Transgenerational Variations in DNA Methylation Induced by Drought Stress in Two Rice Varieties with Distinguished Difference to Drought Resistance

Adverse environmental conditions have large impacts on plant growth and crop production. One of the crucial mechanisms that plants use in variable and stressful natural environments is gene expression modulation through epigenetic modification. In this study, two rice varieties with different drought resistance levels were cultivated under drought stress from tilling stage to seed filling stage for six successive generations. The variations in DNA methylation of the original generation (G0) and the sixth generation (G6) of these two varieties in normal condition (CK) and under drought stress (DT) at seedling stage were assessed by using Methylation Sensitive Amplification Polymorphism (MSAP) method. The results revealed that drought stress had a cumulative effect on the DNA methylation pattern of both varieties, but these two varieties had different responses to drought stress in DNA methylation. The DNA methylation levels of II-32B (sensitive) and Huhan-3 (resistant) were around 39% and 32%, respectively. Genome-wide DNA methylation variations among generations or treatments accounted for around 13.1% of total MSAP loci in II-32B, but was only approximately 1.3% in Huhan-3. In II-32B, 27.6% of total differentially methylated loci (DML) were directly induced by drought stress and 3.2% of total DML stably transmitted their changed DNA methylation status to the next generation. In Huhan-3, the numbers were 48.8% and 29.8%, respectively. Therefore, entrainment had greater effect on Huhan-3 than on II-32B. Sequence analysis revealed that the DML were widely distributed on all 12 rice chromosomes and that it mainly occurred on the gene’s promoter and exon region. Some genes with DML respond to environmental stresses. The inheritance of epigenetic variations induced by drought stress may provide a new way to develop drought resistant rice varieties.     

Inheritance Patterns and Stability of DNA Methylation Variation in Maize Near-Isogenic Lines

DNA methylation is a chromatin modification that contributes to epigenetic regulation of gene expression. The inheritance patterns and trans-generational stability of 962 differentially methylated regions (DMRs) were assessed in a panel of 71 near-isogenic lines (NILs) derived from maize (Zea mays) inbred lines B73 and Mo17. The majority of DMRs exhibit inheritance patterns that would be expected for local (cis) inheritance of DNA methylation variation such that DNA methylation level was coupled to local genotype. There are few examples of DNA methylation that exhibit trans-acting control or paramutation-like patterns. The cis-inherited DMRs provide an opportunity to study the stability of inheritance for DNA methylation variation. There was very little evidence for alterations of DNA methylation levels at these DMRs during the generations of the NIL population development. DNA methylation level was associated with local genotypes in nearly all of the >30,000 potential cases of inheritance. The majority of the DMRs were not associated with small RNAs. Together, our results suggest that a significant portion of DNA methylation variation in maize exhibits locally (cis) inherited patterns, is highly stable, and does not require active programming by small RNAs for maintenance.DNA methylation may contribute to heritable epigenetic information in many eukaryotic genomes. In this study, we have documented the inheritance patterns and trans-generational stability for nearly 1000 DNA methylation variants in a segregating maize population. At most loci studied, the DNA methylation differences are locally inherited and are not influenced by the other allele or other genomic regions. The inheritance of DNA methylation levels across generations is quite robust with almost no examples of unstable inheritance, suggesting that DNA methylation differences can be quite stably inherited, even in segregating populations.     

Transgenic elite Indica rice varieties,resistant to Xanthomonas oryzae pv. oryzae

The agronomically important Indica (group 1) rice varieties IR64, IR72, hybrid restorer line Minghui 63, and BG90-2 were co-transformed by microbombardment of embryogenic suspensions with plasmids that contain the Xa21 gene which confers resistance to Xanthomonas oryzae pv. oryzae and the hph gene for resistance to hygromycin B. Six of the 55 transgenic R0 plant lines containing the Xa21 gene displayed high levels of resistance to the pathogen, and no partial resistance was observed. The trait was stably inherited in subsequent generations, and transgenic plants are currently in field tests. The ability to transfer agronomically important genes into elite Indica rice varieties demonstrates the applicability of genetic engineering for the agronomic improvement of rice.     

Heritable alteration in DNA methylation induced by nitrogen-deficiency stress accompanies enhanced tolerance by progenies to the stress in rice (Oryza sativa L.)

Cytosine methylation is responsive to various biotic- and abiotic-stresses, which may produce heritable epialleles. Nitrogen (N)-deficiency is an abiotic stress being repeatedly experienced by plants. To address possible epigenetic consequences of N-deficiency-stress, we investigated the stability of cytosine methylation in rice (Oryza sativa L.) subsequent to a chronic (a whole-generation) N-deficiency at two levels, moderate (20 mg/L) and severe (10 mg/L), under hydroponic culture. MSAP analysis revealed that locus-specific methylation alteration occurred in leaf-tissue of the stressed plants (S₀) experiencing either level of N-deficiency, which was validated by gel-blotting. Analysis on three non-stressed self-fed progenies (S₁, S₂ and S₃) by gel-blotting indicated that ca. 50% of the altered methylation patterns in somatic cells (leaf) of the stressed S₀ plants were recaptured in S₁, which were then stably inherited to S₂ and S₃. Bisulfite sequencing of two variant MSAP loci with homology to low-copy retrotransposons on one stressed plant (S₀) and its non-stressed progenies (S₁ and S₂) showed that whereas one locus exhibited limited and non-heritable CHH methylation alteration, the other locus manifested dramatic heritable hypermethylation at nearly all cytosine sites within the assayed region. Intriguingly, when two groups of S₂ plants descended from the same N-deficiency-stressed S₀ plant were re-subjected to the stress, the group inheriting the modified methylation patterns showed enhanced tolerance to the N-deficiency-stress compared with the group bearing the original patterns. Our results thus demonstrate heritability of an acquired adaptive trait in rice, which was accompanied by epigenetic inheritance of modified cytosine methylation patterns, implicating an epigenetic basis underlying the inheritance of an acquired trait in plants.     

Inheritance and Variation of Cytosine Methylation in Three Populus Allotriploid Populations with Different Heterozygosity

DNA methylation is an epigenetic mechanism with the potential to regulate gene expression and affect plant phenotypes. Both hybridization and genome doubling may affect the DNA methylation status of newly formed allopolyploid plants. Previous studies demonstrated that changes in cytosine methylation levels and patterns were different among individual hybrid plant, therefore, studies investigating the characteristics of variation in cytosine methylation status must be conducted at the population level to avoid sampling error. In the present study, an F1 hybrid diploid population and three allotriploid populations with different heterozygosity [originating from first-division restitution (FDR), second-division restitution (SDR), and post-meiotic restitution (PMR) 2n eggs of the same female parent] were used to investigate cytosine methylation inheritance and variation relative to their common parents using methylation-sensitive amplification polymorphism (MSAP). The variation in cytosine methylation in individuals in each population exhibited substantial differences, confirming the necessity of population epigenetics. The total methylation levels of the diploid population were significantly higher than in the parents, but those of the three allotriploid populations were significantly lower than in the parents, indicating that both hybridization and polyploidization contributed to cytosine methylation variation. The vast majority of methylated status could be inherited from the parents, and the average percentages of non-additive variation were 6.29, 3.27, 5.49 and 5.07% in the diploid, FDR, SDR and PMR progeny populations, respectively. This study lays a foundation for further research on population epigenetics in allopolyploids.     

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.     

Endosperm-specific hypomethylation,and meiotic inheritance and variation of DNA methylation level and pattern in sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> L.) inter-strain hybrids

Understanding dynamics and inheritance of DNA methylation represents important facets for elucidating epigenetic paradigms in plant development and evolution. Using four sets of sorghum (Sorghum bicolor L.) inter-strain hybrids and their inbred parents, the developmental stability and inheritance of cytosine methylation in two tissues, leaf and endosperm, by MSAP analysis were investigated. It was found that in all lines (inbred and hybrid) studied, endosperm exhibited a markedly reduced level of full methylation of the external cytosine or both cytosines at the CCGG sites relative to leaf, which caused a variable reduction in the estimated total methylation level in endosperm by 6.89–19.69% (11.47% on average). For both tissues, a great majority of cytosine methylation profiles transmitted to F1 hybrids, however, from 1.69 to 3.22% of the profiles showed altered patterns in hybrids. Both inherited and altered methylation profiles can be divided into distinct groups, and their frequencies are variable among the cross-combinations, and between the two tissues. The variations in methylation level and pattern detected in the hybrids were not caused by parental heterozygosity, and they could be either non-random or stochastic among hybrid individuals. Homology analysis of isolated bands that showed endosperm-specific hypomethylation or variation in hybrids indicated that diverse sequences were involved, including known-function cellular genes and mobile elements. RT-PCR analysis of six genes representing endosperm-specific hypomethylation in MSAP profiles indicated that all showed higher expression in endosperm than in leaf, suggesting involvement of methylation state in regulating tissue-specific or tissue-biased expression in sorghum. Analysis on leaf-RNA from 5-azacytidine-treated plants further corroborated this possibility. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Direct and transgenerational impact on Daphnia magna of chemicals with a known effect on DNA methylation

The purpose of this study is to investigate (1) the induction of epigenetic effects in the crustacean Daphnia magna using DNA methylation as an epigenetic mark and (2) the potential stable transfer of such an epigenetic effect to non-exposed subsequent generations. Daphnids were exposed to chemical substances known to affect DNA methylation in mammals: vinclozolin, 5-azacytidine, 2′-deoxy-5-azacytidine, genistein and biochanin A. Effects on overall DNA cytosine methylation, body length and reproduction were evaluated in 21 day experiments. Using a multi-generational experimental design these endpoints were also evaluated in the F₁ and F₂ generation of both exposed and non-exposed offspring from F₀ daphnids exposed to 5-azacytidine, genistein or vinclozolin. A reduction in DNA methylation was consistently observed in daphnids exposed to vinclozolin and 5-azacytidine. Only in organisms exposed to 5-azacytidine was this effect transferred to the two subsequent non-exposed generations. A concurrent reduction in body length at day 7 was observed in these treatments. For the first time, exposure to environmental chemicals was shown to affect DNA methylation in the parental generation of D. magna. We also demonstrated a transgenerational alteration in an epigenetic system in D. magna, which indicates the possibility of transgenerational inheritance of environment-induced epigenetic changes in non-exposed subsequent generations.     

Heritable alteration in DNA methylation pattern occurred specifically at mobile elements in rice plants following hydrostatic pressurization

Intrinsic DNA methylation pattern is an integral component of the epigenetic network in many eukaryotes. Exploring the extent to which DNA methylation patterns can be altered under a specific condition is important for elucidating the biological functions of this epigenetic modification. This is of added significance in plants wherein the newly acquired methylation patterns can be inherited through organismal generations. We report here that DNA methylation patterns of mobile elements but not of cellular genes were specifically altered in rice plants following hydrostatic pressurization. This was evidenced by methylation-sensitive gel-blot analysis, which showed that 10 out of 10 studied low-copy transposons and retrotransposons manifested methylation alteration in at least one of the 8 randomly chosen pressure-treated plants, whereas none of the 16 studied low-copy cellular genes showed any change. Both gel-blotting and genome-wide fingerprinting indicated that the methylation alteration in mobile elements was not accompanied by a general genetic instability. Progeny analysis indicated retention of the altered methylation patterns in most progeny plants, underscoring early occurrence of the alterations, and their faithful epigenetic inheritance.     

云南药用野生稻BIBAC文库混合克隆池制备及筛选

在已构建完成的云南药用野生稻BIBAC( binary bacterial artificial chomosome)文库的基础上,将文库制备成一、二、三级混合克隆池,各级混合池的数量分别为3 360、140和14个.根据Xa21抗病基因序列设计1对特异引物,利用4步PCR法对文库混合克隆池进行逐级筛选,初步确定了3个抗病基因阳性克隆.为今后以PCR法高效利用云南药用野生稻BIBAC文库挖掘其优异基因奠定了良好的基础.     

Epigenetic Differentiation Persists after Male Gametogenesis in Natural Populations of the Perennial Herb Helleborus foetidus (Ranunculaceae)

Despite the importance of assessing the stability of epigenetic variation in non-model organisms living in real-world scenarios, no studies have been conducted on the transgenerational persistence of epigenetic structure in wild plant populations. This gap in knowledge is hindering progress in the interpretation of natural epigenetic variation. By applying the methylation-sensitive amplified fragment length polymorphism (MSAP) technique to paired plant-pollen (i.e., sporophyte-male gametophyte) DNA samples, and then comparing methylation patterns and epigenetic population differentiation in sporophytes and their descendant gametophytes, we investigated transgenerational constancy of epigenetic structure in three populations of the perennial herb Helleborus foetidus (Ranunculaceae). Single-locus and multilocus analyses revealed extensive epigenetic differentiation between sporophyte populations. Locus-by-locus comparisons of methylation status in individual sporophytes and descendant gametophytes showed that ∼75% of epigenetic markers persisted unchanged through gametogenesis. In spite of some epigenetic reorganization taking place during gametogenesis, multilocus epigenetic differentiation between sporophyte populations was preserved in the subsequent gametophyte stage. In addition to illustrating the efficacy of applying the MSAP technique to paired plant-pollen DNA samples to investigate epigenetic gametic inheritance in wild plants, this paper suggests that epigenetic differentiation between adult plant populations of H. foetidus is likely to persist across generations.     

水稻白叶枯病广谱抗性基因Xa21导入两用不育系培矮64S

以克隆的Xa21基因为外源基因,成熟胚愈伤组织为转化受体,应用农杆菌介导法对水稻两用型核不育系培矮64S进行转化,获46株转基因植株。PCR和Southern分析结果表明,Xa21已整合到受体基因组。用稻白叶枯病病原菌(Xanthomonasoryzaepv.oryzae)菲律宾小种6号接种鉴定,结果表明大多数转基因植株获得了抗病性。已整合的Xa21基因能够稳定地遗传,在所检测转基因株系的T1代中,Xa21基因显示3:1的分离。     

Linking inter-individual variability to endocrine disruptors: insights for epigenetic inheritance

Endocrine disrupting chemicals (EDCs) can induce a myriad of adverse health effects. An area of active investigation is the multi- and transgenerational inheritance of EDC-induced adverse health effects referring to the transmission of phenotypes across multiple generations via the germline. The inheritance of EDC-induced adverse health effects across multiple generations can occur independent of genetics, spurring much research into the transmission of underlying epigenetic mechanisms. Epigenetic mechanisms play important roles in the development of an organism and are responsive to environmental exposures. To date, rodent studies have demonstrated that acquired epigenetic marks, particularly DNA methylation, that are inherited following parental EDC exposure can escape embryonic epigenome reprogramming. The acquired epimutations can lead to subsequent adult-onset diseases. Increasing studies have reported inter-individual variations that occur with epigenetic inheritance. Factors that underlie differences among individuals could reveal previously unidentified mechanisms of epigenetic transmission. In this review, we give an overview of DNA methylation and posttranslational histone modification as the potential mechanisms for disease transmission, and define the requirements for multi- and transgenerational epigenetic inheritance. We subsequently evaluate rodent studies investigating how acquired changes in epigenetic marks especially DNA methylation across multiple generations can vary among individuals following parental EDC exposure. We also discuss potential sources of inter-individual variations and the challenges in identifying these variations. We conclude our review discussing the challenges in applying rodent generational studies to humans.

     

玉米细菌性条斑病非寄主抗性基因Rxo1转化水稻的研究

水稻细菌性条斑病是我国重要的水稻病害之一,但是在水稻种质资源中尚未发现抗细菌性条斑病单个主效基因。利用农杆菌介导的转化系统将从玉米中克隆的细菌性条斑病非寄主抗性基因Rxo1转入我国2个杂交稻恢复系和2个常规水稻品种。转基因植株的PCR和Southern分析结果表明Rxo1基因已整合到受体基因组中,Rxo1基因单拷贝整合的转化体在自交T1代呈现抗感3∶1分离。人工接种实验和病菌的生长曲线表明携带Rxo1的转基因植株对水稻细条病菌可以产生过敏性抗病反应。上述结果为利用非寄主抗性基因防治该病害提供了有用的信息。