Exome-wide association study to identify rare variants influencing COVID-19 outcomes: Results from the Host Genetics Initiative.

Host genetics is a key determinant of COVID-19 outcomes. Previously, the COVID-19 Host Genetics Initiative genome-wide association study used common variants to identify multiple loci associated with COVID-19 outcomes. However, variants with the largest impact on COVID-19 outcomes are expected to be rare in the population. Hence, studying rare variants may provide additional insights into disease susceptibility and pathogenesis, thereby informing therapeutics development. Here, we combined whole-exome and whole-genome sequencing from 21 cohorts across 12 countries and performed rare variant exome-wide burden analyses for COVID-19 outcomes. In an analysis of 5,085 severe disease cases and 571,737 controls, we observed that carrying a rare deleterious variant in the SARS-CoV-2 sensor toll-like receptor TLR7 (on chromosome X) was associated with a 5.3-fold increase in severe disease (95% CI: 2.75-10.05, p = 5.41x10-7). This association was consistent across sexes. These results further support TLR7 as a genetic determinant of severe disease and suggest that larger studies on rare variants influencing COVID-19 outcomes could provide additional insights.

DNA Demethylation in Response to Heat Stress in Arabidopsis thaliana.

Environmental stress is one of the most important factors affecting plant growth and development. Recent studies have shown that epigenetic mechanisms, such as DNA methylation, play a key role in adapting plants to stress conditions. Here, we analyzed the dynamics of changes in the level of DNA methylation in Arabidopsis thaliana (L.) Heynh. (Brassicaceae) under the influence of heat stress. For this purpose, whole-genome sequencing of sodium bisulfite-treated DNA was performed. The analysis was performed at seven time points, taking into account the control conditions, heat stress, and recovery to control conditions after the stress treatment was discontinued. In our study we observed decrease in the level of DNA methylation under the influence of heat stress, especially after returning to control conditions. Analysis of the gene ontology enrichment and regulatory pathways showed that genes characterized by differential DNA methylation are mainly associated with stress response, including heat stress. These are the genes encoding heat shock proteins and genes associated with translation regulation. A decrease in the level of DNA methylation in such specific sites suggests that under the influence of heat stress we observe active demethylation phenomenon rather than passive demethylation, which is not locus specific.

Response of rhizospheric and endophytic bacterial communities of white mustard (Sinapis alba) to bioaugmentation of soil with the Pseudomonas sp. H15 strain.

A factor that may significantly increase the efficacy of phytoextraction is soil bioaugmentation with specific bacteria, which can alter the composition of rhizospheric and endophytic bacterial communities. The aim of this study was to compare the effect of soil treatment with living (bioaugmentation) and dead (control) cells of the plant growth-promoting metal-resistant endophytic strain Pseudomonas sp. H15 on the bacterial community composition in the rhizo- and endo-sphere of white mustard during enhanced phytoextraction. The bacterial communities in the rhizosphere were dominated (51.7-68.2%) by Proteobacteria, regardless of the soil treatment or sampling point. A temporary increase in the number of sequences belonging to Gammaproteobacteria (up to 37.3%) was only observed 24 h after the soil treatment with living Pseudomonas sp. H15 cells, whereas for the remaining samples, the relative abundance of this class did not exceed 7.1%. The relative abundance of Proteobacteria in the endosphere of the roots, stems, and leaves of white mustard was higher in the control than in bioaugmented plants. The most pronounced dominance of the Gammaproteobacteria sequences was observed in the stems and leaves of the control plants at the first sampling point, which strongly indicates the ability of the plants to rapidly uptake DNA from soil and translocate it to the aboveground parts of the plants. Additionally, the bioaugmentation of the soil caused a diverse shift in the bacterial communities in the rhizo- and endo-sphere of white mustard compared to control. The most distinct differences, which were dependent on the treatment, were observed in the endosphere of plants at the beginning of the experiment and decreased over time. These results indicate that the rhizo- and endo-biome of white mustard reacts to soil bioaugmentation and may influence the efficiency of bacterial-assisted phytoextraction.

Molecular changes in vitellogenin gene of Spodoptera exigua after long-time exposure to cadmium - Toxic side effect or microevolution?

The reproduction of pest insects is a continuously ongoing issue, especially in the environmental pollution context. Natural or artificial stressing factors enforce a kind of trade-off, most often between growth/survival and reproduction, which improves fitness of the organism. Harmful factors, such as cadmium, can affect the vitellogenesis leading to reduction of yolk synthesis and egg production. The aim of this study was to assess whether 130-generational selection to cadmium in food might have induced change in vitellogenesis of Spodoptera exigua. We analyzed the level of Vg gene expression in S. exigua from the control and the cadmium strain at regular time intervals within 48h after eclosion. The full sequence of Vg gene was also compared between strains. The vitellogenin gene expression in both strains was time-dependent. This dependence was more visible in the control strain. In the cadmium strain the vitellogenin expression was significantly lower, comparing with the control strain in the first day after eclosion but increased significantly in the second day. The sequenced CDS (5286bp long) of the control and the cadmium strains were translated into protein sequences containing both 1761 aa. The protein sequences comparison revealed that there is one amino acid change at aa position 1282. Multiple alignments of six orthologous proteins from different species showed that amino acid change is located in the conserved position. Long-lasting exposure to cadmium resulted in permanent mutation in vitellogenin gene. We do not know yet if the mutation can improve fitness of the cadmium-selected insects. However, we can suppose that the mutation is neutral or even beneficial. The mutation and most probably additional effects of cadmium exposure have an influence on the vitellogenin expression. Some modification in the expression of the vitellogenin receptor are also likely to be important.

Water-deficiency conditions differently modulate the methylome of roots and leaves in barley (Hordeum vulgare L.).

One of the strategies of plant adaptation to stress is the modulation of gene expression, which may result from the regulation of DNA methylation. This study attempted to characterize and compare the barley methylome of leaves and roots under water-deficiency treatment and in the subsequent rewatering phase. Our results, obtained using methylation-sensitive amplification polymorphism sequencing analysis, indicated that the overall DNA methylation level in the barley genome was high and in general stable under water-deficiency conditions. Nevertheless, numerous differentially methylated sites (DMSs) were induced by stress in the leaves and roots. Equal proportions of novel stress-induced methylation and demethylation events were observed within the genes in the leaves, but new methylations dominated in the roots. Repetitive elements preferentially underwent demethylation in the leaves and novel methylations in the roots. Importantly, rewatering and plant recovery resulted in the reversibility of the majority of stress-induced methylation events, but this process was more efficient in the leaves than in the roots. Different biological processes were enriched within the subsets of the DMSs that were identified in the genic regions of leaves and roots. We assume that the organ specificity of the methylome changes in response to water deficiency might be an important regulatory mechanism that leads to multi-level mechanisms of stress tolerance in barley.

Transcriptome analysis reveals the role of the root hairs as environmental sensors to maintain plant functions under water-deficiency conditions.

An important part of the root system is the root hairs, which play a role in mineral and water uptake. Here, we present an analysis of the transcriptomic response to water deficiency of the wild-type (WT) barley cultivar 'Karat' and its root-hairless mutant rhl1.a. A comparison of the transcriptional changes induced by water stress resulted in the identification of genes whose expression was specifically affected in each genotype. At the onset of water stress, more genes were modulated by water shortage in the roots of the WT plants than in the roots of rhl1.a. The roots of the WT plants, but not of rhl1.a, specifically responded with the induction of genes that are related to the abscisic acid biosynthesis, stomatal closure, and cell wall biogenesis, thus indicating the specific activation of processes that are related to water-stress signalling and protection. On the other hand, the processes involved in the further response to abiotic stimuli, including hydrogen peroxide, heat, and high light intensity, were specifically up-regulated in the leaves of rhl1.a. An extended period of severe stress caused more drastic transcriptome changes in the roots and leaves of the rhl1.a mutant than in those of the WT. These results are in agreement with the much stronger damage to photosystem II in the rhl1.a mutant than in its parent cultivar after 10 d of water stress. Taking into account the putative stress sensing and signalling features of the root hair transcriptome, we discuss the role of root hairs as sensors of environmental conditions.

Ancient evolutionary trade-offs between yeast ploidy states.

The number of chromosome sets contained within the nucleus of eukaryotic organisms is a fundamental yet evolutionarily poorly characterized genetic variable of life. Here, we mapped the impact of ploidy on the mitotic fitness of baker's yeast and its never domesticated relative Saccharomyces paradoxus across wide swaths of their natural genotypic and phenotypic space. Surprisingly, environment-specific influences of ploidy on reproduction were found to be the rule rather than the exception. These ploidy-environment interactions were well conserved across the 2 billion generations separating the two species, suggesting that they are the products of strong selection. Previous hypotheses of generalizable advantages of haploidy or diploidy in ecological contexts imposing nutrient restriction, toxin exposure, and elevated mutational loads were rejected in favor of more fine-grained models of the interplay between ecology and ploidy. On a molecular level, cell size and mating type locus composition had equal, but limited, explanatory power, each explaining 12.5%-17% of ploidy-environment interactions. The mechanism of the cell size-based superior reproductive efficiency of haploids during Li(+) exposure was traced to the Li(+) exporter ENA. Removal of the Ena transporters, forcing dependence on the Nha1 extrusion system, completely altered the effects of ploidy on Li(+) tolerance and evoked a strong diploid superiority, demonstrating how genetic variation at a single locus can completely reverse the relative merits of haploidy and diploidy. Taken together, our findings unmasked a dynamic interplay between ploidy and ecology that was of unpredicted evolutionary importance and had multiple molecular roots.

iRootHair: a comprehensive root hair genomics database.

The specialized root epidermis cells of higher plants produce long, tubular outgrowths called root hairs. Root hairs play an important role in nutrient and water uptake, and they serve as a valuable model in studies of plant cell morphogenesis. More than 1,300 articles that describe the biological processes of these unique cells have been published to date. As new fields of root hair research are emerging, the number of new papers published each year and the volumes of new relevant data are continuously increasing. Therefore, there is a general need to facilitate studies on root hair biology by collecting, presenting, and sharing the available information in a systematic, curated manner. Consequently, in this paper, we present a comprehensive database of root hair genomics, iRootHair, which is accessible as a Web-based service. The current version of the database includes information about 153 root hair-related genes that have been identified to date in dicots and monocots along with their putative orthologs in higher plants with sequenced genomes. In order to facilitate the use of the iRootHair database, it is subdivided into interrelated, searchable sections that describe genes, processes of root hair formation, root hair mutants, and available references. The database integrates bioinformatics tools with a focus on sequence identification and annotation. iRootHair is a unique resource for root hair research that integrates the large volume of data related to root hair genomics in a single, curated, and expandable database that is freely available at www.iroothair.org.

Circulating serum miR-362-3p and miR-6721-5p as potential biomarkers for classification patients with adult-type diffuse glioma.

According to the fifth edition of the WHO Classification of Tumours of the Central Nervous System (CNS) published in 2021, grade 4 gliomas classification includes IDH-mutant astrocytomas and wild-type IDH glioblastomas. Unfortunately, despite precision oncology development, the prognosis for patients with grade 4 glioma remains poor, indicating an urgent need for better diagnostic and therapeutic strategies. Circulating miRNAs besides being important regulators of cancer development could serve as promising diagnostic biomarkers for patients with grade 4 glioma. Here, we propose a two-miRNA miR-362-3p and miR-6721-5p screening signature for serum for non-invasive classification of identified glioma cases into the highest-grade 4 and lower-grade gliomas. A total of 102 samples were included in this study, comprising 78 grade 4 glioma cases and 24 grade 2-3 glioma subjects. Using the NanoString platform, seven miRNAs were identified as differentially expressed (DE), which was subsequently confirmed via RT-qPCR analysis. Next, numerous combinations of DE miRNAs were employed to develop classification models. The dual panel of miR-362-3p and miR-6721-5p displayed the highest diagnostic value to differentiate grade 4 patients and lower grade cases with an AUC of 0.867. Additionally, this signature also had a high AUC = 0.854 in the verification cohorts by RT-qPCR and an AUC = 0.842 using external data from the GEO public database. The functional annotation analyses of predicted DE miRNA target genes showed their primary involvement in the STAT3 and HIF-1 signalling pathways and the signalling pathway of pluripotency of stem cells and glioblastoma-related pathways. For additional exploration of miRNA expression patterns correlated with glioma, we performed the Weighted Gene-Co Expression Network Analysis (WGCNA). We showed that the modules most associated with glioma grade contained as many as six DE miRNAs. In conclusion, this study presents the first evidence of serum miRNA expression profiling in adult-type diffuse glioma using a classification based on the WHO 2021 guidelines. We expect that the discovered dual miR-362-3p and miR-6721-5p signatures have the potential to be utilised for grading gliomas in clinical applications.

GestaltMatcher Database - A global reference for facial phenotypic variability in rare human diseases.

The most important factor that complicates the work of dysmorphologists is the significant phenotypic variability of the human face. Next-Generation Phenotyping (NGP) tools that assist clinicians with recognizing characteristic syndromic patterns are particularly challenged when confronted with patients from populations different from their training data. To that end, we systematically analyzed the impact of genetic ancestry on facial dysmorphism. For that purpose, we established the GestaltMatcher Database (GMDB) as a reference dataset for medical images of patients with rare genetic disorders from around the world. We collected 10,980 frontal facial images - more than a quarter previously unpublished - from 8,346 patients, representing 581 rare disorders. Although the predominant ancestry is still European (67%), data from underrepresented populations have been increased considerably via global collaborations (19% Asian and 7% African). This includes previously unpublished reports for more than 40% of the African patients. The NGP analysis on this diverse dataset revealed characteristic performance differences depending on the composition of training and test sets corresponding to genetic relatedness. For clinical use of NGP, incorporating non-European patients resulted in a profound enhancement of GestaltMatcher performance. The top-5 accuracy rate increased by +11.29%. Importantly, this improvement in delineating the correct disorder from a facial portrait was achieved without decreasing the performance on European patients. By design, GMDB complies with the FAIR principles by rendering the curated medical data findable, accessible, interoperable, and reusable. This means GMDB can also serve as data for training and benchmarking. In summary, our study on facial dysmorphism on a global sample revealed a considerable cross ancestral phenotypic variability confounding NGP that should be counteracted by international efforts for increasing data diversity. GMDB will serve as a vital reference database for clinicians and a transparent training set for advancing NGP technology.

Automatic analysis of 2D polyacrylamide gels in the diagnosis of DNA polymorphisms.

INTRODUCTION: The analysis of polyacrylamide gels is currently carried out manually or automatically. In the automatic method, there are limitations related to the acceptable degree of distortion of lane and band continuity. The available software cannot deal satisfactorily with this type of situations. Therefore, the paper presents an original image analysis method devoid of the aforementioned drawbacks. MATERIAL: This paper examines polyacrylamide gel images from Li-Cor DNA Sequencer 4300S resulting from the use of the electrophoretic separation of DNA fragments. The acquired images have a resolution dependent on the length of the analysed DNA fragments and typically it is MG×NG=3806×1027 pixels. The images are saved in TIFF format with a grayscale resolution of 16 bits/pixel. The presented image analysis method was performed on gel images resulting from the analysis of DNA methylome profiling in plants exposed to drought stress, carried out with the MSAP (Methylation Sensitive Amplification Polymorphism) technique. RESULTS: The results of DNA polymorphism analysis were obtained in less than one second for the Intel Core™ 2 Quad CPU Q9300@2.5GHz, 8GB RAM. In comparison with other known methods, specificity was 0.95, sensitivity = 0.94 and AUC (Area Under Curve) = 0.98. CONCLUSIONS: It is possible to carry out this method of DNA polymorphism analysis on distorted images of polyacrylamide gels. The method is fully automatic and does not require any operator intervention. Compared with other methods, it produces the best results and the resulting image is easy to interpret. The presented method of measurement is used in the practical analysis of polyacrylamide gels in the Department of Genetics at the University of Silesia in Katowice, Poland.

Identification of serum miR-1246 and miR-150-5p as novel diagnostic biomarkers for high-grade serous ovarian cancer.

Epithelial ovarian cancer (EOC) is one of the leading cancers in women, with high-grade serous ovarian cancer (HGSOC) being the most common and lethal subtype of this disease. A vast majority of HGSOC are diagnosed at the late stage of the disease when the treatment and total recovery chances are low. Thus, there is an urgent need for novel, more sensitive and specific methods for early and routine HGSOC clinical diagnosis. In this study, we performed miRNA expression profiling using the NanoString miRNA assay in 34 serum samples from patients with HGSOC and 36 healthy women. We identified 13 miRNAs that were differentially expressed (DE). For additional exploration of expression patterns correlated with HGSOC, we performed weighted gene co-expression network analysis (WGCNA). As a result, we showed that the module most correlated with tumour size, nodule and metastasis contained 8 DE miRNAs. The panel including miR-1246 and miR-150-5p was identified as a signature that could discriminate HGSOC patients with AUCs of 0.98 and 1 for the training and test sets, respectively. Furthermore, the above two-miRNA panel had an AUC = 0.946 in the verification cohorts of RT-qPCR data and an AUC = 0.895 using external data from the GEO public database. Thus, the model we developed has the potential to markedly improve the diagnosis of ovarian cancer.

Development of microsatellite markers using pyrosequencing in Galium trifidum (Rubiaceae), a rare species in Central Europe.

We identify a large number of microsatellites from Galium trfidum, a plant species considered rare and endangered in Central and Western Europe. Using a combination of a total enriched genomic library and small-scale 454 pyrosequencing, we determined 9755 contigs with a length of 100 to 6192 bp. Within this dataset, we identified 153 SSR motifs in 144 contigs. Here, we tested 14 microsatellite loci in 2 populations of G. trifidum. The number of alleles and expected heterozygosity were 1-8 (mean 3.2) and 0.00-0.876 (0.549 on average), respectively. The markers described in this study will be useful for evaluating genetic diversity within and between populations, and gene flow between G. trifidum populations. These markers could also be applied to investigate the biological aspects of G. trifidum, such as the population dynamics and clonal structure, and to develop effective conservation programs for the Central European populations of this species.

Isolation and characterization of Simple Sequence Repeats (SSR) Markers from the moss genus Orthotrichum using a small throughput pyrosequencing machine.

Here, we report the results of next-generation sequencing on the GS Junior system to identify a large number of microsatellites from the epiphytic moss Orthotrichum speciosum. Using a combination of a total (non-enrichment) genomic library and small-scale 454 pyrosequencing, we determined 5382 contigs whose length ranged from 103 to 5445 bp. In this dataset we identified 92 SSR (simple sequence repeats) motifs in 89 contigs. Forty-six of these had flanking regions suitable for primer design. We tested PCR amplification, reproducibility, and the level of polymorphism of 46 primer pairs for Orthotrichum speciosum using 40 individuals from two populations. As a result, the designed primers revealed 35 polymorphic loci with more than two alleles detected. This method is cost- and time-effective in comparison with traditional approaches involving cloning and sequencing.

Insights into the Histone Acetylation-Mediated Regulation of the Transcription Factor Genes That Control the Embryogenic Transition in the Somatic Cells of Arabidopsis.

Somatic embryogenesis (SE), which is a process that involves the in vitro-induced embryogenic reprogramming of plant somatic cells, requires dynamic changes in the cell transcriptome. These changes are fine-tuned by many genetic and epigenetic factors, including posttranslational histone modifications such as histone acetylation. Antagonistically acting enzymes, histone acetyltransferases (HATs) and deacetylases (HDACs), which control histone acetylation in many developmental processes, are believed to control SE. However, the function of specific HAT/HDACs and the genes that are subjected to histone acetylation-mediated regulation during SE have yet to be revealed. Here, we present the global and gene-specific changes in histone acetylation in Arabidopsis explants that are undergoing SE. In the TSA (trichostatin A)-induced SE, we demonstrate that H3 and H4 acetylation might control the expression of the critical transcription factor (TF) genes of a vital role in SE, including LEC1, LEC2 (LEAFY COTYLEDON 1; 2), FUS3 (FUSCA 3) and MYB118 (MYB DOMAIN PROTEIN 118). Within the HATs and HDACs, which mainly positively regulate SE, we identified HDA19 as negatively affecting SE by regulating LEC1, LEC2 and BBM. Finally, we provide some evidence on the role of HDA19 in the histone acetylation-mediated regulation of LEC2 during SE. Our results reveal an essential function of histone acetylation in the epigenetic mechanisms that control the TF genes that play critical roles in the embryogenic reprogramming of plant somatic cells. The results implicate the complexity of Hac-related gene regulation in embryogenic induction and point to differences in the regulatory mechanisms that are involved in auxin- and TSA-induced SE.

Case report: Rare among ultrarare-Clinical odyssey of a new patient with Ogden syndrome.

Introduction: The definition of ultra-rare disease in terms of its prevalence varies between the sources, usually amounting to ca. 1 in 1.000.000 births. Nonetheless, there are even less frequent disorders, such as Ogden syndrome, which up to this day was diagnosed in less than 10 patients worldwide. They present typically with a variety of developmental defects, including postnatal growth retardation, psychomotor delay and hypotonia. This disorder is caused by the heterozygous mutations in NAA10 gene, which encodes N-alpha-acetyltransferase 10, involved in protein biosynthesis. Therefore, Ogden syndrome belongs to the broader group of genetic disorders, collectively described as NAA10-related syndrome. Case report: We present a case of a Polish male infant, born in 39. GW with c-section due to the pathological cardiotocography signal. Hypotrophy (2400 g) and facial dysmorphism were noted in the physical examination. From the first minute, the child required mechanical ventilation - a nasal continuous positive airway pressure. For the first 27 days, the patient was treated in a neonatal intensive care unit, where a series of examinations were conducted. On their basis, the presence of the following defects was determined: muscular ventricular septal defects, patent foramen ovale, pectus excavatum, clubfoot and axial hypotonia. Child was then consequently referred to the genetic clinic for counselling. Results of the tests allowed the diagnosis of Ogden syndrome. In the following months the patient's condition worsened due to the numerous pulmonary infections. Despite the advanced treatment including the variety of medications, the patient eventually died at the age of 10 months. Conclusion: This case report presents a tenth patient diagnosed with Ogden syndrome reported worldwide. It expands the morphologic and clinical phenotype, emphasizing the possible severity of pneumonological disorders in these patients, which may pose a greater threat to a child's life than more frequently described cardiovascular dysfunctions associated with this syndrome.

Do Not Miss the (Genetic) Diagnosis of Gaucher Syndrome: A Narrative Review on Diagnostic Clues and Management in Severe Prenatal and Perinatal-Lethal Sporadic Cases.

With a growing number of proved therapies and clinical trials for many lysosomal storage disorders (LSDs), a lot of hope for many patients and families exists. However, there are sometimes cases with poor prognosis, fatal outcomes when our efforts must be directed towards a prompt and correct genetic diagnosis, which offers the only possibility of providing the family with appropriate prevention and treatment. To address this issue, in this article, we present the clinical and genetic hallmarks of the lethal form of Gaucher disease (PLGD) and discuss the potential management. We hope that this will draw attention to its specific manifestations (such as collodion-baby phenotype, ichthyosis, arthrogryposis), which differ from best-known GD complications and ensure appropriate diagnostic assessment to provide families at risk with reliable counselling and treatment to avoid the medical complication of GD.

Aluminum or Low pH - Which Is the Bigger Enemy of Barley? Transcriptome Analysis of Barley Root Meristem Under Al and Low pH Stress.

Aluminum (Al) toxicity is considered to be the most harmful abiotic stress in acidic soils that today comprise more than 50% of the world's arable lands. Barley belongs to a group of crops that are most sensitive to Al in low pH soils. We present the RNA-seq analysis of root meristems of barley seedlings grown in hydroponics at optimal pH (6.0), low pH (4.0), and low pH with Al (10 µM of bioavailable Al3+ ions). Two independent experiments were conducted: with short-term (24 h) and long-term (7 days) Al treatment. In the short-term experiment, more genes were differentially expressed (DEGs) between root meristems grown at pH = 6.0 and pH = 4.0, than between those grown at pH = 4.0 with and without Al treatment. The genes upregulated by low pH were associated mainly with response to oxidative stress, cell wall organization, and iron ion binding. Among genes upregulated by Al, overrepresented were those related to response to stress condition and calcium ion binding. In the long-term experiment, the number of DEGs between hydroponics at pH = 4.0 and 6.0 were lower than in the short-term experiment, which suggests that plants partially adapted to the low pH. Interestingly, 7 days Al treatment caused massive changes in the transcriptome profile. Over 4,000 genes were upregulated and almost 2,000 genes were downregulated by long-term Al stress. These DEGs were related to stress response, cell wall development and metal ion transport. Based on our results we can assume that both, Al3+ ions and low pH are harmful to barley plants. Additionally, we phenotyped the root system of barley seedlings grown in the same hydroponic conditions for 7 days at pH = 6.0, pH = 4.0, and pH = 4.0 with Al. The results correspond to transcriptomic data and show that low pH itself is a stress factor that causes a significant reduction of root growth and the addition of aluminum further increases this reduction. It should be noted that in acidic arable lands, plants are exposed simultaneously to both of these stresses. The presented transcriptome analysis may help to find potential targets for breeding barley plants that are more tolerant to such conditions.

DNA Methylation Analysis in Barley and Other Species with Large Genomes.

Detailed DNA methylation analyses in plant species with large and highly repetitive genomes can be challenging as well as costly. Here, we describe a complete protocol for a high-throughput DNA methylation changes analysis using Methylation-Sensitive Amplification Polymorphism Sequencing (MSAP-Seq; Chwialkowska et al., Front Plant Sci. 8: 2056 (2017)). This method allows detailed information about DNA methylation changes in large and complex genomes to be obtained at a relatively low cost. MSAP-Seq is based on conventional MSAP marker analysis and employs all its basic steps such as restriction cleavage with methylation-sensitive restriction enzyme, ligation of universal adapters, and PCR amplification. However, the traditional gel-based amplicon separation is replaced by direct, global sequencing with next-generation sequencing (NGS) methods. Consequently, MSAP-Seq allows for parallel analysis of hundreds of thousands of different CCGG sites and evaluation of their DNA methylation state. This technique especially targets to genic regions, so it is well suited for large genomes with low gene density, such as barley and other plants with large genomes.