Epigenetic variation predicts regional and local intraspecific functional diversity in a perennial herb

The ecological significance of epigenetic variation has been generally inferred from studies on model plants under artificial conditions, but the importance of epigenetic differences between individuals as a source of intraspecific diversity in natural plant populations remains essentially unknown. This study investigates the relationship between epigenetic variation and functional plant diversity by conducting epigenetic (methylation‐sensitive amplified fragment length polymorphisms, MSAP) and genetic (amplified fragment length polymorphisms, AFLP) marker–trait association analyses for 20 whole‐plant, leaf and regenerative functional traits in a large sample of wild‐growing plants of the perennial herb Helleborus foetidus from ten sampling sites in south‐eastern Spain. Plants differed widely in functional characteristics, and exhibited greater epigenetic than genetic diversity, as shown by per cent polymorphism of MSAP fragments (92%) or markers (69%) greatly exceeding that for AFLP ones (41%). After controlling for genetic structuring and possible cryptic relatedness, every functional trait considered exhibited a significant association with at least one AFLP or MSAP marker. A total of 27 MSAP (13.0% of total) and 12 AFLP (4.4%) markers were involved in significant associations, which explained on average 8.2% and 8.0% of trait variance, respectively. Individual MSAP markers were more likely to be associated with functional traits than AFLP markers. Between‐site differences in multivariate functional diversity were directly related to variation in multilocus epigenetic diversity after multilocus genetic diversity was statistically accounted for. Results suggest that epigenetic variation can be an important source of intraspecific functional diversity in H. foetidus, possibly endowing this species with the capacity to exploit a broad range of ecological conditions despite its modest genetic diversity.     

Comparative analyses of genetic/epigenetic diversities and structures in a wild barley species (Hordeum brevisubulatum) using MSAP, SSAP and AFLP

We analyzed genetic diversity and population genetic structure of four artificial populations of wild barley (Hordeum brevisubulatum); 96 plants collected from the Songnen Prairie in northeastern China were analyzed using amplified fragment length polymorphism (AFLP), specific-sequence amplified polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) markers. Indices of (epi-)genetic diversity, (epi-)genetic distance, gene flow, genotype frequency, cluster analysis, PCA analysis and AMOVA analysis generated from MSAP, AFLP and SSAP markers had the same trend. We found a high level of correlation in the artificial populations between MSAP, SSAP and AFLP markers by the Mantel test (r > 0.8). This is incongruent with previous findings showing that there is virtually no correlation between DNA methylation polymorphism and classical genetic variation; the high level of genetic polymorphism could be a result of epigenetic regulation. We compared our results with data from natural populations. The population diversity of the artificial populations was lower. However, different from what was found using AFLP and SSAP, based on MSAP results the methylation polymorphism of the artificial populations was not significantly reduced. This leads us to suggest that the DNA methylation pattern change in H. brevisubulatum populations is not only related to DNA sequence variation, but is also regulated by other controlling systems.     

Untangling individual variation in natural populations: ecological, genetic and epigenetic correlates of long-term inequality in herbivory

Individual variation in ecologically important features of organisms is a crucial element in ecology and evolution, yet disentangling its underlying causes is difficult in natural populations. We applied a genomic scan approach using amplified fragment length polymorphism (AFLP) markers to quantify the genetic basis of long‐term individual differences in herbivory by mammals at a wild population of the violet Viola cazorlensis monitored for two decades. In addition, methylation‐sensitive amplified polymorphism (MSAP) analyses were used to investigate the association between browsing damage and epigenetic characteristics of individuals, an aspect that has been not previously explored for any wild plant. Structural equation modelling was used to identify likely causal structures linking genotypes, epigenotypes and herbivory. Individuals of V. cazorlensis differed widely in the incidence of browsing mammals over the 20‐year study period. Six AFLP markers (1.6% of total) were significantly related to herbivory, accounting altogether for 44% of population‐wide variance in herbivory levels. MSAP analyses revealed considerable epigenetic variation among individuals, and differential browsing damage was significantly related to variation in multilocus epigenotypes. In addition, variation across plants in epigenetic characteristics was related to variation in several herbivory‐related AFLP markers. Statistical comparison of alternative causal models suggested that individual differences in herbivory are the outcome of a complex causal structure where genotypes and epigenotypes are interconnected and have direct and indirect effects on herbivory. Insofar as methylation states of MSAP markers influential on herbivory are transgenerationally heritable, herbivore‐driven evolutionary changes at the study population will involve correlated changes in genotypic and epigenotypic distributions.     

MSAP markers and global cytosine methylation in plants: a literature survey and comparative analysis for a wild‐growing species

Methylation of DNA cytosines affects whether transposons are silenced and genes are expressed, and is a major epigenetic mechanism whereby plants respond to environmental change. Analyses of methylation‐sensitive amplification polymorphism (MS‐AFLP or MSAP) have been often used to assess methyl‐cytosine changes in response to stress treatments and, more recently, in ecological studies of wild plant populations. MSAP technique does not require a sequenced reference genome and provides many anonymous loci randomly distributed over the genome for which the methylation status can be ascertained. Scoring of MSAP data, however, is not straightforward, and efforts are still required to standardize this step to make use of the potential to distinguish between methylation at different nucleotide contexts. Furthermore, it is not known how accurately MSAP infers genome‐wide cytosine methylation levels in plants. Here, we analyse the relationship between MSAP results and the percentage of global cytosine methylation in genomic DNA obtained by HPLC analysis. A screening of literature revealed that methylation of cytosines at cleavage sites assayed by MSAP was greater than genome‐wide estimates obtained by HPLC, and percentages of methylation at different nucleotide contexts varied within and across species. Concurrent HPLC and MSAP analyses of DNA from 200 individuals of the perennial herb Helleborus foetidus confirmed that methyl‐cytosine was more frequent in CCGG contexts than in the genome as a whole. In this species, global methylation was unrelated to methylation at the inner CG site. We suggest that global HPLC and context‐specific MSAP methylation estimates provide complementary information whose combination can improve our current understanding of methylation‐based epigenetic processes in nonmodel plants.     

Epigenetic variation reflects dynamic habitat conditions in a rare floodplain herb

Variation of DNA methylation is thought to play an important role for rapid adjustments of plant populations to dynamic environmental conditions, thus compensating for the relatively slow response time of genetic adaptations. However, genetic and epigenetic variation of wild plant populations has not yet been directly compared in fast changing environments. Here, we surveyed populations of Viola elatior from two adjacent habitat types along a successional gradient characterized by strong differences in light availability. Using amplified fragment length polymorphisms (AFLP) and methylation‐sensitive amplification polymorphisms (MSAP) analyses, we found relatively low levels of genetic (H′_gen = 0.19) and epigenetic (H′_epi = 0.23) diversity and high genetic (?_ST = 0.72) and epigenetic (?_ST = 0.51) population differentiation. Diversity and differentiation were significantly correlated, suggesting that epigenetic variation partly depends on the same driving forces as genetic variation. Correlation‐based genome scans detected comparable levels of genetic (17.0%) and epigenetic (14.2%) outlier markers associated with site specific light availability. However, as revealed by separate differentiation‐based genome scans for AFLP, only few genetic markers seemed to be actually under positive selection (0–4.5%). Moreover, principal coordinates analyses and Mantel tests showed that overall epigenetic variation was more closely related to habitat conditions, indicating that environmentally induced methylation changes may lead to convergence of populations experiencing similar habitat conditions and thus may play a major role for the transient and/or heritable adjustment to changing environments. Additionally, using a new MSAP‐scoring approach, we found that mainly the unmethylated (?_ST = 0.60) and CG‐methylated states (?_ST = 0.46) of epiloci contributed to population differentiation and putative habitat‐related adaptation, whereas CHG‐hemimethylated states (?_ST = 0.21) only played a marginal role.     

Genetic and epigenetic variations associated with adaptation to heterogeneous habitat conditions in a deciduous shrub

Environmentally induced phenotypic plasticity is thought to play an important role in the adaption of plant populations to heterogeneous habitat conditions, and yet the importance of epigenetic variation as a mechanism of adaptive plasticity in natural plant populations still merits further research. In this study, we investigated populations of Vitex negundo var. heterophylla (Chinese chastetree) from adjacent habitat types at seven sampling sites. Using several functional traits, we detected a significant differentiation between habitat types. With amplified fragment length polymorphisms (AFLP) and methylation‐sensitive AFLP (MSAP), we found relatively high levels of genetic and epigenetic diversity but very low genetic and epigenetic differences between habitats within sites. Bayesian clustering showed a remarkable habitat‐related differentiation and more genetic loci associated with the habitat type than epigenetic, suggesting that the adaptation to the habitat is genetically based. However, we did not find any significant correlation between genetic or epigenetic variation and habitat using simple and partial Mantel tests. Moreover, we found no correlation between genetic and ecologically relevant phenotypic variation and a significant correlation between epigenetic and phenotypic variation. Although we did not find any direct relationship between epigenetic variation and habitat environment, our findings suggest that epigenetic variation may complement genetic variation as a source of functional phenotypic diversity associated with adaptation to the heterogeneous habitat in natural plant populations.     

Variation in DNA methylation transmissibility,genetic heterogeneity and fecundity‐related traits in natural populations of the perennial herb Helleborus foetidus

Inferences about the role of epigenetics in plant ecology and evolution are mostly based on studies of cultivated or model plants conducted in artificial environments. Insights from natural populations, however, are essential to evaluate the possible consequences of epigenetic processes in biologically realistic scenarios with genetically and phenotypically heterogeneous populations. Here, we explore associations across individuals between DNA methylation transmissibility (proportion of methylation‐sensitive loci whose methylation status persists unchanged after male gametogenesis), genetic characteristics (assessed with AFLP markers), seed size variability (within‐plant seed mass variance), and realized maternal fecundity (number of recently recruited seedlings), in three populations of the perennial herb Helleborus foetidus along a natural ecological gradient in southeastern Spain. Plants (sporophytes) differed in the fidelity with which DNA methylation was transmitted to descendant pollen (gametophytes). This variation in methylation transmissibility was associated with genetic differences. Four AFLP loci were significantly associated with transmissibility and accounted collectively for ~40% of its sample‐wide variance. Within‐plant variance in seed mass was inversely related to individual transmissibility. The number of seedlings recruited by individual plants was significantly associated with transmissibility. The sign of the relationship varied between populations, which points to environment‐specific, divergent phenotypic selection on epigenetic transmissibility. Results support the view that epigenetic transmissibility is itself a phenotypic trait whose evolution may be driven by natural selection, and suggest that in natural populations epigenetic and genetic variation are two intertwined, rather than independent, evolutionary factors.     

Epigenetic Variability in the Genetically Uniform Forest Tree Species Pinus pinea L

There is an increasing interest in understanding the role of epigenetic variability in forest species and how it may contribute to their rapid adaptation to changing environments. In this study we have conducted a genome-wide analysis of cytosine methylation pattern in Pinus pinea, a species characterized by very low levels of genetic variation and a remarkable degree of phenotypic plasticity. DNA methylation profiles of different vegetatively propagated trees from representative natural Spanish populations of P. pinea were analyzed with the Methylation Sensitive Amplified Polymorphism (MSAP) technique. A high degree of cytosine methylation was detected (64.36% of all scored DNA fragments). Furthermore, high levels of epigenetic variation were observed among the studied individuals. This high epigenetic variation found in P. pinea contrasted with the lack of genetic variation based on Amplified Fragment Length Polymorphism (AFLP) data. In this manner, variable epigenetic markers clearly discriminate individuals and differentiates two well represented populations while the lack of genetic variation revealed with the AFLP markers fail to differentiate at both, individual or population levels. In addition, the use of different replicated trees allowed identifying common polymorphic methylation sensitive MSAP markers among replicates of a given propagated tree. This set of MSAPs allowed discrimination of the 70% of the analyzed trees.     

Epigenetic instability in genetically stable micropropagated plants of <Emphasis Type="Italic">Gardenia jasminoides</Emphasis> Ellis

Gardenia jasminoides Ellis is an evergreen tropical plant and favorite to gardeners throughout the world. Several studies have documented that in vitro micropropagation can be used for clonal propagation of G. jasminoides Ellis, the efficiency remained low. In addition, no information is available on the genetic and epigenetic fidelity of the micropropagated plants. Here, we report on a simplified protocol for high efficient micropropagation of G. jasminoides Ellis cv. “Kinberly” based on enhanced branching of shoot-tips as explants. The protocol consisted of sequential use of three media, namely, bud-induction, elongation and root-induction. By using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation sensitive amplified polymorphism (MSAP), we analyzed the genetic and DNA methylation pattern stability of 23 morphologically normal plants randomly taken from a sub-population (>100) of micropropagated plants originated from a single shoot-tip. We found that of >1,000 scored AFLP bands across the 23 micropropagated plants, no incident of genetic variation was detected. In contrast, of 750 scored MSAP bands, moderate but clear alteration in several DNA methylation patterns occurred in the majority of the 23 micropropagated plants. The changed methylation patterns involved both CG and CHG sites representing either hyper- or hypo-methylation, which occurred without altering the total methylation levels partly due to concomitant hyper- and hypo-methylation alterations. Our results indicated that epigenetic instability in the form of DNA methylation patterns can be susceptible to the in vitro micropropagation process for G. jasminoides Ellis, and needs to be taken into account in the process of large-scale commercial propagation of this plant.     

Genetic and epigenetic instabilities induced by tissue culture in wild barley (<Emphasis Type="Italic">Hordeum brevisubulatum</Emphasis> (Trin.) Link)

A simple tissue culture protocol was developed for efficient plant regeneration from young inflorescence-derived calli in wild barley, Hordeum brevisubulatum (Trin.) Link, an important pasturage grass. Genetic and epigenetic instabilities in the regenerated plants (regenerants) were assessed by three molecular markers AFLP, S-SAP and MSAP. Two pools of calli derived from young inflorescences of a single donor plant and 44 randomly chosen regenerants were subjected to AFLP analysis. Results showed that 74 out of 793 scored bands were polymorphic among the studied samples, giving rise to a genetic variation frequency of 9.3%. The number of variant bands as compared to the donor plant varied greatly among the regenerants, with a small number of regenerants accumulated a large number of variant bands (maximum 55), while the majority of regenerants showed only 2–3 variant bands. A subset of regenerants together with the two pools of calli were selected for S-SAP and MSAP analysis to detect possible retrotranspositional activity of a prominent retroelement family, BARE-1, in the genomes of Hordem species, and possible alterations in cytosine methylation. S-SAP analysis showed that of the 768 scored bands, 151 were polymorphic among the analyzed samples, giving rise to a genetic variation frequency of 19.7%, albeit no evidence for retrotranspositional event was obtained based on locus-specific PCR amplifications. MSAP analysis revealed that tissue culture has caused cytosine methylation alterations in both level and pattern compared with the donor plant. Sequencing of selected variant bands indicated that both protein-coding genes and transposon/retrotransposons were underlying the genetic and epigenetic variations. Correlation analysis of the genetic and epigenetic instabilities indicated that there existed a significant correlation between MSAP and S-SAP (r = 0.8118, 1,000 permutations, P < 0.05), whereas the correlation between MSAP and AFLP (r = 0.1048) is not statistically significant. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Xiaoling Li and Xiaoming Yu contributed equally to this work.     

猕猴桃倍性混合居群基因组遗传和表观遗传变异

植物倍性混合居群的形成和维系常伴随着明显的基因组遗传及表观遗传变异。利用AFLP和MSAP两种分子标记探讨了中华猕猴桃复合体(Actinidia chinensis)倍性混合居群的遗传变异和结构及其基因组甲基化变异方式。结果表明, 该倍性混合居群具有较高的遗传和表观遗传多样性, 但两者之间没有明显的相关性。种群的遗传多样性与海拔呈显著的负相关(P<0.05), 但表观遗传多样性与海拔不具显著相关性。AMOVA分析显示, 主要的遗传和表观遗传分化出现在倍性小种内部(97.65% vs 99.84%, P<0.05); 同时, AFLP邻接聚类分析显示二者存在一定程度的倍性相关性, MSAP分析则未显示有明显的倍性相关性。进一步研究发现, 中华猕猴桃居群的总甲基化程度为24.86%, 且多倍体具有更多的甲基化位点变异。该研究结果为深入探讨猕猴桃倍性混合居群的形成和维系机制奠定了基础。     

msap: a tool for the statistical analysis of methylation‐sensitive amplified polymorphism data

In this study msap, an R package which analyses methylation‐sensitive amplified polymorphism (MSAP or MS‐AFLP) data is presented. The program provides a deep analysis of epigenetic variation starting from a binary data matrix indicating the banding pattern between the isoesquizomeric endonucleases HpaII and MspI, with differential sensitivity to cytosine methylation. After comparing the restriction fragments, the program determines if each fragment is susceptible to methylation (representative of epigenetic variation) or if there is no evidence of methylation (representative of genetic variation). The package provides, in a user‐friendly command line interface, a pipeline of different analyses of the variation (genetic and epigenetic) among user‐defined groups of samples, as well as the classification of the methylation occurrences in those groups. Statistical testing provides support to the analyses. A comprehensive report of the analyses and several useful plots could help researchers to assess the epigenetic and genetic variation in their MSAP experiments. msap is downloadable from CRAN ( http://cran.r-project.org/ ) and its own webpage ( http://msap.r-forge.R-project.org/ ). The package is intended to be easy to use even for those people unfamiliar with the R command line environment. Advanced users may take advantage of the available source code to adapt msap to more complex analyses.     

红豆杉脱分化过程中的遗传和表观遗传变异

采用扩增片段长度多态性（AFLP）和甲基化敏感扩增多态性（MSAP）技术分析红豆杉脱分化前后基因组DNA和DNA甲基化状态的变化。选用32个AFLP引物组合从红豆杉植株及其愈伤组织分别扩增出1834个片段,无多态性片段产生。这说明红豆杉植株在诱导形成愈伤组织的过程中基因组DNA保持高度的遗传稳定性。另用32个MSAP引物组合从红豆杉植株及其愈伤组织分别扩增出1197个片段,总扩增位点的甲基化水平由脱分化前的12．4％上升为16．2％,表明红豆杉在脱分化过程中的某些位点发生了甲基化。红豆杉脱分化前后的DNA甲基化模式也存在较大差异,说明DNA甲基化对愈伤组织形成有调控作用。     

Jack of all nectars, master of most: DNA methylation and the epigenetic basis of niche width in a flower-living yeast

In addition to genetic differences between individuals as a result of nucleotide sequence variation, epigenetic changes that occur as a result of DNA methylation may also contribute to population niche width by enhancing phenotypic plasticity, although this intriguing possibility remains essentially untested. Using the nectar‐living yeast Metschnikowia reukaufii as study subject, we examine the hypothesis that changes in genome‐wide DNA methylation patterns underlie the ability of this fugitive species to exploit a broad resource range in its heterogeneous and patchy environment. Data on floral nectar characteristics and their use by M. reukaufii in the wild were combined with laboratory experiments and methylation‐sensitive amplified polymorphism (MSAP) analyses designed to detect epigenetic responses of single genotypes to variations in sugar environment that mimicked those occurring naturally in nectar. M. reukaufii exploited a broad range of resources, occurring in nectar of 48% of species and 52% of families surveyed, and its host plants exhibited broad intra‐ and interspecific variation in sugar‐related nectar features. Under experimental conditions, sugar composition, sugar concentration and their interaction significantly influenced the mean probability of MSAP markers experiencing a transition from unmethylated to methylated state. Alterations in methylation status were not random but predictably associated with certain markers. The methylation inhibitor 5‐azacytidine (5‐AzaC) had strong inhibitory effects on M. reukaufii proliferation in sugar‐containing media, and a direct relationship existed across sugar × concentration experimental levels linking inhibitory effect of 5‐AzaC and mean per‐marker probability of genome‐wide methylation. Environmentally induced DNA methylation polymorphisms allowed genotypes to grow successfully in extreme sugar environments, and the broad population niche width of M. reukaufii was largely made possible by epigenetic changes enabling genotype plasticity in resource use.     

A new image of plantain diversity assessed by SSR,AFLP and MSAP markers

Using both SSR and AFLP markers, the genetic diversity of 30 plantains constituting a representative sample of the phenotypic diversity was assessed. The results confirmed a very narrow genetic base of this cultivar group. SSR and AFLP data support the hypothesis that these cultivars may have arisen from vegetative multiplication of a single seed. MSAP were used to survey cytosine methylation status at CCGG sites in order to obtain an alternative source of diversity data. A higher degree of polymorphism was revealed allowing the classification of the samples into three clusters. No correlation was observed between the phenotypic classification and methylation diversity. Implications for breeding programs are discussed.     

Evaluation of Genetic and Epigenetic Modification in Rapeseed （Brassica napus） Induced by Salt Stress

Salinity is an important limiting environmental factor for rapeseed production worldwide. In this study, we assessed the extent and pattern of DNA damages caused by salt stress in rapeseed plants. Amplified fragment length polymorphism （AFLP） analysis revealed dose-related increases in sequence alterations in plantlets exposed to 10-1000 mmol/L sodium chloride. In addition, individual plantlets exposed to the same salt concentration showed different AFLP and selected region amplified polymorphism banding patterns. These observations suggested that DNA mutation in response to salt stress was random in the genome and the effect was dose-dependant. DNA methylation changes in response to salt stress were also evaluated by methylation sensitive amplified polymorphism （MSAP）. Three types of MSAP bands were recovered. Type Ⅰ bands were observed with both isoschizomers Hpa Ⅱ and Msp Ⅰ, while type Ⅱ and type Ⅲ bands were observed only with Hpa Ⅱ and Msp Ⅰ, respectively. Extensive changes in types of MSAP bands after NaCI treatments were observed, including appearance and disappearance of type Ⅰ, Ⅱ and Ⅲ bands, as well as exchanges between either type Ⅰand type Ⅱ or type Ⅰ and type Ⅲ bands. An increase of 0.2-17.6% cytosine methylated CCGG sites were detected in plantlets exposed to 10- 200 mmol/L salt compared to the control, and these changes included both de novo methylation and demethylation events. Nine methylation related fragments were also recovered and sequenced, and one sharing a high sequence homology with the ethylene responsive element binding factor was identified. These results demonstrated clear DNA genetic and epigenetic alterations in planUets as a response to salt stress, and these changes may suggest a mechanism for plants adaptation under salt stress.     

Variation in cytosine methylation patterns during ploidy level conversions in Eragrostis curvula

In many species polyploidization involves rearrangements of the progenitor genomes, at both genetic and epigenetic levels. We analyzed the cytosine methylation status in a ‘tetraploid-diploid-tetraploid’ series of Eragrostis curvula with a common genetic background by using the MSAP (Methylation-sensitive Amplified Polymorphism) technique. Considerable levels of polymorphisms were detected during ploidy conversions. The total level of methylation observed was lower in the diploid genotype compared to the tetraploid ones. A significant proportion of the epigenetic modifications occurring during the tetraploid–diploid conversion reverted during the diploid–tetraploid one. Genetic and expression data from previous work were used to analyze correlation with methylation variation. All genetic, epigenetic and gene expression variation data correlated significantly when compared by pairs in simple Mantel tests. Dendrograms reflecting genetic, epigenetic and expression distances as well as principal coordinate analysis suggested that plants of identical ploidy levels present similar sets of data. Twelve (12) different genomic fragments displaying different methylation behavior during the ploidy conversions were isolated, sequenced and characterized.     

Genetic and DNA Methylation Changes in Cotton (Gossypium) Genotypes and Tissues

In plants, epigenetic regulation is important in normal development and in modulating some agronomic traits. The potential contribution of DNA methylation mediated gene regulation to phenotypic diversity and development in cotton was investigated between cotton genotypes and various tissues. DNA methylation diversity, genetic diversity, and changes in methylation context were investigated using methylation-sensitive amplified polymorphism (MSAP) assays including a methylation insensitive enzyme (BsiSI), and the total DNA methylation level was measured by high-performance liquid chromatography (HPLC). DNA methylation diversity was greater than the genetic diversity in the selected cotton genotypes and significantly different levels of DNA methylation were identified between tissues, including fibre. The higher DNA methylation diversity (CHG methylation being more diverse than CG methylation) in cotton genotypes suggest epigenetic regulation may be important for cotton, and the change in DNA methylation between fibre and other tissues hints that some genes may be epigenetically regulated for fibre development. The novel approach using BsiSI allowed direct comparison between genetic and epigenetic diversity, and also measured CC methylation level that cannot be detected by conventional MSAP.     

Frequencies and variation in cytosine methylation patterns in diploid and tetraploid cytotypes of Paspalum notatum

Paspalum notatum Flügge is a grass species organized as an agamic complex. The objective of the current research was to survey the frequencies and variation of cytosine methylation at CCGG sequences in diploid and tetraploid genotypes, and to determine the occurrence of methylation changes associated with tetraploidization by using methylation-sensitive amplification polymorphism (MSAP) markers. No differences were found in the average proportions of methylated CCGG sites between cytotypes, but methylation patterns were significantly more variable in tetraploids. In both groups of plants, epigenetic and non-epigenetic variation correlated significantly when compared by Mantel tests. The evaluation of 159 common MSAP markers showed that 18.86 % of them differed in their methylation status in the different ploidies. Dendrogram analysis, reflecting epigenetic distances, showed that the four diploids and one experimentally-obtained sexually-reproducing tetraploid, grouped together. MSAP analysis performed on a diploid plant and its autotetraploid derivative showed that new epialleles emerged after tetraploidization. Sequencing of several MASP markers showed homologies with low copy genes, non-coding sequences and transposon/retrotransposon elements.     

Genetic diversity analysis of Jatropha curcas L. (Euphorbiaceae) based on methylation-sensitive amplification polymorphism

Genetic analysis of 56 samples of Jatropha curcas L. collected from Thailand and other countries was performed using the methylation-sensitive amplification polymorphism (MSAP) technique. Nine primer combinations were used to generate MSAP fingerprints. When the data were interpreted as amplified fragment length polymorphism (AFLP) markers, 471 markers were scored. All 56 samples were classified into three major groups: γ-irradiated, non-toxic and toxic accessions. Genetic similarity among the samples was extremely high, ranging from 0.95 to 1.00, which indicated very low genetic diversity in this species. The MSAP fingerprint was further analyzed for DNA methylation polymorphisms. The results revealed differences in the DNA methylation level among the samples. However, the samples collected from saline areas and some species hybrids showed specific DNA methylation patterns. AFLP data were used, together with methylation-sensitive AFLP (MS-AFLP) data, to construct a phylogenetic tree, resulting in higher efficiency to distinguish the samples. This combined analysis separated samples previously grouped in the AFLP analysis. This analysis also distinguished some hybrids. Principal component analysis was also performed; the results confirmed the separation in the phylogenetic tree. Some polymorphic bands, involving both nucleotide and DNA methylation polymorphism, that differed between toxic and non-toxic samples were identified, cloned and sequenced. BLAST analysis of these fragments revealed differences in DNA methylation in some known genes and nucleotide polymorphism in chloroplast DNA. We conclude that MSAP is a powerful technique for the study of genetic diversity for organisms that have a narrow genetic base.