基于MSAP和SSR标记的高粱甲基化连锁群LGC、LGD的构建

以高粱(Sorghum bicolor(L.)Moench)品种‘B_2V_4’和‘1383-2’杂交获得的F_2群体为材料,通过SSR和MSAP标记检测高粱基因组差异,构建其甲基化遗传连锁群。结果显示,高粱甲基化连锁群LGC含有3个SSR标记和23个甲基化标记,覆盖高粱基因组44.3 cM;甲基化连锁群LGD含有4个SSR标记和8个甲基化标记,覆盖高粱基因组46.2 cM。LGC上甲基化位点仅来源于EcoRⅠ/MspⅠ酶切组合,而LGD上有来源于EcoRⅠ/MspⅠ和EcoRⅠ/HpaⅡ两种酶切组合的甲基化位点。在LGC连锁群Xtxp 69附近检测到一个密集的甲基化位点区域。研究结果表明MSAP标记可以快速检测植物基因组甲基化差异,适用于构建甲基化连锁群。     

镉胁迫下萝卜基因组DNA甲基化敏感扩增多态性分析

应用甲基化敏感扩增多态性(MSAP)技术分析了重金属镉(cd)胁迫处理后萝卜基因组DNA甲基化程度的变化。结果表明,经50、250和500mg/L CdCl_2处理后,MSAP比率分别为37%、43%和51%,均高于对照(34%);全甲基化率(双链C~mCGG)分别为23%、25%和27%,而其对照为22%,表明重金属CdCl_2胁迫后,某些位点发生了重新甲基化。萝卜叶片DNA中总甲基化水平的增加与CdCl_2处理浓度呈显著正相关。甲基化变异可分为重新甲基化、去甲基化、不定类型以及与对照相同的甲基化模式等类型,Cd胁迫处理引起的植株基因组DNA甲基化程度的提高主要是重新甲基化。     

五月季竹开花及复壮过程中DNA甲基化的MSAP分析

以五月季竹为材料,采用MSAP技术对其开花及花后无性复壮过程中的DNA甲基化状况进行检测,分析其开花前后的甲基化动态,以揭示竹子开花及复壮过程中的表观遗传变化规律。结果显示:（1）五月季竹开花时其叶片甲基化水平降低,而在无性复壮产生不再开花新竹的过程中其叶片甲基化水平又逐渐回升;（2）与未开花竹株相比,五月季竹开花时有29.09%的甲基化位点发生了变异,其中有17.88%的位点在开花植株中发生了完全的去甲基化,远高于发生甲基化位点的比率;（3）复壮竹株与未开花竹株之间发生变异的位点数和所占比率,尤其是发生去甲基化的位点数和比率,低于开花竹株;（4）开花五月季竹花器官的甲基化水平低于叶片,同时有28.58%的位点发生了甲基化状态的改变,且同样以去甲基化为主。     

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.     

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.     

Genomic Change,Retrotransposon Mobilization and Extensive Cytosine Methylation Alteration in Brassica napus Introgressions from Two Intertribal Hybridizations

Hybridization and introgression represent important means for the transfer and/or de novo origination of traits and play an important role in facilitating speciation and plant breeding. Two sets of introgression lines in Brassica napus L. were previously established by its intertribal hybridizations with two wild species and long-term selection. In this study, the methods of amplified fragment length polymorphisms (AFLP), sequence-specific amplification polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) were used to determine their genomic change, retrotransposon mobilization and cytosine methylation alteration in these lines. The genomic change revealed by the loss or gain of AFLP bands occurred for ∼10% of the total bands amplified in the two sets of introgressions, while no bands specific for wild species were detected. The new and absent SSAP bands appeared for 9 out of 11 retrotransposons analyzed, with low frequency of new bands and their total percentage of about 5% in both sets. MSAP analysis indicated that methylation changes were common in these lines (33.4–39.8%) and the hypermethylation was more frequent than hypomethylation. Our results suggested that certain extents of genetic and epigenetic alterations were induced by hybridization and alien DNA introgression. The cryptic mechanism of these changes and potential application of these lines in breeding were also discussed.     

Generality and characteristics of genetic and epigenetic changes in newly synthesized allotetraploid wheat lines

Previous studies have shown rapid and extensive genomic instability associated with early stages of allopolyploidization in wheat.However, these studies are based on either a few pre-selected genomic loci or genome-wide analysis of a single plant individual for a given cross combination, thus making the extent and generality of the changes uncertain.To further study the generality and characteristics of allopolyploidization-induced genomic instability in wheat, we investigated genetic and epigenetic changes from a genome-wide perspective (by using the AFLP and MSAP markers) in four sets of newly synthesized allotetraploid wheat lines with various genome constitutions, each containing three randomly chosen individual plants at the same generation.We document that although general chromosomal stability was characteristic of all four sets of allotetraploid wheat lines, genetic and epigenetic changes at the molecular level occurred in all these plants, with both kinds of changes classifiable into two distinct categories, i.e., stochastic and directed.The abundant type of genetic change is loss of parental bands while the prevalent cytosine methylation pattern alteration is hypermethylation at the CHG sites.Our results have extended previous studies regarding allopolyploidization-induced genomic dynamics in wheat by demonstrating the generality of both genetic and epigenetic changes associated with multiple nascent allotetraploid wheat lines, and providing novel insights into the characteristics of the two kinds of induced genomic instabilities.     

Analysis of DNA methylation during the germination of wheat seeds

DNA methylation is known to play a crucial role in regulating plant development and organ or tissue differentiation. Here, we focused on the DNA methylation dynamics during the germination of wheat seeds using the adapted AFLP technique so called methylation-sensitive amplified polymorphism (MSAP). The MSAP profiles of genomic DNA in embryo and endosperm tissues of germinating seeds, as well as dry seeds were characterized and notable changes of cytosine methylation were detected. Comparisons of MSAP profiles in different tissues tested showed that the methylation level in dry seeds is the highest. The alteration analysis of cytosine methylation displayed that the number of demethylation events were three times higher than that of de novo methylation, which indicated that the demethylation was predominant in germinating wheat seeds, though the methylation events occurred as well. Sixteen differentially displayed DNA fragments in MSAP profiles were cloned and the sequencing analysis confirmed that nine of them contained CCGG sites. The further BLAST search showed that four of the cloned sequences were located in coding regions. Interestingly, three of the sixteen candidates were homologous to retrotransposons, which indicated that switches between DNA methylation and demethylation occurred in retrotransposon elements along with the germination of wheat seeds.     

Genetic and epigenetic instability of amplification-prone sequences of a novel B chromosome induced by tissue culture in <Emphasis Type="Italic">Plantago lagopus</Emphasis> L.

Gene amplification is prevalent in many eukaryotes and has been found linked to various phenomena such as ontogenesis, carcinogenesis, in vitro culturing, neoplasia and drug resistance. Earlier, we reported a novel B chromosome in Plantago lagopus L., which was found to have arisen as a result of massive amplification of 5S rDNA. In addition, the chromosome is also composed of 45S rDNA and transposable elements. While the importance of gene amplification cannot be underestimated, its mechanism of origin is still unclear. Therefore, the aim of the present study was to determine whether amplification can be reactivated in the novel B chromosome. For this purpose, in vitro culture was used as stress. Three modes of tissue culture, i.e., direct, indirect and somatic embryogenesis were used for raising in vitro cultures. The variations due to genetic and epigenetic mechanisms were assessed in regenerants using molecular techniques, namely, PCR-RFLP, SSAP and MSAP. The retrotransposon-based molecular markers were applied to detect the polymorphism within transposable elements of in vitro regenerated and mother plants. We detected the variations that may be due to genetic changes either because of element recombination or activation of transposable elements which can lead to increase in the copy number. MSAP analysis revealed the differences in the DNA methylation pattern of the regenerants derived from novel chromosome bearing mother plants. Some regenerated plants were associated with increase and decrease in DNA methylation of both internal and external cytosine of the CCGG sequence.     

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.     

Detection of changes in DNA methylation induced by low-energy ion implantation in Arabidopsis thaliana

This study evaluated changes in DNA methylation in Arabidopsis thaliana plants grown from seeds implanted with low-energy N(+) and Ar(+) ions. Methylation-sensitive amplified polymorphism (MSAP) testing revealed altered DNA methylation patterns after ion implantation at doses of 1 × 10(14) to 1 × 10(16) ions/cm(2). Comparison of the MSAP electrophoretic profiles revealed nine types of polymorphisms in ion-implanted seedlings relative to control seedlings, among which four represented methylation events, three represented demethylation events, and the methylation status of two was uncertain. The diversity of plant DNA methylation was increased by low-energy ion implantation. At the same time, total genomic DNA methylation levels at CCGG sites were unchanged by ion implantation. Moreover, a comparison of polymorphisms seen in N(+) ion-implanted, Ar(+) ion-implanted, and control DNA demonstrated that the species of incident ion influenced the resulting DNA methylation pattern. Sequencing of eight isolated fragments that showed different changing patterns in implanted plants allowed their mapping onto variable regions on one or more of the five Arabidopsis chromosomes; these segments included protein-coding genes, transposon and repeat DNA sequence. A further sodium bisulfite sequencing of three fragments also displayed alterations in methylation among either different types or doses of incident ions. Possible causes for the changes in methylation are discussed.     

Variation and Patterns of DNA Methylation in Maize C-type CMS Lines and their Maintainers

DNA methylation plays an important role in gene expression regulation during biological development in plants. This study adopted methylation sensitive amplification polymorphism (MSAP) to compare the levels and patterns of cytosine methylation at CCGG sites in maize genome. The tissues assayed included seedlings and tassels of C-type cytoplasmic male sterility (C Huang Zao Si, C 48-2) and its maintainer lines. For each tissue, both C Huang Zao Si and C 48-2 were more methylated than their corresponding maintainers not only on MSAP ratios, but also on the full methylation levels. In different nuclear backgrounds, the two tissues were more methylated in Huang Zao Si than in 48-2, although the two lines shared the same cytoplasm. Full methylation of internal cytosine was the dominant type in the maize genome. In addition, four different classes of methylation patterns were identified in tassels between C-CMS lines and their maintainer lines; these were specific-methylation, demethylation, hypo-methylation, and hyper-methylation. The results obtained demonstrated the power of the MSAP technique for large-scale DNA methylation detection in the maize genome, and suggested the possible association between DNA methylation polymorphism and C-type cytoplasmic male sterility.     

Dedifferentiation-mediated changes in transposition behavior make the Activator transposon an ideal tool for functional genomics in rice

There is an inverse relationship between the level of cytosine methylation in genomic DNA and the activity of plant transposable elements. Increased transpositional activity is seen during early plant development when genomic methylation patterns are first erased and then reset. Prolonging the period of hypomethylation might therefore result in an increased transposition frequency, which would be useful for rapid genome saturation in transposon-tagged plant lines. We tested this hypothesis using transgenic rice plants containing Activator (Ac) from maize. R₁ seeds from an Ac-tagged transgenic rice line were either directly germinated and grown to maturity, or induced to dedifferentiate in vitro, resulting in cell lines that were subsequently regenerated into multiple mature plants. Both populations were then analyzed for the presence, active reinsertion and amplification of Ac. Plants from each population showed excision-reinsertion events to both linked and unlinked sites. However, the frequency of transposition in plants regenerated from cell lines was more than nine-fold greater than that observed in plants germinated directly from seeds. Other aspects of transposon behavior were also markedly affected. For example, we observed a significantly larger proportion of transposition events to unlinked sites in cell line-derived plants. The tendency for Ac to insert into transcribed DNA was not affected by dedifferentiation. The differences in Ac activity coincided with a pronounced reduction in the level of genomic cytosine methylation in dedifferentiated cell cultures. We used the differential transposon behavior induced by dedifferentiation in the cell-line derived population for direct applications in functional genomics and validated the approach by recovering Ac insertions in a number of genes. Our results demonstrate that obtaining multiple Ac insertions is useful for functional annotation of the rice genome.These authors contributed equally to the work     

Musa methylated DNA sequences associated with tolerance toMycosphaerella fijiensis toxins

DNA methylation is an epigenetic phenomenon associated with gene silencing in transgenic plants, retrotransposons and virus infection. Expression analysis of specific genes in Arabidopsis methylation mutants showed an association between DNA methylation and gene expression. To determine whether DNA methylation is associated with resistance to black Sigatoka (BS) andMycosphaerella fijiensis (MF), we used anin vitro assay of mesophyll cell suspensions of reference cultivars with known resistance to BS. Methylation of CC^mGG sequences was evaluated by methylation-sensitive amplification polymorphism (MSAP) markers of reference cultivars and somaclonal variants to identify molecular markers associated with resistance to MF toxins and BS. Four MSAP markers were associated with resistance (MAR) to MF toxins. The MSAP markers show a high degree of sequence similarity with resistance gene analog and with retrotransposon sequences. The MSAP markers are useful as molecular indicators of tolerance to MF toxins and resistance to BS.     

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.     

Detection of DNA methylation pattern in thidiazuron-induced blueberry callus using methylation-sensitive amplification polymorphism

During the normal developmental process, programmed gene expression is an essential phenomenon in all organisms. In eukaryotes, DNA methylation plays an important role in the regulation of gene expression. The extent of cytosine methylation polymorphism was evaluated in leaf tissues collected from the greenhouse grown plants and in in vitro-derived callus of three lowbush and one hybrid blueberry genotypes, using methylation-sensitive amplification polymorphism (MSAP) technique. Callus formation started from the leaf segments after 4 weeks of culture on a thidiazuron (TDZ) containing medium. Maximum callus formation (98 %) was observed in the hybrid blueberry at 1.0 mg dm^-3 TDZ. Although noticeable changes in cytosine methylation pattern were detected within the MSAP profiles of both leaf and callus tissues, methylation events were more polymorphic in calli than in leaf tissues. The number of methylated CCGG sites varied significantly within the genotypes ranging from 75 to 100 in leaf tissues and from 215 to 258 in callus tissues. Differences in the methylation pattern were observed not only in a tissue-specific manner but also within the genotype in a treatment specific manner. These results demonstrated the unique effect of TDZ and the tissue culture process on DNA methylation during callus development.     

Analysis of DNA methylation polymorphism in a set of stable chromosome translocation lines

Interspecific hybridization is associated with the origin of novel traits and confers increased vigor compared with the parent lines, although its molecular basis is poorly understood. We report here the identification of genetic and epigenetic changes in a set of wheat–rye translocation lines (R59, R57, and R25) which exhibited novel heritable morphological characteristics compared with the parent lines (MY11 and L155). Genome in situ hybridization and amplified fragment length polymorphism analyses revealed no obvious variations in the primary structure of the genome in different translocation lines, with the exception of the same 1RS chromosome translocation. Global assessment of the extent and pattern of cytosine methylation alterations by methylation-sensitive amplified polymorphism (MSAP) analyses revealed differences in the extent of genomic DNA methylation between the rye and wheat parent lines. Fully-methylated sites were significantly increased and hemi-methylated sites were markedly decreased in the genome of translocation lines compared with the wheat parental cultivar MY11. Comparisons of different MSAP patterns revealed both monomorphic and polymorphic sites between translocation lines and wheat parents. Sequencing of 44 isolated fragments that showed methylation alterations indicated that cellular genes and especially transposable elements were targets for methylation alterations in translocation lines. The present study provides further understanding of the rules governing the distribution and existence of DNA methylation variations induced in the wheat genome during alien germplasm introduction. Furthermore, our study provides insights into the relationship between DNA methylation and hybrid vigor as well as a theoretical basis for further fundamental research and breeding application.     

Somaclonal variation in rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.) analyzed using polymorphic and sequenced AFLP markers

Amplified fragment length polymorphism (AFLP) analysis of 24 in vitro regenerated rye plants was performed in order to evaluate the somaclonal variation rate in this species and to identify rye genomic regions where mutations are preferentially promoted by in vitro culture processes. Regenerated plants were obtained from cell lines derived from immature embryos and plants were regenerated by somatic embryogenesis. Twenty-three regenerants showed variation when compared against sibling plants obtained from the same cell line. A total number of 887 AFLP markers were scored, and 8.8% identified the same polymorphism in plants obtained independently from different cell lines, revealing putative mutational hot spots. Using controlled crossings and analysis of the corresponding progenies, we were able to verify the genetic stability in the next generation for only five of these polymorphisms. The nucleotide sequence of the AFLP amplicon of four of the polymorphic markers was obtained, but only the sequence of two markers was clearly identified in the databases. The sequence of marker A1-303 was identified as part of a tandemly repeated sequence, the 120-bp family, which is located at telomeric regions and is widely distributed among rye chromosomes. The marker A5-375 showed high similarity with regions of Angela retrotransposons.     

Cytosine methylation at CG and CNG sites is differential during the development of triploid black poplar

It has been widely shown that polyploidization can result in changes in cytosine methylation. However, little is known regarding how cytosine methylation changes in polyploids development, especially in polyploid trees. In this study, we investigated drifting changes of DNA methylation status at 5′-CCGG sites in the apical bud, young and mature leaf tissues of triploid black poplar (Populus. euramericana) with methylation-sensitive amplification polymorphism (MSAP) and assessed the expression of multiple DNA methyltransferases (MTases) and DNA demethylase during different developmental stages. MSAP analysis detected methylation levels at CG and CNG sites of diploid tissues reduced during development from bud to leaves, while for the triploid, methylation at CNG sites increased during development, but levels of methylation at CG sites first decreased in young leaves before increasing in mature leaves. MTase genes related to CG or CNG methylation were respectively preferential in different triploid tissues with high CG or CNG methylation levels. High expression of DNA demethylase was observed in tissue with high demethylation trends. These finding suggest CG and CNG methylation and their related enzymes are involved with different biological functions and networks of gene regulation in different developmental stages of triploid.     

Tissue culture‐induced genomic alteration in maize (Zea mays) inbred lines and F1 hybrids

Genomic alteration is a common phenomenon associated with plant tissue culture, which often encompasses genetic changes and epigenetic modifications (e.g. cytosine methylation). Here, we studied genomic alteration in maize by assessing calli and regenerated plants derived from three inbred lines (M17, J7 and JC) and two pairs of reciprocal F1 hybrids (pair I: M17/J7 and J7/M17 and pair II: M17/JC and JC/M17). By employing two molecular markers, the amplified fragment length polymorphism and methylation‐sensitive amplified polymorphism, we found that both types of genomic alterations occurred in calli and regenerated plants of all the studied maize inbred lines and F1 hybrids, but the extent and pattern of changes varied substantially across the genotypes. Among the three inbred lines, M17 showed markedly higher frequencies of both genetic (from 2.1% to 3.8%) and methylation alterations (from 6.5% to 9.9%, by adding up the various patterns) than the other two lines which showed similar frequencies for both types of alterations (genetic: 0.5–1.8%, methylation: 2.1–3.7%). Of the two F1 hybrid pairs, while pair I showed genetic variation frequencies similar to that of the inbred parent with lower changing frequency and pair II was intermediate of those of the parents, both pairs showed frequencies of methylation alteration more or less intermediate of those of their inbred parental lines. Parent‐of‐origin effects in both genetic and methylation changes were detected in only one of the hybrid pairs (primarily pair II) for a given changing pattern. Statistical testing confirmed the genotypic difference in both genetic and methylation (hypomethylation) alterations among the regenerants. Taken together, it could be concluded that the frequency and pattern of both genetic and cytosine methylation alterations in maize tissue culture were largely genetic context‐dependent traits, but stochasticity also played an important part. F1 hybrids were not significantly more stable than their inbred parental lines under tissue culture conditions.