- This analysis showed that 77.0% of the contexts of DMSs were mCHH, while only 9.1% and 13.9% were mCHG and mCG, respectively. - However, collectively, the level of the DNA methylation in the gene body slightly decreased in response to salinity treatment. - In general, DNA methylation of the promoter regions often leads to reduced gene expression [17]. - Moreover plants can regulate the methylation level through demethylases of the DEMETER family such as those found in Arabidopsis [28]. - However, the global methylomic landscape, including the identity of the affected genes, the degree of methylation and the redistribution of each sequence context in the genome in response to salinity stress, were yet to be identified.. - analysis of the methylated genes of M. - Each pool of the DNA sample was considered as a single biological replicate which was used for WGBS following the previously implemented strategy in other plant species . - Bisulfite treatment of the fragments was performed using the EZ DNA Methylation- Lightning Kit (ZR, Cat. - After removal of the duplicate reads, unique best alignments were retained and the methylation ratios from the alignment file across the gene and the promoter regions were calculated. - In general, sites were categorized as hypermethylated if a site within the genome of the salinity- treated plant was significantly (p <. - 0.05) more methylated than the control, and were categorized as hypomethylated if a site within the genome of the salinity-- treated plant was significantly less methylated than the control.. - If the methylation ratio of a DMS of the control sample was higher than the methylation ratio of the same DMS of the NaCl-treated sample then the DMS was considered as a hypomethylated site in response to salinity treatment. - However, if the methylation ratio of a DMS of the control sample was lower than the methylation ratio of the same DMS of the NaCl-treated sample then. - The change in methylation level of each DMS for each sequence contexts was calculated separately based on the following formula: The methylation ratio of the control sample (C) was subtracted from the methylation ratio of the NaCl-treated sample (T) and the resulted number was divided by the methylation ratio of the control sample (C),. - The top 2000 sites with the highest methylation changes including strongly hyper- or hypomethylated at the mCG, mCHG and mCHH sites were functionally anno- tated based on the similarity of the protein-coding mRNA sequence to other proteins available in the Gen- Bank databases. - Mapping of the coding protein sequences within the metabolic path- ways was carried out using the Kyoto Encyclopedia of Genes and Genomes (KEGG) [38] tools, implemented within the Blast2GO PRO software.. - Identification of differentially methylated regions (DMRs) Pairwise comparison of the methylation profiles for the control and treated samples was carried out for different annotated gene regions. - The DMRs were calculated by subtracting the methylation ratio of a region within the genome of the control sample from the methylation ra- tio of the same region within the genome of the salinity treated sample. - In addition to the Nanodrop spectrophotometry, the quality and the quantity of the RNA were checked using 1% TAE agarose gel electrophor- esis. - To determine the statistical significance of the gene expression ratios, the qPCR data was analyzed using the online statistical analysis tool BootstRatio [45].. - The analysis resulted in a total of read pairs and unique mCGs obtained from sequencing DNA samples extracted from root tissues of the control and salinity-treated plants. - Differential methylation status of the mCG, mCHG and mCHH sites. - Genome-wide analysis of the 5-mCs was screened and scored based on the methylation ratios. - The analysis also showed that three-quarters of the identified context sequences were mCHH, while the rest were either mCHG or mCHH (Fig. - The statistical analysis showed that the majority (77.0%) of the significantly (p <. - Regard- less of the methylated sequence context, the DMSs showed. - This increase ranged between 3.8 to 10.2% in the methyla- tion level of the significantly (p <. - To obtain information about the identity of the strongly methylated/demethylated genes to a significant level (p <. - An overview of the DNA methylation levels using box plots showed that the methylation ratio at the mCG was the highest among the other methylated sequence con- texts (Fig. - However, the methy- lation levels of the mCHG at the coding regions were lower than those found in the promoters (Fig. - It was observed that the methylation ratios at the mCG of the promoters and the coding regions were slightly reduced in response to salinity (Fig. - This minor reduction in response to salinity was also observed in the mCHG and the mCHH of the promoter region (Fig. - The level of the 5-mCs within the putative functional genes and their promoters was collectively assessed in the genome extracted from NaCl-treated and control root tis- sues. - The results also showed that the DNA methylation level of the mCG was the highest among the sequence contexts, followed by mCHG and then mCHH. - The methylation profile of the promoter regions (upstream from the TSS) revealed that the DNA methylation for the mCG sequence context was relatively high, and this level was consistent with the flanked region of the transcript, yet this level de- creased in the center of the transcript before incrementally increasing to the topmost level in the region downstream from the TES region (Fig. - In comparison with the transcript region, the promoter regions at the mCHG and mCHH sites were relatively hypomethylated, yet the level of DNA methylation dramatically increased at the TSS region and subsequently declined in the rest of the transcript and toward the downstream regions of the TES (Fig. - In order to determine the distribution of the 5-mCs across the M. - truncatula genome and the effect of the salinity treatment on the level of DNA methylation, mCG, mCHG and mCHH sequence contexts of the eight chromosomes were estimated based on the ratio of reads reported as 5-mCs. - 1 The percentage of the total methylated sequence of each context (a) and the percentage of the significantly (p <. - acquired the highest level of methylation, while mCHH showed the lowest methylation level among the sequence contexts as indicated by the heights of the peaks in the scatterplot. - The whole chromosome plot of the mCG and mCHG sites also revealed that the level of DNA methylation reached the maximum level around the center of each chromosome however, mCHH showed an almost constant level of methylation across the chromosomes. - In this analysis, pairwise comparison of the methylation profiles did not show clear differences in the methylation levels between saline- treated and control samples at the chromosomal level.. - Circos plot demonstration of the hypo and hyper- methylated mCG, mCHG and mCHH regions within the exons, introns and promoters of the eight chromosomes showed that the mCG methylation was distributed at the highest densities and peaks heights among the other sequence contexts followed by mCHG (Fig. - The cir- cos plot showed also that the number of the strongly methylated DMRs localized within promoters was lower than those regions found in the exons and the intron re- gions (Fig. - Indeed, the sequence analysis of the DMRs showed that the number of strongly methylated mCG regions was higher than the regions embraced other sequence contexts (Table 3). - Since global methylation analysis of the genes and pro- moter region is unlikely to reveal a particular relation- ship between salinity treatment and DNA methylation changes, two groups of genes embraced highly DMSs (top 100 or 2000) were further investigated.. - Cluster analysis of the top 100 methylated sites. - 0.05) hyper- methylated in the genome extracted from plants grown under control conditions, the majority of the mCHH sites were hypermethylated in the genome extracted from plants exposed to salinity stress (Additional file 2:. - The results revealed that the mCHH context was the main methylated site within the promoters of the genes when plants were grown under normal conditions, while the mCG context was the main in the same region for those plants grown under salinity stress (Fig. - The methylation context mCG occupied the highest percentage among the contexts found in the exons of the DNA when plants were grown under normal conditions, while the methylated mCHG and mCHH contexts occupied the highest percentages among the contexts found in the exon regions in when exposed to salinity. - Regardless of the salinity treatment, the intron regions were enriched with mCHG sites (Fig. - However, for the majority of the annotated genes, the methylation level and distribution varied among the gene structures and methylation contexts. - For example, 18.2% of the hypomethylated mCG sites were located within the promoter, 33.6% within the exon and 25.2%. - Meanwhile, the position of 25.9% of the hypermethylated mCG was found within Table 2 A summary of the methylome data obtained from genome-wide profiling. - 35.5% of the sites were hypermethy- lated in both the promoter and exon regions (Fig. - On the other hand, 27.8% of the annotated genes were hypomethylated at the mCHG site located within the promoter, 19% within the exon and 26% within the intron region. - However, 38.4% of the genes were hyper- methylated in the exon region in response to salinity stress (Fig. - Interestingly, the highest percentage of hypomethylated promoter regions was within the mCHH context, where it accounted for 38.3% of the studied genes (Fig. - Functional annotation of the top 2000 methylated sites To obtain a comprehensive account of the function of those genes that were significantly altered by DNA methylation when M. - The functional an- notation results showed that most of the coding genes. - belonged to the functions of metabolism, oxidation- reduction and regulation of the transcription process (Additional file 3: Figure S2), and were involved in integral components of the membrane, nucleus, plasma and vacuolar membranes and endoplasmic reticulum (Additional file 4: Figure S3), mainly functioning in cellular protein, ion, ATP and nucleic acid-binding activ- ities (Additional file 5: Figure S4).. - This analysis was carried out separately for each of the mCG, mCHG and mCHH methylation contexts. - hypomethylated mCHG was enriched in the majority of the gene ontologies except DNA recombination and response to organonitrogen compound catabolic, hydrogen peroxide metabolic and reactive oxygen species metabolic processes (Fig. - However, hypermethylated mCHH was enriched in the majority of the gene ontologies except protein catabolic, terpenoid biosynthetic and cellular protein catabolic processes, and the mitochondrial protein complex cellular component (Fig. - 4 Chromosome-level view of the methylation percentage of different cytosine sequence contexts. - Functional annotation analysis of the significantly meth- ylated sequences showed that these genes encoded 1511 enzymes, including 532, 540 and 439 enzymes, mCG, mCHG and mCHH, respectively (Additional file 6: Tables S2, Additional file 7: Tables S3, Additional file 8: Tables S4). - Classification of the differentially methylated genes based on enzyme categories revealed these genes belonged to the hydrolase, isomerase, ligase, lyase, oxidoreductase and transferase enzyme classes (Additional file 9: Figure S5). - Therefore, there was a significant (p ≤ 0.05) increase of about 19.4% in 5mdC in the genomic DNA extracted from roots of the plants exposed to salinity.. - DNA methylation is part of the stress reaction that takes place in plants in response to suboptimal environmental Table 3 The number of DMRs observed after salinity treatment. - however, little information was provided regarding the identity of the methylated genes in response to salinity stress [31]. - 7 Pie distribution of the DMSs among the gene features of the top 2000 altered genes by methylation for the mCG (a), mCHG (b) and mCHH (c) sequence contexts. - Additionally, the align- ment is only as good as the reference genome and the quality of the mapped reads. - For example, mass spectrometry analysis of the Arabidopsis thaliana leaf samples grown under control environmental conditions revealed that 14.0% of total cytosines were methylated [51].. - Regardless of plant conditions, the WGBS analysis revealed that the number of the mCHH is highest among the methylated sequence contexts found in the M.. - For example, the relatively abundant proportion of the different methylated sequence contexts in M. - Global analysis of the chromosomes and the gene body methylation levels of the 5-mC showed that the mCG and mCHG sequence contexts had a higher ratio of methylation than the mCHH, but these methylation levels slightly reduced only at the gene body level in all sequence contexts in response to salinity treatment (Fig.. - For example, the presence of a high level of DNA methylation in the putative centro- mere and pericentromere regions of the chromosomes is consistent with other plant species, such as Arabidopsis [14, 59] and black cottonwood [60]. - related to the high probability of occurrence in the genome of the M. - truncatula due to the redundancy of the asymmetric CHH motif sequence. - However, these alterations are influenced by the location of the methylated region across the gene structures. - While hypermethylation at the mCG sites in promoter regions are known as gene silencing marks in eukaryotes, the mCHH sequence contexts are associated with the enrichment of the 24-nt small RNA species (sRNAs) and gene expression [13, 65]. - truncatula, based on the analysis of the top 2000 DMSs sites. - In addition, a fluctuation in the methylation status was observed at the mCHH sites of the promoter sequence of the ETHYLENE RESPONSIVE FACTOR 6, SUPPRESSION OF RVS 161 DELTA 4 and 3-KETOACYL-COA SYNTHASE 13 genes, which control cotton fiber growth [67]. - On the other hand, gene expres- sion analysis of methylated genes on mCG sites of the promoters and exons did not show a clear correlation between methylation status and transcriptome abundance (Fig. - This could be due to inappropriate sites of methy- lation within the promoter, which may not affect the affinity of the transcription factor to the binding site.. - The enrichment analysis of the top. - However, the gene ontology terms of the mCHG DMSs were enriched with various gene categories, whether the plants grew under control or saline conditions. - While the number of the mCG sites was the least and the num- ber of the mCHH sites was the most among the 5-mC identified in the M. - truncatula genome, the level of the methylation was inversely correlated with the number of each 5-mC sequence context found within the genome.. - Oligonucleotides used in the qPCR and the methylation status of the corresponding gene on different component.. - Clustering heat map analysis of the top 100 methylated sites based on DNA methylation levels of mCG, mCHG and mCHH DMSs. - Functional annotations of DMSs of the top 2000 altered genes for the mCG sequence context. - The annotations and gene ontologies were classified based on the biological process (A), cellular components (B) and molecular functions (C) of the annotated genes. - Functional annotations of the DMSs of the top 2000 altered genes for the mCHG sequence context. - Functional annotations of the DMSs of the top 2000 altered genes for the mCHH sequence context. - Classification of the enzymes coded by gene-harbored DMSs for the mCG (A), mCHG (B) and mCHH (C) contexts.. - Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. - A genome-wide identification of the miRNAome in response to salinity stress in date palm (Phoenix dactylifera L. - Medicago truncatula, a model plant for studying the molecular genetics of the Rhizobium-legume symbiosis. - Shotgun Bisulfite sequencing of the Betula Platyphylla genome reveals the Tree's DNA Methylation patterning.
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