Methylation-sensitive Amplification Polymorphism Research Articles

Soybean Methylation Analysis during Strontium Stress using Methylation-Sensitive Amplified Polymorphism

The effect of strontium stress on the pattern and degree of DNA methylation in soybean seedlings was analysed using the methylation-sensitive amplified polymorphism (MSAP) method. The growth traits were inhibited by SrCl2 treatments. A total of 167 loci were determined and evaluated for DNA methylation after different treatments. The level of cytosine methylation initially decreased and then increased with increasing Sr concentration. Methylation was lowest after 10 mmol/l SrCl2 treatment. Strontium stress resulted in a 57.48% alteration of DNA methylation patterns in 5′-CCGG-3′ loci. The pattern variation initially decreased and then increased along with increasing strontium concentration. There was a positive correlation between the total methylation and full methylation induced by strontium stress, and weight and length of shoots and roots in soybean. Overall, the changes in the pattern and degree of methylation may be a key regulatory mechanism for soybean adaptation to strontium.

Genomic and epigenetic alterations in diploid gynogenetic hybrid fish

Gynogenesis, as an effective technology to manipulate genome, is used fairly widely in aquaculture-related research. However, the knowledge regarding gynogenesis in allodiploid hybrid fish with the hybrid genome producing haploid gametes is sparse. This study focused on the analyses on the genomic and epigenetic alterations of the gynogenetic hybrids with improved trait. The mature eggs of the fourth generation (BTF4, 2n = 48) of allodiploid hybrid lineage of blunt snout bream (BSB, 2n = 48, Megalobrama amblycephala, ♀) × topmouth culter (TC, 2n = 48, Culter alburnus, ♂) were activated by UV-irradiated sperm from koi carp (Koi, 2n = 100, Cyprinus carpio haematopterus, ♂) and doubled by cold shock. The analyses of the DNA content and morphological observation confirmed that all of the progenies were gynogenetic diploids (GBTF4, 2n = 48), suggesting that the eggs of BTF4 possessed the hybrid genome. A genomic survey resulted from microsatellite indicated that 87.72% of the GBTF4 genome was the same as that of BTF4, 12.28% of the GBTF4 genome exhibited polymorphism compared with that of BTF4, and 0.03% of the GBTF4 genome was inherited from Koi. On the other hand, the results of methylation sensitive amplification polymorphism (MSAP) showed that the DNA methylation level (38.61%) in the GBTF4 genome was lower than that (40.99%) in the BTF4 genome. In addition, MSAP profiling also showed 30.1% methylation pattern changes in GBTF4 compared with BTF4 at 588 randomly selected CCGG sites. These results enriched our knowledge regarding the combination of hybridization and gynogenesis and the genomic and epigenetic alterations in allodiploid hybrid fish following process of gynogenesis, providing new insights into fish genetic breeding.

MSAP analysis of epigenetic changes reveals the mechanism of bicolor petal formation in Paeonia suffruticosa 'Shima Nishiki'.

Paeonia suffruticosa 'Shima Nishiki' is a very valuable bicolor cultivar because of its distinctive and colorful flowers. However, our understanding of the mechanisms underlying bicolor petal formation is limited. In this study, we used the methylation-sensitive amplified polymorphism (MSAP) method to assess the levels and pattern of cytosine methylation in different-colored petals during floral development. Our data showed differences in the methylation levels of red and pink petals. The methylation rate of the red petals was consistently higher than that of the pink petals, with maximum values of 58.45% (red petals) and 44.36% (pink petals) during the S2 developmental stage. However, obvious differences were not observed in the patterns of cytosine methylation in different-colored petals; methylation and demethylation occurred simultaneously and the proportions were similar. In addition, we isolated and sequenced the differentially methylated fragments and found that one fragment was homologous to the bHLH1 gene of P. suffruticosa 'Luoyang Hong'; its expression pattern suggested that the bHLH1 gene may be involved in the regulation of the formation of bicolor flowers in P. suffruticosa 'Shima Nishiki'. These results will provide a valuable resource for further investigation of the genetic mechanisms underlying bicolor petal formation in P. suffruticosa 'Shima Nishiki'.

Agromorphologic, genetic and methylation profiling of Dioscorea and Musa species multiplied under three micropropagation systems.

Plant in vitro vegetative propagation using classical semi-solid culture medium is limited due to the low degree of automation, suboptimal nutrient availability and induced physiological stress which often reduce its efficiency. Temporary Immersion System (TIS) emerged as an innovative approach to optimize and eliminate the drawbacks associated with the conventional system of micropropagation. In this study, both Dioscorea and Musa spp. were subjected to conventional semi-solid culture media, complete immersion in shaking liquid culture media and TIS using RITA bioreactor. In vitro grown plantlets were screened for possible vegetative changes using agro-morphological descriptors while genetic and methylation differences were assessed using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP). In vitro results showed that the number of shoots produced in Musa spp. varied significantly (P≤0.001) with the type of culture system. The highest mean shoot produced was observed with TIS (28.40) and the least using semi-solid culture medium (1.13). For Dioscorea spp., there was no significant interaction between the hormone combination and the culture system. However, the lowest mean shoot value (1.55) was observed in the semi-solid culture medium. Genetic analysis via AFLP using 15 primer pair combinations revealed that the 3 culture systems maintained genetic variation for Musa and Dioscorea spp. under in vitro and field conditions. Results showed 99% and 91% of the total bands were polymorphic under in vitro and field conditions respectively for Musa and 100% polymorphism for Dioscorea under in vitro and field conditions. Methylation investigation via MSAP using 12 primer pair combinations showed 25% and 46% polymorphic methylated-sensitive loci, 100% and 78% of non-methylated loci of the total bands generated under in vitro and field conditions respectively. Unmethylated (HPA+/MSP+) levels were highest in TIS (0.0842) as compared to CI (0.0227) and SS (0.0161) while full methylation or absence of target (HPA-/MSP-) was lowest in TIS (0.5890) and highest in SS (0.7138). For Dioscorea, 52% and 53% methylated sensitive loci and 100% non-methylated loci were polymorphic under in vitro and field conditions respectively. Although in vitro plant tissue culture techniques led to methylation at some loci of both species, there were no observable changes in the phenotype of both crops under field conditions. This also confirmed that not all methylation events lead to phenotypic changes.

Common garden experiment reveals altered nutritional values and DNA methylation profiles in micropropagated three elite Ghanaian sweet potato genotypes.

Micronutrient deficiency is the cause of multiple diseases in developing countries. Staple crop biofortification is an efficient means to combat such deficiencies in the diets of local consumers. Biofortified lines of sweet potato (Ipomoea batata L. Lam) with enhanced beta-carotene content have been developed in Ghana to alleviate Vitamin A Deficiency. These genotypes are propagated using meristem micropropagation to ensure the generation of virus-free propagules. In vitro culture exposes micropropagated plants to conditions that can lead to the accumulation of somaclonal variation with the potential to generate unwanted aberrant phenotypes. However, the effect of micropropagation induced somaclonal variation on the production of key nutrients by field-grown plants has not been previously studied. Here we assessed the extent of in vitro culture induced somaclonal variation, at a phenotypic, compositional and genetic/epigenetic level, by comparing field-maintained and micropropagated lines of three elite Ghanaian sweet potato genotypes grown in a common garden. Although micropropagated plants presented no observable morphological abnormalities compared to field maintained lines, they presented significantly lower levels of iron, total protein, zinc, and glucose. Methylation Sensitive Amplification Polymorphism analysis showed a high level of in vitro culture induced molecular variation in micropropagated plants. Epigenetic, rather than genetic variation, accounts for most of the observed molecular variability. Taken collectively, our results highlight the importance of ensuring the clonal fidelity of the micropropagated biofortified lines in order to reduce potential losses in the nutritional value prior to their commercial release.

The DNA methylation level is associated with the superior growth of the hybrid fry in snakehead fish (Channa argus × Channa maculata)

Hybrid vigour, or heterosis, refers to the increased productivity and growth rate of hybrid offsprings relative to the parents. Various heterosis have been well exploited in fish for fisheries. However, the molecular mechanisms underlying heterosis are largely unknown in fish. In this study, two inbred and hybrid lines between the northern snakehead (NS, Channa argus) and blotched snakehead (BS, Channa maculata) were generated. The analysis on various growth traits, including body length, head length, and body height, showed that hybrid fry obviously exhibited a spontaneous growth heterosis over the inbred. Moreover, the methylation-sensitive amplification polymorphism (MSAP) analysis revealed that the DNA methylation levels were negatively related to the body growth in all fry. Especially, the DNA methylation levels in the hybrid fry were significantly lower than those in the inbred. Additionally, qRT-PCR showed that the snakehead fish Dnmt3a mRNA was initially detectable in embryos at 12 hpf and gradually increased as developing. Intriguingly, the level of Dnmt3a mRNA expression was found to be closely correlated to the DNA methylation level in embryos/fry. The results of this study firstly demonstrated the correlations between growth heterosis, DNA methylation level and Dnmt3a mRNA expression in fish fry. The findings of this study implied that the hybrids' heterosis formation is probably accompanied by DNA methylation alterations and modulated by Dnmt3a gene in fish. This study would provide new clues for further investigations on mechanisms behind heterosis formation in fish hybrid.

Cadmium stress alters cytosine methylation status and expression of a select set of genes in Nicotiana benthamiana

In this paper, we evaluated the genotoxicity of cadmium (Cd) in plants by performing a methylation-sensitive amplification polymorphism (MSAP) on the model plant Nicotiana benthamiana. Among 255 loci examined, 14 genes were found to show altered cytosine methylation patterns in response to Cd stress. Four of those genes (NbMORC3, NbHGSNAT, NbMUT, and NbBG) were selected for further analysis due to their predicted roles in plant development. Cd-induced changes of cytosine methylation status in MSAP fragments of selected genes were confirmed using bisulfite sequencing polymerase chain reaction (BSP). In addition, the expression levels of these genes were found to correlate with cadmium dosage, and a knock-down of these four genes via virus-induced genes silencing (VIGS) led to abnormal development and elevated sensitivity to cadmium stress. Silencing of these four genes resulted in altered cadmium accumulation in different parts of the experimental plants. Our data indicate that cadmium exposure causes dramatic changes in the cytosine methylation status of the plant genome, thus affecting the expression of many genes that are vital for plant growth and are involved in cadmium stress response.

Chilling-stress modifies DNA methylation level in cucumber (Cucumis sativus L.) seedling radicle to regulate elongation rate

Seedlings have been used as an experimental model to understand plant response to chilling-stress. In this study, we investigated if the occurrence or severity of chilling injury correlated with genomic methylation state in cucumber radicles, using Methylation-Sensitive Amplification Polymorphism (MSAP) analysis. Radicles were chilled or treated with a methylation inhibitor, 5-azacytidine (AZA), or a combination thereof. Chilling cucumber radicles at 2.5 °C was marked by an immediate cessation in elongation and higher levels of genomic methylation. Rewarming was associated with a partial reversal of the methylation caused by chilling-stress, a decrease in genomic methylation to the same level as that before chilling, and to a resumption in radicle growth, but only to 18.6% of that without chilling. This cessation and recovery of elongation during and after chilling was also observed when radicles were treated with AZA. Under normal and rewarming conditions, 60% and 74% suppression of elongation were detected in AZA-treated radicles, respectively. Growth rate of the radicles was slowest in AZA-treated radicles after exposure to chilling-stress, suggesting a “double” suppressive effect due to the treatment combination. This was paralleled by methylation modifications at novel sites, with no change in the global methylation level, although the latter was expected to decrease as an effect of AZA. This work showed that DNA methylation is associated with, and may partially regulate, radicle elongation rate under chilling-stress, through a dynamic alteration of methylation pattern.

Analysis of genomic DNA methylation level in foxtail millet by Methylation Sensitive Amplified Polymorphism

DNA methylation is an important type of epigenetic modification in eukaryotes. In order to research genome-wide methylation levels and patterns in foxtail millet (Setaria italica), the Methylation Sensitive Amplified Polymorphism (MSAP) analysis (employing double digestion with EcoR I and Hpa II/Msp I) was established and applied in two foxtail millet cultivars (Chaogu 58 and Yugu 1). The results showed that 32 pairs of MSAP primers were selected from 100 MSAP primers, and 1 615 and 1 482 clearly distinguishable and reproducible bands were amplified from Chaogu 58 and Yugu 1 respectively, including 3 types of methylation patterns. Cytosine methylation levels of CCGG context in Chaogu 58 and Yugu 1 were characterized as 6.93% and 8.77% respectively. Such different genomic DNA methylation levels between two foxtail millet varieties may provide a preliminary reference for the cultivation of this crop from a novel epigenetic viewpoint.

Physiological, epigenetic and genetic regulation in some olive cultivars under salt stress

Cultivated olive, a typical fruit crop species of the semi-arid regions, could successfully face the new scenarios driven by the climate change through the selection of tolerant varieties to salt and drought stresses. In the present work, multidisciplinary approaches, including physiological, epigenetic and genetic studies, have been applied to clarify the salt tolerance mechanisms in olive. Four varieties (Koroneiki, Royal de Cazorla, Arbequina and Picual) and a related form (O. europaea subsp. cuspidata) were grown in a hydroponic system under different salt concentrations from zero to 200 mM. In order to verify the plant response under salt stress, photosynthesis, gas exchange and relative water content were measured at different time points, whereas chlorophyll and leaf concentration of Na+, K+ and Ca2+ ions, were quantified at 43 and 60 days after treatment, when stress symptoms became prominent. Methylation sensitive amplification polymorphism (MSAP) technique was used to assess the effects of salt stress on plant DNA methylation. Several fragments resulted differentially methylated among genotypes, treatments and time points. Real time quantitative PCR (RT-qPCR) analysis revealed significant expression changes related to plant response to salinity. Four genes (OePIP1.1, OePetD, OePI4Kg4 and OeXyla) were identified, as well as multiple retrotransposon elements usually targeted by methylation under stress conditions.

DNA METHYLATION AND FESOD GENE EXPRESSION AFFECTED BY PLANT DENSITY IN ZEA MAYS L.

Methylation Sensitive Amplification Polymorphism (MSAP) was used to characterize the alterations in DNA methylation in maize (Zea mays L.) inbred lines and their half-daillels affected by plant densities (213333 plant h-1 and 13333 plant h-1). The two restriction was enzymes ( HpaII and MspI) succeeded in diagnosing a total of 23 specific loci, most of (22 loci) were Methylation Sensitive Loci (MSL), while the only one NML (No Methylated Loci) was monomorphic. Thirteen out of 22 MSL loci polymorphic, recording a were polymorphism percentage of 59%. Results of FeSOD gene expression cleared the different response of maize inbreds and hybrids to high plant density stress. Generally, the expression of the targeted gene was increased in plants submitted to high plant density stress compared with low density. The inbred 3 and its single hybrid 1×3 achieved the highest level of gene expression under high planting density (5505.7 and 21098.6 copy, respectively), meanwhile, inbred 5 and it's single hybrid 4×5 gained the maximum level of FeSOD expression at the low plant density (8317.6 and 6862.1 copy, respectively). The response reached to its maximum limit in many of those genotypes, some other genotypes showed relatively steady performance along with higher stress, such as parent 1, that gave the lowest number of gene copies in both, high and low plant density (1375.8 and 1569.5 copy, respectively).

Changes in grapevine DNA methylation and polyphenols content induced by solar ultraviolet-B radiation, water deficit and abscisic acid spray treatments

Environment and crop management shape plant's phenotype. Argentinean high-altitude vineyards are characterized by elevated solar ultraviolet-B radiation (UVB) and water deficit (D) that enhance enological quality for red winemaking. These signals promote phenolics accumulation in leaves and berries, being the responses mediated by abscisic acid (ABA). DNA methylation is an epigenetic mechanism that regulates gene expression and may affect grapevine growth, development and acclimation, since methylation patterns are mitotically heritable. Berry skins low molecular weight polyphenols (LMWP) were characterized in field grown Vitis vinifera L. cv. Malbec plants exposed to contrasting UV-B, D, and ABA treatments during one season. The next season early fruit shoots were epigenetically (methylation-sensitive amplification polymorphism; MSAP) and biochemically (LMWP) characterized. Unstable epigenetic patterns and/or stochastic stress-induced methylation changes were observed. UV-B and D were the treatments that induced greater number of DNA methylation changes respect to Control; and UV-B promoted global hypermethylation of MSAP epiloci. Sequenced MSAP fragments associated with UV-B and ABA showed similarities with transcriptional regulators and ubiquitin ligases proteins activated by light. UV-B was associated with flavonols accumulation in berries and with hydroxycinnamic acids in the next season fruit shoots, suggesting that DNA methylation could regulate the LMWP accumulation and participate in acclimation mechanisms.

Methylation-sensitive amplified polymorphism analysis of resistant and susceptible interactions of cucumber with Podosphaera xanthii

Cytosine methylation alterations play key roles in gene regulation in plants interacting with biotrophic pathogens. For resistant and susceptible interactions of cucumber with the powdery mildew pathogen, Podosphaera xanthii, methylation of DNA cytosines were monitored by methylation-sensitive amplified polymorphism (MSAP) analysis from immediately prior to 96 h post inoculation (hpi). In both resistant and susceptible interactions, methylation levels significantly changed over time, but a significant association was observed for decreasing number of loci with demethylated CCGG motif in the susceptible cultivar compared to increasing numbers in the resistant cultivar. Sequencing of six demethylated MSAP fragments from the resistant cultivar and one from susceptible cultivar showed that all contained promoter and cis-acting regulatory elements (CAREs). For the resistant cultivar, three of these were in possible regulatory regions located within or near tRNA coding genes, and those three plus two others contained CAREs associated with response to biotic stress and defense hormones SA, MeJA, ABA and ET. One possible reason for demethylation in the resistant genomic tRNA genes may be that demethylation can de-suppress gene expression resulting in increasing the availability of certain amino acids for defense proteins.

Changes of DNA methylation of Isoetes sinensis under Pb and Cd stress.

To investigate the molecular response of ancient plants to heavy metal stress and to explore the feature of DNA methylation in endangered plants after exposure to heavy metals, the Isoetes sinensis, an endangered plant, was stressed with three different concentrations of two heavy metals lead (Pb) and cadmium (Cd), respectively. Then the degrees and the patterns of DNA methylation in the leaves were measured on the 14th day using Methylation Sensitive Amplified Polymorphism (MSAP) technique. The results showed that the DNA methylated profile of I. sinensis was affected by Pb and Cd stress. There was no significant difference in the amount of DNA methylation among control check (CK), Pb stress group, and Cd stress group (CK 46.96%, Pb 48.23%, and Cd 48.1%). However, full-methylation level of Pb stress group (28.34%) and Cd stress group (20.25%) was lower than control (33.91%), in contrast, hemi-methylation level Pb stress group (19.89%) and Cd stress group (27.85%) were higher than control (13.04%). The change of patterns from no methylation or hemi-methylation of internal and external cytosines into full-methylation of internal and external cytosines accounted for a large proportion in enhanced methylation aspects. The full-methylation into no methylation or hemi- or full-methylation of internal and external cytosines occupied most of demethylation. The proportion of DNA methylation (including hypermethylation) by both Pb and Cd stresses is nearly equal (39.04% and 39.71%), but the proportion of DNA demethylation by Cd is higher than that by Pb (46.86% than 33.92%).

Increased population epigenetic diversity of the clonal invasive species Alternanthera philoxeroides in response to salinity stress.

Epigenetic modification can change the pattern of gene expression without altering the underlying DNA sequence, which may be adaptive in clonal plant species. In this study, we used MSAP (methylation-sensitive amplification polymorphism) to examine epigenetic variation in Alternanthera philoxeroides, a clonal invasive species, in response to salinity stress. We found that salinity stress could significantly increase the level of epigenetic diversity within a population. This effect increased with increasing stress duration and was specific to particular genotypes. In addition, the epigenetic modification of young plants seems less sensitive to salinity than that of mature plants. This elevated epigenetic diversity in response to environmental stress may compensate for genetic impoverishment and contribute to evolutionary potential in clonal species.

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.

The variation of berry development and DNA methylation after treatment with 5-azaC on ‘Kyoho’ grape

DNA methylation has been proved to function in fruit ripening. The different concentrations of azacitidine (5-azaC), an inhibitor of methyltransferase, were sprayed on ‘Kyoho’ grape berries to explore the changes of DNA methylation, the effect of 5-azaC on berry ripening, and the relationship between DNA methylation and reactive oxygen species (ROS) metabolism during the grape berry development. The results showed that the treatment of 100 μM 5-azaC could promote the berry ripening of ‘Kyoho’ 20 days earlier than the control. The assessment of methylation by methylation sensitive amplification polymorphism (MSAP) markers after the treatment of 100 μM 5-azaC, demonstrated that the methylation rate of the treatment was all lower than the control at any berry development periods. 5-azaC treatments influenced the ROS metabolic activities and related physiological indexes of berries. The sucrose and soluble solid content of 5-azaC-treated berries were significantly higher than control at 75 dpa. The change trend of H2O2 content of the control was similar with the 100 μM 5-azaC treatment. All treatments enhanced rates of superoxide anion (O2−) production. Superoxide dismutase (SOD) activity in treated berries were higher than the control and had significant difference with the control at 35 dpa. The results demonstrated that 5-azaC could accelerate grape berries ripening, and there was a connection between ROS metabolism and DNA methylation during the grape berry development.

DNA methylation in lowbush blueberry (Vaccinium angustifolium Ait.) propagated by softwood cutting and tissue culture

Plant DNA methylation is one of the frequent epigenetic variations induced by tissue culture. Global DNA methylation was evaluated in lowbush blueberry (Vaccinium angustifolium Ait.) wild clone QB9C and cultivar Fundy propagated by conventional softwood cutting (SC) and tissue culture (TC) using the methylation-sensitive amplification polymorphism (MSAP) technique. In all, 106 and 107 DNA fragments were amplified using 16 selective primer combinations in SC plants of QB9C and Fundy, respectively. In micropropagated QB9C and Fundy plants, there were 105 and 109 amplified fragments, respectively. Overall, 25% of restriction sites were methylated at the cytosine nucleotide in QB9C plants propagated by SC compared with 19% in Fundy. In contrast, a total of 29% and 20% of restriction sites were methylated at cytosine in micropropagated QB9C and Fundy plants, respectively. Tissue culture plants demonstrated higher methylation events than SC plants in both genotypes. Previously, methylation polymorphism has been detected in TC plants but not in SC counterparts. Different patterns of DNA methylation and polymorphism in the plants propagated in in vitro and in vivo conditions suggest the possibility of involvement of these fragments in the processes of regulating plant growth and development under prevailing growth conditions.

Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence.

The geographical expansion of invasive species usually leads to temporary and/or permanent changes at multiple levels (genetics, epigenetics, gene expression, etc.) to acclimatize to abiotic and/or biotic stresses in novel environments. Epigenetic variation such as DNA methylation is often involved in response to diverse local environments, thus representing one crucial mechanism to promote invasion success. However, evidence is scant on the potential role of DNA methylation variation in rapid environmental response and invasion success during biological invasions. In particular, DNA methylation patterns and possible contributions of varied environmental factors to methylation differentiation have been largely unknown in many invaders, especially for invasive species in marine systems where extremely complex interactions exist between species and surrounding environments. Using the methylation‐sensitive amplification polymorphism (MSAP) technique, here we investigated population methylation structure at the genome level in two highly invasive model ascidians, Ciona robusta and C. intestinalis, collected from habitats with varied environmental factors such as temperature and salinity. We found high intrapopulation methylation diversity and significant population methylation differentiation in both species. Multiple analyses, such as variation partitioning analysis, showed that both genetic variation and environmental factors contributed to the observed DNA methylation variation. Further analyses found that 24 and 20 subepiloci were associated with temperature and/or salinity in C. robusta and C. intestinalis, respectively. All these results clearly showed significant methylation divergence among populations of both invasive ascidians, and varied local environmental factors, as well as genetic variation, were responsible for the observed DNA methylation patterns. The consistent findings in both species here suggest that DNA methylation, coupled with genetic variation, may facilitate local environmental adaptation during biological invasions, and DNA methylation variation molded by local environments may contribute to invasion success.

The influence of Al3+ on DNA methylation and sequence changes in the triticale (\xd7 Triticosecale Wittmack) genome

Abiotic stressors such as drought, salinity, and exposure to heavy metals can induce epigenetic changes in plants. In this study, liquid chromatography (RP-HPLC), methylation amplified fragment length polymorphisms (metAFLP), and methylation-sensitive amplification polymorphisms (MSAP) analysis was used to investigate the effects of aluminum (Al) stress on DNA methylation levels in the crop species triticale. RP-HPLC, but not metAFLP or MSAP, revealed significant differences in methylation between Al-tolerant (T) and non-tolerant (NT) triticale lines. The direction of methylation change was dependent on phenotype and organ. Al treatment increased the level of global DNA methylation in roots of T lines by approximately 0.6%, whereas demethylation of approximately 1.0% was observed in NT lines. DNA methylation in leaves was not affected by Al stress. The metAFLP and MSAP approaches identified DNA alterations induced by Al3+ treatment. The metAFLP technique revealed sequence changes in roots of all analyzed triticale lines and few mutations in leaves. MSAP showed that demethylation of CCGG sites reached approximately 3.97% and 3.75% for T and NT lines, respectively, and was more abundant than de novo methylation, which was observed only in two tolerant lines affected by Al stress. Three of the MSAP fragments showed similarity to genes involved in abiotic stress.