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。     

Utility of the methylation-sensitive amplified polymorphism (MSAP) marker for detection of DNA methylation polymorphism and epigenetic population structure in a wild barley species (<Emphasis Type="Italic">Hordeum brevisubulatum</Emphasis>)

We report here that by using a modified scoring criterion, the methylation-sensitive amplified polymorphism or MSAP marker can be used effectively to detect polymorphism in DNA methylation patterns within and among populations of a perennial wild barley species, Hordeum brevisubulatum. Twenty-four selected individual genotypes representing four natural populations of H. brevisubulatum distributed in the Songnen Prairie in northeastern China were studied. The utility of MSAP was evidenced by its detection of high levels of polymorphism in DNA methylation patterns between individuals within a given population, and the clear inter-population differentiation in methylation patterns (methylation-based epigenetic population structure) revealed among the four populations. The resolving power of MSAP to detect DNA methylation polymorphism was found to be comparable with that of a retrotransposon-based sequence-specific amplified polymorphism marker, or SSAP, to detect genetic polymorphism in the same set of plants, suggesting that MSAP with a modified scoring criterion can be used efficiently to detect DNA methylation polymorphism and assess epigenetic population structure in natural plant populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

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.     

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.     

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.     

Genetic and epigenetic divergence between disturbed and undisturbed subpopulations of a Mediterranean shrub: a 20‐year field experiment

Little is known on the potential of ecological disturbance to cause genetic and epigenetic changes in plant populations. We take advantage of a long‐term field experiment initiated in 1986 to study the demography of the shrub Lavandula latifolia, and compare genetic and epigenetic characteristics of plants in two adjacent subplots, one experimentally disturbed and one left undisturbed, 20 years after disturbance. Experimental setup was comparable to an unreplicated ‘Before‐After‐Control‐Impact’ (BACI) design where a single pair of perturbed and control areas were compared. When sampled in 2005, plants in the two subplots had roughly similar ages, but they had established in contrasting environments: dense conspecific population (‘Undisturbed’ subpopulation) versus open area with all conspecifics removed (‘Disturbed’ subpopulation). Plants were characterized genetically and epigenetically using amplified fragment length polymorphism (AFLP) and two classes of methylation‐sensitive AFLP (MSAP) markers. Subpopulations were similar in genetic diversity but differed in epigenetic diversity and multilocus genetic and epigenetic characteristics. Epigenetic divergence between subpopulations was statistically unrelated to genetic divergence. Bayesian clustering revealed an abrupt linear boundary between subpopulations closely coincident with the arbitrary demarcation line between subplots drawn 20 years back, which supports that genetic and epigenetic divergence between subpopulations was caused by artificial disturbance. There was significant fine‐scale spatial structuring of MSAP markers in both subpopulations, which in the Undisturbed one was indistinguishable from that of AFLP markers. Genetic differences between subpopulations could be explained by divergent selection alone, while the concerted action of divergent selection and disturbance‐driven appearance of new methylation variants in the Disturbed subpopulation is proposed to explain epigenetic differences. This study provides the first empirical evidence to date suggesting that relatively mild disturbances could leave genetic and epigenetic signatures on the next adult generation of long‐lived plants.     

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.     

Identification of exotic genetic components and DNA methylation pattern analysis of three cotton introgression lines from <Emphasis Type="Italic">Gossypium bickii</Emphasis>

The impact of alien DNA fragments on plant genome has been studied in many species. However, little is known about the introgression lines of Gossypium. To study the consequences of introgression in Gossypium, we investigated ∼2000 genomic and ∼800 epigenetic sites in three typical cotton introgression lines, as well as their cultivar (Gossypium hirsutum) and wild parents (Gossypium bickii), by amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP). The results demonstrate that an average of 0.5% of exotic DNA segments from wild cotton is transmitted into the genome of each introgression line, with the addition of other forms of genetic variation. In total, an average of 0.7% of genetic variation sites is identified in introgression lines. Simultaneously, the overall cytosine methylation level in each introgression line is very close to that of the upland cotton parent (an average of 22.6%). Further dividing patterns reveal that both hypomethylation and hypermethylation occurred in introgression lines in comparison with the upland cotton parent. Sequencing of nine methylation polymorphism fragments showed that most (7 of 9) of the methylation alternations occurred in the noncoding sequences. The molecular evidence of introgression from wild cotton into introgression lines in our study is identified by AFLP. Moreover, the causes of petal variation in introgression lines are discussed.     

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。     

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.     

Genetically stable regeneration of apple plants from slow growth

Shoot-tips of apple cultivar `Gala' were stored in vitrousing a low temperature slow-growth culture method. All shoot-tips survived 1-year storage, with a significant height increment over that period. Eight `Gala' single-bud sibling lines were established for genetic analysis. Although cytological examination detected chromosomal variation in plants recovered from slow growth culture, the ploidy remained genetically stable relative to the before-storage cultures. An amplified fragment length polymorphism (AFLP) assay was performed to detect DNA sequence variation. No differences in the DNA fragment patterns were observed using 20 primer combinations between the before-storage and the stored samples. In addition, a methylation sensitive amplified polymorphism (MSAP) assay was performed to investigate the DNA methylation status in both the before-storage and stored samples. It was found that the slow-growth storage resulted in a significant DNA methylation change in the stored shoots compared with the before-storage samples.     

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.     

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.     

Genetic and epigenetic stability of cryopreserved and cold-stored hops (Humulus lupulus L.)

Conventional cold storage and cryopreservation methods for hops (Humulus lupulus L.) are available but, to our knowledge, the genetic and epigenetic stability of the recovered plants have not been tested. This study analyzed 51 accessions of hop using the molecular techniques, Random Amplified DNA Polymorphism (RAPD) and Amplified Fragment Length Polymorphism (AFLP), revealing no genetic variation among greenhouse-grown controls and cold stored or cryopreserved plants. Epigenetic stability was evaluated using Methylation Sensitive Amplified Polymorphism (MSAP). Over 36% of the loci were polymorphic when the cold and cryo-treated plants were compared to greenhouse plants. The main changes were demethylation events and they were common to the cryopreserved and cold stored plants indicating the possible effect of the in vitro establishment process, an essential step in both protocols. Protocol-specific methylation patterns were also detected indicating that both methods produced epigenetic changes in plants following cold storage and cryopreservation.     

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.     

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.     

组织培养导致的草莓DNA甲基化变异

以草莓品种‘丰香’和‘全明星’为材料,用甲基化敏感扩增多态性（MSAP）技术研究组织培养对草莓DNA甲基化的影响。结果表明,与普通苗相比,组织培养导致草莓试管苗的DNA甲基化水平下降,甲基化模式的变异以去甲基化为主。组织培养导致的DNA甲基化变异不稳定,在田间无性繁殖过程中,试管苗的无性繁殖后代DNA甲基化水平逐渐升高,仅部分变异的甲基化模式能够在试管苗的无性繁殖后代中稳定传递。两个品种之间,纽织培养对DNA甲基化变异程度的影响不同。     

Comparative analysis of DNA methylation changes in two contrasting wheat genotypes under water deficit

DNA methylation is one of the epigenetic mechanisms regulating gene expression in plants in response to environmental conditions. In this study, analysis of methylation patterns was carried out in order to assess the effect of water stress in two contrasting wheat genotypes using methylation-sensitive amplified polymorphism (MSAP). The results revealed that demethylation was higher in drought-tolerant genotype (C306) as compared to drought-sensitive genotype (HUW468) after experiencing drought stress. Comparisons of different MSAP patterns showed a high percentage of polymorphic bands between tolerant and susceptible wheat genotypes (from 74.79 % at anthesis to 88.89 % at tillering). Furthermore, differential DNA methylation in roots and leaves also revealed tissue-specific methylation of genomic DNA. Interestingly, 54 developmental stage-specific bands and 23 bands that were found contrasting between these two wheat genotypes were detected. Furthermore, a few sites with stable DNA methylation differences were identified between drought-tolerant and drought-sensitive cultivars, thus providing genotype-specific epigenetic markers. These results not only provide data on differences in DNA methylation changes but also contribute to dissection of molecular mechanisms of drought response and tolerance in wheat.