Use of Combined MSAP and NGS Techniques to Identify Differentially Methylated Regions in Somaclones: A Case Study of Two Stable Somatic Wheat Mutants.

The appearance of somaclonal variability induced by in vitro cultivation is relatively frequent and can, in some cases, provide a valuable source of new genetic variation for crop improvement. The cause of this phenomenon remains unknown; however, there are a number of reports suggesting that epigenetics, including DNA methylations, are an important factor. In addition to the non-heritable DNA methylation changes caused by transient and reversible stress-responsive gene regulation, recent evidence supports the existence of mitotically and meiotically inherited changes. The induction of phenotypes via stable DNA methylation changes has occasionally great economical value; however, very little is known about the genetic or molecular basis of these phenotypes. We used a novel approach consisting of a standard MSAP analysis followed by deep amplicon sequencing to better understand this phenomenon. Our models included two wheat genotypes, and their somaclones induced using in vitro cultivation with a changed heritable phenotype (shortened stem height and silenced high molecular weight glutenin). Using this novel procedure, we obtained information on the dissimilarity of DNA methylation landscapes between the standard cultivar and its respective somaclones, and we extracted the sequences and genome regions that were differentially methylated between subjects. Transposable elements were identified as the most likely factor for producing changes in somaclone properties. In summary, the novel approach of combining MSAP and NGS is relatively easy and widely applicable, which is a rather unique feature compared with the currently available techniques in the epigenetics field.

Dynamics and Reversibility of the DNA Methylation Landscape of Grapevine Plants (Vitis vinifera) Stressed by In Vitro Cultivation and Thermotherapy.

There is relatively little information concerning long-term alterations in DNA methylation following exposure of plants to environmental stress. As little is known about the ratio of non-heritable changes in DNA methylation and mitotically-inherited methylation changes, dynamics and reversibility of the DNA methylation states were investigated in grapevine plants (Vitis vinifera) stressed by in vitro cultivation. It was observed that significant part of induced epigenetic changes could be repeatedly established by exposure to particular planting and stress conditions. However, once stress conditions were discontinued, many methylation changes gradually reverted and plants returned to epigenetic states similar to those of maternal plants. In fact, in the period of one to three years after in vitro cultivation it was difficult to distinguish the epigenetic states of somaclones and maternal plants. Forty percent of the observed epigenetic changes disappeared within a year subsequent to termination of stress conditions ending and these probably reflect changes caused by transient and reversible stress-responsive acclimation mechanisms. However, sixty percent of DNA methylation diversity remained after 1 year and probably represents mitotically-inherited epimutations. Sequencing of regions remaining variable between maternal and regenerant plants revealed that 29.3% of sequences corresponded to non-coding regions of grapevine genome. Eight sequences (19.5%) corresponded to previously identified genes and the remaining ones (51.2%) were annotated as "hypothetical proteins" based on their similarity to genes described in other species, including genes likely to undergo methylation changes following exposure to stress (V. vinifera gypsy-type retrotransposon Gret1, auxin-responsive transcription factor 6-like, SAM-dependent carboxyl methyltransferase).

Genetic changes in grapevine genomes after stress induced by in vitro cultivation, thermotherapy and virus infection, as revealed by AFLP.

The Amplification Fragment Length Polymorphism (AFLP) technique was employed to study genetic variations which can be induced in vines by the stress occurring during different aspects of viticulture (in vitro cultivation, in vitro thermotherapy and virus infection). Analysis of AFLP banding patterns, generated by using 15 primer combinations, pointed to negligible genetic variation among plants exposed to individual stress. The average of similarity coefficients between differently stressed plants of the cultivars Müller Thurgau and Riesling were 0.984 and 0.991, respectively, as revealed by AFLP analysis. The low incidence of observed polymorphism demonstrates the high level of genome uniformity in plants reproduced by in vitro micropropagation via nodes, those subjected to in vitro thermotherapy and virus-infected plants.

Genetic changes in grapevine genomes after stress induced by in vitro cultivation, thermotherapy and virus infection, as revealed by AFLP

The Amplification Fragment Length Polymorphism (AFLP) technique was employed to study genetic variations which can be induced in vines by the stress occurring during different aspects of viticulture (in vitro cultivation, in vitro thermotherapy and virus infection). Analysis of AFLP banding patterns, generated by using 15 primer combinations, pointed to negligible genetic variation among plants exposed to individual stress. The average of similarity coefficients between differently stressed plants of the cultivars Müller Thurgau and Riesling were 0.984 and 0.991, respectively, as revealed by AFLP analysis. The low incidence of observed polymorphism demonstrates the high level of genome uniformity in plants reproduced by in vitro micropropagation via nodes, those subjected to in vitro thermotherapy and virus-infected plants.