Miroslav Baránek

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.

Comprehensive Virus Detection Using Next Generation Sequencing in Grapevine Vascular Tissues of Plants Obtained from the Wine Regions of Bohemia and Moravia (Czech Republic)

Comprehensive next generation sequencing virus detection was used to detect the whole spectrum of viruses and viroids in selected grapevines from the Czech Republic. The novel NGS approach was based on sequencing libraries of small RNA isolated from grapevine vascular tissues. Eight previously partially-characterized grapevines of diverse varieties were selected and subjected to analysis: Chardonnay, Laurot, Guzal Kara, and rootstock Kober 125AA from the Moravia wine-producing region; plus Müller-Thurgau and Pinot Noir from the Bohemia wine-producing region, both in the Czech Republic. Using next generation sequencing of small RNA, the presence of 8 viruses and 2 viroids were detected in a set of eight grapevines; therefore, confirming the high effectiveness of the technique in plant virology and producing results supporting previous data on multiple infected grapevines in Czech vineyards. Among the pathogens detected, the Grapevine rupestris vein feathering virus and Grapevine yellow speckle viroid 1 were recorded in the Czech Republic for the first time.

Screening of differentially expressed genes during the end of endogenous dormancy of flower buds in Prunus armeniaca L.

An insufficient endogenous dormancy period of apricot flower buds can result in reduced frost resistance. This is one of the causes of lower fruit yields in certain years. The aim of this work was the analysis of gene expression profiles in flower buds of four apricot cultivars (‘Sundrop’, ‘Stark Early Orange’, ‘Vestar’ and ‘Betinka’) that differ in the duration of their dormancy periods. The investigation was carried out before and during exit of buds from endogenous dormancy. Transcriptomes from bud tissues were analyzed by cDNA amplified fragment length polymorphism. Amplified fragments showing a significant change in their intensity within obtained expression profiles were considered to be derived from genes associated with the exit of flower buds from endogenous dormancy. All selected fragments were sequenced and compared with sequences recorded in public databases, yielding 147 sequences with similarity to previously described genes. The majority of identified genes match with theoretical expectations for events happening in dormant tissues. Moreover, some of the identified genes are included in the category of cellular or physiological processes previously reported to directly influence the exit from endogenous dormancy (e.g. aquaporin, GTP-binding proteins, elongation factor 1-alpha, ATP-dependent ubiquitin, xyloglucan endotransglycosylase hydrolase, and EXGT-A1).

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.

Genetic Diversity Assessment in Amaranth Germplasm using AFLP and ISSR Markers

The genetic potential of and relationships among amaranth (Amaranthus spp.) genotypes conserved in gene banks are generally unknown. In this study, nine amplified fragment length polymorphism (AFLP) and 14 inter simple sequence repeats (ISSR) primers were used to study genetic diversity among amaranth species. A total of 92 ISSR loci, of which 86 were polymorphic, were detected. The bands ranged from 300 bp to 2000 bp with polymorphic information content (PIC) of 047. The AFLP primer combinations revealed a total of 648 bands, with 489 as the number of polymorphic loci. The fragments ranged from 52 bp to 423 bp in length with PIC of 0.48. The correlation between genetic similarity based on ISSR and AFLP was 0.77: thus, 59% of the relationship was explained. The cluster analysis revealed distinct groups of amaranth genotypes. Therefore, the use of molecular markers would be valuable for effective utilization of amaranth in breeding programs.

GFLV coat protein constructs based on local isolates from the Czech Republic

Coat protein genes of grapevine fanleaf virus local strains isolated in South-Moravia, Czech Republic were sequenced, and artificial coat protein gene was designed and synthesized. It retains typical sequence features of local strains but is free of mRNA destabilizing sequences. Three variants of the synthetic gene were cloned into Agrobacterium plant expression vector and their function was tested after potato protoplasts transformation, assaying transient mRNA and coat protein production.