MSAP Marker Based Epigenetic Regulation in Symptomatic and Asymptomatic Floral Malady of Soybean (Glycine max)

Abstract: It has learned that soybean is being affected by a floral disorder known as floral malady where plants fail to develop pod and do notattendfull maturity. For this floral disorder, we present a new methylation-sensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics of non-methylation, hyper-methylation, hemi-methylation, and full methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied a technique to analyze alterations in the cytosine methylation a popular Indian soybean (Glycine max L.) genotype, JS-335.The result revealed that in the symptomatic plant, out of 392 MSAP sites, 281 (71.68%), 33 (8.41%),38 (9.69%), and 40(10.20%) found to beun-methylated, hemi-methylated, fully methylated and hyper-methylated, respectively. Whereas, the MSAP profile of asymptomatic plants revealedout of 402MSAP sites, 330 (81.28%) was un-methylated, 22(5.41%) hemi-methylated,29(7.14%) fully methylatedand 25 (6.15%) hypermethylated. In comparison with asymptomatic(18.71%) plant, approximately 10% increased methylation was noted in symptomatic(28.31%) plantprofiles. The increased levels of methylation was recorded in the symptomatic plants about 28.31%and18.71% in asymptomatic. The study showed a higher epigenetic influence on JS-335 genotype of floral malady symptomatic than same genotype of asymptomatic plant. No pod formation in symptomatic plant induce genome wide changes either in promoter or coding region of gene(s) and DNA fragments showing polymorphism related to differences in pattern and extent of methylation associated with floral malady.

Vineyard environments influence Malbec grapevine phenotypic traits and DNA methylation patterns in a clone-dependent way.

KEY MESSAGE: By studying three cv. Malbec clones cultivated in two vineyards with contrasting environmental conditions, we demonstrated that DNA methylation has an important role in the phenotypic plasticity and that epigenetic modulation is clone-dependent. Clonal selection and vegetative propagation determine low genetic variability in grapevine cultivars, although it is common to observe diverse phenotypes. Environmental signals may induce epigenetic changes altering gene expression and phenotype. The range of phenotypes that a genotype expresses in different environments is known as phenotypic plasticity. DNA methylation is the most studied epigenetic mechanism, but only few works evaluated this novel source of variability in grapevines. In the present study, we analyzed the effects on phenotypic traits and epigenome of three Vitis vinifera cv. Malbec clones cultivated in two contrasting vineyards of Mendoza, Argentina. Anonymous genome regions were analyzed using methylation-sensitive amplified polymorphism (MSAP) markers. Clone-dependent phenotypic and epigenetic variability between vineyards were found. The clone that presented the clearer MSAP differentiation between vineyards was selected and analyzed through reduced representation bisulfite sequencing. Twenty-nine differentially methylated regions between vineyards were identified and associated to genes and/or promoters. We discuss about a group of genes related to hormones homeostasis and sensing that could provide a hint of the epigenetic role in the determination of the different phenotypes observed between vineyards and conclude that DNA methylation has an important role in the phenotypic plasticity and that epigenetic modulation is clone-dependent.

CleanBSequences: an efficient curator of biological sequences in R.

This work presents a new method and tool to solve a common problem of molecular biologists and geneticists who use molecular markers in their scientific research and developments: curation of sequences. Omic studies conducted by molecular biologists and geneticists usually involve the use of molecular markers. AFLP, cDNA-AFLP, and MSAP are examples of markers that render information at the genomics, transcriptomics, and epigenomics levels, respectively. These three types of molecular markers use adaptors that are the template for PCR amplification. The sequences of the adaptors have to be eliminated for the analysis of the results. Since a large number of sequences are usually obtained in these studies, this clean-up of the data could demand long time and work. To automate this work, an R package, named CleanBSequences, was created that allows the sequences to be curated massively, quickly, without errors and can be used offline. The curating is performed by aligning the forward and/or reverse primers or ends of cloning vectors with the sequences to be removed. After the alignment, new subsequences are generated without biological fragments not desired by the user, i.e., sequences needed by the techniques. In conclusion, the CleanBSequences tool facilitates the work of researchers, reducing time, effort, and working errors. Therefore, the present tool would respond to the problems related to the curation of sequences obtained from the use of some types of molecular markers. In addition to the above, being an open source, CleanBSequences is a flexible tool that has the potential to be used in future improvements to respond to new problems.

Changes in grapevine DNA methylation and polyphenols content induced by solar ultraviolet-B radiation, water deficit and abscisic acid spray treatments.

Environment and crop management shape plant's phenotype. Argentinean high-altitude vineyards are characterized by elevated solar ultraviolet-B radiation (UVB) and water deficit (D) that enhance enological quality for red winemaking. These signals promote phenolics accumulation in leaves and berries, being the responses mediated by abscisic acid (ABA). DNA methylation is an epigenetic mechanism that regulates gene expression and may affect grapevine growth, development and acclimation, since methylation patterns are mitotically heritable. Berry skins low molecular weight polyphenols (LMWP) were characterized in field grown Vitis vinifera L. cv. Malbec plants exposed to contrasting UV-B, D, and ABA treatments during one season. The next season early fruit shoots were epigenetically (methylation-sensitive amplification polymorphism; MSAP) and biochemically (LMWP) characterized. Unstable epigenetic patterns and/or stochastic stress-induced methylation changes were observed. UV-B and D were the treatments that induced greater number of DNA methylation changes respect to Control; and UV-B promoted global hypermethylation of MSAP epiloci. Sequenced MSAP fragments associated with UV-B and ABA showed similarities with transcriptional regulators and ubiquitin ligases proteins activated by light. UV-B was associated with flavonols accumulation in berries and with hydroxycinnamic acids in the next season fruit shoots, suggesting that DNA methylation could regulate the LMWP accumulation and participate in acclimation mechanisms.

Comparison of the phenolic contents and epigenetic and genetic variability of wild and cultivated watercress (Rorippa nasturtium var. aquaticum L.)

Background: Epigenetic modifications are key factors modulating the expression of genes involved in the synthesis of phytochemicals. The knowledge of plant epigenetic and genetic variations can contribute to enhance the production of bioactive compounds. These issues have been little explored thus far in Rorippa nasturtium var. aquaticum L. (watercress), an edible and medicinal plant. The aim of the current study was to determine and compare the phenolic composition and epigenetic and genetic variations between wild and cultivated watercress. Results: Significant differences were found in the quantitative phenolic composition between wild and cultivated watercress. The eight primer combinations used in the methylation-sensitive amplification polymorphism (MSAP) method revealed different epigenetic status for each watercress type, the cultivated one being the most epigenetically variable. The genetic variability revealed by the EcoRI/MspI amplification profile and also by eight inter-simple sequence repeat (ISSR) primers was different between the two types of watercress. The results of the Mantel test showed that the correlation between genetic and epigenetic variations has diminished in the cultivated type. Cluster analyses showed that the epigenetic and genetic characterizations clearly discriminated between wild and cultivated watercress. Conclusions: Relevant chemical, epigenetic, and genetic differences have emerged between wild and cultivated watercress. These differences can contribute to fingerprint and develop quality control tools for the integral and safety use and the commercialization of watercress. The richness of epialleles could support the development of tools to manipulate the watercress epigenome to develop high bioproduct­producing cultivars

Effect of nickel chloride on Arabidopsis genomic DNA and methylation of 18S rDNA

Background In recent years, nickel (Ni) has been widely applied in industrial and agricultural production and has become a kind of environmental pollution. In this study, the effect of nickel chloride (NiCl2) with different concentrations on Arabidopsis genomic stability and DNA methylation has been demonstrated. The nucleolus variation and 18S rDNA methylation after NiCl2 treatment have been analyzed. Results The results are as follows: (1) The NiCl2 could result in heritable genomic methylation variations. The genomic DNA methylation variations have been detected by methylation-sensitive amplified polymorphism (MSAP) molecular markers, and the result showed that after NiCl2 treatment, there was methylation variation in T0 generation seedlings, and partial site changes maintained in T1 generation, which suggested that the effects of NiCl2 on DNA methylation could be heritable in offspring. (2) NiCl2 brought deformity and damage to nucleolar structure in Arabidopsis root tip cells, and the damage was positively correlated with the NiCl2 concentration. 3. In the nucleolus, there was an increased cytosine methylation in 18S rDNA. The plant nucleolus variation and 18S rDNA methylation may be used as an examination indicator for Ni pollution in soil or plant. Conclusions NiCl2 application caused variation of DNA methylation of the Arabidopsis genomic and offspring's. NiCl2 also resulted in nucleolar injury and deformity of root tip cells. The methylation rate of 18S rDNA also changed by adding NiCl2.

Analysis of genetic and epigenetic variation in in vitro propagated potato somatic hybrid by AFLP and MSAP marker

Background: Genetic and epigenetic changes (DNA methylation) were examined in the tissue-culture propagated interspecific potato somatic hybrids between dihaploid Solanum tuberosum and S. pinnatisectum. Amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) were applied to detect the genetic and epigenetic changes, respectively in the somatic hybrids mother plants (1st cycle) and their regenerants (30th cycles sub-cultured). Results: To detect genetic changes, eight AFLP primer combinations yielded a total of 329 scorable bands of which 49 bands were polymorphic in both mother plants and regenerants. None of the scorable bands were observed in term of loss of original band of mother plant or gain of novel band in their regenerants. AFLP profiles and their cluster analysis based on the Jaccard’s similarity coefficient revealed 100% genetic similarity among the mother plant and their regenerants. On the other hand, to analyze epigenetic changes, eight MSAP primer pair combinations detected a few DNA methylation patterns in the mother plants (0 to 3.4%) and their regenerants (3.2 to 8.5%). Out of total 2320 MSAP sites in the mother plants, 2287 (98.6%) unmethylated, 21 (0.9%) fully methylated and 12 (0.5%) hemi-methylated, and out of total 2494 MSAP sites in their regenerants, 2357 (94.5%) unmethylated, 79 (3.1%) fully methylated and 58 (2.3%) hemi-methylated sites were amplified. Conclusion: The study concluded that no genetic variations were observed among the somatic hybrids mother plants and their regenerants by eight AFLP markers. However, minimum epigenetic variations among the samples were detected ranged from 0 to 3.4% (mother plants) and 3.2 to 8.5% (regenerants) during the tissue culture process.