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.     

Detection of epigenetic variation in tissue-culture-derived plants of <Emphasis Type="Italic">Doritaenopsis</Emphasis> by methylation-sensitive amplification polymorphism (MSAP) analysis

Somaclones exhibiting variations with flower characteristics were recovered from the tissue-culture-derived plants of Doritaenopsis. Two molecular techniques, random amplified polymorphic DNA (RAPD) and methylation-sensitive amplification polymorphism (MSAP) analyses, were used to characterize the somaclones. RAPD analysis, using 100 randomly selected primers, failed to differentiate variants and normal plants, even though some primers (six out of 100 primers) exhibited 6–10 distinct banding patterns. However, MSAP analysis revealed the differences in the DNA methylation patterns in the normal and variant plants which were correlated with phenotypic variation. In all, 311, 337, 366, and 343 fragments were obtained with normal and V1, V2, and V3 variant plants, respectively; each representing recognition site cleaved by either or both of the isoshizomers were amplified using 12 combination of primers. A total of 36 (11.6%), 77 (22.9%), 73 (19.9%), and 47 (13.7%) sites were found to be methylated at cytosine in the genomes of normal and V1, V2, and V3 variant Doritaenopsis plants. This study demonstrates usefulness of MSAP to detect DNA methylation events in tissue cultured Doritaenopsis plants.     

Epigenetic Differentiation Persists after Male Gametogenesis in Natural Populations of the Perennial Herb Helleborus foetidus (Ranunculaceae)

Despite the importance of assessing the stability of epigenetic variation in non-model organisms living in real-world scenarios, no studies have been conducted on the transgenerational persistence of epigenetic structure in wild plant populations. This gap in knowledge is hindering progress in the interpretation of natural epigenetic variation. By applying the methylation-sensitive amplified fragment length polymorphism (MSAP) technique to paired plant-pollen (i.e., sporophyte-male gametophyte) DNA samples, and then comparing methylation patterns and epigenetic population differentiation in sporophytes and their descendant gametophytes, we investigated transgenerational constancy of epigenetic structure in three populations of the perennial herb Helleborus foetidus (Ranunculaceae). Single-locus and multilocus analyses revealed extensive epigenetic differentiation between sporophyte populations. Locus-by-locus comparisons of methylation status in individual sporophytes and descendant gametophytes showed that ∼75% of epigenetic markers persisted unchanged through gametogenesis. In spite of some epigenetic reorganization taking place during gametogenesis, multilocus epigenetic differentiation between sporophyte populations was preserved in the subsequent gametophyte stage. In addition to illustrating the efficacy of applying the MSAP technique to paired plant-pollen DNA samples to investigate epigenetic gametic inheritance in wild plants, this paper suggests that epigenetic differentiation between adult plant populations of H. foetidus is likely to persist across generations.     

Epigenetic correlates of plant phenotypic plasticity: DNA methylation differs between prickly and nonprickly leaves in heterophyllous Ilex aquifolium (Aquifoliaceae) trees

Phenotypic plasticity is central to the persistence of populations and a key element in the evolution of species and ecological interactions, but its mechanistic basis is poorly understood. This article examines the hypothesis that epigenetic variation caused by changes in DNA methylation are related to phenotypic plasticity in a heterophyllous tree producing two contrasting leaf types. The relationship between mammalian browsing and the production of prickly leaves was studied in a population of Ilex aquifolium (Aquifoliaceae). DNA methylation profiles of contiguous prickly and nonprickly leaves on heterophyllous branchlets were compared using a methylation‐sensitive amplified polymorphism (MSAP) method. Browsing and the production of prickly leaves were correlated across trees. Within heterophyllous branchlets, pairs of contiguous prickly and nonprickly leaves differed in genome‐wide DNA methylation. The mean per‐marker probability of methylation declined significantly from nonprickly to prickly leaves. Methylation differences between leaf types did not occur randomly across the genome, but affected predominantly certain specific markers. The results of this study, although correlative in nature, support the emerging three‐way link between herbivory, phenotypic plasticity and epigenetic changes in plants, and also contribute to the crystallization of the consensus that epigenetic variation can complement genetic variation as a source of phenotypic variation in natural plant populations. © 2012 The Linnean Society of London     

Natural epigenetic variation in the female great roundleaf bat (Hipposideros armiger) populations

Epigenetic modifications are considered to have an important role in evolution. DNA methylation is one of the best studied epigenetic mechanisms and methylation variability is crucial for promoting phenotypic diversification of organisms in response to environmental variation. A critical first step in the assessment of the potential role of epigenetic variation in evolution is the identification of DNA methylation polymorphisms and their relationship with genetic variations in natural populations. However, empirical data is scant in animals, and particularly so in wild mammals. Bats are considered as bioindicators because of their sensitivity to environmental perturbations and they may present an opportunity to explore epigenetic variance in wild mammalian populations. Our study is the first to explore these questions in the female great roundleaf bat (Hipposideros armiger) populations using the methylation-sensitive amplified polymorphism (MSAP) technique. We obtained 868 MSAP sites using 18 primer combinations and found (1) a low genomic methylation level (21.3?% on average), but extensive DNA methylation polymorphism (90.2?%) at 5'-CCGG-3' sites; (2) epigenetic variation that is structured into distinct between- (29.8?%) and within- (71.2?%) population components, as does genetic variation; and (3) a significant correlation between epigenetic and genetic variations (P?相似文献   

Changes in DNA methylation fingerprint of Quercus ilex trees in response to experimental field drought simulating projected climate change

Rapid genetic changes in plants have been reported in response to current climate change. We assessed the capacity of trees in a natural forest to produce rapid acclimation responses based on epigenetic modifications. We analysed natural populations of Quercus ilex, the dominant tree species of Mediterranean forests, using the methylation‐sensitive amplified polymorphism (MSAP) technique to assess patterns and levels of methylation in individuals from unstressed forest plots and from plots experimentally exposed to drought for 12 years at levels projected for the coming decades. The percentage of hypermethylated loci increased, and the percentage of fully methylated loci clearly decreased in plants exposed to drought. Multivariate analyses exploring the status of methylation at MSAP loci also showed clear differentiation depending on stress. The PCA scores for the MSAP profiles clearly separated the genetic from the epigenetic structure, and also significantly separated the samples within each group in response to drought. Changes in DNA methylation highlight the large capacity of plants to rapidly acclimate to changing environmental conditions, including trees with long life spans, and our results demonstrate those changes. These changes, although unable to prevent the decreased growth and higher mortality associated with this experimental drought, occurred together with a dampening in such decreases as the long‐term treatment progressed.     

微生境对鼎湖山锥表观遗传变异的影响

为了解种群内水平上影响植物的适应机制与空间格局关系的因素,对鼎湖山2个演替阶段林分锥(Castanopsis chinensis)种群通过DNA甲基敏感扩增片段多态性进行表观遗传特征分析,结果表明,微生境对表观遗传变异的贡献由成熟林的20.2%降低为过熟林的15.7%,但地形因素的影响却增大,同时微生境中具体起显著作用的环境因素在两个林分也不同。因此,微生境特征对种群适应机制和分布格局有显著影响,对演替阶段也有影响。     

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。     

Analysis of DNA methylation patterns of PLBs derived from <Emphasis Type="Italic">Cymbidium hybridium</Emphasis> based on MSAP

The best known and most thoroughly studied epigenetic phenomenon is DNA methylation, which plays an important role in regulating gene expression during plant regeneration and development. In this study, the methylation-sensitive amplified polymorphism (MSAP) technique was carried out to determine differences in methylation profiles between two forms of protocorm-like bodies (PLBs), continuously proliferating PLBs (cPLBs) and spontaneously-differenting PLBs (sdPLBs), derived from cultures of Cymbidium hybridium. A total of 72 selective primer combinations were used to assess the status of cytosine methylation of DNA in these tissues. Of 4,440 fragments obtained 911 fragments, each representing a recognition site cleaved by one or both of the isoschizomers (Hpa II and Msp I), were amplified and were significantly different between the two forms of PLBs. Frequency of total and full-methylation of cPLBs and sdPLBs were 26.7/12.2%, 24.1/11.1%, respectively. In addition, 14 types of MSAP patterns detected in the two forms of PLBs belonged to two classes, type I and II. Sequencing of 14 differentially methylated fragments and their subsequent blast search revealed that cytosine methylated 5′-CCGG-3′ sequences were equally distributed in the coding and non-coding regions. Southern blotting was conducted to verify the methylation polymorphism.     

Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum

Interspecific hybridization is known for triggering genetic and epigenetic changes, such as modifications on DNA methylation patterns and impact on phenotypic plasticity and ecological adaptation. Wild potatoes (Solanum, section Petota) are adapted to multiple habitats along the Andes, and natural hybridizations have proven to be a common feature among species of this group. Solanum × rechei, a recently formed hybrid that grows sympatrically with the parental species S. kurtzianum and S. microdontum, represents an ideal model for studying the ecologically and evolutionary importance of hybridization in generating of epigenetic variability. Genetic and epigenetic variability and their correlation with morphological variation were investigated in wild and ex situ conserved populations of these three wild potato species using amplified fragment length polymorphism (AFLP) and methylation‐sensitive amplified polymorphism (MSAP) techniques. We observed that novel methylation patterns doubled the number of novel genetic patterns in the hybrid and that the morphological variability measured on 30 characters had a higher correlation with the epigenetic than with the genetic variability. Statistical comparison of methylation levels suggested that the interspecific hybridization induces genome demethylation in the hybrids. A Bayesian analysis of the genetic data reveled the hybrid nature of S. × rechei, with genotypes displaying high levels of admixture with the parental species, while the epigenetic information assigned S. × rechei to its own cluster with low admixture. These findings suggested that after the hybridization event, a novel epigenetic pattern was rapidly established, which might influence the phenotypic plasticity and adaptation of the hybrid to new environments.     

Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation‐sensitive amplified polymorphism

Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal‐contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation‐sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal‐contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal‐contaminated site compared to uncontaminated populations. Other genotypes from a different metal‐contaminated site within the same region appear to be recalcitrant to metal‐induced DNA alterations even ≥30 years of tree life exposure to nickel and copper . MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal‐contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.     

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.     

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。     

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.     

甲基化敏感扩增多态性技术(MSAP)在生态学中的应用

DNA甲基化是生物体内最为重要的表观遗传修饰形式之一,在生态学上的应用越来越广泛。在收集、整理生态表观遗传学相关文献的基础上,介绍了甲基化敏感扩增多态性技术(MSAP)的原理、优势与局限性及其在生态学上的应用和展望。MSAP因其应用广泛、操作简便等优点成为研究DNA甲基化水平的有力工具,特别是在探究生物体如何快速适应生境变化以及外来入侵生物如何突破遗传瓶颈等问题上。MSAP技术能够很好地揭示生物种群内部或种群之间的表观遗传差异,是对遗传多样性、遗传变异研究的有力补充。     

Genome‐wide DNA methylation alterations of Alternanthera philoxeroides in natural and manipulated habitats: implications for epigenetic regulation of rapid responses to environmental fluctuation and phenotypic variation

Alternanthera philoxeroides (alligator weed) is an invasive weed that can colonize both aquatic and terrestrial habitats. Individuals growing in different habitats exhibit extensive phenotypic variation but little genetic differentiation in its introduced range. The mechanisms underpinning the wide range of phenotypic variation and rapid adaptation to novel and changing environments remain uncharacterized. In this study, we examined the epigenetic variation and its correlation with phenotypic variation in plants exposed to natural and manipulated environmental variability. Genome‐wide methylation profiling using methylation‐sensitive amplified fragment length polymorphism (MSAP) revealed considerable DNA methylation polymorphisms within and between natural populations. Plants of different source populations not only underwent significant morphological changes in common garden environments, but also underwent a genome‐wide epigenetic reprogramming in response to different treatments. Methylation alterations associated with response to different water availability were detected in 78.2% (169/216) of common garden induced polymorphic sites, demonstrating the environmental sensitivity and flexibility of the epigenetic regulatory system. These data provide evidence of the correlation between epigenetic reprogramming and the reversible phenotypic response of alligator weed to particular environmental factors.     

叶锈菌胁迫下的小麦基因组MSAP分析

内源DNA甲基化是真核生物表观遗传调控的重要组成部分, 在真核生物的基因表达调控中具有重要的作用。生物胁迫为植物提供一种内在的表观遗传进化动力。研究生物胁迫下DNA甲基化的变异模式, 有助于全面理解DNA甲基化的表观调控生物学功能。小麦近等基因系TcLr19、TcLr41及其感病亲本Thatcher在苗期对叶锈菌生理小种THTT、TKTJ分别表现为小种特异性抗病反应和感病反应。文章利用甲基化敏感扩增多态性(Methylation-sensitive amplified polymorphism, MSAP)技术分析了小麦的甲基化水平, 同时比较了苗期在生物胁迫前后基因组DNA胞嘧啶甲基化模式。用60对MSAP引物对接种前后的小麦DNA进行全基因组筛选, 没有直接分离得到接菌前后的甲基化模式的差异, 结果初步表明, 叶锈菌并没有诱导稳定且特异的植物基因组DNA胞嘧啶位点的甲基化模式变化, 但发现TcLr41及其感病亲本Thatcher之间存在表观遗传学差异。