Methylation-sensitive Amplified Fragment Length Polymorphism Research Articles

Analysis of DNA methylation in different maize tissues

DNA methylation plays an important role in gene expression regulation during biological development and tissue differentiation in plants. This study adopted methylation-sensitive Amplified fragment length polymorphism (AFLP) to compare the levels of DNA cytosine methylation at CCGG sites in tassel, bracteal leaf, and ear leaf from maize inbred lines, 18 White and 18 Red, respectively, and also examined specific methylation patterns of the three tissues. Significant differences in cytosine methylation level among the three tissues and the same changing tendency in two inbred lines were detected. Both MSAP (methylation sensitive amplification polymorphism) ratio and full methylation level were the highest in bracteal leaf, and the lowest in tassel. Meanwhile, different methylation levels were observed in the same tissue from the inbred lines, 18 White and 18 Red. Full methylation of internal cytosine was the dominant type in the maize genome. The differential methylation patterns in the three tissues were observed. In addition, sequencing of nine differentially methylated fragments and the subsequent blast search revealed that the cytosine methylated 5′ -CCGG-3′ sequences were distributed in repeating sequences, in the coding and noncoding regions. Southern hybridization was used to verify the methylation polymorphism. These results clearly demonstrated the power of the MSAP technique for large-scale DNA methylation detection in the maize genome, and the complexity of DNA methylation change during plant growth and development. The different methylation levels may be related to specific gene expression in various tissues.

Natural Homologous Triploidization and DNA Methylation in SARII-628, a Twin-seedling Line of Rice ( Oryza sativa)

A total of five pairs of diploid-triploid twin-seedlings (a diploid seedling and a triploid seedling emerged from a grain) were selected out from 4500 pairs of seedlings from SARII-628, a twin-seedling rice line. SSR analysis indicated that no difference between the diploid seedling and corresponding triploid seedling in a twin-seedling was found at the 310 loci, indicating that there was no obvious change in DNA primary structure. A modified AFLP technique ‘MSAP (methylation-sensitive AFLP)’ was used to analyze methylation mutation. Although no methylation mutation was noted among the five diploids, 29 methylation mutation loci were found from the corresponding triploids. This suggested that methylation mutation happened rapidly on M 0 generation after natural homologous triploidization. The mutations were classified into 10 types, including 3 increased types, 3 decreased types and 4 undecided types of methylation-degrees. The bands of 22 loci were sequenced and then those sequences were searched through website. The result showed that the methylation mutation involved into the whole rice genome and the 12 pairs of chromosomes. The mutation trend was site-related and there were different mutation loci for different triploids, which foretold that SARII-628 would have different evolution fates after natural homologous triploidization.

New Insights into a Controversial Topic: The Methylation–Cancer Connection

New Insights into a Controversial Topic: The Methylation–Cancer Connection

Quantification of the tissue-culture induced variation in barley (Hordeum vulgare L.)

BackgroundWhen plant tissue is passaged through in vitro culture, many regenerated plants appear to be no longer clonal copies of their donor genotype. Among the factors that affect this so-called tissue culture induced variation are explant genotype, explant tissue origin, medium composition, and the length of time in culture. Variation is understood to be generated via a combination of genetic and/or epigenetic changes. A lack of any phenotypic variation between regenerants does not necessarily imply a concomitant lack of genetic (or epigenetic) change, and it is therefore of interest to assay the outcomes of tissue culture at the genotypic level.ResultsA variant of methylation sensitive AFLP, based on the isoschizomeric combinations Acc65I/MseI and KpnI/MseI was applied to analyze, at both the sequence and methylation levels, the outcomes of regeneration from tissue culture in barley. Both sequence mutation and alteration in methylation pattern were detected. Two sets of regenerants from each of five DH donor lines were compared. One set was derived via androgenesis, and the other via somatic embryogenesis, developed from immature embryos. These comparisons delivered a quantitative assessment of the various types of somaclonal variation induced. The average level of variation was 6%, of which almost 1.7% could be accounted for by nucleotide mutation, and the remainder by changes in methylation state. The nucleotide mutation rates and the rate of epimutations were substantially similar between the andro- and embryo-derived sets of regenerants across all the donors.ConclusionWe have developed an AFLP based approach that is capable of describing the qualitative and quantitative characteristics of the tissue culture-induced variation. We believe that this approach will find particular value in the study of patterns of inheritance of somaclonal variation, since non-heritable variation is of little interest for the improvement of plant species which are sexually propagated. Of significant biological interest is the conclusion that the mode of regeneration has no significant effect on the balance between sequence and methylation state change induced by the tissue culture process.

Infraspecific DNA Methylation Polymorphism in Cotton (Gossypium hirsutum L.)

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.

Patterns of DNA cytosine methylation between haploids and corresponding diploids in rice

Eighteen pairs of diploid-haploid twin-seedlings were identified and screened out from special rice SARII-628 population. Five pairs of them were selected and randomly designated as A, B, C, D and E. Simple sequence repeats (SSR) analysis showed that there was no difference among 310 sites, which indicated that there was no base mutation on DNA primary structure. DNA methylation plays an important role in gene expression regulation during growth and development stages in eukaryotes. A modified AFLP technique (methylation-sensitive AFLP, MSAP) was employed to detect the DNA methylation patterns in the 5′-CCGG sites of the five pairs of twin-seedlings. Although no methylation mutation was detected among the five diploids, forty-three methylation mutation sites were found from the corresponding haploids. The MSAP ratios, which were the ratios of MSAP sites to the total amplified sites, in five haploids were 18.79%, 19.35%, 18.49%, 18.45% and 18.75%, respectively. And corresponding full methylation levels (5′-CmCGG in double strands) of those haploids were 10.58%, 11.3%, 10.11%, 10.09% and 10.34%, respectively. Both MSAP and full methylation levels in the five haploids were higher than that of their corresponding diploids, which suggested that hypermethylation occurred in some 5′-CCGG sites. Five types of MASP patterns among the five pairs of twin-seedlings were detected as follows: (1) no changes, methylation levels were the same in both haploids and diploids; (2) demethylation, diploid was methylated but no methylation in the same site in haploid; (3) hypermethylation, the methylation level in haploid was higher than those in diploid; (4) hypomethylation, methylation in haploid was lower than those in diploid; (5) undecided pattern, change trend of methylation levels in haploids was not decided. The bands of 18 sites were reclaimed, then sequenced and searched on website to determine the sites of those sequences on rice chromosomes. The result showed that the methylation mutation involved the whole rice genome and 12 pairs of chromosomes. The mutation was site-related and there were different mutation sites for different haploids. Compared to diploids, the higher methylation level in haploids might be a readjusting reaction to the decrease in ploidy for the sake of survival.

Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage

We studied the relationships between genetic and epigenetic alterations in gastrointestinal cancer by integrating DNA copy number changes determined by arbitrarily primed PCR (AP-PCR) with DNA methylation variations estimated by methylation-sensitive amplified fragment length polymorphism (MS-AFLP). We analyzed about 100 different chromosomal regions by AP-PCR and over 150 random CpG loci by MS-AFLP in human colon and gastric carcinomas. DNA hypomethylation and hypermethylation alterations distributed gradually and increased with cancer patient age, in contrast with the age-independent genomic alterations. Increased DNA hypomethylation and hypermethylation correlated with increased genomic damage, but only hypomethylation was highly significant in multivariate analyses. We conclude that age-dependent accumulation of DNA demethylation precedes diploidy loss in a significant subset of gastrointestinal cancers.

Analysis of DNA methylation related to rice adult plant resistance to bacterial blight based on methylation-sensitive AFLP (MSAP) analysis

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. The rice cultivar Wase Aikoku 3 becomes resistant to the blight pathogen Xanthomonas oryzae pv. oryzae at the adult stage. Using methylation-sensitive amplified polymorphism (MSAP) analysis, we compared the patterns of cytosine methylation in seedlings and adult plants of the rice cultivar Wase Aikoku 3 that had been inoculated with the pathogen Xanthomonas oryzae pv. oryzae, subjected to mock inoculation or left untreated. In all, 2000 DNA fragments, each representing a recognition site cleaved by either or both of two isoschizomers, were amplified using 60 pairs of selective primers. A total of 380 sites were found to be methylated. Of these, 45 showed differential cytosine methylation among the seedlings and adult plants subjected to different treatments, and overall levels of methylation were higher in adult plants than in seedlings. All polymorphic fragments were sequenced, and six showed homology to genes that code for products of known function. Northern analysis of three fragments indicated that their expression varied with methylation pattern, with hypermethylation being correlated with repression of transcription, as expected. The results suggest that significant differences in cytosine methylation exist between seedlings and adult plants, and that hypermethylation or hypomethylation of specific genes may be involved in the development of adult plant resistance (APR) in rice plants.

Genomic Targeting and High-Resolution Mapping of the Gene in Wheat

Tan spot, caused by the fungal pathogen Pyrenophora tritici-repentis (Died.) Drechs. causes severe yield losses in wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) and durum (T. turgidum L., 2n = 4x = 28, AABB). The Tsn1 gene acts dominantly to confer sensitivity to a host-selective proteinaceous toxin (Ptr ToxA) produced by the fungus. Our objectives were to: (i) target markers to the Tsn1 genomic region and (ii) develop a high-resolution map of the Tsn1 locus. The techniques of methylation-sensitive AFLP, traditional AFLP, and cDNA-AFLP were combined with bulked segregant analysis (BSA) using various wheat and durum cytogenetic stocks to target markers to the Tsn1 genomic region. Over 500 primer combinations were screened resulting in the identification of 18 low-copy markers closely linked to Tsn1. High-resolution mapping of the markers delineated the Tsn1 gene to a 0.2 cM interval in a hexaploid wheat population consisting of 1266 gametes, and to 0.8 cM in a durum wheat population consisting of 1860 gametes. Comparisons with rice BAC/PAC sequences indicated the lack of colinearity within the Tsn1 genomic region. Tsn1 was located within a gene-rich recombination hot spot region, and the physical distance separating the flanking markers may be as little as 200 kb. Therefore, these markers will serve as a basis for the map-based cloning of Tsn1 The isolation of Tsn1 will further our knowledge of wheat–tan spot interactions as well as host–pathogen interactions in general.

DNA METHYLATION VS. SOMACLONAL VARIATION IN HIGHER PLANTS: OIL PALM AS A CASE STUDY

In the aim of better understanding molecular phenomena underlying the mantled somaclonal variation in oil palm, our research work has focused on the role of genomic DNA methylation. Two complementary methods, aimed at evaluating the methylation rate at the genome-wide scale, have previously demonstrated the occurrence of a highly significant DNA hypomethylation in leaves and calli from abnormal regenerants, compared to their normal counterparts. Investigations are now aimed at targeting the individual sequences which epigenetic misregulation could account for the onset or for the maintenance of the mantled' phenotype. We are currently trying, through a wide range of techniques, to focus our methylation studies on the only genes that are relevant with regard to the mantled phenotype. In this aim, we have undertaken methylation-sensitive RFLP and AFLP studies, involving the isoschizomeric enzymes Mspl and HpaII. RFLPs were envisaged primarily in order to screen a pool of oil palm cDNA, while the aim of MSAP (methylation-sensitive amplified polymorphism) investigations was to generate a large number of relevant markers, exhibiting a differential methylation pattern depending on the normal/mantled phenotype but independent of the genetic origin of clones. Hypomethylating drugs were used in embryogenic cell suspensions in order to evaluate the impact of DNA demethylation on the genotype of regenerants and to simultaneously assess the set of methylation-sensitive markers previously developed. By studying oil palm DNA-methyltransferases, it will be possible to determine whether DNA methylation is the only epigenetic regulator affected, and which regions of the genome are specifically modified in variants. (Resume d'auteur)

Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers

Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers

Transient decreases in methylation at 5'-cCGG-3' sequences in potato (Solanum tuberosum L.) meristem DNA during progression of tubers through dormancy precede the resumption of sprout growth.

The 5-methylcytosine (5mC) content in DNA of tuber meristems isolated from field-grown potatoes (Solanum tuberosum L.) was determined during a 7-month storage period at 3 degrees C for three growing/postharvest seasons. No significant changes in 5mC levels were noted genome-wide or within 5'-CG-3' dinucleotide sequences, 5'-CG-3' islands or 5'-CA(T)G-3' trinucleotide sequences during storage. However, a consistent but transient 50-70% decrease in methylation at both cytosines within 5'-CCGG-3' sequences was detected that peaked 112-194 days after harvest. This result was corroborated by methylation-sensitive amplified fragment length polymorphism analysis of meristem DNA. Similar to tuber meristems undergoing progression through natural dormancy, premature chemical termination of dormancy resulted in rapid, transient 5'-CCGG-3' demethylation in meristem DNA. Minimum methylation levels at this sequence preceded initiation of high levels of de novo DNA synthesis by two days. Cytosine methylation status was also followed in in vitro-generated potato microtubers during 7 months of post-harvest storage. As in DNA from tuber bud meristems, no changes in genome-wide 5mC content or methylation at 5'-CA(T)G-3' or 5'-CG-3' island sequences were noted in microtuber DNA. However, there was a transient 46% drop in methylation at 5'-CG-3' dinucleotides concomitant with minimum levels of 5'-CCGG-3' methylation (30-60% below those in dormant microtubers) 57-98 days after harvest. As microtubers exited dormancy, there were sustained three- and seven-fold increases in RNA and DNA synthesis rates, peaking on or after 98 days of storage, respectively. Together, these data demonstrate that demethylation of 5'-CCGG-3' sequences occurs independently of tuber age during dormancy progression and precedes transcriptional activation of genes leading to cell division and meristem growth in potatoes.

Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers.

AFLP analysis using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites has been used to analyse the methylation state of anonymous CCGG sequences in Arabidopsis thaliana. The technique was modified to improve the quality of fingerprints and to visualise larger numbers of scorable fragments. Sequencing of amplified fragments indicated that detection was generally associated with non-methylation of the cytosine to which the isoschizomer is sensitive. Comparison of EcoRI/ HpaII and EcoRI/ MspI patterns in different ecotypes revealed that 35-43% of CCGG sites were differentially digested by the isoschizomers. Interestingly, the pattern of digestion among different plants belonging to the same ecotype is highly conserved, with the rate of intra-ecotype methylation-sensitive polymorphisms being less than 1%. However, pairwise comparisons of methylation patterns between samples belonging to different ecotypes revealed differences in up to 34% of the methylation-sensitive polymorphisms. The lack of correlation between inter-ecotype similarity matrices based on methylation-insensitive or methylation-sensitive polymorphisms suggests that whatever the mechanisms regulating methylation may be, they are not related to nucleotide sequence variation.

Notl-Msell methylation-sensitive amplied fragment length polymorhism for DNA methylation analysis of human cancers.

We have applied a methylation-sensitive restriction endonuclease, NotI, to the existing amplified fragment length polymorphism (AFLP) method and developed NotI-MseI methylation-sensitive-AFLP (MS-AFLP). NotI-MseI MS-AFLP allows the analysis of DNA methylation alterations at the NotI sites scattered over the genome. Hypermethylation and hypomethylation are visualized by the decrease and increase in the band intensity of DNA fingerprints. Identification of consistent changes can be facilitated through parallel electrophoresis of multiple samples. DNA fragments exhibiting alterations can be cloned from fingerprint bands by amplification of gel-eluted DNA with the same pair of primers used for radioactive fingerprint presentation. Fluorescent NotI-MseI MS-AFLP offers a safer method of studying the alterations in DNA methylation, and may be applied to the hybridization of DNA microarrays in the future. Using NotI-MseI MS-AFLP, we observed frequent hypomethylation of a satellite DNA repeat sequence in a majority of breast tumors.

Variation in oil palm (Elaeis guineensis Jacq.) tissue culture-derived regenerants revealed by AFLPs with methylation-sensitive enzymes

Tissue culture-derived plants of oil palm (Elaeis guineensis Jacq.) can develop abnormal flowers in which stamen primordia are converted into carpel-like tissues (mantled fruit). This abnormality can be heritable; individual palms may show variation in mantling and reversion to the normal phenotype over time has been observed. Four sets of ortets (mother plant used as tissue source) and ramets (regenerated plants) were compared using standard amplified fragment length polymorphism (AFLP) analysis and AFLPs using methylation-sensitive enzymes. No polymorphisms were found when standard AFLPs were produced with ten different primer combinations. In contrast, when methylation-sensitive AFLPs were used, polymorphisms were detectable. Polymorphisms appeared as new bands in the ramets, suggesting that a reduction in methylation had occurred during tissue culture. The highest number of polymorphic bands (0.3%) was obtained when HpaII was used as the restriction endonuclease, indicating that the loss of methylation had occurred most frequently at the internal C within the HpaII recognition sequence 5’-CCGG-3’. Conversion of nine of the polymorphic bands into probes for Southern analysis confirmed that these were not due to partial digestion of the AFLP templates and showed that the majority were single-copy sequences. The exceptions were fragments showing homology to 25S ribosomal RNA genes and the chalcone synthase gene family. Examination of the Southern blots suggested that most of the single-copy sequences were partially de-methylated, and one example was found in which de-methylation affected only one allele. No polymorphism was consistently different between normal and abnormal clones in all the sets. This suggests that, whilst this method is an effective way of detecting variation in tissue culture-derived plants, different approaches will be required to identify the causal basis of the mantled fruit abnormality.

Surveying CpG methylation at 5'-CCGG in the genomes of rice cultivars.

We investigated the CpG methylation status of the sequence CCGG in the rice genome by using methylation-sensitive AFLP and subsequent Southern analyses with the isolated AFLP fragments as probes. CpGs located in single- or low-copy-sequence regions could be grouped into two classes on the basis of their methylation status: methylation status at the class 1 CpG sites was conserved among genetically diverse rice cultivars, whereas cultivar-specific differential methylation was frequently detected among the cultivars at the class 2 CpG sites. The frequency of occurrence of methylation polymorphism between a pair of cultivars was not related to the genetic distance between the two. Through mapping, five class 2 CpG sites were localized on different chromosomes and were not clustered together in the genome. Segregation analysis of the cultivar-specific methylations with their target sites indicated that the differential methylation was stably inherited in a Mendelian fashion over 6 generations, although alterations in the methylation status at the class 2 CpG sites were observed with a low frequency.