Genetic and epigenetic evaluations of citrus calluses recovered from slow-growth culture

The embryogenic callus of "Red Marsh" grapefruit was stored in vitro by slow growth culture method for one year, and survived with a significant weight increment over that period. The survivers regenerated somatic embryos more easily than the controls. Eight callus lines were used for genetic analyses. Although chromosome number variations were verified by cytological examination both in the controls and the stored samples, the ploidy level remained relatively stable during the storage period. Randomly amplified polymorphic DNA (RAPD) analysis was performed to detect DNA sequence variation. No difference in RAPD pattern was found with the 102 primers used. However, a methylation sensitive amplified polymorphism (MSAP) assay showed DNA methylation changes in the stored samples compared with the controls.     

Effect of cryopreservation on apple genetic resources at morphological, chromosomal, and molecular levels.

Shoot tips of three apple genotypes, namely, Malus pumila cv. M26, Gala, and Hokkaido No. 9, were successfully cryopreserved using a modified encapsulation-dehydration method. As a result, in addition to a high survival rate and regeneration rate, the capacity of shoots regenerated from cryopreserved samples to root was enhanced. Eight M26 single-bud sibling lines were used to assess genetic stability. Although cytological examination revealed a ploidy difference in the noncryopreserved control, the ploidy constitution remained relatively stable during the period of cryopreservation. Amplified fragment length polymorphism (AFLP) assay was performed to detect DNA-level variation. No change in DNA fragment pattern and number was observed between the control and the cryopreserved samples. In addition, methylation-sensitive amplified polymorphism (MSAP) assay was carried out to investigate the DNA methylation status during the period of cryopreservation. It was found that cryopreservation induced a decrease in DNA methylation level.     

In vitro storage of cedar shoot cultures under minimal growth conditions

We developed procedures for slow-growth storage of Cedrus atlantica and Cedrus libani microcuttings of juvenile and adult origin, noting factors favouring the extension of subculture intervals. Microcuttings could be stored effectively up to 6 months at 4°C and reduced light intensity, provided that they were grown on a diluted modified MS medium. The addition of 6% mannitol to the storage media affected negatively survival and multiplication capacity of the cultures. The slow-growth storage conditions used in our experiments did not induce remarkable effects on both RAPD variability and average DNA methylation in the species.     

Genetic diversity analysis of Jatropha curcas L. (Euphorbiaceae) based on methylation-sensitive amplification polymorphism

Genetic analysis of 56 samples of Jatropha curcas L. collected from Thailand and other countries was performed using the methylation-sensitive amplification polymorphism (MSAP) technique. Nine primer combinations were used to generate MSAP fingerprints. When the data were interpreted as amplified fragment length polymorphism (AFLP) markers, 471 markers were scored. All 56 samples were classified into three major groups: γ-irradiated, non-toxic and toxic accessions. Genetic similarity among the samples was extremely high, ranging from 0.95 to 1.00, which indicated very low genetic diversity in this species. The MSAP fingerprint was further analyzed for DNA methylation polymorphisms. The results revealed differences in the DNA methylation level among the samples. However, the samples collected from saline areas and some species hybrids showed specific DNA methylation patterns. AFLP data were used, together with methylation-sensitive AFLP (MS-AFLP) data, to construct a phylogenetic tree, resulting in higher efficiency to distinguish the samples. This combined analysis separated samples previously grouped in the AFLP analysis. This analysis also distinguished some hybrids. Principal component analysis was also performed; the results confirmed the separation in the phylogenetic tree. Some polymorphic bands, involving both nucleotide and DNA methylation polymorphism, that differed between toxic and non-toxic samples were identified, cloned and sequenced. BLAST analysis of these fragments revealed differences in DNA methylation in some known genes and nucleotide polymorphism in chloroplast DNA. We conclude that MSAP is a powerful technique for the study of genetic diversity for organisms that have a narrow genetic base.     

Assessment of genetic and epigenetic stability in long-term in vitro shoot culture of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

In vitro clonal propagation of plants should generate identical copies of the selected genotype. However, associated stress might result in a breakdown of control mechanisms and consequent instability of the genome. We have used several molecular methods to assess the genetic stability of long-term propagated (24 years) multiple shoot in vitro culture of pea (Pisum sativum L.). We focused on assessing the stability of repetitive sequences, such as simple sequence repeats (SSR) and retrotransposons, both comprising a large part of genome. No differences were found when seedlings (Co-2004) or original seed (Co-1982) controls and long-term or newly established in vitro (one subculture cycle) samples were investigated by the SSR, inter-repeats (ISSR) or inter-retrotransposon amplified polymorphism (IRAP) method. However, the more global amplified fragment length polymorphism (AFLP) and particularly the methylation sensitive MSAP methods detected 11 and 18% polymorphism among samples, respectively. Interestingly, investigation of the global cytosine methylation status by HPCE measurement revealed no statistically significant differences. Some evidence of retrotransposon re-arrangement was observed by sequence-specific amplification polymorphism. This occurred mostly in the abundant Ty3-gypsy type Cyclop element and to a smaller extent in the Ogre element. Alternatively, no polymorphism was detected among the PDR-1 element of the Ty1-copia type retrotransposon. Based on these results, multiple shoot culture of pea maintained over a long period may be considered as a true to type multiplication method of the original genotype.     

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     

DNA methylation in the termite <Emphasis Type="Italic">Coptotermes lacteus</Emphasis>

Social insects are key examples of organisms that display polyphenism. Their genomes encode instructions for the development of multiple phenotypes, known as castes, which typically have highly divergent morphology, physiology and behaviour. DNA methylation, an epigenetic mechanism associated with modulation of gene expression in various eukaryotes, has recently been shown to provide a key link between environmental cues and caste-specific gene expression in honey bees (Hymenoptera). In termites—a major social insect group phylogenetically distant from Hymenoptera—the existence of DNA methylation has not, to our knowledge, been reported to date. Since genes encoding key DNA methylation enzymes are known to be absent in the genomes of a number of insect species, we sought to test whether termites are able to methylate their DNA, and, if so, whether caste-specific patterns of DNA methylation exist. We performed methylation-specific amplified fragment length polymorphism on the termite Coptotermes lacteus, and found evidence for DNA methylation. However, a comparison of methylation levels in different castes did not reveal any significant differences in methylation levels. The demonstration of DNA methylation in termites sets the stage for future epigenetic studies in these important social insects.     

Comparative analyses of genetic/epigenetic diversities and structures in a wild barley species (Hordeum brevisubulatum) using MSAP, SSAP and AFLP

We analyzed genetic diversity and population genetic structure of four artificial populations of wild barley (Hordeum brevisubulatum); 96 plants collected from the Songnen Prairie in northeastern China were analyzed using amplified fragment length polymorphism (AFLP), specific-sequence amplified polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) markers. Indices of (epi-)genetic diversity, (epi-)genetic distance, gene flow, genotype frequency, cluster analysis, PCA analysis and AMOVA analysis generated from MSAP, AFLP and SSAP markers had the same trend. We found a high level of correlation in the artificial populations between MSAP, SSAP and AFLP markers by the Mantel test (r > 0.8). This is incongruent with previous findings showing that there is virtually no correlation between DNA methylation polymorphism and classical genetic variation; the high level of genetic polymorphism could be a result of epigenetic regulation. We compared our results with data from natural populations. The population diversity of the artificial populations was lower. However, different from what was found using AFLP and SSAP, based on MSAP results the methylation polymorphism of the artificial populations was not significantly reduced. This leads us to suggest that the DNA methylation pattern change in H. brevisubulatum populations is not only related to DNA sequence variation, but is also regulated by other controlling systems.     

Utility of the methylation-sensitive amplified polymorphism (MSAP) marker for detection of DNA methylation polymorphism and epigenetic population structure in a wild barley species (<Emphasis Type="Italic">Hordeum brevisubulatum</Emphasis>)

We report here that by using a modified scoring criterion, the methylation-sensitive amplified polymorphism or MSAP marker can be used effectively to detect polymorphism in DNA methylation patterns within and among populations of a perennial wild barley species, Hordeum brevisubulatum. Twenty-four selected individual genotypes representing four natural populations of H. brevisubulatum distributed in the Songnen Prairie in northeastern China were studied. The utility of MSAP was evidenced by its detection of high levels of polymorphism in DNA methylation patterns between individuals within a given population, and the clear inter-population differentiation in methylation patterns (methylation-based epigenetic population structure) revealed among the four populations. The resolving power of MSAP to detect DNA methylation polymorphism was found to be comparable with that of a retrotransposon-based sequence-specific amplified polymorphism marker, or SSAP, to detect genetic polymorphism in the same set of plants, suggesting that MSAP with a modified scoring criterion can be used efficiently to detect DNA methylation polymorphism and assess epigenetic population structure in natural plant populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">In vitro</Emphasis> germplasm conservation of <Emphasis Type="Italic">Podophyllum peltatum</Emphasis> L. under slow growth conditions

Germplasm conservation of Podophyllum peltatum L. was attempted by using synthetic seed technology and media supplemented with osmotic agents. Excised buds from in vitro cultures were encapsulated in calcium alginate beads and cultured on different substrates then stored at 5, 10, and 25°C for up to 8 mo. Survival and vigor in re-growth were the parameters used to evaluate the germplasm storage conditions. Vigor in re-growth was measured by number of buds induced after storage, which was achieved on a substrate containing water solidified with 1% w/v agar under 10°C. In vitro storage of shoot cultures was also evaluated by supplementing osmotic agents, mannitol, or sorbitol to the media. Such treatment had a negative impact on post-storage re-growth (at 25°C), even though the inclusion of 2% w/v sorbitol and mannitol each to the media increased plantlet survival during 10°C storage treatment. A deleterious effect was noticed among cultures in re-growth when higher concentrations of these supplements were added to the media. Genetic stability was assessed following 8 mo of storage using a PCR-based multilocus DNA fingerprinting technique, amplified fragment-length polymorphism. No differences in the DNA fragment patterns were observed using eight primer combinations in stored clones. However, a polymorphic band was noticed in the accession that served as explant source, suggesting that the mutation has occurred prior to this study perhaps during the 9 years of in vitro cultivation.     

Epigenetic regulation of photoperiodic flowering

The cytidine analogue 5-azacytidine, which causes DNA demethylation, induced flowering in the non-vernalization-requiring plants Perilla frutescens var. crispa, Silene armeria and Pharbitis nil (synonym Ipomoea nil) under non-inductive photoperiodic conditions, suggesting that the expression of photoperiodic flowering-related genes is regulated epigenetically by DNA methylation. The flowering state induced by DNA demethylation was not heritable. Changes in the genome-wide methylation state were examined by methylation-sensitive amplified fragment length polymorphism analysis. This analysis indicated that the DNA methylation state was altered by the photoperiodic condition. DNA demethylation also induced dwarfism, and the induced dwarfism of P. frutescens was heritable.Key words: 5-azacytidine, DNA methylation, photoperiodic flowering, epigenetics, methylation-sensitive amplified fragment length polymorphism, CpG island, dwarfism     

AFLP-Based detection of DNA methylation

By using the isoschizomersHpa II andMsp I which display differential sensitivity to cytosine methylation, a modified amplified fragment length polymorphism (AFLP) technique has been developed to investigate DNA methylation profiles in eukaryotic organims. Genomic DNA was digested with a mixture ofEcoR I and one of the isoschizomers, and ligated to oligonucleotide adapters. After two rounds of selective PCR amplification, followed by DNA separation on a Long Ranger gel electrophoresis, a subset of restriction fragments can be displayed on an X-ray film. Comparison of AFLP banding patterns betweenHpa II andMsp I revealed the extent of DNA methylation. The technique has been successfully applied in this study to investigate DNA methylation profiles of apple (Malus domestica cv. Gala) genomic DNA extracted from leaves of field-grown adult trees andin vitro-grown shoot cultures. The results showed that up to 25 percent of AFLP bands were derived from methylated sequences, and among those, a few bands unique to either adult trees orin vitro shoots were observed. These results demonstrated that this protocol is effective in identifying methylated DNA profiles. Both first authors have contributed equally to this work.     

Morphological and molecular analysis of genetic stability in micropropagated Fragaria×Ananassa cv. pocahontas

Summary Micropropagated strawberry plants (Fragaria×ananassa L.) grown on 5 μM and 15 μM BA medium or cold-stored were grown in the field to examine morphological variation. Except for plant height, morphological characteristics did not differ for field-grown plants micropropagated on 5 μM and 15 μM BA medium. Cold-stored plants were less vigorous, both vegetatively and reproductively, than BA-treated plants. Random amplified polymorphic DNA (RAPD) markers were used to determine if cold storage or supraoptimal levels of N⁶-benzyladenine (BA) in the culture medium caused genetic changes leading to somaclonal variation. No mutations were observed in 246 loci amplified by the 29 primers tested. Possible changes in methylation patterns of ribosomal DNA genes of strawberries were also examined. Changes in methylation patterns were observed in only one DNA sample from plants grown on 15 μM BA medium and in one of the cold-stored plants. Length polymorphism was observed in two samples from plantlets derived from one explant. The low levels of RAPD variation and methylation observed, and the apparently epigenetic changes in morphological characteristics in plants used in this study, indicated that mutations had not occurred. Part of a thesis submitted by M. B. K. in partial fulfillment of the requirements for the MS degree. The use of trade names in this publication does not imply endorsement by the U.S. Department of Agriculture or Oregon State University.     

微生境对鼎湖山锥表观遗传变异的影响

为了解种群内水平上影响植物的适应机制与空间格局关系的因素,对鼎湖山2个演替阶段林分锥(Castanopsis chinensis)种群通过DNA甲基敏感扩增片段多态性进行表观遗传特征分析,结果表明,微生境对表观遗传变异的贡献由成熟林的20.2%降低为过熟林的15.7%,但地形因素的影响却增大,同时微生境中具体起显著作用的环境因素在两个林分也不同。因此,微生境特征对种群适应机制和分布格局有显著影响,对演替阶段也有影响。     

Specificity of DNA Methylation Changes During Fungal Dimorphism and Its Relationship to Polyamines

We utilized our modification of the amplified fragment length polymorphism technique for the determination of changes occurring in the DNA methylation patterns during the dimorphic transition of the fungi Mucor rouxii, Yarrowia lipolytica, and Ustilago maydis. To determine the specificity of differential methylation in regards to dimorphism, we obtained the yeast-like form of the three fungi under conditions that induced mycelial growth, by addition of 1,4-diaminobutanone (DAB), an inhibitor of ornithine decarboxylase in the case of M. rouxii and Y. lipolytica. In an odc null mutant of U. maydis, repression of the dimorphic transition was brought about by limitation in the amounts of exogenous putrescine. Yeasts from the three fungi thus obtained conserved a significant number of the differential DNA fragments with the methylation pattern displayed by normal yeasts, indicating their true correlation with dimorphism. Our results also confirm a role of polyamines in differential DNA methylation and fungal dimorphic transition.     

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.     

Genome methylation patterns across castes and generations in a parasitoid wasp

Environmental influences shape phenotypes within and across generations, often through DNA methylations that modify gene expression. Methylations were proposed to mediate caste and task allocation in some eusocial insects, but how an insect's environment affects DNA methylation in its offspring is yet unknown. We characterized parental effects on methylation profiles in the polyembryonic parasitoid wasp Copidosoma koehleri, as well as methylation patterns associated with its simple caste system. We used methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) to compare methylation patterns, among (1) reproductive and soldier larvae; and (2) offspring (larvae, pupae, and adults) of wasps that were reared at either high or low larval density and mated in the four possible combinations. Methylation frequencies were similar across castes, but the profiles of methylated fragments differed significantly. Parental rearing density did not affect methylation frequencies in the offspring at any developmental stage. Principal coordinate analysis indicated no significant differences in methylation profiles among the four crossbreeding groups and the three developmental stages. Nevertheless, a clustering analysis, performed on a subset of the fragments, revealed similar methylation patterns in larvae, pupae, and adults in two of the four parental crosses. Nine fragments were methylated at two cytosine sites in all larvae, and five others were methylated at two sites in all adults. Thus, DNA methylations correlate with within‐generation phenotypic plasticity due to caste. However, their association with developmental stage and with transgenerational epigenetic effects is not clearly supported.     

Flowering and dwarfism induced by DNA demethylation in Pharbitis nil

Flowering and dwarfism induced by 5‐azacytidine and zebularine, which both cause DNA demethylation, were studied in a short‐day (SD) plant Pharbitis nil (synonym Ipomoea nil), var. Violet whose photoinduced flowering state does not last for a long period of time. The DNA demethylating reagents induced flowering under non‐inductive long‐day (LD) conditions. The flower‐inducing effect of 5‐azacytidine did not last for a long period of time, and the plants reverted to vegetative growth. The progeny of the plants that were induced to flower by DNA demethylation did not flower under the non‐inductive photoperiodic conditions. These results suggest that the flowering‐related genes were activated by DNA demethylation and then remethylated again in the progeny. The DNA demethylation also induced dwarfism. The dwarfism did not last for a long period of time, was not heritable and was overcome by gibberellin A₃ but not by t‐zeatin or kinetin. The change in the genome‐wide methylation state was examined by methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. The analysis detected many more polymorphic fragments between the DNA samples isolated from the cotyledons treated with SD than from the cotyledons under LD conditions, indicating that the DNA methylation state was altered by photoperiodic conditions. Seven LD‐specific fragments were extracted from the gel of the MS‐AFLP and were sequenced. One of these fragments was highly homologous with the genes encoding ribosomal proteins.     

Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants

The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has been proposed as a mechanism that may explain at least a part of these changes. In the present study, the methylation of tomato callus DNA was compared with that of leaf DNA, from control or regenerated plants, at MspI/HpaII sites around five middle-repetitive sequences. Although the methylation of the internal cytosine in the recognition sequence CCGG varied from zero to nearly full methylation, depending on the probe used, no differences were found between callus and leaf DNA. For the external cytosine, small differences were revealed between leaf and callus DNA with two probes, but no polymorphisms were detected among DNA samples of calli or DNA samples of leaves of regenerated plants. When callus DNA cut with HindIII was studied with one of the probes, H9D9, most of the signal was found in high-molecular-weight DNA, as opposed to control leaf DNA where almost all the signal was in a fragment of 530 bp. Also, an extra fragment of 630 bp was found in the callus DNA that was not present in control leaf DNA. Among leaves of plants regenerated from tissue culture, the 630-bp fragment was found in 10 of 68 regenerated plants. This 630-bp fragment was present among progeny of only 4 of these 10 plants after selfing, i.e. it was partly inherited. In these cases, the fragment was not found in all progeny plants, indicating heterozygosity of the regenerated plants. The data are interpreted as indicating that a HindIII site becomes methylated in callus tissue, and that some of this methylation persists in regenerated plants and is partly transmitted to their progeny.     

DNA methylation analysis using bisulfite treatment and PCR-single-strand conformation polymorphism in colorectal cancer showing microsatellite instability.

The combination of bisulfite treatment and PCR-single-strand DNA conformation polymorphism (SSCP) analysis is proposed for quantitative methylation assay. We applied this procedure to the methylation analysis of the hMLH1 promoter region in colorectal cancer. An analysis of mixtures of known amounts of methylated and unmethylated DNA revealed a linear relation. Using a calibration curve, proportions of methylated DNA were calculated. The hMLH1 promoter region was highly methylated in about 80% of microsatellite instability (MSI) (+) colorectal cancers, but in none of the MSI(-) colorectal cancers. A significant correlation existed between hypermethylation of the hMLH1 promoter and MSI, as in previous reports. In conclusion, bisulfite-PCR-SSCP (BiPS) analysis could be applied to the rapid identification of methylation status in multiple samples, quantification of methylation differences, and detection of methylation heterogeneity in amplified DNA fragments.