Epigenetic diversity of Saccharum spp. accessions assessed by methylation-sensitive amplification polymorphism (MSAP).
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ABSTRACT:
SUBMITTER: Martins AA
PROVIDER: S-EPMC7246279 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Regeneration of protoplasts into de novo plants was reported for a large number of seaweed species. The regeneration of protoplasts into different morphotypes as a result of epigenetic variations was discussed for the first time in this study. The loci assessed for methylation modifications in normal filamentous thalli showed a frequency of 32.43% as unmethylated DNA, 24.32% as a hemimethylated, and 20.27% as a methylation of internal cytosine at both the strands. The corresponding methylation values for disk-type thalli were 27.02%, 32.43%, and 14.86%, respectively. The hypermethylation condition was apparent in the disk-type thalli with methylation ratio of 72.97% compared to that of normal filamentous thalli with 67.56%. The frequency of methylation polymorphic sites among the two morphotypes was 53%. The present study reveals the distinct expression of cytosine methylation and is thus correlated to differential morphogenesis of plants regenerated from cultured cells. The number of protoplasts regenerating into filamentous thalli declined with increasing temperature from 15°C, 20°C, 25°C, and 30°C. The disk-type variant had higher thermal stability at 30°C over normal filamentous thalli. Further, this variant could maintain itself for more than a year in the laboratory indicating its suitability for in vitro germplasm maintenance and propagation.
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Project description:BACKGROUND: DNA methylation plays a key role in development, contributes to genome stability, and may also respond to external factors supporting adaptation and evolution. To connect different types of stimuli with particular biological processes, identifying genome regions with altered 5-methylcytosine distribution at a genome-wide scale is important. Many researchers are using the simple, reliable, and relatively inexpensive Methylation Sensitive Amplified Polymorphism (MSAP) method that is particularly useful in studies of epigenetic variation. However, electrophoretic patterns produced by the method are rather difficult to interpret, particularly when MspI and HpaII isoschizomers are used because these enzymes are methylation-sensitive, and any C within the CCGG recognition motif can be methylated in plant DNA. RESULTS: Here, we evaluate MSAP patterns with respect to current knowledge of the enzyme activities and the level and distribution of 5-methylcytosine in plant and vertebrate genomes. We discuss potential caveats related to complex MSAP patterns and provide clues regarding how to interpret them. We further show that addition of combined HpaII?+?MspI digestion would assist in the interpretation of the most controversial MSAP pattern represented by the signal in the HpaII but not in the MspI profile. CONCLUSIONS: We recommend modification of the MSAP protocol that definitely discerns between putative hemimethylated mCCGG and internal CmCGG sites. We believe that our view and the simple improvement will assist in correct MSAP data interpretation.
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Project description:Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes.
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Project description:The stress-induced epimutations could be inherited over generations and play important roles in plant adaption to stressful environments. Upland rice has been domesticated in water-limited environments for thousands of years and accumulated drought-induced epimutations of DNA methylation, making it epigenetically differentiated from lowland rice. To study the epigenetic differentiation between upland and lowland rice ecotypes on their drought-resistances, the epigenetic variation was investigated in 180 rice landraces under both normal and osmotic conditions via methylation-sensitive amplified polymorphism (MSAP) technique. Great alterations (52.9~54.3% of total individual-locus combinations) of DNA methylation are recorded when rice encountering the osmotic stress. Although the general level of epigenetic differentiation was very low, considerable level of ?ST (0.134~0.187) was detected on the highly divergent epiloci (HDE). The HDE detected in normal condition tended to stay at low levels in upland rice, particularly the ones de-methylated in responses to osmotic stress. Three out of four selected HDE genes differentially expressed between upland and lowland rice under normal or stressed conditions. Moreover, once a gene at HDE was up-/down-regulated in responses to the osmotic stress, its expression under the normal condition was higher/lower in upland rice. This result suggested expressions of genes at the HDE in upland rice might be more adaptive to the osmotic stress. The epigenetic divergence and its influence on the gene expression should contribute to the higher drought-resistance in upland rice as it is domesticated in the water-limited environment.
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Project description:Sugarcane is an important crop for food and energy security, providing sucrose and bioethanol from sugar content and bioelectricity from lignocellulosic bagasse. In order to evaluate the diversity and genetic structure of the Brazilian Panel of Sugarcane Genotypes (BPSG), a core collection composed by 254 accessions of the Saccharum complex, eight TRAP markers anchored in sucrose and lignin metabolism genes were evaluated. A total of 584 polymorphic fragments were identified and used to investigate the genetic structure of BPSG through analysis of molecular variance (AMOVA), principal components analysis (PCA), a Bayesian method using STRUCTURE software, genetic dissimilarity and phylogenetic tree. AMOVA showed a moderate genetic differentiation between ancestors and improved accessions, 0.14, and the molecular variance was higher within populations than among populations, with values of 86%, 95% and 97% when constrasting improved with ancestors, foreign with ancestors and improved with foreign, respectively. The PCA approach suggests clustering in according with evolutionary and Brazilian breeding sugarcane history, since improved accessions from older generations were positioned closer to ancestors than improved accessions from recent generations. This result was also confirmed by STRUCTURE analysis and phylogenetic tree. The Bayesian method was able to separate ancestors of the improved accessions while the phylogenetic tree showed clusters considering the family relatedness within three major clades; the first being composed mainly by ancestors and the other two mainly by improved accessions. This work can contribute to better management of the crosses considering functional regions of the sugarcane genome.
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Project description:With the use of the isoschizomeric restriction endonucleases HpaII and MspI, we found that mouse tumour ornithine decarboxylase (ODC; EC 4.1.1.17) genes are extensively methylated. ODC genes in L1210 mouse leukaemia cells were apparently more methylated than in Ehrlich ascites carcinoma, as revealed by the use of HpaII endonuclease, yet the digestion of genomic DNA isolated from these two murine tumour cell lines with MspI, which cleaves at a CCGG sequence, also with internally methylated cytosine, resulted in an apparently identical restriction pattern. It is possible that the amplification of ODC genes in Ehrlich ascites-carcinoma cells in response to 2-difluoromethylornithine (DFMO) was associated with hypomethylation, or that less-methylated genes were amplified. A human myeloma (Sultan) cell line only revealed three separate hybridization signals when cleaved with HpaII. One of these signals was amplified under the pressure of DFMO. When cleaved with MspI, these three HpaII fragments disappeared and were replaced by a double signal of 2.3-2.4 kilobase-pairs (kbp) in size. The amplified ODC sequences in the Sultan myeloma cell line apparently originated from chromosome 2, as indicated by a unique hybridization signal in a 5.8 kbp HindIII fragment specific for the human ODC locus on chromosome 2. A comparison of different human cells, the Sultan myeloma, a lymphocytic B-cell leukaemia (Ball), normal mononuclear leucocytes and leucocytes obtained from leukaemia patients, revealed interesting differences in the methylation of ODC genes. The use of two restriction endonucleases (HpaII and CfoI), the cleavage site for both of which contains a CG sequence and which only cleave when cytosine is unmethylated, indicated that ODC genes in the lymphocytic leukaemia cells were much less methylated than those in the normal leucocytes or in the Sultan cells.
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Project description:BackgroundMalaria is one of the most important infectious diseases in the world. Although most cases are found distributed in the tropical regions of Africa, Asia, Central and South Americas, there is in Europe a significant increase in the number of imported cases in non-endemic countries, in particular due to the higher mobility in today's society.MethodsThe prevalence of a possible asymptomatic infection with Plasmodium species was assessed using Nucleic Acid Sequence Based Amplification (NASBA) assays on clinical samples collected from 195 study cases with no clinical signs related to malaria and coming from sub-Saharan African regions to Southern Italy. In addition, base-line demographic, clinical and socio-economic information was collected from study participants who also underwent a full clinical examination.ResultsSixty-two study subjects (31.8%) were found positive for Plasmodium using a pan Plasmodium specific NASBA which can detect all four Plasmodium species causing human disease, based on the small subunit 18S rRNA gene (18S NASBA). Twenty-four samples (38%) of the 62 18S NASBA positive study cases were found positive with a Pfs25 mRNA NASBA, which is specific for the detection of gametocytes of Plasmodium falciparum. A statistically significant association was observed between 18S NASBA positivity and splenomegaly, hepatomegaly and leukopaenia and country of origin.ConclusionThis study showed that a substantial proportion of people originating from malaria endemic countries harbor malaria parasites in their blood. If transmission conditions are available, they could potentially be a reservoir. Therefore, health authorities should pay special attention to the health of this potential risk group and aim to improve their health conditions.
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