The influence of Al<Superscript>3+</Superscript> on DNA methylation and sequence changes in the triticale (× <Emphasis Type="Italic">Triticosecale</Emphasis> Wittmack) genome

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

Flow Cytometry,HPLC-RP,and metAFLP Analyses to Assess Genetic Variability in Somatic Embryo-Derived Plantlets of <Emphasis Type="BoldItalic">Gentiana pannonica</Emphasis> Scop

Cytometric and molecular techniques were used to verify genetic uniformity among somatic embryo-derived plantlets of Gentiana pannonica Scop. Cytometric analysis of regenerants revealed absence of chromosomal changes and alterations in ploidy. However, reverse phase high pressure liquid chromatography detected higher levels of methylation in regenerated plants than those of control plants. These changes were further investigated using a quantitative molecular marker-based approach. This revealed that numerous tissue culture-induced variations, ∼3% (epi)mutations, were observed, including sequence variation and changes in methylation patterns. Moreover, complex patterns of variation, including combinations of genetic and epigenetic changes, were relatively high (ca. 9%). Overall, tissue culture-induced variation reached 16%; while, demethylation was lower than de novo methylation in heterozygotic material and similar in all regenerated plantlets.     

DNA methylation changes in triticale due to in vitro culture plant regeneration and consecutive reproduction

Doubled haploids of triticale are of interest for plant breeders due to hybrid breeding programs based on cytoplasmic male sterility Tt phenomenon. However, (epi)mutations appearing during in vitro culture regeneration may lead to a phenotypic variation that makes the uniformity of plant materials questionable. Using RP-HPLC genomic DNA methylation of donor doubled haploid plants utilized as a source of tissues for the in vitro regeneration (via androgenesis and somatic embryogenesis) of triticale cv. Bogo and their consecutive generative progeny was evaluated. It was demonstrated that in vitro cultures induced a decrease of the DNA methylation of the regenerants independently of the approach used for plant regeneration. The decrease in DNA methylation of genomic DNA proceeded up to the first/second successive generations followed by the beginning of its reestablishment. Moreover, somatic embryogenesis resulted in a higher level of genomic DNA demethylation in regenerants than androgenesis and the process of methylation seems to be affected by donor plant. It is being speculated that long term changes in genomic DNA methylation may be a source of off-type individuals that may spontaneously arise during plant breeding.     

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。     

Cryopreservation enhances embryogenic capacity of Gentiana cruciata (L.) suspension culture and maintains (epi)genetic uniformity of regenerants

The embryogenic cell suspension culture of Gentiana cruciata, cryopreserved by the encapsulation/dehydration method, survived both short- (48 h) and long-term (1.5 years) cryostorage with more than 80% viability. To assess the influence of cryotreatments on the embryogenic potential, a proembryogenic mass was encapsulated and exposed to the following treatments: (1) osmotic dehydration (OD), (2) OD + air desiccation (AD) and (3) OD + AD + cryostorage (LN). The somatic embryogenesis efficiency increased ten times after osmotic dehydration. The AD and LN cryotreatments did not cause any significant alterations in somatic embryo production. We monitored the (epi)genetic stability of 288 regenerants derived from: non-cryotreated, short-term, and long-term cryostored tissue using metAFLP markers and ten primer combinations. Changes in the sequence and DNA methylation levels were studied by subjecting the DNA to digestion with two pairs of isoschisomer restriction enzymes (KpnI/MseI and Acc65I/MseI). Two new AFLP unique DNA fragments at the DNA sequence level, with no differences at the methylation level, were found between regenerants derived from cryopreserved tissue, compared with the non-cryotreated controls. The Acc65I/MseI methylation levels for the three groups of regenerants were not significantly different. Cluster analysis was capable of identifying a number of sub-clusters. Only one of the sub-clusters comprises almost all regenerants derived from non-cryotreated and short-term cryostored tissue. Plantlets derived from long-term cryostored tissue were grouped into separate clusters. The observed AFLP alterations did not appear to be associated with the use of cryopreservation, but were probably related to the process of in vitro culture.     

Consistent and Heritable Alterations of DNA Methylation Are Induced by Tissue Culture in Maize

Plants regenerated from tissue culture and their progenies are expected to be identical clones, but often display heritable molecular and phenotypic variation. We characterized DNA methylation patterns in callus, primary regenerants, and regenerant-derived progenies of maize using immunoprecipitation of methylated DNA (meDIP) to assess the genome-wide frequency, pattern, and heritability of DNA methylation changes. Although genome-wide DNA methylation levels remained similar following tissue culture, numerous regions exhibited altered DNA methylation levels. Hypomethylation events were observed more frequently than hypermethylation following tissue culture. Many of the hypomethylation events occur at the same genomic sites across independent regenerants and cell lines. The DNA methylation changes were often heritable in progenies produced from self-pollination of primary regenerants. Methylation changes were enriched in regions upstream of genes and loss of DNA methylation at promoters was associated with altered expression at a subset of loci. Differentially methylated regions (DMRs) found in tissue culture regenerants overlap with the position of naturally occurring DMRs more often than expected by chance with 8% of tissue culture hypomethylated DMRs overlapping with DMRs identified by profiling natural variation, consistent with the hypotheses that genomic stresses similar to those causing somaclonal variation may also occur in nature, and that certain loci are particularly susceptible to epigenetic change in response to these stresses. The consistency of methylation changes across regenerants from independent cultures suggests a mechanistic response to the culture environment as opposed to an overall loss of fidelity in the maintenance of epigenetic states.     

Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications

In vitro cell and tissue-based systems have tremendous potential in fundamental research and for commercial applications such as clonal propagation, genetic engineering and production of valuable metabolites. Since the invention of plant cell and tissue culture techniques more than half a century ago, scientists have been trying to understand the morphological, physiological, biochemical and molecular changes associated with tissue culture responses. Establishment of de novo developmental cell fate in vitro is governed by factors such as genetic make-up, stress and plant growth regulators. In vitro culture is believed to destabilize the genetic and epigenetic program of intact plant tissue and can lead to chromosomal and DNA sequence variations, methylation changes, transposon activation, and generation of somaclonal variants. In this review, we discuss the current status of understanding the genomic and epigenomic changes that take place under in vitro conditions. It is hoped that a precise and comprehensive knowledge of the molecular basis of these variations and acquisition of developmental cell fate would help to devise strategies to improve the totipotency and embryogenic capability in recalcitrant species and genotypes, and to address bottlenecks associated with clonal propagation.     

Methylation patterns of the human apoA-I/C-III/A-IV gene cluster in adult and embryonic tissues suggest dynamic changes in methylation during development.

We describe here a detailed analysis of the methylation patterns of the apoC-III and apoA-IV genes in adult and embryonic tissues. Together with previously reported data on the human apoA-I gene (4), the results presented here constitute a comprehensive study on the methylation pattern of the apoA-I/C-III/A-IV gene cluster. The two genes (apoC-III and apoA-IV) display tissue-specific methylation patterns that correlate with their activity. This gene-specific methylation pattern indicates that the apoA-I/C-III/A-IV gene cluster is not one entity with respect to methylation. The cluster is almost entirely methylated in tissues that do not express any of the genes; however, individual gene regions are unmethylated in the tissue of expression. A comparison of the observed methylation patterns in adult tissues with those in embryonic tissues suggests that the mature tissue-specific methylation patterns are a result of an interplay between demethylation and de novo methylation events in the embryo. These changes in DNA methylation include demethylation in the early embryo followed by de novo methylation at later stages. A second round of tissue-specific demethylation and methylation de novo occurs in the late embryo as well. Evidence presented here supports the idea that CpG islands are protected in general from methylation de novo by a built-in signal and not by CpG density per se.     

马铃薯（Solanum tuberosum L.）茎尖的超低温保存及其遗传变异的初步观察

研究马铃薯茎尖超低温保存技术的结果表明,4℃低温下锻炼6d,在添加二甲基亚砜（DMSO）和乙酰胺的培养基中预培养5d,60％PVS2于室温下装载30min,0℃下PVS2脱水40min时,茎尖成活率最高（71．6％）,再生植株生长分化正常。进一步对再生植株进行AFLP分析,6对引物组合共扩增出385条带,超低温保存前后的材料之间未见到明显差的异带,但用MSAP技术分析超低温保存前后植株甲基化的结果显示：超低温保存后的材料均有不同程度的甲基化。在扩增的624条带中,处理与否之间完全一致的带型为584条;有变化的带型为40条,处理2（茎尖经过完整的超低温保存过程,区别于处理1,增加了冷冻、解冻和洗涤后恢复培养）有13个位点的甲基化增加,21个位点去甲基化。     

Changes in genomic methylation patterns during the formation of triploid asexual dandelion lineages

DNA methylation is an epigenetic mechanism that has the potential to affect plant phenotypes and that is responsive to environmental and genomic stresses such as hybridization and polyploidization. We explored de novo methylation variation that arises during the formation of triploid asexual dandelions from diploid sexual mother plants using methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. In dandelions, triploid apomictic asexuals are produced from diploid sexual mothers that are fertilized by polyploid pollen donors. We asked whether the ploidy level change that accompanies the formation of new asexual lineages triggers methylation changes that contribute to heritable epigenetic variation within novel asexual lineages. Comparison of MS‐AFLP and AFLP fragment inheritance in a diploid × triploid cross revealed de novo methylation variation between triploid F₁ individuals. Genetically identical offspring of asexual F₁ plants showed modest levels of methylation variation, comparable to background levels as observed among sibs in a long‐established asexual lineage. Thus, the cross between ploidy levels triggered de novo methylation variation between asexual lineages, whereas it did not seem to contribute directly to variation within new asexual lineages. The observed background level of methylation variation suggests that considerable autonomous methylation variation could build up within asexual lineages under natural conditions.     

Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer.

Cytosine methylation at CpG dinucleotides is thought to cause more than one-third of all transition mutations responsible for human genetic diseases and cancer. We investigated the methylation status of the CpG dinucleotide at codon 248 in exon 7 of the p53 gene because this codon is a hot spot for inactivating mutations in the germ line and in most human somatic tissues examined. Codon 248 is contained within an HpaII site (CCGG), and the methylation status of this and flanking CpG sites was analyzed by using the methylation-sensitive enzymes CfoI (GCGC) and HpaII. Codon 248 and the CfoI and HpaII sites in the flanking introns were methylated in every tissue and cell line examined, indicating extensive methylation of this region in the p53 gene. Exhaustive treatment of an osteogenic sarcoma cell line, TE85, with the hypomethylating drug 5-aza-2'-deoxycytidine did not demethylate codon 248 or the CfoI sites in intron 6, although considerable global demethylation of the p53 gene was induced. Constructs containing either exon 7 alone or exon 7 and the flanking introns were transfected into TE85 cells to determine whether de novo methylation would occur. The presence of exon 7 alone caused some de novo methylation to occur at codon 248. More extensive de novo methylation of the CfoI sites in intron 6, which contains an Alu sequence, occurred in cells transfected with a vector containing exon 7 and flanking introns. With longer time in culture, there was increased methylation at the CfoI sites, and de novo methylation of codon 248 and its flanking HpaII sites was observed. These de novo-methylated sites were also resistant to 5-aza-2'-deoxycytidine-induced demethylation. The frequent methylation of codon 248 and adjacent Alu sequence may explain the enhanced mutability of this site as a result of the deamination of the 5-methylcytosine.     

Methylation analysis revealed salicylic acid affects pearl millet defense through external cytosine DNA demethylation

The cytosine DNA methylation and demethylation have a role in regulating plant responses to the environment by affecting the promoter regions of most plant defense-related genes through the CpG islands or the CCGG motifs. Salicylic acid, a defense and signaling plant hormone, is seen playing crucial role in the variation of the methylome. In this study, the effects of salicylic acid and feeding of the millet headminer (Heliocheilus albipunctella de Joannis) on pearl millet DNA methylome changes were evaluated through MSAP epigenotyping during panicle development. The results showed that millet headminer feeding increased the level of genomic methylation while application of salicylic acid caused DNA demethylation occurring mostly at external cytosine and accompanied by a decrease of the number of larvae per panicle. This suggests that hemimethylation (external cytosine methylation) has key role in regulating defense responses and conferring tolerance to pearl millet through salicylic acid application.     

Tissue culture-induced locus-specific alteration in DNA methylation and its correlation with genetic variation in <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> Benth. et Hook. f

We have reported recently that tissue culture induced a high level of genetic variation at the primary nucleotide sequence in regenerants of medicinal plant Codonopsis lanceolata. It is not known, however, whether epigenetic variation in the form of alteration in DNA methylation also occurred in these plants. Here, we investigated possible alterations in level and pattern of cytosine methylation at the CCGG sites in the same set of regenerants relative to the donor plant, by the MSAP method employing a pair of isoschizomers, HpaII and MspI, which recognize the same restriction site but are differentially sensitive to cytosine methylation at the CCGG sites. A total of 1,674 MSAP profiles were resolved using 39 primer combinations. Of these, 177 (10.5%) profiles were polymorphic among the regenerants and/or between the regenerant(s) and the donor plant, in EcoRI + HpaII or EcoRI + MspI digest but not in both, indicating alteration in cytosine methylation patterns of specific loci, though their estimated total level of methylation remained more or less the same as the donor plant. Gel blot analysis validated most of the variant MSAP profiles as bona fide alteration in methylation patterns. Correlation analysis between the MSAP data and the previously reported ISSR and RAPD data revealed significant correlations, suggesting their possible intrinsic interrelatedness. Thirty-seven typical variant MSAP profiles were isolated and sequenced, of which 5 showed significant homology to known-function genes, 2 to chloroplast sequences, whilst the rest 30 did not find a match in the database. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. W. L. Guo and R. Wu contributed equally to this work.     

Sex-specific dynamics of global chromatin changes in fetal mouse germ cells

Mammalian germ cells undergo global reprogramming of DNA methylation during their development. Global DNA demethylation occurs around the time when the primordial germ cells colonize the embryonic gonads and this coincides with dynamic changes in chromatin composition. Global de novo DNA methylation takes place with remarkably different dynamics between the two sexes, prospermatogonia attaining methylation during fetal stages and oocytes attaining methylation postnatally. Our hypothesis was that dynamic changes in chromatin composition may precede or accompany the wave of global DNA de novo methylation as well. We used immunocytochemistry to measure global DNA methylation and chromatin components in male and female mouse fetal germ cells compared to control somatic cells of the gonad. We found that global DNA methylation levels sharply increased in male germ cells at 17.5 days post coitum, but remained low in female germ cells at all fetal stages. Global changes in chromatin composition: i, preceded global DNA methylation in fetal germ cells; ii, sex specifically occurred in male but not in female germ cells; iii, affected active and repressive histone marks and iv, included histone tail and histone globular domain modifications. Our data suggest that dynamic changes of chromatin composition may provide a framework for the pattern of male-specific de novo DNA methylation in prospermatogonia.     

DNA methylation in mouse gametogenesis

DNA methylation is involved in many biological processes and is particularly important for both development and germ cell differentiation. Several waves of demethylation and de novo methylation occur during both male and female germ line development. This has been found at both the gene and all genome levels, but there is no demonstrated correlation between them. During the postnatal germ line development of spermatogenesis, we found very complex and drastic DNA methylation changes that we could correlate with chromatin structure changes. Thus, detailed studies focused on localization and expression pattern of the chromatin proteins involved in both DNA methylation, histone tails modification, condensin and cohesin complex formation, should help to gain insights into the mechanisms at the origin of the deep changes occurring during this particular period.     

Physiological aspects of genome variability in tissue culture. I. Growth phase-dependent differential DNA methylation of the carrot genome (Daucus carota L.) during primary culture

Investigations were performed on growth phase-dependent EcoRII site-specific DNA methylation of the carrot genome during primary culture to elucidate physiological aspects of genome DNA variability in tissue culture. While DNA methylation of the root cambium and the secondary phloem and petioles of carrot leaves were strikingly different, the methylation level of the secondary phloem seemed to be independent of cultivar origin, the age of the plants and the extent of secondary root growth. As was shown earlier a change in the differentiated state of the secondary phloem by tissue culture leads to changes in genome modification. Whereas de novo methylation was observed during the first 2 weeks of growth initiation, the results presented demonstrate genome de-methylation during the transition to stationary growth indicating differential nome methylation during different phases of culture. The presence of kinetin in the nutrient medium of the primary culture was found to be antagonistic to changes in genome modification in general. De novo methylation and subsequent de-methylation of the carrot genome are discussed as gross changes obviously essential to molecular genome differentiation during tissue culture.