existing assemblies in terms of contiguity, integrity, and genome completeness.

CRISPR/Cas9-mediated mutagenesis of the mediator complex subunits MED5a and MED5b genes impaired secondary metabolite accumulation in hop (Humulus lupulus).

Hop (Humulus lupulus L.) is an important commercial crop known for the biosynthesis of valuable specialized secondary metabolites in glandular trichomes (lupulin glands), which are used for the brewing industry. To achieve burgeoning market demands is the essentiality of comprehensive understanding of the mechanisms of biosynthesis of secondary metabolites in hop. Over the past year, several studies using structural biology and functional genomics approaches have shown that Mediator (MED) serves as an integrative hub for RNAP II-mediated transcriptional regulation of various physiological and cellular processes, including involvement of MED5a and MED5b in hyperaccumulation of phenylpropanoid in A. thaliana. In the present work, an unprecedented attempt was made to generate Hlmed5a/med5b double loci mutant lines in hop using a CRISPR/Cas9-based genome editing system. The Hlmed5a/med5b double loci mutant lines showed reduced expression of structural genes of the flavonoid, humulone, and terpenoid biosynthetic pathways, which was more pronounced in the lupulin gland compared to leaf tissue and was consistent with their reduced accumulation. Phenotypic and anatomical observations revealed that Hlmed5a/med5b double loci mutant line exhibited robust growth, earlier flowering, earlier cone maturity, reduced cone size, variations in floral structure patterns, and distorted lupulin glands without any remarkable changes in leaf morphology, intensity of leaf color, and chlorophyll content. Comparative transcriptome analysis of leaf and lupulin gland tissues indicates that the expression of enzymatic genes related to secondary metabolite biosynthesis, phytohormone biosynthesis, floral organs, flowering time, and trichome development, including other genes related to starch and sucrose metabolism and defense mechanisms, were differentially modulated in the Hlmed5a/med5b lines. The combined results from functional and transcriptomic analyses illuminates the pivotal function of HlMED5a and HlMED5b in homeostasis of secondary meatbolites accumulation in hop.

The multifaceted roles of R2R3 transcription factor HlMYB7 in the regulation of flavonoid and bitter acids biosynthesis, development and biotic stress tolerance in hop (Humulus lupulus L.).

Hop (Humulus lupulus) biosynthesizes the highly economically valuable secondary metabolites, which include flavonoids, bitter acids, polyphenols and essential oils. These compounds have important pharmacological properties and are widely implicated in the brewing industry owing to bittering flavor, floral aroma and preservative activity. Our previous studies documented that ternary MYB-bHLH-WD40 (MBW) and binary WRKY1-WD40 (WW) protein complexes transcriptionally regulate the accumulation of bitter acid (BA) and prenylflavonoids (PF). In the present study, we investigated the regulatory functions of the R2R3-MYB repressor HlMYB7 transcription factor, which contains a conserved N-terminal domain along with the repressive motif EAR, in regulating the PF- and BA-biosynthetic pathway and their accumulation in hop. Constitutive expression of HlMYB7 resulted in transcriptional repression of structural genes involved in the terminal steps of biosynthesis of PF and BA, as well as stunted growth, delayed flowering, and reduced tolerance to viroid infection in hop. Furthermore, yeast two-hybrid and transient reporter assays revealed that HlMYB7 targets both PF and BA pathway genes and suppresses MBW and WW protein complexes. Heterologous expression of HlMYB7 leads to down-regulation of structural genes of flavonoid pathway in Arabidopsis thaliana, including a decrease in anthocyanin content in Nicotiana tabacum. The combined results from functional and transcriptomic analyses highlight the important role of HlMYB7 in fine-tuning and balancing the accumulation of secondary metabolites at the transcriptional level, thus offer a plausible target for metabolic engineering in hop.

Genome Assembly of a Relict Arabian Species of Daphnia O. F. Müller (Crustacea: Cladocera) Adapted to the Desert Life.

The water flea Daphnia O.F. Müller 1776 (Crustacea: Cladocera) is an important model of recent evolutionary biology. Here, we report a complete genome of Daphnia (Ctenodaphnia) arabica (Crustacea: Cladocera), recently described species endemic to deserts of the United Arab Emirates. In this study, genome analysis of D. arabica was carried out to investigate its genomic differences, complexity as well as its historical origins within the subgenus Daphnia (Ctenodaphnia). Hybrid genome assembly of D. arabica resulted in ~116 Mb of the assembled genome, with an N50 of ~1.13 Mb (BUSCO score of 99.2%). From the assembled genome, in total protein coding, 5374 tRNA and 643 rRNA genes were annotated. We found that the D. arabica complete genome differed from those of other Daphnia species deposited in the NCBI database but was close to that of D. cf. similoides. However, its divergence time estimate sets D. arabica in the Mesozoic, and our demographic analysis showed a great reduction in its genetic diversity compared to other Daphnia species. Interestingly, the population expansion in its diversity occurred during the megadrought climate around 100 Ka ago, reflecting the adaptive feature of the species to arid and drought-affected environments. Moreover, the PFAM comparative analysis highlights the presence of the important domain SOSS complex subunit C in D. arabica, which is missing in all other studied species of Daphnia. This complex consists of a few subunits (A, B, C) working together to maintain the genome stability (i.e., promoting the reparation of DNA under stress). We propose that this domain could play a role in maintaining the fitness and survival of this species in the desert environment. The present study will pave the way for future research to identify the genes that were gained or lost in this species and identify which of these were key factors to its adaptation to the harsh desert environment.

Tapping into Plant-Microbiome Interactions through the Lens of Multi-Omics Techniques.

This review highlights the pivotal role of root exudates in the rhizosphere, especially the interactions between plants and microbes and between plants and plants. Root exudates determine soil nutrient mobilization, plant nutritional status, and the communication of plant roots with microbes. Root exudates contain diverse specialized signaling metabolites (primary and secondary). The spatial behavior of these metabolites around the root zone strongly influences rhizosphere microorganisms through an intimate compatible interaction, thereby regulating complex biological and ecological mechanisms. In this context, we reviewed the current understanding of the biological phenomenon of allelopathy, which is mediated by phytotoxic compounds (called allelochemicals) released by plants into the soil that affect the growth, survival, development, ecological infestation, and intensification of other plant species and microbes in natural communities or agricultural systems. Advances in next-generation sequencing (NGS), such as metagenomics and metatranscriptomics, have opened the possibility of better understanding the effects of secreted metabolites on the composition and activity of root-associated microbial communities. Nevertheless, understanding the role of secretory metabolites in microbiome manipulation can assist in designing next-generation microbial inoculants for targeted disease mitigation and improved plant growth using the synthetic microbial communities (SynComs) tool. Besides a discussion on different approaches, we highlighted the advantages of conjugation of metabolomic approaches with genetic design (metabolite-based genome-wide association studies) in dissecting metabolome diversity and understanding the genetic components of metabolite accumulation. Recent advances in the field of metabolomics have expedited comprehensive and rapid profiling and discovery of novel bioactive compounds in root exudates. In this context, we discussed the expanding array of metabolomics platforms for metabolome profiling and their integration with multivariate data analysis, which is crucial to explore the biosynthesis pathway, as well as the regulation of associated pathways at the gene, transcript, and protein levels, and finally their role in determining and shaping the rhizomicrobiome.

Microbiome of Citrullus colocynthis (L.) Schrad. Reveals a Potential Association with Non-Photosynthetic Cyanobacteria.

Citrullus colocynthis grows in the sandy desert soil of the Arabian Peninsula with limited access to water, aside from occasional precipitation or dew. Understanding its ability to produce water-filled fruit and nutrient-rich seeds despite the harsh environment, can be useful for agricultural applications. However, information regarding the microbiome of C. colocynthis is lacking. We hypothesized that C. colocynthis associates with bacteria that aid its survival, like what has been observed in other desert plants. Here, we used 16S rRNA gene data to gain insight into the microbiome of C. colocynthis to identify its associated bacteria. In total, 9818 and 6983 OTUs were generated from root, soil, and leaf samples combined. Overall, bulk soils had the highest alpha diversity, followed by rhizosphere and root zone soils. Furthermore, C. colocynthis is associated with known plant-growth-promoting bacteria (including Acidobacteria, Bacterioidetes, and Actinobacteria), and interestingly a class of non-photosynthetic Cyanobacteria (Melainabacteria) that is more abundant on the inside and outside of the root surface than control samples, suggesting its involvement in the rhizophagy process. This study will provide a foundation for functional studies to further understand how C. colocynthis-microbes interactions help them grow in the desert, paving the path for possible agricultural applications.

The Genome of the Mimosoid Legume Prosopis cineraria, a Desert Tree.

The mimosoid legumes are a clade of ~40 genera in the Caesalpinioideae subfamily of the Fabaceae that grow in tropical and subtropical regions. Unlike the better studied Papilionoideae, there are few genomic resources within this legume group. The tree Prosopis cineraria is native to the Near East and Indian subcontinent, where it thrives in very hot desert environments. To develop a tool to better understand desert plant adaptation mechanisms, we sequenced the P. cineraria genome to near-chromosomal assembly, with a total sequence length of ~691 Mb. We predicted 77,579 gene models (76,554 CDS, 361 rRNAs and 664 tRNAs) from the assembled genome, among them 55,325 (~72%) protein-coding genes that were functionally annotated. This genome was found to consist of over 58% repeat sequences, primarily long terminal repeats (LTR-)-retrotransposons. We find an expansion of terpenoid metabolism genes in P. cineraria and its relative Prosopis alba, but not in other legumes. We also observed an amplification of NBS-LRR disease-resistance genes correlated with LTR-associated retrotransposition, and identified 410 retrogenes with an active burst of chimeric retrogene creation that approximately occurred at the same time of divergence of P. cineraria from a common lineage with P. alba~23 Mya. These retrogenes include many biotic defense responses and abiotic stress stimulus responses, as well as the early Nodulin 93 gene. Nodulin 93 gene amplification is consistent with an adaptive response of the species to the low nitrogen in arid desert soil. Consistent with these results, our differentially expressed genes show a tissue specific expression of isoprenoid pathways in shoots, but not in roots, as well as important genes involved in abiotic salt stress in both tissues. Overall, the genome sequence of P. cineraria enriches our understanding of the genomic mechanisms of its disease resistance and abiotic stress tolerance. Thus, it is a very important step in crop and legume improvement.

Salt flat microbial diversity and dynamics across salinity gradient.

Sabkhas are hypersaline, mineral-rich, supratidal mudflats that harbor microbes that are adapted to high salt concentration. Sabkha microbial diversity is generally studied for their community composition, but less is known about their genetic structure and heterogeneity. In this study, we analyzed a coastal sabkha for its microbial composition using 16S rDNA and whole metagenome, as well as for its population genetic structure. Our 16S rDNA analysis show high alpha diversity in both inner and edge sabkha than outer sabkha. Beta diversity result showed similar kind of microbial composition between inner and edge sabkha, while outer sabkha samples show different microbial composition. At phylum level, Bacteroidetes (~ 22 to 34%), Euryarchaeota (~ 18 to ~ 30%), unclassified bacteria (~ 24 to ~ 35%), Actinobacteria (~ 0.01 to ~ 11%) and Cyanobacteria (less than 1%) are predominantly found in both inside and edge sabkha regions, whereas Proteobacteria (~ 92 to ~ 97%) and Parcubacteria (~ 1 to ~ 2%) are predominately found in outer sabkha. Our 225 metagenomes assembly from this study showed similar bacterial community profile as observed in 16S rDNA-based analysis. From the assembled genomes, we found important genes that are involved in biogeochemical cycles and secondary metabolite biosynthesis. We observed a dynamic, thriving ecosystem that engages in metabolic activity that shapes biogeochemical structure via carbon fixation, nitrogen, and sulfur cycling. Our results show varying degrees of horizontal gene transfers (HGT) and homologous recombination, which correlates with the observed high diversity for these populations. Moreover, our pairwise population differentiation (Fst) for the abundance of species across the salinity gradient of sabkhas identified genes with strong allelic differentiation, lower diversity and elevated nonsynonymous to synonymous ratio of variants, which suggest selective sweeps for those gene variants. We conclude that the process of HGT, combined with recombination and gene specific selection, constitute the driver of genetic variation in bacterial population along a salinity gradient in the unique sabkha ecosystem.

Patterns of Volatile Diversity Yield Insights Into the Genetics and Biochemistry of the Date Palm Fruit Volatilome.

Volatile organic compounds are key components of the fruit metabolome that contribute to traits such as aroma and taste. Here we report on the diversity of 90 flavor-related fruit traits in date palms (Phoenix dactylifera L.) including 80 volatile organic compounds, which collectively represent the fruit volatilome, as well as 6 organic acids, and 4 sugars in tree-ripened fruits. We characterize these traits in 148 date palms representing 135 varieties using headspace solid-phase microextraction gas chromatography. We discovered new volatile compounds unknown in date palm including 2-methoxy-4-vinylphenol, an attractant of the red palm weevil (Rhynchophorus ferrugineus Olivier), a key pest that threatens the date palm crop. Associations between volatile composition and sugar and moisture content suggest that differences among fruits in these traits may be characterized by system-wide differences in fruit metabolism. Correlations between volatiles indicate medium chain and long chain fatty acid ester volatiles are regulated independently, possibly reflecting differences in the biochemistry of fatty acid precursors. Finally, we took advantage of date palm clones in our analysis to estimate broad-sense heritabilities of volatiles and demonstrate that at least some of volatile diversity has a genetic basis.

Signatures of selection underpinning rapid coral adaptation to the world's warmest reefs.

Coral populations in the world's warmest reefs, the Persian/Arabian Gulf (PAG), represent an ideal model system to understand the evolutionary response of coral populations to past and present environmental change and to identify genomic loci that contribute to elevated thermal tolerance. Here, we use population genomics of the brain coral Platygyra daedalea to show that corals in the PAG represent a distinct subpopulation that was established during the Holocene marine transgression, and identify selective sweeps in their genomes associated with thermal adaptation. We demonstrate the presence of positive and disruptive selection and provide evidence for selection of differentially methylated haplotypes. While demographic analyses suggest limited potential for genetic rescue of neighboring Indian Ocean reefs, the presence of putative targets of selection in corals outside of the PAG offers hope that loci associated with thermal tolerance may be present in the standing genetic variation.

The genomes of ancient date palms germinated from 2,000 y old seeds.

Seven date palm seeds (Phoenix dactylifera L.), radiocarbon dated from the fourth century BCE to the second century CE, were recovered from archaeological sites in the Southern Levant and germinated to yield viable plants. We conducted whole-genome sequencing of these germinated ancient samples and used single-nucleotide polymorphism data to examine the genetics of these previously extinct Judean date palms. We find that the oldest seeds from the fourth to first century BCE are related to modern West Asian date varieties, but later material from the second century BCE to second century CE showed increasing genetic affinities to present-day North African date palms. Population genomic analysis reveals that by ∼2,400 to 2,000 y ago, the P. dactylifera gene pool in the Eastern Mediterranean already contained introgressed segments from the Cretan palm Phoenix theophrasti, a crucial genetic feature of the modern North African date palm populations. The P. theophrasti introgression fraction content is generally higher in the later samples, while introgression tracts are longer in these ancient germinated date palms compared to modern North African varieties. These results provide insights into crop evolution arising from an analysis of plants originating from ancient germinated seeds and demonstrate what can be accomplished with the application of a resurrection genomics approach.

Complete Genome Sequences of Pseudomonas atacamensis Strain SM1 and Pseudomonas toyotomiensis Strain SM2, Isolated from the Date Palm Rhizosphere.

Here, we announce the complete genome sequences of two phosphate-solubilizing rhizobacteria, Pseudomonas atacamensis strain SM1 (genome size, ∼5.9 Mb) and Pseudomonas toyotomiensis strain SM2 (genome size, ∼5.2 Mb), isolated from the rhizosphere of date palms growing in the oasis agroecosystem of the United Arab Emirates (UAE).

Large-scale genome sequencing reveals the driving forces of viruses in microalgal evolution.

Being integral primary producers in diverse ecosystems, microalgal genomes could be mined for ecological insights, but representative genome sequences are lacking for many phyla. We cultured and sequenced 107 microalgae species from 11 different phyla indigenous to varied geographies and climates. This collection was used to resolve genomic differences between saltwater and freshwater microalgae. Freshwater species showed domain-centric ontology enrichment for nuclear and nuclear membrane functions, while saltwater species were enriched in organellar and cellular membrane functions. Further, marine species contained significantly more viral families in their genomes (p = 8e-4). Sequences from Chlorovirus, Coccolithovirus, Pandoravirus, Marseillevirus, Tupanvirus, and other viruses were found integrated into the genomes of algal from marine environments. These viral-origin sequences were found to be expressed and code for a wide variety of functions. Together, this study comprehensively defines the expanse of protein-coding and viral elements in microalgal genomes and posits a unified adaptive strategy for algal halotolerance.

Complete Genome Sequence of Phytobacter diazotrophicus Strain UAEU22, a Plant Growth-Promoting Bacterium Isolated from the Date Palm Rhizosphere.

Here, we present a complete circular genome (5.4 Mb) and a plasmid (104,972 bp) of the plant growth-promoting bacterium Phytobacter diazotrophicus strain UAEU22, isolated from date palm rhizosphere in the United Arab Emirates (UAE). Annotation of the genome resulted in 5,229 predicted genes.

Complete Genome Sequence of the Plant Growth-Promoting Bacterium Pantoea agglomerans Strain UAEU18, Isolated from Date Palm Rhizosphere Soil in the United Arab Emirates.

We report the complete genome sequence of the plant growth-promoting bacterium Pantoea agglomerans strain UAEU18. Genome assembly of P. agglomerans strain UAEU18 resulted in a single gapless circular genome of 4.04 Mb, three associated plasmids (plasmid 1, 513,383 bp; plasmid 2, 86,850 bp; and plasmid 3, 184,488 bp), and a total of 4,556 gene models.

Genome-wide association mapping of date palm fruit traits.

Date palms (Phoenix dactylifera) are an important fruit crop of arid regions of the Middle East and North Africa. Despite its importance, few genomic resources exist for date palms, hampering evolutionary genomic studies of this perennial species. Here we report an improved long-read genome assembly for P. dactylifera that is 772.3 Mb in length, with contig N50 of 897.2 Kb, and use this to perform genome-wide association studies (GWAS) of the sex determining region and 21 fruit traits. We find a fruit color GWAS at the R2R3-MYB transcription factor VIRESCENS gene and identify functional alleles that include a retrotransposon insertion and start codon mutation. We also find a GWAS peak for sugar composition spanning deletion polymorphisms in multiple linked invertase genes. MYB transcription factors and invertase are implicated in fruit color and sugar composition in other crops, demonstrating the importance of parallel evolution in the evolutionary diversification of domesticated species.

Cross-species hybridization and the origin of North African date palms.

Date palm (Phoenix dactylifera L.) is a major fruit crop of arid regions that were domesticated ∼7,000 y ago in the Near or Middle East. This species is cultivated widely in the Middle East and North Africa, and previous population genetic studies have shown genetic differentiation between these regions. We investigated the evolutionary history of P. dactylifera and its wild relatives by resequencing the genomes of date palm varieties and five of its closest relatives. Our results indicate that the North African population has mixed ancestry with components from Middle Eastern P. dactylifera and Phoenix theophrasti, a wild relative endemic to the Eastern Mediterranean. Introgressive hybridization is supported by tests of admixture, reduced subdivision between North African date palm and P. theophrasti, sharing of haplotypes in introgressed regions, and a population model that incorporates gene flow between these populations. Analysis of ancestry proportions indicates that as much as 18% of the genome of North African varieties can be traced to P. theophrasti and a large percentage of loci in this population are segregating for single-nucleotide polymorphisms (SNPs) that are fixed in P. theophrasti and absent from date palm in the Middle East. We present a survey of Phoenix remains in the archaeobotanical record which supports a late arrival of date palm to North Africa. Our results suggest that hybridization with P. theophrasti was of central importance in the diversification history of the cultivated date palm.

Potential for Heightened Sulfur-Metabolic Capacity in Coastal Subtropical Microalgae.

The activities of microalgae support nutrient cycling that helps to sustain aquatic and terrestrial ecosystems. Most microalgal species, especially those from the subtropics, are genomically uncharacterized. Here we report the isolation and genomic characterization of 22 microalgal species from subtropical coastal regions belonging to multiple clades and three from temperate areas. Halotolerant strains including Halamphora, Dunaliella, Nannochloris, and Chloroidium comprised the majority of these isolates. The subtropical-based microalgae contained arrays of methyltransferase, pyridine nucleotide-disulfide oxidoreductase, abhydrolase, cystathionine synthase, and small-molecule transporter domains present at high relative abundance. We found that genes for sulfate transport, sulfotransferase, and glutathione S-transferase activities were especially abundant in subtropical, coastal microalgal species and halophytic species in general. Our metabolomics analyses indicate lineage- and habitat-specific sets of biomolecules implicated in niche-specific biological processes. This work effectively expands the collection of available microalgal genomes by ∼50%, and the generated resources provide perspectives for studying halophyte adaptive traits.

Mapping of HKT1;5 Gene in Barley Using GWAS Approach and Its Implication in Salt Tolerance Mechanism.

Sodium (Na+) accumulation in the cytosol will result in ion homeostasis imbalance and toxicity of transpiring leaves. Studies of salinity tolerance in the diploid wheat ancestor Triticum monococcum showed that HKT1;5-like gene was a major gene in the QTL for salt tolerance, named Nax2. In the present study, we were interested in investigating the molecular mechanisms underpinning the role of the HKT1;5 gene in salt tolerance in barley (Hordeum vulgare). A USDA mini-core collection of 2,671 barley lines, part of a field trial was screened for salinity tolerance, and a Genome Wide Association Study (GWAS) was performed. Our results showed important SNPs that are correlated with salt tolerance that mapped to a region where HKT1;5 ion transporter located on chromosome four. Furthermore, sodium (Na+) and potassium (K+) content analysis revealed that tolerant lines accumulate more sodium in roots and leaf sheaths, than in the sensitive ones. In contrast, sodium concentration was reduced in leaf blades of the tolerant lines under salt stress. In the absence of NaCl, the concentration of Na+ and K+ were the same in the roots, leaf sheaths and leaf blades between the tolerant and the sensitive lines. In order to study the molecular mechanism behind that, alleles of the HKT1;5 gene from five tolerant and five sensitive barley lines were cloned and sequenced. Sequence analysis did not show the presence of any polymorphism that distinguishes between the tolerant and sensitive alleles. Our real-time RT-PCR experiments, showed that the expression of HKT1;5 gene in roots of the tolerant line was significantly induced after challenging the plants with salt stress. In contrast, in leaf sheaths the expression was decreased after salt treatment. In sensitive lines, there was no difference in the expression of HKT1;5 gene in leaf sheath under control and saline conditions, while a slight increase in the expression was observed in roots after salt treatment. These results provide stronger evidence that HKT1;5 gene in barley play a key role in withdrawing Na+ from the xylem and therefore reducing its transport to leaves. Given all that, these data support the hypothesis that HKT1;5 gene is responsible for Na+ unloading to the xylem and controlling its distribution in the shoots, which provide new insight into the understanding of this QTL for salinity tolerance in barley.