Methylation-sensitive Amplified Polymorphism Analysis Research Articles

The Mutational, Epigenetic, and Transcriptional Effects Between Mixed High-Energy Particle Field (CR) and 7Li-Ion Beams (LR) Radiation in Wheat M1 Seedlings.

Ionizing radiation (IR) is an effective approach for mutation breeding. Understanding the mutagenesis and transcriptional profiles induced by different mutagens is of great significance for improving mutation breeding efficiency. Here, using RNA sequencing and methylation-sensitive amplification polymorphism (MSAP) approaches, we compared the genetic variations, epigenetics, and transcriptional responses induced by the mixed high-energy particle field (CR) and 7Li-ion beam (LR) radiation in M1 seedlings of two wheat genotypes (Yangmai 18 and Yangmai 20). The results showed that, in both wheat genotypes, CR displayed significantly a higher mutation efficiency (1.79 × 10–6/bp) than that by LR (1.56 × 10–6/bp). The induced mutations were not evenly distributed across chromosomes and varied across wheat genotypes. In Y18 M1, the highest number of mutations were detected on Chr. 6B and Chr. 6D, whilst in Y20 M1, Chr. 7A and Chr. 3A had the highest mutations. The transcript results showed that total of 4,755 CR-regulated and 1,054 LR-regulated differentially expressed genes (DEGs) were identified in the both genotypes. Gene function enrichment analysis of DEGs showed that these DEGs overlapped or diverged in the cascades of molecular networks involved in “phenylpropanoid biosynthesis” and “starch and sucrose metabolism” pathways. Moreover, IR type specific responses were observed between CR an LR irradiation, including specific TFs and response pathways. MSAP analysis showed that DNA methylation level increased in LR treatment, while decreased at CR. The proportion of hypermethylation was higher than that of hypomethylation at LR, whereas a reverse pattern was observed at CR, indicating that DNA methylation plays critical roles in response to IR irradiation. All these results support that the response to different IRs in wheat includes both common and unique pathways, which can be served as a useful resource to better understand the mechanisms of responses to different IRs in other plants.

The impact of environment on genetic and epigenetic variation in Trifolium pratense populations from two contrasting semi-natural grasslands.

Central European grasslands, such as calcareous grasslands and oat-grass meadows, are characterized by diverse environmental conditions and management regimes. Therefore, we aimed to determine potential differences in genetic and epigenetic variation patterns between the contrasting habitats and to identify the drivers of genetic and epigenetic variation. We investigated the genetic and epigenetic variation of the ecologically variable plant species Trifolium pratense L. applying amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses. We observed low levels of genetic and epigenetic differentiation among populations and between habitat types. Genetic and epigenetic variations were not interdependent. Thus, genetic variation was significantly isolated by habitat dissimilarity, whereas epigenetic variation was affected by environment. More specifically, we observed a significant correlation of epigenetic diversity with soil moisture and soil pH (the latter potentially resulting in phosphorus limitation). Genetic variation was, therefore, affected more strongly by habitat-specific environmental conditions induced by land use-related disturbance and gene flow patterns, while epigenetic variation was driven by challenging environmental conditions.

Exploring japonica rice epigenetic diversity in the main production regions of Heilongjiang Province

As a major epigenetic modification, DNA methylation plays an important role in coordinating plant responses to environmental changes. Methylation-sensitive amplified polymorphism (MSAP) technology was used in this study to investigate the epigenetic diversity of fifty japonica rice samples from five regions in Heilongjiang Province, China. In addition, the phenotypic indicators of japonica rice samples and the environmental conditions of the sampling sites were investigated and analysed. Based on the MSAP analysis technique, using eight pairs of selective primers, we identified a total of 551 amplified loci, of which 267 (48.5%) were classified as methylation loci. The methylation status and levels of the japonica rice genome in different regions differed significantly (p < 0.05). The results of the analysis of molecular variance (AMOVA) revealed that most of the molecular variation (91%) came from within the groups (regions) and was caused by individual variation within the region. Furthermore, the results of principal coordinates analysis (PCoA), cluster analysis, and population structure analysis indicated that there was no obvious correlation between the epigenetic differences and geographical locations, which may have been due to the limited range of sampling sites. When environmental factors, phenotypic indicators, and epigenetic data analysis are combined, it is easy to conclude that japonica rice grown in the same latitudinal region has increased epigenetic and phenotypic similarities due to similar climatic conditions and production practices.

Ultraviolet B Radiation Triggers DNA Methylation Change and Affects Foraging Behavior of the Clonal Plant Glechoma longituba.

Clonal plants in heterogeneous environments can benefit from their habitat selection behavior, which enables them to utilize patchily distributed resources efficiently. It has been shown that such behavior can be strongly influenced by their memories on past environmental interactions. Epigenetic variation such as DNA methylation was proposed to be one of the mechanisms involved in the memory. Here, we explored whether the experience with Ultraviolet B (UV-B) radiation triggers epigenetic memory and affects clonal plants’ foraging behavior in an UV-B heterogeneous environment. Parental ramets of Glechoma longituba were exposed to UV-B radiation for 15 days or not (controls), and their offspring ramets were allowed to choose light environment enriched with UV-B or not (the species is monopodial and can only choose one environment). Sizes and epigenetic profiles (based on methylation-sensitive amplification polymorphism analysis) of parental and offspring plants from different environments were also analyzed. Parental ramets that have been exposed to UV-B radiation were smaller than ramets from control environment and produced less and smaller offspring ramets. Offspring ramets were placed more often into the control light environment (88.46% ramets) than to the UV-B light environment (11.54% ramets) when parental ramets were exposed to UV-B radiation, which is a manifestation of “escape strategy.” Offspring of control parental ramets show similar preference to the two light environments. Parental ramets exposed to UV-B had lower levels of overall DNA methylation and had different epigenetic profiles than control parental ramets. The methylation of UV-B-stressed parental ramets was maintained among their offspring ramets, although the epigenetic differentiation was reduced after several asexual generations. The parental experience with the UV-B radiation strongly influenced foraging behavior. The memory on the previous environmental interaction enables clonal plants to better interact with a heterogeneous environment and the memory is at least partly based on heritable epigenetic variation.

Variations of Cytosine Methylation Patterns between Staminate and Perfect Flowers within Andromonoecious Taihangia rupestris (Rosaceae) Revealed by Methylation-Sensitive Amplification Polymorphism

DNA methylation is an epigenetic modification that plays important roles in flower development through regulation of gene expression. Taihangia rupestris is an andromonoecious plant species, which can produce staminate and perfect flowers within the same individual. In this study, we employed methylation-sensitive amplified polymorphism (MSAP) to investigate cytosine methylation patterns in staminate and perfect flowers within the andromonoecious T. rupestris. Our data showed the cytosine methylation patterns varied in staminate and perfect flowers at early and late developmental stages. The level of cytosine methylation was slightly higher in perfect flowers than in staminate flowers, and an increase of cytosine methylation levels was found alongside developmental stages in both staminate and perfect flowers. Based on MSAP profiles, a total of 116 differentially methylated fragments (DMFs) were obtained and sequenced. Of these obtained DMFs, 42 fragments showed sequence similarity to functional genes involved in a wide range of biological processes such as flower development, signal transduction, and cell proliferation, and transcription regulation. By integrative analysis of MSAP and transcriptome data, we found transcript levels correlated with cytosine methylation patterns for majority of DMF-associated genes. This study revealed that DNA methylation could play important roles in regulation of gene expression during establishment and development of staminate and perfect flower within andromonoecious T. rupestris and would be helpful to shed light on the underlying molecular mechanisms responsible for flower formation at epigenetic scale in andromonoecious sexual system.

Assessment of epigenetic methylation changes in hop (Humulus lupulus) plants obtained by meristem culture

In vitro meristem cultures have been used for the production of hop (Humulus lupulus L.) virus-free rootstocks worldwide, because multipropagation is considered to preserve the genetic stability of the produced plantlet. Nevertheless, in vitro tissue cultures can cause genetic and epigenetic changes. Therefore, we studied the genetic and epigenetic variability of Saaz Osvald’s clones, Sládek and Premiant cultivars on the DNA methylation level by methylation-sensitive amplification polymorphism (MSAP). In vitro propagated plants, acclimatised glasshouse rootstocks as well as derived mericlones and control plants under field conditions were used for the analyses. A total of 346 clearly and highly reproducible amplified products were detected in the MSAP analyses within the studied hop plants. We found 16 polymorphic products (4.6% of products) and 64 products with methylation changes (18.5% of products) in the analyses. The demethylation events were comparable to the de novo methylation events. Most demethylation changes were found in the in vitro plants, but only a few of them were found in the derived mericlones under field conditions. In contrast, the de novo methylation changes persisted in the acclimatised plants under glasshouse or field conditions. A hierarchical cluster analysis was used for the evaluation of the molecular genetic variability within the individual samples. The dendrogram showed that the individual samples of the same variety, more or less, clustered together. Because the methylation status varied during the virus-free rootstock production process, we suppose that de/methylation process is a natural tool of epigenetics and evolution in vegetatively propagated plants.

Epigenetic Analysis through MSAP-NGS Coupled Technology: The Case Study of White Poplar Monoclonal Populations/Stands.

Over the last several decades, several lines of evidence have shown that epigenetic modifications modulate phenotype and mediate an organism’s response to environmental stimuli. Plant DNA is normally highly methylated, although notable differences exist between species. Many biomolecular techniques based on PCR have been developed to analyse DNA methylation status, however a qualitative leap was made with the advent of next-generation sequencing (NGS). In the case of large, repetitive, or not-yet-sequenced genomes characterised by a high level of DNA methylation, the NGS analysis of bisulphite pre-treated DNA is expensive and time consuming, and moreover, in some cases data analysis is a major challenge. Methylation-sensitive amplification polymorphism (MSAP) analysis is a highly effective method to study DNA methylation. The method is based on the comparison of double DNA digestion profiles (EcoRI-HpaII and EcoRI-MspI) to reveal methylation pattern variations. These are often attributable to pedoclimatic and stress conditions which affect all organisms during their lifetime. In our study, five white poplar (Populus alba L.) specimens were collected from different monoclonal stands in the Maltese archipelago, and their DNA was processed by means of an innovative approach where MSAP analysis was followed by NGS. This allowed us to identify genes that were differentially methylated among the different specimens and link them to specific biochemical pathways. Many differentially methylated genes were found to encode transfer RNAs (tRNAs) related to photosynthesis or light reaction pathways. Our results clearly demonstrate that this combinatorial method is suitable for epigenetic studies of unsequenced genomes like P. alba (at the time of study), and to identify epigenetic variations related to stress, probably caused by different and changing pedoclimatic conditions, to which the poplar stands have been exposed.

Methylation-sensitive amplification polymorphism analysis reveals the DNA methylation dynamics during capitulum development in Chrysanthemum lavandulifolium

Chrysanthemum lavandulifolium (Fischer ex Trautv.) Makino is a diploid plant belonging to the Asteraceae family, with typical capitula composed of female ray florets and bisexual disc florets. The differentiation and development of these two types of florets have long been important research focuses; however, the potential epigenetic mechanisms governing these processes have not been elucidated. In the present study, methylation-sensitive amplification polymorphism method was used to trace the dynamic changes of DNA methylation during capitulum development in C. lavandulifolium. DNA methylation patterns and levels were detected in the whole capitula during seven developmental stages, and the obtained results revealed that DNA demethylation was dominant during this process. In addition, DNA methylation patterns and levels showed significant differences between ray and disc florets. Moreover, the expression patterns of candidate genes potentially involved in the development processes of two types of florets were analyzed by real-time quantitative reverse transcription polymerase chain reaction, and correlation analysis indicated that the expression levels of ClPI, ClAG2, ClSEP1, ClCYC2c, ClCYC2d, and ClCYC2e were highly correlated with DNA methylation levels. These results indicate that DNA methylation may be involved in the differentiation and development of ray and disc florets. This study provides epigenetic insights into the capitulum development in C. lavandulifolium.

Rapid Genomic and Epigenetic Alterations in Gynogenetic Carassius auratus Red Var. Derived from Distant Hybridization.

Gynogenesis is an important reproductive mode in fish and is used fairly widely in genetic breeding. Gynogenetic offspring (2n = 100, abbreviated as GRCC) were generated through the distant hybridization of Carassius auratus red var. (2n = 100, RCC) (♀) × Megalobrama amblycephala (2n = 48, BSB) (♂), in which male and female individual both had normal gonadal development. To better understand genomic and epigenetic consequences of GRCC, fluorescence in situ hybridization, amplified fragment length polymorphism, and methylation-sensitive amplification polymorphism analysis were performed on GRCC and RCC. GRCC possess two sets of RCC-derived chromosomes and one to three microchromosomes, in which 30.44% of bands inherit these patterns from red crucian carp and blunt snout bream, and 24.12% of novel bands were found by amplified fragment length polymorphism analysis. In terms of methylation, the DNA methylation level of GRCC was lower than that of their parents, and 45.29% of methylation patterns in GRCC were altered compared with their parents. GRCC show a special genetic composition in the genome, in which genome-wide changes and the adjustment of DNA methylation levels and patterns occurred. The result revealed that genetic and epigenetic changes were rapidly triggered in gynogenetic fish that were derived from distant hybridization, showing a special genetic composition in the genome. This study provides new insights into fish genetic breeding and the evolutionary patterns of the vertebrate genome.

MSAP analysis reveals diverse epigenetic statuses in opium poppy varieties with different benzyisoquinoline alkaloid content.

DNA methylation is one of the major epigenetic modifications influencing the regulation of gene expression. The opium poppy is an important medicinal plant. Its latex contains opium, which is a rich source of pharmaceutical benzyisoquinoline alkaloids (BIA). Here, the methylation-sensitive amplification polymorphism (MSAP) profiling technique using 21 MSAP molecular markers was applied in order to compare levels of DNA methylation between 6 opium poppy varieties. MSAP profiling reflected the different methylation statuses among opium poppy varieties having divergent BIA content. Moreover, different organ-specific epigenetic profiles were observed between the samples. Differential epigenetic profiles of capsules and shoots from the leaves pointed to the impact of methylation on BIA biosynthesis. The data implied that the different DNA methylation status may have important biological significance, in the case of alkaloid content in opium poppy in particular.

Complex relationship between DNA methylation and gene expression due to Lr28 in wheat-leaf rust pathosystem.

Differential DNA methylation due to Lr28 was examined in susceptible (S) wheat cv. HD2329 and its resistant (R) near isogenic line (NIL) (HD2329+Lr28) using two approaches: methylation sensitive amplified polymorphism (MSAP) and methylated DNA immunoprecipitation (MeDIP). S/R lines each had a large number of hypomethylated genes and relatively fewer hypermethylated genes at 96 hai (hours after inoculation) relative to 0 hbi (hours before inoculation), suggesting activation of many genes during the passage of time (96 hai), although identity of genes may differ in S and R lines. When R NIL was compared with S cultivar, there were many hypermethylated and fewer hypomethylated genes in R NIL relative to S cultivar, suggesting that many genes that are active in S cultivar are silenced in R NIL, both at 0 hbi and at 96 hai. Level of methylation was generally abundant in intergenic regions followed by that in promoters, transcription terminationsites (TTSs) and exons/introns. Hypermethylation in promoter and gene body regions was not always associated with inhibition of gene expression and vice-versa, indicating that more than one regulatory mechanisms may control theexpression of genes due to pathogen attack in presence and absence of Lr28. MSAP analysis also showed abundance of mCG methylation in Scultivar and that of mCCG methylation in R NIL(at 96 hai), suggesting differences in methylation context in NILs with and without Lr28. The results of the present study improved our understanding of the epigenetic control of leaf rust resistance in wheat.

Induction, identification and genetics analysis of tetraploid Actinidia chinensis.

Actinidia chinensis is a commercially important fruit, and tetraploid breeding of A. chinensis is of great significance for economic benefit. In order to obtain elite tetraploid cultivars, tetraploid plants were induced by colchicine treatment with leaves of diploid A. chinensis ‘SWFU03’. The results showed that the best treatment was dipping leaves 30 h in 60 mg l−1 colchicine solutions, with induction rate reaching 26%. Four methods, including external morphology comparison, stomatal guard cell observation, chromosome number observation and flow cytometry analysis were used to identify the tetraploid of A. chinensis. Using the induction system and flow cytometry analysis methods, 187 tetraploid plants were identified. Three randomly selected tetraploid plants and their starting diploid plants were further subjected to transcriptome analysis, real-time quantitative polymerase chain reaction (RT-qPCR) and methylation-sensitive amplification polymorphism (MSAP) analysis. The transcriptome analysis results showed that there were a total of 2230 differentially expressed genes (DEG) between the diploid and tetraploid plants, of which 660 were downregulated and 1570 upregulated. The DEGs were mainly the genes involved in growth and development, stress resistance and antibacterial ability in plants. RT-qPCR results showed that the gene expression levels of the growth and stress resistance of tetraploid plants were higher than those of diploid ones at the transcriptome level. MSAP analysis of DNA methylation results showed that tetraploid plants had lower methylation ratio than diploid ones. The present results were valuable to further explore the epigenetics of diploid and tetraploid kiwifruit plants.

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'.

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.

Comparison of physiological and methylational changes in resynthesized Brassica napus and diploid progenitors under drought stress

Brassica napus is a polyploid of certain research and economical value. Resynthesizing B. napus with diploid B. rapa and B. oleracea is essential for Brassica research because of the limited genetic background of B. napus. Considering that polyploids possess better agronomic traits and resistance compared with the corresponding diploids, we investigated drought tolerance after polyploidization of B. napus and revealed the epigenetic differences between polyploids and diploids. After drought stress, B. rapa and first-generation of synthesized hybrids (F1) were more wilted than B. oleracea and F2–F4 generations. However, the relative water content and water retention in F1 were better than others after drought stress. The increased number of partially opened and closed stomata in F1 was not as significant as that in F2 and F3, but stomata density in F1 was lower than F2, and the stomatal size in F1 was significantly reduced than F3. Physiological parameters varied among different generations of B. napus and diploid parents, and most of these parameters in hybrids were higher than B. rapa and lower than B. oleracea. However, the peroxidase activity in F3 and F4 was significantly higher than both parents, and the malondialdehyde content in F3 and F4 was lower than both parents, indicating that F3 and F4 might be more adaptive to oxidative stresses than other generations. DNA methylation level was decreased in F2 and F3 compared with F1, and then increased in F4. Methylation-sensitive amplified polymorphism analysis revealed that DNA methylation and demethylation broadly happened after drought stress. The methylation and demethylation level was F1 > F4 > B. oleracea > F2 > F3 > B. rapa and B. rapa > F4 > F3 > F2 > B. oleracea > F1, respectively. The epigenetic changes under drought stress might be related to the different stress tolerances during B. napus polyploidization.

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.

Comparison of DNA methylation in developing seeds of Ricinus communis L. using MSAP analysis

ABSTRACTDNA methylation plays an important role in endosperm development and is involved in the epigenetic regulation of gene expression. However, the epigenetic regulation of castor seed development has not yet been reported. In this study, methylation-sensitive amplification polymorphism (MSAP) analysis was used to investigate the methylation level in castor. In 20-day-old castor seeds, 41.17% of the fragments were fully methylated and 15.61% were hemimethylated. In 60 day-old seed, the full methylation and hemimethylation rates were 19.77% and 7.56%, respectively, suggesting that some methylation level changes existed during the castor seed development. A total of 647 bands were identified with different methylation or demethylation patterns during the seed maturation. However, only 37 sequences were homologous to well-known proteins, such as phospholipid-transporting ATPase, phosphatases and methyltransferase. Therefore, these results clearly demonstrated that the MSAP technique is very useful for the analysis of the global DNA methylation state in castor and the complexity of DNA methylation changes during seed development. The different methylation levels may be correlated with the specific gene expression in different seed development stages.

New Insights into the Methylation Status of Virulent Diuraphis noxia (Hemiptera: Aphididae) Biotypes.

Epigenetic modifications provide a means for aphid biotype development that a lack of genetic variation, owing to an anholocyclic reproduction lifecycle, fails to do. Here we present data on the DNA methylation status in four South African Russian wheat aphids (RWA), Diuraphis noxia (Kurdjomov) (Hemiptera: Aphididae) biotypes expressing different levels of virulence against its host, Triticum aestivum L. (Poales: Poaceae, Triticeae). The DNA methylation status of these biotypes was determined through the use of methylation-sensitive amplification polymorphism analysis, restriction site-specific fluorescence labeling-a novel technique, and measuring relative global DNA methylation and hydroxymethylation. The least virulent RWA biotype, SA1, was methylated, while biotypes displaying intermediate virulence, SA2 and SA3, exhibited intermediate levels of hemimethylation. The genome of the most virulent RWA biotype, SAM, seems to be hypomethylated, which is likely attained through the process of demethylation.

Epigenetic Differentiation of Natural Populations of Lilium bosniacum Associated with Contrasting Habitat Conditions.

Epigenetic variation in natural populations with contrasting habitats might be an important element, in addition to the genetic variation, in plant adaptation to environmental stress. Here, we assessed genetic, epigenetic, and cytogenetic structure of the three Lilium bosniacum populations growing on distinct habitats. One population was growing under habitual ecological conditions for this species and the other two were growing under stress associated with high altitude and serpentine soil. Amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses revealed that the three populations did not differentiate genetically, but were clearly separated in three distinct clusters according to DNA methylation profiles. Principal coordinate analysis showed that overall epigenetic variation was closely related to habitat conditions. A new methylation-sensitive amplification polymorphism scoring approach allowed identification of mainly unmethylated (φST = 0.190) and fully CpG methylated (φST = 0.118) subepiloci playing a role in overall population differentiation, in comparison with hemimethylated sites (φST = 0.073). In addition, unusual rDNA repatterning and the presence of B chromosomes bearing 5S rDNA loci were recorded in the population growing on serpentine soil, suggesting dynamic chromosome rearrangements probably linked to global genome demethylation, which might have reactivated some mobile elements. We discuss our results considering our earlier data on morphology and leaf anatomy of several L. bosniacum populations, and suggest a possible role of epigenetics as a key element in population differentiation associated with environmental stress in these particular lily populations.

Detection of changes in DNA methylation patterns in Pyropia haitanensis under high-temperature stress using a methylation-sensitive amplified polymorphism assay

DNA methylation is the most important epigenetic regulatory strategy that is involved in various biological processes, including environmental stress response. In the present study, the potential contribution of DNA methylation-mediated gene regulation in a high-temperature-tolerant strain of Pyropia haitanensis (Z-61) was investigated by using the methylation-sensitive amplification polymorphism (MSAP) assay. With high temperature, the level of global DNA methylation (5-mC) in Z-61 decreased from 2.21% (control) to 1.36% (12 h) and 0.60% (144 h). MSAP analysis indicated that the total methylation ratio (%) decreased from 30.19% (control) to 15.82% (12 h) and 16.89% (144 h) and that the demethylation of fully methylated fragments was the main contributor of the DNA methylation alterations. A total of 55 polymorphic bands representing fragments that were generated during methylation/demethylation events in different samples were randomly selected and sequenced, of which 29 fragments were situated within a gene, whereas the others were located within noncoding regions or promoters. The 29 MSAP fragments consisted of 18 fragments that were highly expressed as indicated by RNA-seq analysis and 11 genes that exhibited either very low or no expression at all time points. BLASTN analysis indicated that the methylated fragments situated within genes are homologous to those involved in numerous processes, including abiotic stress response, programmed cell death, photosynthesis, energy metabolism, signal transduction, protein synthesis, and transposon activation. To the best of our knowledge, this is the first report on changes in methylation patterns in macroalgae under environmental stress, and our findings provide insights into the elucidation of the mechanism underlying stress adaptation in P. haitanensis.