Methylation-sensitive Amplification Polymorphism Research Articles

Physiological and DNA methylation analysis provides epigenetic insights into kenaf cadmium tolerance heterosis

Soil heavy metal pollution is one of the most challenging problems. Kenaf is an important natural fiber crop with strong heterosis and a higher tolerance to heavy metals. However, little is known about the molecular mechanisms of kenaf heavy metal tolerance, especially the mechanism of genomic DNA methylation regulating heterosis. In this study, kenaf cultivars CP085, CP089, and their hybrid F1 seedlings were subjected to 300 µM cadmium stress and found obvious heterosis of cadmium resistance in morphology and antioxidant enzyme activity of F1 hybrid seedlings. Through methylation-sensitive amplification polymorphism (MSAP) analysis, we highlighted that the total DNA methylation level under cadmium decreased by 16.9 % in F1 and increased by 14.0 % and 3.0 % in parents CP085 and CP089, respectively. The hypomethylation rate was highest (21.84 %), but hypermethylation was lowest (17.24 %) in F1 compared to parent cultivars. In particular, principal coordinates analysis (PCoA) indicates a significant epigenetic differentiation between F1 and its parents under cadmium. Furthermore, 21 differentially methylated DNA fragments (DMFs) were analyzed. Especially, the expression of NPF2.7, NADP-ME, NAC71, TPP-D, LRR-RLKs, and DHX51 genes were changed due to cadmium stress and related to cytosine methylation regulation. Finally, the knocked-down of the differentially methylated gene NPF2.7 by virus-induced gene silencing (VIGS) resulted in increased sensitivity of kenaf seedlings under cadmium stress. It is speculated that low DNA methylation levels can regulate gene expression that led to the heterosis of cadmium tolerance in kenaf.

Investigation of antifungal activities of myrcene on Fusarium reference strains.

Antifungal effects of myrcene, the plant-based naturel compound, were investigated on Fusarium graminearum PH-1 and Fusarium culmorum FcUK99 references, for the first time. Minimum inhibitory concentration (MIC) and half of MIC (MIC50) of both Fusarium strains against myrcene were found as 25µg/µl and 12.5µg/µl, respectively. MIC50 application decreased the cell viabilities in the ratios of 34.90% and 33.91% in PH-1 and FcUK99, respectively (p < 0.01). The significantly increased catalase (CAT) activity was recorded in MIC50 treated strains (p < 0.01). Apoptosis-like process and cellular oxidative stress were also monitored with acridine orange/ethidium bromide (Ao/Eb) dual staining and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) staining. The genomic template stability (GTS) percentages were calculated as 79% for PH-1 and 71% for FcUK99 via random amplified polymorphic DNA (RAPD). Methylation polymorphism values were calculated as 53.8% and 50.6% in PH-1 and 40.4% and 39% in FcUK99 for HapII and MspI, respectively by coupled restriction enzyme digestion-random amplification (CRED-RA). Methylation-sensitive amplified polymorphism (MSAP) revealed that myrcene caused both type I and type III epigenetic modifications in both genomes. MIC50 dose caused up to 13.86 ± 0.42-fold changes in the expressions of cat, mst20, and stuA, whereas downregulation in tri5 was recorded. Myrcene application did not change the retrotransposon movement in both species by the amplifying of idiomorphic retrotransposon patterns through inter-retrotransposon polymorphism-polymerase chain reaction (IRAP-PCR). This study demonstrated that myrcene is an effective compound in the management of phytopathogenic Fusarium species by causing morphological, genetic, epigenetic, and cellular alterations, and has a potential to utilize as an antifungal agent.

Physiological, Biochemical, and Epigenetic Reaction of Maize (Zea mays L.) to Cultivation in Conditions of Varying Soil Salinity and Foliar Application of Silicon.

Soil salinity is one of the basic factors causing physiological, biochemical and epigenetic changes in plants. The negative effects of salt in the soil environment can be reduced by foliar application of silicon (Si). The study showed some positive effects of Si on maize plants (Zea mays L.) grown in various salinity conditions. At high soil salinity (300 and 400 mM NaCl), higher CCI content was demonstrated following the application of 0.2 and 0.3% Si. Chlorophyll fluorescence parameters (PI, FV/F0, Fv/Fm and RC/ABS) were higher after spraying at 0.3 and 0.4% Si, and plant gas exchange (Ci, PN, gs, E) was higher after spraying from 0.1 to 0.4% Si. Soil salinity determined by the level of chlorophyll a and b, and carotenoid pigments caused the accumulation of free proline in plant leaves. To detect changes in DNA methylation under salt stress and in combination with Si treatment of maize plants, the methylation-sensitive amplified polymorphism (MSAP) technique was used. The overall DNA methylation level within the 3'CCGG 5' sequence varied among groups of plants differentially treated. Results obtained indicated alterations of DNA methylation in plants as a response to salt stress, and the effects of NaCl + Si were dose-dependent. These changes may suggest mechanisms for plant adaptation under salt stress.

Effects of TDZ and IBA on DNA methylation during callus induction in pear (&lt;i&gt;Pyrus ussuriensis&lt;/i&gt; Maxim)

One of the essential epigenetic processes in higher eukaryotes, DNA methylation is essential for maintaining genome integrity and influencing gene expression. There aren't many research on how plant growth regulators (PGR) affect DNA methylation during the pear callus formation process. In this work, By using methylation-sensitive amplification polymorphism (MSAP), DNA methylation in pear callus caused by various quantities of thidiazuron (TDZ) and indole-3-butyric acid (IBA) was compared. The concentrations of TDZ and IBA could affect the callus induction rate and methylation level. It was discovered that induction rate and methylation were negatively correlated. The rate of pear callus induction was highest (54%) while the medium suppled with 3.0 mg·L^-1 TDZ and 4.0 mg·L^-1 IBA, although the matching DNA methylation level was lowest (27.96%). Additionally, there were significant difference in th level of genes' expression between different treatments and Both methylation and demethylation are regulated by these genes. As for genes related to auxin and cytokinin, gene expression analysis revealed that their levels of expression after different concentrations of TDZ combined with IBA present in differences significantly. Our findings provide light on DNA methylation mechanisms of plant tissue culture (PTC) dedifferentiation.

Effect of Silicon on Oat Salinity Tolerance: Analysis of the Epigenetic and Physiological Response of Plants

Environmental conditions are the primary factor determining the growth and yield of plants. As a result of climate change, the negative impact of abiotic factors is intensifying. One of them is salt stress. Soil salinity is one of the major problems in agriculture in the world and affects many cultivar species. The aim of this study was to evaluate the effect of silicon foliar application on the physiological and epigenetic reaction of oats (Avena sativa L.) under salt stress. The pot experiment was carried out in controlled conditions. Oat plants were subject to sodium chloride (NaCl) at a concentration of 200 mM and applied to the soil. Three concentrations of Optysil (200 g∙L−1 SiO2) were used for foliar fertilization. Measurements were made of the relative chlorophyll content in the leaves, the selected chlorophyll fluorescence parameters, and the gas exchange parameters. In this study, methylation-sensitive amplification polymorphisms (MSAP) analysis was used to investigate the effect of Si application during salinity stress on the DNA methylation level in oat plants. The results of this study indicated that the exogenous application of silicon improved the tolerance of the oat plants to salinity. The doses of 0.1% and 0.2% Optysil had the greatest effect on alleviating the impact of salt stress on the oat plants. In this research, the epigenetic as well as the physiological response of plants to the applied experimental factors were analyzed, which is a broad coverage of the research topic on the effects of salinity and silicon on plants.

Epigenetic and Genetic Variability in Contrasting Latitudinal Fagus sylvatica L. Provenances

The adaptive capacity of a species and its population is determined by both genetic and epigenetic variation, which defines the potential for adaptive evolution and plastic response to environmental changes. In this study, we used Methylation Sensitive Amplified Polymorphism (MSAP) and Amplified Fragment Length Polymorphism (AFLP), similar genome-wide profiling techniques, to analyze the epigenetic and genetic variability in European beech provenances from Germany (DE), Spain (ES) and Sweden (SE), representing the latitudinal distribution of the species. In addition, we evaluated the effect of moderate water stress on cytosine methylation dynamics by comparing two latitudinal contrasting provenances. Our analysis revealed that trees from ES showed lower values of epigenetic and genetic diversity than those from DE and SE. Analyses of molecular variance for MSAPs and AFLPs showed that 16% and 15% of the among population variations were associated with epigenetic and genetic variation, respectively. The study of the effect of water stress on cytosine methylation dynamics in seedlings from ES and SE revealed no significant levels of epigenetic differentiation between well-watered and stressed plants. Only 2% of the epigenetic variation was explained by the watering regime in ES without changes in SE. The results support that DNA methylation may play a role in the local adaptation of Fagus sylvatica to environmental variation.

Genetic and epigenetic variation separately contribute to range expansion and local metalliferous habitat adaptation during invasions of Chenopodium ambrosioides into China.

Invasive plants often colonize wide-ranging geographical areas with various local microenvironments. The specific roles of epigenetic and genetic variation during such expansion are still unclear. Chenopodium ambrosioides is a well-known invasive alien species in China that can thrive in metalliferous habitats. This study aims to comprehensively understand the effects of genetic and epigenetic variation on the successful invasion of C. ambrosioides. We sampled 367 individuals from 21 heavy metal-contaminated and uncontaminated sites with a wide geographical distribution in regions of China. We obtained environmental factors of these sampling sites, including 13 meteorological factors and the contents of four heavy metals in soils. Microsatellite markers were used to investigate the demographic history of C. ambrosioides populations in China. We also analysed the effect of epigenetic variation on metalliferous microhabitat adaptation using methylation-sensitive amplified polymorphism (MSAP) markers. A common garden experiment was conducted to compare heritable phenotypic variations among populations. Two distinct genetic clusters that diverged thousands of years ago were identified, suggesting that the eastern and south-western C. ambrosioides populations in China may have originated from independent introduction events without recombination. Genetic variation was shown to be a dominant determinant of phenotypic differentiation relative to epigenetic variation, and further affected the geographical distribution pattern of invasive C. ambrosioides. The global DNA unmethylation level was reduced in metalliferous habitats. Dozens of methylated loci were significantly associated with the heavy metal accumulation trait of C. ambrosioides and may contribute to coping with metalliferous microenvironments. Our study of C. ambrosioides highlighted the dominant roles of genetic variation in large geographical range expansion and epigenetic variation in local metalliferous habitat adaptation.

Long term environmental variability modulates the epigenetics of maternal traits of kelp crabs in the coast of Chile

The methylation of DNA is an environmentally inducible epigenetic mechanism reflecting the short‐term ecological and environmental background of populations. Marine invertebrate populations, which spread along a latitudinal cline, are particularly suitable for profiling DNA methylation, due to the heterogenous environmental conditions experienced. We used the MSAP (Methylation Sensitive Amplified Polymorphism) technique to investigate the natural variation in DNA methylation of different female’s tissues (muscle, gonads, and gills) and early-stage eggs from five populations of the kelp crab Taliepus dentatus, distributed along a latitudinal cline in the coast of Chile. We assessed whether, (1) the distribution of DNA methylation profiles can be associated with the temporal variability of long term (18 years) climatologies (sea surface temperature, turbidity and productivity) and (2) the epigenetic diversity of eggs is related to the population-level phenotypic variability of several maternal investment traits (egg volume, egg weight, egg lipids and fecundity). The DNA methylation of eggs correlated positively and negatively with the long term variability in productivity and sea surface temperature, respectively. Furthermore, the diversity of DNA methylation of eggs correlated positively with the population-level phenotypic variability of several maternal investment traits, suggesting a key role of epigenetic mechanisms in generating phenotypic variability at population level for this species. We provide evidence of a strong link between the temporal variability of long term climatologies with the epigenetic profiles of key early ontogenetic traits associated with the maternal investment of kelp crabs. These modulating mechanisms can hence contribute early to phenotypic variability at population levels in response to local and past environmental fluctuation.

Epigenetics for crop adaptation to climate change

Epigenetics for crop adaptation to climate change

Global Methylation Analysis Using MSAP Reveals Differences in Chilling-Associated DNA Methylation Changes during Dormancy Release in Contrasting Sweet Cherry Varieties

Dormancy is an adaptive strategy developed by temperate perennial crops to protect overwinter tissues from unfavorable environmental conditions. Sweet cherry (Prunus avium L.), a member of the Rosaceae family, requires chilling to overcome dormancy. The time of harvest is directly correlated with chilling requirements in sweet cherries. Consequently, early and late season varieties have low and high chilling requirements, respectively. There is evidence that the expression of dormancy-related genes is regulated by DNA methylation. In this work, methylation-sensitive amplified polymorphism (MSAP) was applied to study genome-wide DNA methylation changes associated with dormancy in two low-chill varieties, ‘Royal Dawn’ and ‘Glen Red’, and one high-chill variety, ‘Kordia’. Our primary results suggest that the occurrence of progressive DNA demethylation is associated with chilling accumulation during dormancy in the three varieties, independent of their chilling requirements. Genes were identified with different methylation status changes, detected by MSAP, related to cell wall remodeling and energy metabolism. Several MSAP profiles among the varieties were observed, suggesting that fine epigenetic control is required to coordinate hormonal and environmental signals that induce dormancy and its release.

Methyl-Sensitive Amplification Polymorphism (MSAP) Analysis Provides Insights into the DNA Methylation Underlying Heterosis in Kenaf (Hibiscus Cannabinus L.) Drought Tolerance

ABSTRACT Kenaf (Hibiscus cannabinus L.) is an industrial fiber crop with significant heterosis and tolerance to abiotic stresses. However, the mechanism underlying heterosis in kenaf under drought conditions is completely unknown. In this study, F1 hybrids were remarkably tolerant to drought stress. Through methylation-sensitive amplification polymorphism (MSAP) analysis, we highlighted that total DNA methylation level under drought decreased by 11.2% in F1 and increased by 29.7% and 13.1% in parents CP079 and CP071, respectively. Unlike parents, the hypomethylation rate was 38% higher than hypermethylation in F1, the overall change in methylation pattern was higher in F1 (1.6%) than parents (0.4%). Furthermore, 35 key DNA fragments showing different methylation patterns were analyzed and found that the cytosine methylation status of DnaJ, ERF5, ZIP2, and PATL3 revealed changes in DNA methylation that may affect their respective expression level. Subsequently, virus-induced gene silencing (VIGS) mediated knockdown of the DnaJ, significantly increased the sensitivity of kenaf seedlings to drought. Overall, this study provides an epigenetic mechanism that could be used to improve the breeding strategy in kenaf.

Production and characterization of a novel interspecific somatic hybrid combining drought tolerance and high quality of sweet potato and Ipomoea triloba L.

A novel interspecific somatic hybrid combining drought tolerance and high quality of sweet potato and Ipomoea triloba L. was obtained and its genetic and epigenetic variations were studied. Somatic hybridization can be used to overcome the cross-incompatibility between sweet potato (Ipomoea batatas (L.) Lam.) and its wild relatives and transfer useful and desirable genes from wild relatives to cultivated plants. However, most of the interspecific somatic hybrids obtained to date cannot produce storage roots and do not exhibit agronomic characters. In the present study, a novel interspecific somatic hybrid, named XT1, was obtained through protoplast fusion between sweet potato cv. Xushu 18 and its wild relative I. triloba. This somatic hybrid produced storage roots and exhibited significantly higher drought tolerance and quality compared with its cultivated parent Xushu 18. Transcriptome and real-time quantitative PCR (qRT-PCR) analyses revealed that the well-known drought stress-responsive genes in XT1 and I. triloba were significantly up-regulated under drought stress. The genomic structural reconstructions between the two genomes of the fusion parents in XT1 were confirmed using genomic in situ hybridization (GISH) and specific nuclear and cytoplasmic DNA markers. The DNA methylation variations were characterized by methylation-sensitive amplified polymorphism (MSAP). This study not only reveals the significance of somatic hybridization in the genetic improvement of sweet potato but also provides valuable materials and knowledge for further investigating the mechanism of storage root formation in sweet potato.

Epigenomic insight of lingonberry and health-promoting traits during micropropagation

Epigenetic variation plays a role in developmental gene regulation and responses to the environment. An efficient interaction of zeatin-induced cytosine methylation and secondary compounds has been displayed for the first time in tissue-culture shoots/plants of lingonberry (Vaccinium vitis-idaea L.) cultivar Erntedank in vitro (NC1, in a liquid medium; NC2, on a semi-solid medium), ex vitro (NC3, node culture-derived plants; LC1, leaf culture-derived plants) and its cutting-propagated (ED) plants. Through methylation-sensitive amplification polymorphism (MSAP) assay, we observed highest methylated sites in leaf regenerants (LC1) from all primer combinations (108 bands), along with the highest secondary metabolites. The four types of tissue culture-derived shoots/plants (NC1, NC2, NC3, LC1) showed higher methylation bands than cutting propagated donor plants (ED) that exhibited 79 bands of methylation, which is comparatively low. Our study showed more methylation in micropropagated shoots/plants than those derived from ED plants. On the contrary, we observed higher secondary metabolites in ED plants but comparatively less in micropropagated shoots (NC1, NC2) and plants (NC3, LC1).

Modification of Gene Expression, DNA Methylation and Small RNAs Expression in Rice Plants under In Vitro Culture

Tissue culture is an important experimental technique widely used for plant transformation and can induce somaclonal variation that is shown to be associated with genetic and epigenetic changes. However, the molecular basis of somaclonal variation and plant cell response to tissue culture has yet to be fully understood. In this study, we investigated gene expression, DNA methylation, and small RNA changes in regenerated lines (RL) compared with the wild-type progenitor plants (WT) of rice cv. Hitomebore. Using microarray, we identified many genes that were differentially expressed in the shoot-tip tissue and showed that TEs were generally activated in RL. Methylation Sensitive Amplification Polymorphism (MSAP) analysis of 5′CCGG sites combined with bisulfite sequencing detected a generally reduced DNA methylation in the RL lines. Small RNA sequencing analysis detected widespread changes in small RNA accumulation between RL and WT. In particular, repeat and TE-associated 24-nt size class of small RNAs, the inducer of RNA-directed DNA methylation, was in general down-regulated in RL, consistent with reduced CHG and CHH methylation at some of the differentially methylated TE loci. A large number of differentially expressed miRNAs were identified in RL and WT lines, including known and novel miRNAs. The expression of some of these miRNAs exhibited inverse correlation with the predicted target genes, suggesting a regulatory function. The RL plants looked similar to WT plants under normal conditions but showed significant phenotypic alterations under abiotic stress conditions. The widespread changes in DNA methylation, small RNA accumulation and gene expression in regenerated plants supports the role of epigenetic changes in tissue culture-induced somaclonal variation.

Effect of chronic radiation on the flax (Linum usitatissimum L.) genome grown for six consecutive generations in the radioactive Chernobyl area.

The growth of plants under chronic radiation stress in the Chernobyl area may cause changes in the genome of plants. To assess the extent of genetic and epigenetic changes in nuclear DNA, seeds of the annual crop flax (Linum usitatissimum L.) of the Kyivskyi variety, sown 21 years after the accident and grown for six generations in radioactive (RAD) and remediated (REM) fields were analysed. Flaxseed used for sowing first generation, which served as a reference (REF), was also analysed. The AFLP (Amplified Fragment Length Polymorphism) revealed a higher number of specific EcoRI-MseI loci (3.4-fold) in pooled flaxseed samples harvested from the RAD field compared with the REM field, indicating a link between the mutation process in the flax genome and the ongoing adaptation process. MSAP (Methylation-Sensitive Amplified Polymorphism) detecting EcoRI-MspI and EcoRI-HpaII loci in flax nuclear DNA genome showed no significant differences in methylation level, reaching about 33% in each of the groups studied. On the other hand, significant changes in the DNA methylation pattern of flaxseed samples harvested from the RAD field compared with controls were detected. Pairwise FST comparison revealed within both, EcoRI-MspI and transformed methylation-Sensitive data sets more than a 3-fold increase of genetic divergence in the RAD field compared with both controls. These results indicate that the nuclear genome of flax exposed to chronic radiation for six generations has more mutations and uses DNA methylation as one of the adaptation mechanisms for sustainability under adverse conditions.

Unexpected Low DNA Methylation in Transposable Elements at the 5′-CCGG Sites in Three Fruit Tree Cultivars

DNA methylation of three cultivars, each of the fruit tree species pear, plum and apple, was analyzed by the methylation-sensitive amplified polymorphism (MSAP) marker. All three fruit tree cultivars were found to contain apparently lower levels of methylation at the 5′-CCGG sites than all other plant species, such as rice and wheat, studied by the same method. Sequencing of the representative loci isolated from the MSAP profiles indicated that both protein-coding genes and transposable elements (TEs) were involved in low methylation. Gel blotting using isolated MSAP fragments and fragment mixtures representing two major types of TEs (copia- and gypsy-like) as hybridization probes confirmed the unexpected low DNA methylation levels at the 5′-CCGG sites in these three fruit tree genomes. Our results suggest that the three asexually propagated perennial fruit trees may indeed contain unusual lower levels of DNA methylation, especially in TEs at the 5′-CCGG sites. Additionally, our results may also suggest that the often used MSAP marker, which targets only one kind of specific methylation-sensitive sites recognized by a pair of isoschizomers (e.g., 5′-CCGG by HpaII/MspI), is not always representative of other cytosine sites (e.g., CHH) or CG sites other than those of 5′-CCGGs in some plant species.

The Mutational, Epigenetic, and Transcriptional Effects Between Mixed High-Energy Particle Field (CR) and 7Li-Ion Beams (LR) Radiation in Wheat M1 Seedlings.

Ionizing radiation (IR) is an effective approach for mutation breeding. Understanding the mutagenesis and transcriptional profiles induced by different mutagens is of great significance for improving mutation breeding efficiency. Here, using RNA sequencing and methylation-sensitive amplification polymorphism (MSAP) approaches, we compared the genetic variations, epigenetics, and transcriptional responses induced by the mixed high-energy particle field (CR) and 7Li-ion beam (LR) radiation in M1 seedlings of two wheat genotypes (Yangmai 18 and Yangmai 20). The results showed that, in both wheat genotypes, CR displayed significantly a higher mutation efficiency (1.79 × 10–6/bp) than that by LR (1.56 × 10–6/bp). The induced mutations were not evenly distributed across chromosomes and varied across wheat genotypes. In Y18 M1, the highest number of mutations were detected on Chr. 6B and Chr. 6D, whilst in Y20 M1, Chr. 7A and Chr. 3A had the highest mutations. The transcript results showed that total of 4,755 CR-regulated and 1,054 LR-regulated differentially expressed genes (DEGs) were identified in the both genotypes. Gene function enrichment analysis of DEGs showed that these DEGs overlapped or diverged in the cascades of molecular networks involved in “phenylpropanoid biosynthesis” and “starch and sucrose metabolism” pathways. Moreover, IR type specific responses were observed between CR an LR irradiation, including specific TFs and response pathways. MSAP analysis showed that DNA methylation level increased in LR treatment, while decreased at CR. The proportion of hypermethylation was higher than that of hypomethylation at LR, whereas a reverse pattern was observed at CR, indicating that DNA methylation plays critical roles in response to IR irradiation. All these results support that the response to different IRs in wheat includes both common and unique pathways, which can be served as a useful resource to better understand the mechanisms of responses to different IRs in other plants.

The impact of environment on genetic and epigenetic variation in Trifolium pratense populations from two contrasting semi-natural grasslands.

Central European grasslands, such as calcareous grasslands and oat-grass meadows, are characterized by diverse environmental conditions and management regimes. Therefore, we aimed to determine potential differences in genetic and epigenetic variation patterns between the contrasting habitats and to identify the drivers of genetic and epigenetic variation. We investigated the genetic and epigenetic variation of the ecologically variable plant species Trifolium pratense L. applying amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses. We observed low levels of genetic and epigenetic differentiation among populations and between habitat types. Genetic and epigenetic variations were not interdependent. Thus, genetic variation was significantly isolated by habitat dissimilarity, whereas epigenetic variation was affected by environment. More specifically, we observed a significant correlation of epigenetic diversity with soil moisture and soil pH (the latter potentially resulting in phosphorus limitation). Genetic variation was, therefore, affected more strongly by habitat-specific environmental conditions induced by land use-related disturbance and gene flow patterns, while epigenetic variation was driven by challenging environmental conditions.

Adaptive Divergence without Distinct Species Relationships Indicate Early Stage Ecological Speciation in Species of the Rhododendronpseudochrysanthum Complex Endemic to Taiwan.

The testing association of environmental variables with genetic and epigenetic variation could be crucial to deciphering the effects of environmental factors playing roles as selective drivers in ecological speciation. Although ecological speciation may occur in closely related species, species boundaries may not be established over a short evolutionary timescale. Here, we investigated the genetic and epigenetic variations using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP), respectively, and tested their associations with environmental variables in populations of four closely related species in the R. pseudochrysanthum complex. No distinctive species relationships were found using genetic clustering analyses, neighbor-joining tree, and neighbor-net tree based on the total AFLP variation, which is suggestive of the incomplete lineage sorting of ancestral variation. Nonetheless, strong isolation-by-environment and adaptive divergence were revealed, despite the significant isolation-by-distance. Annual mean temperature, elevation, normalized difference vegetation index, and annual total potential evapotranspiration were found to be the most important environmental variables explaining outlier genetic and epigenetic variations. Our results suggest that the four closely related species of the R. pseudochrysanthum complex share the polymorphism of their ancestor, but reproductive isolation due to ecological speciation can occur if local environmental divergence persists over time.

Sodium dehydroacetate treatment prolongs the shelf-life of ‘Kyoho’ grape by regulating oxidative stress and DNA methylation

In this study, we tested the ability of sodium dehydroacetate (SD) to extend the shelf-life of ‘Kyoho’ grape. Among the different concentrations of SD tested (0, 0.01, 0.1 and 1.0 mmol L–1), 0.01 mmol L–1 SD was the most effective in prolonging the shelf-life of ‘Kyoho’ grape. Compared with the control, the weight loss rate, browning index and hydrogen peroxide (H2O2) and malonaldehyde contents were significantly lower in the 0.01 mmol L–1 SD treatment, whereas the healthy berry rate, berry firmness, total soluble solids (TSS) content, ascorbic acid content and superoxide dismutase (SOD) activity were significantly higher. In addition, an analysis of ‘Kyoho’ grape DNA using methylation sensitive amplification polymorphism (MSAP) markers showed that the average DNA methylation level was significantly higher in the 0.01 mmol L–1 SD treatment than in the control. Together, these results indicate that 0.01 mmol L–1 SD could be used to extend the shelf-life of ‘Kyoho’ grape. Moreover, a strong connection between reactive oxygen species (ROS) metabolism and DNA methylation change during storage was revealed.