Cytosine Methylation Changes in Rice Centromeric and Telomeric Sequences Induced by Foreign DNA Introgression

Cytosine methylation changes (hyper- or hypomethylation) in centromeric and telomeric sequences were observed in all three studied rice introgression lines containing DNA from wild rice, Zizania latifolia Griseb. The changed genomic Southern hybridization patterns were complex and non-concordant between a pair of isoschizomers (HpaII/MspI) digests, indicating methylation modifications at both the inner and outer cytosines of the CCGG sites. The changed patterns were inherited through generations. Possible mechanism for the methylation changes and their potential implications for the phenotypic variation and genome organization are discussed.     

Patterns of cytosine methylation in an elite rice hybrid and its parental lines, detected by a methylation-sensitive amplification polymorphism technique

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we assessed the extent and pattern of cytosine methylation in the rice genome, using the technique of methylation-sensitive amplified polymorphism (MSAP), which is a modification of the amplified fragment length polymorphism (AFLP) method that makes use of the differential sensitivity of a pair of isoschizomers to cytosine methylation. The tissues assayed included seedlings and flag leaves of an elite rice hybrid, Shanyou 63, and the parental lines Zhenshan 97 and Minghui 63. In all, 1076 fragments, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified using 16 pairs of selective primers. A total of 195 sites were found to be methylated at cytosines in one or both parents, and the two parents showed approximately the same overall degree of methylation (16.3%), as revealed by the incidence of differential digestion by the isoschizomers. Four classes of patterns were identified in a comparative assay of cytosine methylation in the parents and hybrid; increased methylation was detected in the hybrid compared to the parents at some of the recognition sites, while decreased methylation in the hybrid was detected at other sites. A small proportion of the sites was found to be differentially methylated in seedlings and flag leaves; DNA from young seedlings was methylated to a greater extent than that from flag leaves. Almost all of the methylation patterns detected by MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrate that the MSAP technique is highly efficient for large-scale detection of cytosine methylation in the rice genome. We believe that the technique can be adapted for use in other plant species. Received: 23 October 1998 / Accepted: 11 January 1999     

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.     

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.     

DNA-methylation changes in grapevine somaclones following in vitro culture and thermotherapy

The methylation-sensitive amplified polymorphism (MSAP) technique using HpaII and MspI isoschizomers was used to analyse DNA-methylation alterations in stressed grapevine plants. The stress used was in vitro propagation via nodal segments and in vitro thermotherapy for virus elimination. A set of pertinent grapevine plants derived from two cultivars (18 plants each for Müller Thurgau and Riesling) was used as stressed variants for analyses. A total of 695 and 700 MSAP bands were recognised and evaluated as present/absent for all analysed variants derived from both cvs. Müller Thurgau and Riesling. Average computed similarity of MSAP banding between analysed variants (Dice/Nei and Li coefficient) was 0.935 for both cultivars. Clustering of variants within resulting dendrograms showed significant differences between woody cuttings despite originating from the one plant. Further, there was a strong ‘donor’ effect of maternal plants on future arrays of DNA methylation in their regenerants. The ‘donor’ effect even seemed to prevail in the effect of stress on final DNA-methylation state in stressed regenerants. Additional MSAP evaluation suggests that thermotherapy induced an additional array of methylation changes when compared with stress caused by in vitro cultivation. From the viewpoint of whether methylation of CCGG loci increased/decreased due to stress, the results showed moderate prevalence for decreasing CCGG loci methylation.     

Variation of cytosine methylation in 57 sweet orange cultivars

Sweet orange is an important group of citrus cultivars, which includes a number of bud sport cultivars. Little is known about the CpG methylation status of the CCGG sequences in the orange genome. In this study, methylation-sensitive amplification polymorphism (MSAP), based on the application of isoschizomers (Hpa II and Msp I), was first used to analyze cytosine methylation patterns in 57 orange cultivars that were not fully differentiated by regular DNA molecular markers. Three types of bands were generated from ten primer pairs. Type I bands were present following restriction with Eco RI + Hpa II and Eco RI + Msp I; type II or type III were present only following restriction with either Eco RI + Hpa II or with Eco RI + Msp I. The total number of these three types of bands was 802, 72, and 157, respectively. Among these, the number of polymorphic bands were 244 (30.2%), 23 (31.9%), and 32 (20.4%), in type I, II and III, respectively. The methylation patterns of these 57 cultivars are discussed and assessed by dendrograms derived from the analysis of polymorphic MSAP bands. The distribution of polymorphic bands of the above three types demonstrate the methylation patterns and frequency at the cytosine loci. We suggest that methylation events could be more frequent than demethylation events, and that the methylation patterns maybe associated with phenotypic traits.     

Mobilized retrotransposon Tos17 of rice by alien DNA introgression transposes into genes and causes structural and methylation alterations of a flanking genomic region

Tos17 is a copia-like endogenous retrotransposon of rice, which can be activated by various stresses such as tissue culture and alien DNA introgression. To confirm element mobilization by introgression and to study possible structural and epigenetic effects of Tos17 insertion on its target sequences, we isolated all flanking regions of Tos17 in an introgressed rice line (Tong35) that contains minute amount of genomic DNA from wild rice (Zizania latifolia). It was found that there has been apparent but limited mobilization of Tos17 in this introgression line, as being reflected by increased but stable copy number of the element in progeny of the line. Three of the five activated copies of the element have transposed into genes. Based on sequence analysis and Southern blot hybridization with several double-enzyme digests, no structural change in Tos17 could be inferred in the introgression line. Cytosine methylation status at all seven CCGG sites within Tos17 was also identical between the introgression line and its rice parent (Matsumae)-all sites being heavily methylated. In contrast, changes in structure and cytosine methylation patterns were detected in one of the three low-copy genomic regions that flank newly transposed Tos17, and all changes are stably inherited through selfed generations.     

Differential DNA methylation between two wing phenotypes adults of Sogatella furcifera

Sogatella furcifera is a major rice pest with wing dimorphism . DNA methylation is an important epigenetic modification that plays a role in gene regulation and phenotype variation in most organisms. The objective of the current research was to survey the frequencies and variation of cytosine methylation at CCGG sequences in macropterous female adults (MFA) and brachypterous female adults (BFA) of S. furcifera, and to determine the occurrence of methylation changes associated with wing phenotypes by using methylation‐sensitive amplification polymorphism (MSAP). No differences were found in the average proportions of methylated CCGG sites between MFA and BFA, but there were significant differences for methylation patterns between MFA and BFA. The fully methylated ratio was 5.81% in BFA, much higher than 2.40% in MFA; while the hemi‐methylated ratio was 4.35% in BFA, much lower than 8.35% in MFA. These results provide circumstantial evidence that DNA methylation might be related to wing phenotype variation in S. furcifera. We also cloned and got 14 satisfactory sequences, which displayed variable cytosine methylation patterns between MFA and BFA. All these data will facilitate the researches on the epigenetic mechanisms of insect wing polymorphism. genesis 51:819–826. © 2013 Wiley Periodicals, Inc.     

Foreign DNA introgression caused heritable cytosine demethylation in ribosomal RNA genes of rice

Significant cytosine demethylation in ribosomal RNA genes (18S or 25S) were detected in all four studied rice lines containing introgressed DNA from wild rice, Zizania latifolia Griseb. In each line, the changed RFLP (restriction fragment length polymorphism) patterns produced with the methylation-sensitive enzyme (HpaII) were identical between two randomly selected individual plants both within and between generations. This indicates that the methylation changes are non-random and stably inherited. Cytosine demethylation in ribosomal RNA genes could be a major cause for the drastically altered phenotypic variations observed in the introgression lines.     

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.     

Identification of exotic genetic components and DNA methylation pattern analysis of three cotton introgression lines from Gossypium bickii

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.     

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.     

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.     

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

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

Genetic and epigenetic status of triple exotic consanguinity cotton introgression lines

Introgression lines are some of the most important germplasm for breeding applications and other research conducted on cotton crops. The DNA methylation level among 10 introgression lines of cotton (Gossypium hirsutum) and three exotic parental species (G. arboreum, G. thurberi and G. barbadense) were assessed by methylation-sensitive amplified polymorphism (MSAP) technology. The methylation level in the introgression lines ranged from 33.3 to 51.5%. However, the lines PD0111 and PD0113 had the lowest methylation level (34.6 and 33.3%, respectively) due to demethylation of most non-coding sequences. Amplified fragment length polymorphism (AFLP) was used to evaluate the genetic polymorphism in the cotton introgression lines. A high degree of polymorphism was observed in all introgression lines (mean 47.2%) based on AFLP and MSAP analyses. This confirmed the effects of genetic improvement on cotton introgression lines. The low methylation varieties, PD0111 and PD0113 (introgression lines), clustered outside of the introgression lines based on MSAP data, which was incongruent with an AFLP-based dendrogram. This phenomenon could be caused by environmental changes or introgression of exotic DNA fragments.     

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.     

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.     

异源DNA导入水稻诱发活跃反转子Tos17发生可遗传DNA甲基化变异

用水稻(Oryza sativa L.)内源反转座子Tos17为探针,经Southern杂交在5种含有野生稻(Zizania latifolia Griseb)(菰)DNA片段的水稻渐渗杂交系中检测到了可遗传DNA甲基化变异.在分析的4种甲基化敏感限制性内切酶中,每种酶切都发生了亲本杂交片段的消失和新片段的出现.发生甲基化变异的位点包括对称和不对称的胞嘧啶碱基,也包括腺嘌呤碱基.序列分析表明,与水稻亲本比较,所研究的5种渐渗杂交系在Tos17的2个重要区域(5'-LTR和RT)均未发生序列变异.但甲基化敏感-序列特异性PCR分析证实,每种渐渗杂交系在这2个区域内均发生了广泛的DNA甲基化变异.而且,在2种渐渗杂交系中发现5'-LTR和RT区域的甲基化变异存在协同性.甲基化变异可稳定遗传给后代.因为已有的研究表明,在这5种渐渗杂交系中异源DNA导入均导致了Tos17的激活和转座,因此可以推测DNA甲基化在调控Tos17活性中可能具有一定作用.但反转座子激活和甲基化变异之间的确切关系尚有待进一步研究.