DNA methylation changes in photoperiod-thermo-sensitive male sterile rice PA64S under two different conditions

Epigenetic modification can occur at a high frequency in crop plants and might generate phenotypic variation without changes in DNA sequences. DNA methylation is an important epigenetic modification that may contribute to environmentally-induced phenotypic variations by regulating gene expression. Rice Photoperiod-Thermo-Sensitive Genic Male Sterile (PTGMS) lines can transform from sterility to fertility under lower temperatures and short-day (SD) conditions during anther development. So far, little is known about the DNA methylation variation of PTGMS throughout the genome in rice. In this study, we investigated DNA cytosine methylation alterations in the young panicles of PTGMS line PA64S under two different conditions using methylation sensitive amplified polymorphism (MSAP) method. Compared with the DNA methylation level of PA64S under lower temperatures and SD conditions (fertility), higher methylation was observed in PA64S (sterility). The sequences of 25 differentially amplified fragments were successfully obtained and annotated. Three methylated fragments, which are homologous to D2, NAD7 and psaA, were confirmed by bisulfite sequencing and their expression levels were also evaluated by qPCR. Real time quantitative PCR analysis revealed that five of the six selected methylated genes were downregulated in PA64S (sterility). These results suggested that DNA methylation may be involved in the sterility–fertility transition of PA64S under two different environmental conditions.     

Cryopreservation of shoot tips,evaluations of vegetative growth,and assessments of genetic and epigenetic changes in cryo-derived plants of Actinidia spp.

A droplet-vitrification protocol was described for cryopreservation of shoot tips of kiwifruit ‘Yuxiang’ (Actinidia chinensis var. deliciosa). No significant differences were found in root formation and shoot growth between the in vitro-derived shoots (the control) and cryo-derived ones when cultured in vitro. No significant differences were detected in survival and vegetative growth between the in vitro-derived plants (the control) and cryo-derived ones after re-establishment in greenhouse conditions. Inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-establishment in greenhouse conditions. These data indicate rooting ability, vegetative growth and genetic stability are maintained in the cryo-derived kiwifruit plants recovered from the droplet-vitrification cryopreservation. Methylation sensitive amplification polymorphism (MSAP) detected 12.8% and 1.6% DNA methylation in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-established in greenhouse conditions, respectively. This droplet-vitrification was applied to five cultivars and three rootstocks belonging to A. chinensis var. deliciosa, A. chinensis var. chinensis, A. macrosperma, A. polygama and A. valvata. The highest (68.3%) and lowest (22.5%) shoot regrowth were obtained in A. macrosperma and A. chinensis var. chinensis ‘Jinmi’, respectively, with an average of 46.4% shoot regrowth obtained across the eight genotypes. The droplet-vitrification protocol described here can be considered the most applicable cryopreservation method so far reported for the genus Actinidia. Results reported here provide theoretical and technical supports for setting up cryo-banks of genetic resources of Actinidia spp.     

Variation of cytosine methylation in response to water availability in two contrasting growth types of an amphibious plant Alternanthera philoxeroides

We used methylation-sensitive amplified polymorphisms (MSAP) to examine the alterations of cytosine methylation in two contrasting growth types of an amphibious plant Alternanthera philoxeroides in response to change of water availability. Using 34 pairs of selective primer combinations, we amplified 1026 and 1128 clear and reproducible bands in root and leaf of A. philoxeroides, respectively. When the aquatic types of plants were transplanted into drought culture, we found a decrease in the overall DNA methylation. When the terrestrial types of plants were transferred into flood culture, we detected a higher frequency of methylation than demethylation events. Alterations of DNA methylation were more evident in root than in leaf in response to change of water availability. When the confounding effects of variable environmental factors were removed, differences of cytosine methylation profiles were observed between two growth types of plants under common growth conditions.     

基于MSAP和SSR标记的高粱甲基化连锁群LGC、LGD的构建

以高粱(Sorghum bicolor(L.)Moench)品种‘B_2V_4’和‘1383-2’杂交获得的F_2群体为材料,通过SSR和MSAP标记检测高粱基因组差异,构建其甲基化遗传连锁群。结果显示,高粱甲基化连锁群LGC含有3个SSR标记和23个甲基化标记,覆盖高粱基因组44.3 cM;甲基化连锁群LGD含有4个SSR标记和8个甲基化标记,覆盖高粱基因组46.2 cM。LGC上甲基化位点仅来源于EcoRⅠ/MspⅠ酶切组合,而LGD上有来源于EcoRⅠ/MspⅠ和EcoRⅠ/HpaⅡ两种酶切组合的甲基化位点。在LGC连锁群Xtxp 69附近检测到一个密集的甲基化位点区域。研究结果表明MSAP标记可以快速检测植物基因组甲基化差异,适用于构建甲基化连锁群。     

Analysis of Cytosine Methylation Pattern in Response to Water Deficit in Pea Root Tips

Abstract: The correlation between environmental stress and DNA methylation has been studied by following the methylation status of cytosine residues in the DNA of pea root tips exposed to water deficit. DNA methylation was evaluated by two complementary approaches: (i) immunolabelling by means of a monoclonal antibody against 5-methylcytosine; (ii) MSAP (Methylation-Sensitive Amplified Polymorphism) to verify if methylation and de-methylation in response to water deficit may be related to specific DNA sequences. Immunolabelling showed that water stress induces cytosine hypermethylation in the pea genome. Regarding the CCGG target sequence, an increase in methylation specifically in the second cytosine (about 40 % of total site investigated) was revealed by MSAP analyses. In addition, MSAP band profile detected in three independent repetitions was highly reproducible suggesting that, at least for the CCGG target sequence, methylation was addressed to specific DNA sequences.     

组织培养导致的草莓DNA甲基化变异

以草莓品种‘丰香’和‘全明星’为材料,用甲基化敏感扩增多态性（MSAP）技术研究组织培养对草莓DNA甲基化的影响。结果表明,与普通苗相比,组织培养导致草莓试管苗的DNA甲基化水平下降,甲基化模式的变异以去甲基化为主。组织培养导致的DNA甲基化变异不稳定,在田间无性繁殖过程中,试管苗的无性繁殖后代DNA甲基化水平逐渐升高,仅部分变异的甲基化模式能够在试管苗的无性繁殖后代中稳定传递。两个品种之间,纽织培养对DNA甲基化变异程度的影响不同。     

喜旱莲子草MSAP分析技术反应体系的建立

目的：建立一个适于研究喜旱莲子草DNA甲基化的MSAP分析体系。方法：以喜旱莲子草为材料,采用改良CTAB法提取基因组DNA,并对影响MSAP分析的关键步骤包括基因组DNA酶切反应时间、模板DNA连接产物稀释倍数、预扩增产物稀释倍数等进行了优化。结果：改良CTAB方法提取的DNA质量较好,酶切反应时间5h,酶切连接产物稀释10倍进行预扩增,预扩产物稀释10倍进行选择性扩增,所得PCR产物经6%聚丙烯酰胺电泳,条带清晰可辨,并能有效检测喜旱莲子草基因组DNA甲基化的程度和状态。结论：为研究喜旱莲子草表观遗传适应机制奠定了基础。     

Analysis of DNA methylation patterns of PLBs derived from <Emphasis Type="Italic">Cymbidium hybridium</Emphasis> based on MSAP

The best known and most thoroughly studied epigenetic phenomenon is DNA methylation, which plays an important role in regulating gene expression during plant regeneration and development. In this study, the methylation-sensitive amplified polymorphism (MSAP) technique was carried out to determine differences in methylation profiles between two forms of protocorm-like bodies (PLBs), continuously proliferating PLBs (cPLBs) and spontaneously-differenting PLBs (sdPLBs), derived from cultures of Cymbidium hybridium. A total of 72 selective primer combinations were used to assess the status of cytosine methylation of DNA in these tissues. Of 4,440 fragments obtained 911 fragments, each representing a recognition site cleaved by one or both of the isoschizomers (Hpa II and Msp I), were amplified and were significantly different between the two forms of PLBs. Frequency of total and full-methylation of cPLBs and sdPLBs were 26.7/12.2%, 24.1/11.1%, respectively. In addition, 14 types of MSAP patterns detected in the two forms of PLBs belonged to two classes, type I and II. Sequencing of 14 differentially methylated fragments and their subsequent blast search revealed that cytosine methylated 5′-CCGG-3′ sequences were equally distributed in the coding and non-coding regions. Southern blotting was conducted to verify the methylation polymorphism.     

Untangling individual variation in natural populations: ecological, genetic and epigenetic correlates of long-term inequality in herbivory

Individual variation in ecologically important features of organisms is a crucial element in ecology and evolution, yet disentangling its underlying causes is difficult in natural populations. We applied a genomic scan approach using amplified fragment length polymorphism (AFLP) markers to quantify the genetic basis of long‐term individual differences in herbivory by mammals at a wild population of the violet Viola cazorlensis monitored for two decades. In addition, methylation‐sensitive amplified polymorphism (MSAP) analyses were used to investigate the association between browsing damage and epigenetic characteristics of individuals, an aspect that has been not previously explored for any wild plant. Structural equation modelling was used to identify likely causal structures linking genotypes, epigenotypes and herbivory. Individuals of V. cazorlensis differed widely in the incidence of browsing mammals over the 20‐year study period. Six AFLP markers (1.6% of total) were significantly related to herbivory, accounting altogether for 44% of population‐wide variance in herbivory levels. MSAP analyses revealed considerable epigenetic variation among individuals, and differential browsing damage was significantly related to variation in multilocus epigenotypes. In addition, variation across plants in epigenetic characteristics was related to variation in several herbivory‐related AFLP markers. Statistical comparison of alternative causal models suggested that individual differences in herbivory are the outcome of a complex causal structure where genotypes and epigenotypes are interconnected and have direct and indirect effects on herbivory. Insofar as methylation states of MSAP markers influential on herbivory are transgenerationally heritable, herbivore‐driven evolutionary changes at the study population will involve correlated changes in genotypic and epigenotypic distributions.