Methylation-sensitive Amplified Polymorphism Technique Research Articles

Beyond genetic differences: epigenetic variation in common bottlenose dolphins Tursiops truncatus from contrasting marine ecosystems

Recent genetic and morphological studies have indicated an incipient ecological divergence between 2 ecotypes of common bottlenose dolphinTursiops truncatusin the Southwestern Atlantic. However, genetic variation is not the only molecular mechanism that alters the phenotype of these animals: epigenetics can also influence phenotypic plasticity, as well as the ecological adaptation and divergence of natural populations. Nevertheless, very little is known about the role that epigenetics plays in the population ecology of marine mammals. In this work, we tested whether there are differences in DNA methylation patterns between a coastal and an offshore ecotype of common bottlenose dolphin. Methylation patterns were analyzed using the methylation-sensitive amplified polymorphism technique on biopsy samples collected from animals of both ecotypes. We found consistent differences in DNA methylation patterns between coastal and offshore individuals. We also confirmed the genetic differences described in previous studies, indicating that the divergence between ecotypes has both genetic and epigenetic components. Our data show that it is possible to differentiate animals from the coastal and offshore ecotypes using DNA methylation markers, supporting the hypothesis that contrasting environments—which are decisive for the ecological divergence of these populations—lead to epigenetic modifications in common bottlenose dolphins.

Application of the MSAP Technique to Evaluate Epigenetic Changes in Plant Conservation.

Epigenetic variation, and particularly DNA methylation, is involved in plasticity and responses to changes in the environment. Conservation biology studies have focused on the measurement of this variation to establish demographic parameters, diversity levels and population structure to design the appropriate conservation strategies. However, in ex situ conservation approaches, the main objective is to guarantee the characteristics of the conserved material (phenotype and epi-genetic). We review the use of the Methylation Sensitive Amplified Polymorphism (MSAP) technique to detect changes in the DNA methylation patterns of plant material conserved by the main ex situ plant conservation methods: seed banks, in vitro slow growth and cryopreservation. Comparison of DNA methylation patterns before and after conservation is a useful tool to check the fidelity of the regenerated plants, and, at the same time, may be related with other genetic variations that might appear during the conservation process (i.e., somaclonal variation). Analyses of MSAP profiles can be useful in the management of ex situ plant conservation but differs in the approach used in the in situ conservation. Likewise, an easy-to-use methodology is necessary for a rapid interpretation of data, in order to be readily implemented by conservation managers.

Epigenetic variation among natural populations of the South African sandhopper Talorchestia capensis

Ecological epigenetics is gaining importance within the field of Molecular Ecology, because of its novel evolutionary implications. Linking population ecology to the variation in epigenetic profiles can help explain the effect of environmental conditions on phenotypic differences among populations. While epigenetic changes have largely been investigated through the examination of DNA methylation under laboratory conditions, there is a limited understanding of the extent of DNA methylation variation in wild populations. Assuming that epigenetic variation is important in nature, the conditions experienced by different conspecific populations should result in levels of DNA methylation that are independent of their genetic differentiation. To test this, we investigated levels of DNA methylation among populations of the sandhopper Talorchestia capensis that show phenotypic (physiological) differences in their response to environmental conditions, at the same time evaluating their genetic relationships. Given the high levels of inter-individual physiological variation observed within populations, we further hypothesised that inter-individual differences in methylation would be high. Levels of genetic and epigenetic variation were assessed within and among populations from five localities using the methylation sensitive amplified polymorphism technique. Population differentiation was higher for epigenetics than genetics, with no clear geographical pattern or any relation to biogeography. Likewise, individuals showed greater variability in their epigenetic than their genetic profiles. Four out of five populations showed significant negative relationships between epigenetic and genetic diversity. These results show uncoupling between epigenetic and genetic variation and suggest that: (1) epigenetics are more responsive to local, site-specific environmental conditions than genetics and (2) individual differences in epigenetic profiles drive phenotypic variation within (and most likely among) natural populations. Within populations, epigenetics could offer a level of phenotypic flexibility beyond genetic constraint that allows rapid responses to variable or unpredictable environments, potentially compensating for low genetic variability.

Natural epigenetic variation in bats and its role in evolution.

When facing the challenges of environmental change, such as habitat fragmentation, organisms have to adjust their phenotype to adapt to various environmental stresses. Recent studies show that epigenetic modifications could mediate environmentally induced phenotypic variation, and this epigenetic variance could be inherited by future generations, indicating that epigenetic processes have potential evolutionary effects. Bats living in diverse environments show geographic variations in phenotype, and the females usually have natal philopatry, presenting an opportunity to explore how environments shape epigenetic marks on the genome and the evolutionary potential of epigenetic variance in bat populations for adaptation. We have explored the natural epigenetic diversity and structure of female populations of the great roundleaf bat (Hipposideros armiger), the least horseshoe bat (Rhinolophus pusillus) and the eastern bent-winged bat (Miniopterus fuliginosus) using a methylation-sensitive amplified polymorphism technique. We have also estimated the effects of genetic variance and ecological variables on epigenetic diversification. All three bat species have a low level of genomic DNA methylation and extensive epigenetic diversity that exceeds the corresponding genetic variance. DNA sequence divergence, epigenetic drift and environmental variables contribute to the epigenetic diversities of each species. Environment-induced epigenetic variation may be inherited as a result of both mitosis and meiosis, and their potential roles in evolution for bat populations are also discussed in this review.

Cytoplasmic effects on DNA methylation between male sterile lines and the maintainer in wheat (Triticum aestivum L.)

Male sterile cytoplasm plays an important role in hybrid wheat, and three-line system including male sterile (A line), its maintainer (B line) and restoring (R line) has played a major role in wheat hybrid production. It is well known that DNA methylation plays an important role in gene expression regulation during biological development in wheat. However, no reports are available on DNA methylation affected by different male sterile cytoplasms in hybrid wheat. We employed a methylation-sensitive amplified polymorphism technique to characterize nuclear DNA methylation in three male sterile cytoplasms. A and B lines share the same nucleus, but have different cytoplasms which is male sterile for the A and fertile for the B. The results revealed a relationship of DNA methylation at these sites specifically with male sterile cytoplasms, as well as male sterility, since the only difference between the A lines and B line was the cytoplasm. The DNA methylation was markedly affected by male sterile cytoplasms. K-type cytoplasm affected the methylation to a much greater degree than T-type and S-type cytoplasms, as indicated by the ratio of methylated sites, ratio of fully methylated sites, and polymorphism between A lines and B line for these cytoplasms. The genetic distance between the cytoplasm and nucleus for the K-type is much greater than for the T- and S-types because the former is between Aegilops genus and Triticum genus and the latter is within Triticum genus between Triticum spelta and Triticum timopheevii species. Thus, this difference in genetic distance may be responsible for the variation in methylation that we observed.

Variations of DNA methylation and genes expression for brown fiber during its development process

Brown cotton is a kind of naturally colored cotton which accumulates brown pigment in fiber. In the experiment, the variations of DNA methylation and genes expression were studied during the development processes from 5 DPA to 25 DPA for brown fiber. By using the methylation-sensitive amplified polymorphism technique, we choose 66 pairs of selective-amplification primers to assess the status and levels of cytosine methylation. The hemimethylation of the external cytosine and the full methylation of the internal cytosine were scored. As a result, with the development of fiber from 5 DPA to 25 DPA, the ratios of the external hemimethylation and internal full methylation were gradually increased. In detail, at the stages of 5, 10, 15 and 25 DPA, the percentages of external hemimethylation were 8.29, 8.81, 9.77 and 10.09 %, the ratios of internal full methylation were 17.91, 19.20, 20.02 and 20.78 %, respectively. The development of brown fiber triggered the increase of methylated loci in the whole genome. For further analysis, we used cDNA-AFLP protocol to identify the specially expressed genes for further analysis. Totally, 30 polymorphic transcription-derived fragments (TDFs) were isolated and sequenced. By homologous alignment analysis, 19 TDFs were found similar with genes from cotton. Among them, the homologous genes of TDF5, TDF6, TDF10, TDF12, TDF17, TDF22, TDF23 and TDF25 were all from fiber. Furthermore, 13 TDFs were found to be homologous with reported functional genes.

Stable MSAP Markers for the Distinction of Vitis vinifera cv Pinot Noir Clones

Grapevine is one of the most economically important fruit crops. Molecular markers have been used to study grapevine diversity. For instance, simple sequence repeats are a powerful tool for identification of grapevine cultivars, while amplified fragment length polymorphisms have shown their usefulness in intra-varietal diversity studies. Other techniques such as sequence-specific amplified polymorphism are based on the presence of mobile elements in the genome, but their detection lies upon their activity. Relevant attention has been drawn toward epigenetic sources of variation. In this study, a set of Vitis vinifera cv Pinot noir clones were analyzed using the methylation-sensitive amplified polymorphism technique with isoschizomers MspI and HpaII. Nine out of fourteen selective primer combinations were informative and generated two types of polymorphic fragments which were categorized as "stable" and "unstable." In total, 23 stable fragments were detected and they discriminated 92.5% of the studied clones. Detected stable polymorphisms were either common to several clones, restricted to a few clones or unique to a single clone. The identification of these stable epigenetic markers will be useful in clonal diversity studies. We highlight the relevance of stable epigenetic variation in V. vinifera clones and analyze at which level these markers could be applicable for the development of forthright techniques for clonal distinction.

DNA methylation affected by male sterile cytoplasm in rice (Oryza sativa L.)

Male sterile cytoplasm plays an important role in hybrid rice, and cytoplasmic effects are sufficiently documented. However, no reports are available on DNA methylation affected by male sterile cytoplasm in hybrid rice. We used a methylation-sensitive amplified polymorphism technique to characterize DNA methylation in four male sterile cytoplasms that are widely commercialized in China. In total, 12 pairs of selective primers in combinations of EcoRI and MspI/HpaII amplified 350 bands among four male sterile (A) lines and the corresponding maintainer (B) lines. Sites b1 and b3 were fully methylated only in all the B lines, while b2 was fully methylated only in all the A lines. These results implied a relationship of DNA methylation at these sites specifically with male sterile cytoplasms, as well as male sterility, since the only difference between the A and B lines was the cytoplasm. The DNA methylation was markedly affected by male sterile cytoplasms. WA-type and Yinshui-type cytoplasms affected the methylation to a much greater degree than G-type and D-type cytoplasms, as indicated by the number and degree of methylated sites, ratio of methylated sites, number of fully methylated sites, ratio of fully methylated sites, and polymorphism between A and B lines for these cytoplasms. The genetic distance between the cytoplasm and nucleus for the WA-type is much greater than for G- and D-types because the former is between wild and cultivated species and the latter is within indica subspecies between African and Asian cultivars. This difference in genetic distance may be responsible for the variation in methylation which we observed.

Variation of DNA methylation and phenotypic traits following unilateral sexual polyploidization in Medicago

Sexual hybridization is an important generator of biodiversity and a powerful breeding tool. Hybridization can also overcome ploidy barriers when it involves 2n gametes, as in the case of unilateral sexual polyploidization (USP) that has been utilized in several crops, among which alfalfa. This research was aimed at gaining insights into the effects of USP on genome methylation and on phenotypic traits in alfalfa, an important forage species. The Methylation-Sensitive Amplified Polymorphism technique was used to estimate the cytosine methylation changes occurring in a tetraploid (2n = 4x = 32) USP progeny from crosses between a diploid Medicago sativa subsp. falcata genotype that produces 2n eggs and a cultivated tetraploid Medicago sativa subsp. sativa variety. De novo methylation or demethylation in the USP progeny were observed for 13% of the detected genomic sites, indicating that methylation changes can be relevant. USP plants showed larger surface area of the leaf epidermis cells than both parents, but this did not result in larger leaf size or higher plant biomass. They displayed significant higher ovule sterility than the tetraploid parent, but normal fertility was observed in crosses with unrelated male testers. We conclude that hybridization and sexual polyploidization resulted in novel variation in terms of remodeling of the methylation landscape as well as changes in phenotypic traits in alfalfa.

Detection of DNA methylation changes during seed germination in rapeseed (Brassica napus)

DNA methylation is known to play a crucial role in regulating plant development and organ or tissue differentiation. In this study, we assessed the extent and pattern of cytosine methylation during rapeseed (Brassica napus L.) seed germination, and compared the methylation level of various tissues in seedling, using the techniques of methylation-sensitive amplified polymorphism (MSAP) and HPLC separation and quantification of nucleosides. In all, 484 bands, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified by 12 pairs of selective primers in DNA obtained from dry seeds. A total of 76 sites were found to be differentially digested by the isoschizomers, indicating that approximately 15.7% of 5′-CCGG-3′ sites in the genome were cytosine methylated. Four classes of patterns were observed in a comparative assay of cytosine methylation in the dry and germinating seeds; a small number of hypermethylation events occurred at 5′-CCGG-3′ sites in germinating seeds compared with dry seeds, while many more hypomethylation events were detected after seed germination. Differences in DNA methylation level in various tissues were also detected; radicel was less methylated than hypocotyl and cotyledon. These observations were further confirmed by HPLC analysis. In addition, sequencing of eleven differentially methylated fragments and the subsequent blast search revealed that cytosine methylated 5′-CCGG-3′ sequences were equally distributed between coding and non-coding regions. These results clearly demonstrate the power of MSAP technique for large-scale DNA methylation detection in rapeseed genome, and the complexity of DNA methylation change during seed germination. DNA Hypomethylation going with seed germination appears to be a necessary step toward transcriptional activation in gene expression, and may well contribute to the developmental gene regulation.

Relationship between individual DNA methylation percentage differences and carcass traits in pigs

AbstractIn order to investigate the effects of DNA methylation on pig carcass traits, the methylation-sensitive amplified polymorphism technique (MSAP) was adopted to amplify DNA from blood samples taken from 77 F1hybrids of Large White×Meishan crosses. A total of 15 carcass traits were tested. Results showed that means of internal fat rate (IFR), lean meat percentage (LMP) and ratio of lean meat versus fat meat (RLF) were significantly different (P<0.05) among five levels of general individual methylation difference (GIMDP); means of carcass weight (CW), IFR, backfat thickness at buttock (BFT3), average backfat thickness (ABF), carcass length to the first rib (CLR) and carcass length to the first neck vertebra (CLN) were also significantly different (P<0.05) among five levels of special individual methylation percentage difference (SIMDP), while means of 15 carcass traits were not significantly different (P>0.05) among five levels of neutral individual methylation percentage difference (NIMDP). Of all traits that were significantly affected by SIMPD, CW, ABF and BFT3increased, IFR and backfat thickness at shoulder (BFT1) decreased, while CLR and bone percentage (BP) fluctuated with the SIMDP increase. The regressions between SIMDP and IFR, BFT1, BFT3and ABF were significant (P<0.05). It is concluded that DNA methylation can be applied as a marker to related studies in pigs; positive methylation sites were superior to negative methylation sites in predicting hybrid performance, and methylation differences should be maintained at specific levels for different traits to improve the productivity of pigs.

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.