Unveiling the population genetic structure of Iranian horses breeds by whole-genome resequencing analysis.

Preserving genetic diversity is pivotal for enhancing genetic improvement and facilitating adaptive responses to selection. This study focuses on identifying key genetic variants, including single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (INDELs), and copy number variants (CNVs), while exploring the genomic evolutionary connectedness among seven Iranian horses representing five indigenous breeds: Caspian, Turkemen, DareShuri, Kurdish, and Asil. Using whole-genome resequencing, we generated 2.7 Gb of sequence data, with raw reads ranging from 1.2 Gb for Caspian horses to 0.38 Gb for Turkoman horses. Post-filtering, approximately 1.9 Gb of reads remained, with ~ 1.5 Gb successfully mapped to the horse reference genome (EquCab3.0), achieving mapping rates between 76.4% (Caspian) and 98.35% (Turkoman). We identified 2,909,816 SNPs in Caspian horses, constituting around 0.1% of the genome. Notably, 71% of these SNPs were situated in intergenic regions, while 8.5 and 6.8% were located upstream and downstream, respectively. A comparative analysis of SNPs between Iranian and non-Iranian horse breeds showed that Caspian horses had the lowest number of shared SNPs with Turkoman horses. Instead, they showed a closer genetic relationship with DareShuri, Quarter, Arabian, Standardbred, and Asil breeds. Hierarchical clustering highlighted Caspian horses as a distinct cluster, underscoring their distinctive genomic signature. Caspian horses exhibit a unique genetic profile marked by an enrichment of private mutations in neurological genes, influencing sensory perception and awareness. This distinct genetic makeup shapes mating preferences and signifies a separate evolutionary trajectory. Additionally, significant non-synonymous single nucleotide polymorphisms (nsSNPs) in reproductive genes offer intervention opportunities for managing Caspian horses. These findings reveal the population genetic structure of Iranian horse breeds, contributing to the advancement of knowledge in areas such as conservation, performance traits, climate adaptation, reproduction, and resistance to diseases in equine science.

Comparative metabolomics of root-tips reveals distinct metabolic pathways conferring drought tolerance in contrasting genotypes of rice.

BACKGROUND: The mechanisms underlying rice root responses to drought during the early developmental stages are yet unknown. RESULTS: This study aimed to determine metabolic differences in IR64, a shallow-rooting, drought-susceptible genotype, and Azucena, a drought-tolerant and deep-rooting genotype under drought stress. The morphological evaluation revealed that Azucena might evade water stress by increasing the lateral root system growth, the root surface area, and length to access water. At the same time, IR64 may rely mainly on cell wall thickening to tolerate stress. Furthermore, significant differences were observed in 49 metabolites in IR64 and 80 metabolites in Azucena, for which most metabolites were implicated in secondary metabolism, amino acid metabolism, nucleotide acid metabolism and sugar and sugar alcohol metabolism. Among these metabolites, a significant positive correlation was found between allantoin, galactaric acid, gluconic acid, glucose, and drought tolerance. These metabolites may serve as markers of drought tolerance in genotype screening programs. Based on corresponding biological pathways analysis of the differentially abundant metabolites (DAMs), biosynthesis of alkaloid-derivatives of the shikimate pathway, fatty acid biosynthesis, purine metabolism, TCA cycle and amino acid biosynthesis were the most statistically enriched biological pathway in Azucena in drought response. However, in IR64, the differentially abundant metabolites of starch and sucrose metabolism were the most statistically enriched biological pathways. CONCLUSION: Metabolic marker candidates for drought tolerance were identified in both genotypes. Thus, these markers that were experimentally determined in distinct metabolic pathways can be used for the development or selection of drought-tolerant rice genotypes.

Lignocellulose degradation by rumen bacterial communities: New insights from metagenome analyses.

Ruminant animals house a dense and diverse community of microorganisms in their rumen, an enlarged compartment in their stomach, which provides a supportive environment for the storage and microbial fermentation of ingested feeds dominated by plant materials. The rumen microbiota has acquired diverse and functionally overlapped enzymes for the degradation of plant cell wall polysaccharides. In rumen Bacteroidetes, enzymes involved in degradation are clustered into polysaccharide utilization loci to facilitate coordinated expression when target polysaccharides are available. Firmicutes use free enzymes and cellulosomes to degrade the polysaccharides. Fibrobacters either aggregate lignocellulose-degrading enzymes on their cell surface or release them into the extracellular medium in membrane vesicles, a mechanism that has proven extremely effective in the breakdown of recalcitrant cellulose. Based on current metagenomic analyses, rumen Bacteroidetes and Firmicutes are categorized as generalist microbes that can degrade a wide range of polysaccharides, while other members adapted toward specific polysaccharides. Particularly, there is ample evidence that Verrucomicrobia and Spirochaetes have evolved enzyme systems for the breakdown of complex polysaccharides such as xyloglucans, peptidoglycans, and pectin. It is concluded that diversity in degradation mechanisms is required to ensure that every component in feeds is efficiently degraded, which is key to harvesting maximum energy by host animals.

Proteomic analysis of the meristematic root zone in contrasting genotypes reveals new insights in drought tolerance in rice.

Drought is responsible for major losses in rice production. Root tips contain meristematic and elongation zones that play major roles in determination of root traits and adaptive strategies to drought. In this study we analysed two contrasting genotypes of rice: IR64, a lowland, drought-susceptible, and shallow-rooting genotype; and Azucena, an upland, drought-tolerant, and deep-rooting genotype. Samples were collected of root tips of plants grown under control and water deficit stress conditions. Quantitative proteomics analysis resulted in the identification of 7294 proteins from the root tips of IR64 and 6307 proteins from Azucena. Data are available via ProteomeXchange with identifier PXD033343. Using a Partial Least Square Discriminant Analysis on 4170 differentially abundant proteins, 1138 statistically significant proteins across genotypes and conditions were detected. Twenty two enriched biological processes showing contrasting patterns between two genotypes in response to stress were detected through gene ontology enrichment analysis. This included identification of novel proteins involved in root elongation with specific expression patterns in Azucena, including four Expansins and seven Class III Peroxidases. We also detected an antioxidant network and a metallo-sulfur cluster assembly machinery in Azucena, with roles in reactive oxygen species and iron homeostasis, and positive effects on root cell cycle, growth and elongation.

Proteomics study reveals the molecular mechanisms underlying cryotolerance induced by mild sublethal stress in human sperm.

The preconditioning of human sperm with sublethal nitrosative stress before cryopreservation can potentially improve the thawed sperm quality. However, the underlying mechanisms behind this protective strategy are not entirely understood. We compared the cryosurvival of human sperm exposed to 0.01 µM nitric oxide (NO) throughout the cryopreservation and used multiplexed quantitative proteomics approach to identify changes in the proteome profile of preconditioned sperm cells. Semen samples were obtained from 30 normospermia donors and then each sample was divided into three equal parts: fresh (F), frozen-control (C), and frozen exposed to nitric oxide (NO). The sperm undergoing mild sublethal stress showed higher values for motility and viability compared to the frozen control sperm. Moreover, out of 2912 identified proteins, 248 proteins were detected as differentially abundant proteins (DAPs) between cryopreserved groups and fresh group (F) (p < 0.05). Gene ontology (GO) analysis of differentially abundant proteins indicated that the abundance of proteins associated with glycolysis, gluconeogenesis, and fertilization processes was reduced while oxidative phosphorylation pathway was increased in abundance in cryopreserved sperm compared to the fresh sperm. Moreover, redox protein such as thioredoxin 17 was increased in abundance in the NO group compared to the control freezing group. Therefore, the pre-conditioning of sperm prior to cryopreservation may play an important role in maintaining the redox balance in mitochondria of sperm after freezing. Overall, our results indicate that arylsulfatase A (ARSA), serine protease 37 (PRSS37), and sperm surface protein (SP17) may potentially serve as protein biomarkers associated with screening the fertilization potential of the thawed sperm.

Changes in root microbiome during wheat evolution.

BACKGROUND: Although coevolutionary signatures of host-microbe interactions are considered to engineer the healthy microbiome of humans, little is known about the changes in root-microbiome during plant evolution. To understand how the composition of the wheat and its ancestral species microbiome have changed over the evolutionary processes, we performed a 16S rRNA metagenomic analysis on rhizobacterial communities associated with a phylogenetic framework of four Triticum species T. urartu, T. turgidum, T. durum, and T. aestivum along with their ancestral species Aegilops speltoides, and Ae. tauschii during vegetative and reproductive stages. RESULTS: In this study, we illustrated that the genome contents of wild species Aegilops speltoides and Ae. tauschii can be significant factors determining the composition of root-associated bacterial communities in domesticated bread wheat. Although it was found that domestication and modern breeding practices might have had a significant impact on microbiome-plant interactions especially at the reproductive stage, we observed an extensive and selective control by wheat genotypes on associated rhizobacterial communities at the same time. Our data also showed a strong genotypic variation within species of T. aestivum and Ae. tauschii, suggesting potential breeding targets for plants surveyed. CONCLUSIONS: This study performed with different genotypes of Triticum and Aegilops species is the first study showing that the genome contents of Ae. speltoides and Ae. tauschii along with domestication-related changes can be significant factors determining the composition of root-associated bacterial communities in bread wheat. It is also indirect evidence that shows a very extensive range of host traits and genes are probably involved in host-microbe interactions. Therefore, understanding the wheat root-associated microbiome needs to take into consideration of its polygenetic mosaic nature.

The quest of cell surface markers for stem cell therapy.

Stem cells and their derivatives are novel pharmaceutics that have the potential for use as tissue replacement therapies. However, the heterogeneous characteristics of stem cell cultures have hindered their biomedical applications. In theory and practice, when cell type-specific or stage-specific cell surface proteins are targeted by unique antibodies, they become highly efficient in detecting and isolating specific cell populations. There is a growing demand to identify reliable and actionable cell surface markers that facilitate purification of particular cell types at specific developmental stages for use in research and clinical applications. The identification of these markers as very important members of plasma membrane proteins, ion channels, transporters, and signaling molecules has directly benefited from proteomics and tools for proteomics-derived data analyses. Here, we review the methodologies that have played a role in the discovery of cell surface markers and introduce cutting edge single cell proteomics as an advanced tool. We also discuss currently available specific cell surface markers for stem cells and their lineages, with emphasis on the nervous system, heart, pancreas, and liver. The remaining gaps that pertain to the discovery of these markers and how single cell proteomics and identification of surface markers associated with the progenitor stages of certain terminally differentiated cells may pave the way for their use in regenerative medicine are also discussed.

Mouse model of Alzheimer's disease demonstrates differential effects of early disease pathology on various brain regions.

Different parts of the brain are affected distinctively in various stages of the Alzheimer's disease (AD) pathogenesis. Identifying the biochemical changes in specific brain regions is key to comprehend the neuropathological mechanisms in early pre-symptomatic phases of AD. Quantitative proteomics profiling of four distinct areas of the brain of young APP/PS1 mouse model of AD was performed followed by biochemical pathway enrichment analysis. Findings revealed fundamental compositional and functional shifts even in the early stages of the disease. This novel study highlights unique proteome and biochemical pathway alterations in specific regions of the brain that underlie the early stages of AD pathology and will provide a framework for future longitudinal studies. The proteomics data were deposited into the ProteomeXchange Consortium via PRIDE with the identifier PXD019192.

Bi-functionalized aminoguanidine-PEGylated periodic mesoporous organosilica nanoparticles: a promising nanocarrier for delivery of Cas9-sgRNA ribonucleoproteine.

BACKGROUND: There is a great interest in the efficient intracellular delivery of Cas9-sgRNA ribonucleoprotein complex (RNP) and its possible applications for in vivo CRISPR-based gene editing. In this study, a nanoporous mediated gene-editing approach has been successfully performed using a bi-functionalized aminoguanidine-PEGylated periodic mesoporous organosilica (PMO) nanoparticles (RNP@AGu@PEG1500-PMO) as a potent and biocompatible nanocarrier for RNP delivery. RESULTS: The bi-functionalized MSN-based nanomaterials have been fully characterized using electron microscopy (TEM and SEM), nitrogen adsorption measurements, thermogravimetric analysis (TGA), X-ray powder diffraction (XRD), Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), and dynamic light scattering (DLS). The results confirm that AGu@PEG1500-PMO can be applied for gene-editing with an efficiency of about 40% as measured by GFP gene knockdown of HT1080-GFP cells with no notable change in the morphology of the cells. CONCLUSIONS: Due to the high stability and biocompatibility, simple synthesis, and cost-effectiveness, the developed bi-functionalized PMO-based nano-network introduces a tailored nanocarrier that has remarkable potential as a promising trajectory for biomedical and RNP delivery applications.

Plant isomiRs: origins, biogenesis, and biological functions.

MicroRNAs (miRNAs) are small endogenous non-coding RNAs in eukaryotes which regulate the expression of numerous genes post-transcriptionally, thereby playing critical roles in cells and organismal development. The high-throughput sequencing technologies enable the effective detection and annotation of miRNAs. Several miRNA variants with heterogeneous ends, lengths, and sequences can be generated from a single miRNA locus. Discovery of these miRNA variants, also known as miRNA isoforms or isomiRs, has made our understanding of the cells' miRNome deeper than previously pictured. Despite their wide presence in multiple datasets, the different possible origins and true biological significance of isomiRs are yet to be uncovered. Several recent emerging studies suggest that isomiRs are biologically active and non-randomly formed. This review aims to provide a comprehensive insight into the origins and biological importance of isomiRs, highlighting the enormous complexity of miRNA regulatory networks which broadens our knowledge about the post-transcriptional gene regulation in plants.

Human Proteome Project and Human Pluripotent Stem Cells: Odd Bedfellows or a Perfect Match?

The Chromosome-Centric Human Proteome Project (C-HPP) aims at the identification of missing proteins (MPs) and the functional characterization of functionally unannotated PE1 (uPE1) proteins. A major challenge in addressing this goal is that many human proteins and MPs are silent in adult cells. A promising approach to overcome such challenge is to exploit the advantage of novel tools such as pluripotent stem cells (PSCs), which are capable of differentiation into three embryonic germ layers, namely, the endoderm, mesoderm, and ectoderm. Here we present several examples of how the Human Y Chromosome Proteome Project (Y-HPP) benefited from this approach to meet C-HPP goals. Furthermore, we discuss how integrating CRISPR engineering, human-induced pluripotent stem cell (hiPSC)-derived disease modeling systems, and organoid technologies provides a unique platform for Y-HPP and C-HPP for MP identification and the functional characterization of human proteins, especially uPE1s.

SOX2 protein transduction directly converts human fibroblasts into oligodendrocyte-like cells.

Oligodendrocyte precursor cells (OPCs) are ideal therapeutic cells for treatment of spinal cord injuries and diseases that affect myelin. However, it is necessary to generate a cell population with a low risk of teratoma formation and oncogenesis from a patient's somatic cells. In this study, we investigated the direct reprogramming of fibroblasts to oligodendrocyte-like cells in one step with a safe non-genetic delivery method that used protein transduction. Cell morphology and the lineage-specific marker expression profile indicated that human foreskin fibroblasts (HFFs) were converted into oligodendrocyte-like cells by the application of pluripotency factors and the use of a permissible induction medium. Our data demonstrated that SOX2 was sufficient to directly drive OPC fate conversion from HFF by a genetic-free approach. Therefore, this work has provided a strategy to OPC reprogramming by a non-integrating approach for future use in disease modeling and may ultimately provide applications for patient-specific cell-based regenerative medicine.

Proteome analysis of endometrial tissue from patients with PCOS reveals proteins predicted to impact the disease.

Polycystic ovary syndrome (PCOS) is a complex disease that causes an ovulatory infertility in approximately 10% of reproductive-age women. We searched for candidate proteins that might contribute to endometrial receptivity defects in PCOS patients, and result in adverse reproductive outcomes. Shotgun proteomics approach was used to investigate the proteome profile of the endometrium at the luteal phase in PCOS patients compared to healthy fertile individuals. Biological process and pathway analyses were conducted to categorize the proteins with differential expressions. Confirmation was performed for a number of proteins via immunoblotting in new samples. 150 proteins with higher abundance, and 46 proteins with lower abundance were identified in the endometrial tissue from PCOS patients compared to healthy fertile individuals. The proteins with higher abundance were enriched in protein degradation, cell cycle, and signaling cascades. Proteins with lower abundance in PCOS patients were enriched in extracellular matrix (ECM) composition and function, as well as the salvage pathway of purine biosynthesis. Metabolism was the most affected biological process with over 100 up-regulated, and approximately 30 down-regulated proteins. Our results indicate significant imbalances in metabolism, proteasome, cell cycle, ECM related proteins, and signaling cascades in endometrial tissue of PCOS, which may contribute to poor reproductive outcomes in these patients. We postulate that the endometria in PCOS patients may not be well-differentiated and synchronized for implantation. Possible roles of the above-mentioned pathways that underlie implantation failure in PCOS will be discussed. Our findings need to be confirmed in larger populations.

Discovery of Novel Cell Surface Markers for Purification of Embryonic Dopamine Progenitors for Transplantation in Parkinson's Disease Animal Models.

Despite the progress in safety and efficacy of cell replacement therapy with pluripotent stem cells (PSCs), the presence of residual undifferentiated stem cells or proliferating neural progenitor cells with rostral identity remains a major challenge. Here we report the generation of a LIM homeobox transcription factor 1 alpha (LMX1A) knock-in GFP reporter human embryonic stem cell (hESC) line that marks the early dopaminergic progenitors during neural differentiation to find reliable membrane protein markers for isolation of midbrain dopaminergic neurons. Purified GFP positive cells in vitro exhibited expression of mRNA and proteins that characterized and matched the midbrain dopaminergic identity. Further quantitative proteomics analysis of enriched LMX1A+ cells identified several membrane-associated proteins including a polysialylated embryonic form of neural cell adhesion molecule (PSA-NCAM) and contactin 2 (CNTN2), enabling prospective isolation of LMX1A+ progenitor cells. Transplantation of human-PSC-derived purified CNTN2+ progenitors enhanced dopamine release from transplanted cells in the host brain and alleviated Parkinson's disease-related phenotypes in animal models. This study establishes an efficient approach for purification of large numbers of human-PSC-derived dopaminergic progenitors for therapeutic applications.

Plant-Microbe Symbiosis: What Has Proteomics Taught Us?

Beneficial microbes have a positive impact on the productivity and fitness of the host plant. A better understanding of the biological impacts and underlying mechanisms by which the host derives these benefits will help to address concerns around global food production and security. The recent development of omics-based technologies has broadened our understanding of the molecular aspects of beneficial plant-microbe symbiosis. Specifically, proteomics has led to the identification and characterization of several novel symbiosis-specific and symbiosis-related proteins and post-translational modifications that play a critical role in mediating symbiotic plant-microbe interactions and have helped assess the underlying molecular aspects of the symbiotic relationship. Integration of proteomic data with other "omics" data can provide valuable information to assess hypotheses regarding the underlying mechanism of symbiosis and help define the factors affecting the outcome of symbiosis. Herein, an update is provided on the current and potential applications of symbiosis-based "omic" approaches to dissect different aspects of symbiotic plant interactions. The application of proteomics, metaproteomics, and secretomics as enabling approaches for the functional analysis of plant-associated microbial communities is also discussed.

Down-Regulation of a Male-Specific H3K4 Demethylase, KDM5D, Impairs Cardiomyocyte Differentiation.

Despite the small number of Y chromosome genes, their adequate expression is required for regulation of transcription, translation, and protein stability in males, not just for sex determination. In addition to the role in male fertility, the Y chromosome has a significant role in the development and sexual dimorphism of healthy and disease phenotypes. We observed that KDM5D along with its X-counterpart, KDM5C, are up-regulated during the cardiac mesoderm stage of development. Down-regulation of KDM5D using siRNA resulted in accumulation of differentiating cells in the S-phase of the cell cycle and impaired progression to cardiomyocytes as reflected by an altered expression pattern of cardiac progenitor specific markers. Furthermore, while control cells started spontaneous beating at a normal physiological range on day 7 of differentiation induction, no spontaneous beating was observed in KDM5D down-regulated cells. Interestingly, the knockdown of KDM5D had no significant effect on the expression level of its X-counterpart, KDM5C. Thus, we suggest that KDM5D, in cooperation with its X homologue as a dose-sensitive gene, may have an important role in cardiomyocyte differentiation. Our study presents further evidence on the contribution of Y chromosome genes to sex-dependent development outside of sex determination.

Inhibition of Human Y Chromosome Gene, SRY, Promotes Naïve State of Human Pluripotent Stem Cells.

Although males and females have a variety of sexually dimorphic features related to hormonal effects, the genetic basis of dimorphism relies on early embryo development. Two pluripotent states, naïve and primed, emerge during early mammalian development. Identification of signaling pathways that induce differences between these two states can help to modulate conversion of primed cells to naïve cells. Naïve cells have a shorter doubling time and longer survival than their primed counterparts when passaged as single cells. In this study, we sought to explore the role of Y chromosome genes on human pluripotent stem cells (hPSCs) by investigating differential expressions of the male-specific region of the Y chromosome (MSY) genes in primed and naïve cells. Interestingly, we found that several MSY genes, including SRY, showed higher expression levels in primed compared to naïve human embryonic stem cells (hESCs). We hypothesize that SRY prevents WNT/ß-catenin signaling by its interaction and inhibition of ß-catenin activation in the nucleus. Results of the loss-of-function approach conducted by depletion of SRY indicated increased expressions of pluripotency marker genes and alkaline phosphatase (ALP) activity in the primed cells. SRY reduction was associated with overexpression of WNT signaling target genes AXIN2, Brachury, TCF1, TBX2, and TBX3. We suggest that inhibition of SRY may result in activation of ß-catenin and up-regulation of the WNT signaling pathway, both of which are important to naïve conversion.

200+ Protein Concentrations in Healthy Human Blood Plasma: Targeted Quantitative SRM SIS Screening of Chromosomes 18, 13, Y, and the Mitochondrial Chromosome Encoded Proteome.

This work continues the series of the quantitative measurements of the proteins encoded by different chromosomes in the blood plasma of a healthy person. Selected Reaction Monitoring with Stable Isotope-labeled peptide Standards (SRM SIS) and a gene-centric approach, which is the basis for the implementation of the international Chromosome-centric Human Proteome Project (C-HPP), were applied for the quantitative measurement of proteins in human blood plasma. Analyses were carried out in the frame of C-HPP for each protein-coding gene of the four human chromosomes: 18, 13, Y, and mitochondrial. Concentrations of proteins encoded by 667 genes were measured in 54 blood plasma samples of the volunteers, whose health conditions were consistent with requirements for astronauts. The gene list included 276, 329, 47, and 15 genes of chromosomes 18, 13, Y, and the mitochondrial chromosome, respectively. This paper does not make claims about the detection of missing proteins. Only 205 proteins (30.7%) were detected in the samples. Of them, 84, 106, 10, and 5 belonged to chromosomes 18, 13, and Y and the mitochondrial chromosome, respectively. Each detected protein was found in at least one of the samples analyzed. The SRM SIS raw data are available in the ProteomeXchange repository (PXD004374, PASS01192).

Salinity-associated microRNAs and their potential roles in mediating salt tolerance in rice colonized by the endophytic root fungus Piriformospora indica.

Piriformospora indica (P. indica), an endophytic root fungus, supports the growth and enhanced tolerance of plants to biotic and abiotic stresses. Several recent studies showed the significant role of small RNA (sRNA) molecules including microRNAs (miRNAs) in plant adaption to environmental stress, but little is known concerning the symbiosis-mediated salt stress tolerance regulated at miRNAs level. The overarching goal of this research is to elucidate the impact of miRNAs in regulating the P. indica-mediated salt tolerance in rice. Applying sRNA-seq analysis led to identify a set of 547 differentially abundant miRNAs in response to P. indica inoculation and salt stress. These included 206 rice-specific and 341 previously known miRNAs from other plant species. In silico analysis of miRNAs predictions of the differentially abundant miRNAs led to identifying of 193 putatively target genes, most of which were encoded either genes or transcription factors involved in nutrient uptake, sodium ion transporters, growth regulators, and auxin- responsive proteins. The rice-specific miRNAs targeted the transcription factors involved in the import of potassium ions into the root cells, the export of sodium ions, and plant growth and development. Interestingly, P. indica affected the differential abundance of miRNAs regulated genes and transcription factors linked to salt stress tolerance. Our data helps to understand the molecular basis of salt stress tolerance mediated by symbionts in plant and the potential impact of miRNAs for genetic improvement of rice varieties for tolerance to salt stress.

Transcriptomic analysis of Aegilops tauschii during long-term salinity stress.

Aegilops tauschii is the diploid progenitor of the bread wheat D-genome. It originated from Iran and is a source of abiotic stress tolerance genes. However, little is known about the molecular events of salinity tolerance in Ae. tauschii. This study investigates the leaf transcriptional changes associated with long-term salt stress. Total RNA extracted from leaf tissues of control and salt-treated samples was sequenced using the Illumina technology, and more than 98 million high-quality reads were assembled into 255,446 unigenes with an average length of 1398 bp and an N50 of 2269 bp. Functional annotation of the unigenes showed that 93,742 (36.69%) had at least a significant BLAST hit in the SwissProt database, while 174,079 (68.14%) showed significant similarity to proteins in the NCBI nr database. Differential expression analysis identified 4506 salt stress-responsive unigenes. Bioinformatic analysis of the differentially expressed unigenes (DEUs) revealed a number of biological processes and pathways involved in the establishment of ion homeostasis, signaling processes, carbohydrate metabolism, and post-translational modifications. Fine regulation of starch and sucrose content may be important features involved in salt tolerance in Ae. tauschii. Moreover, 82% of DEUs mapped to the D-subgenome, including known QTL for salt tolerance, and these DEUs showed similar salt stress responses in other accessions of Ae. tauschii. These results could provide fundamental insight into the regulatory process underlying salt tolerance in Ae. tauschii and wheat and facilitate identification of genes involved in their salt tolerance mechanisms.