The methylation-sensitive amplified polymorphism (MSAP) technique using HpaII and MspI isoschizomers was used to analyse DNA-methylation alterations in stressed grapevine plants. The stress used was in vitro propagation via nodal segments and in vitro thermotherapy for virus elimination. A set of pertinent grapevine plants derived from two cultivars (18 plants each for Müller Thurgau and Riesling) was used as stressed variants for analyses. A total of 695 and 700 MSAP bands were recognised and evaluated as present/absent for all analysed variants derived from both cvs. Müller Thurgau and Riesling. Average computed similarity of MSAP banding between analysed variants (Dice/Nei and Li coefficient) was 0.935 for both cultivars. Clustering of variants within resulting dendrograms showed significant differences between woody cuttings despite originating from the one plant. Further, there was a strong 'donor' effect of maternal plants on future arrays of DNA methylation in their regenerants. The 'donor' effect even seemed to prevail in the effect of stress on final DNA-methylation state in stressed regenerants. Additional MSAP evaluation suggests that thermotherapy induced an additional array of methylation changes when compared with stress caused by in vitro cultivation. From the viewpoint of whether methylation of CCGG loci increased/decreased due to stress, the results showed moderate prevalence for decreasing CCGG loci methylation.

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).

Eichmeier A., Baránek M., Pidra M. (2010): Analysis of genetic diversity and phylogeny of partial coat protein domain in Czech and Italian GFLV isolates. Plant Protect. Sci., 46: 145-148.The genetic diversity of Grapevine fanleaf virus (GFLV) was evaluated in 4 isolates sampled from naturally infected grapevines from South Moravia (Czech Republic) and 2 Italian isolates from Bari (Italy). Conserved regions within sequences in databases were found and new primers corresponding to these regions were designed and tested for RT-PCR amplification of the CP codifying region. After sequencing of obtained amplicons the similarity of isolates was analysed via alignments of sequences and by means of dendrograms.

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<p style="text-align: justify;">In this study we analyzed 51 grapevine cultivars registered in the Czech Republic using six highly informative SSR loci, and determined SSR profiles typical for each cultivar. All cultivars were easily distinguished on the basis of SSR allele length, except for berry color mutants (Pinot and Chasselas group). The average number of detected alleles per locus was 13.2, while observed heterozygosity is between 0.745 (VrZAG 79) and 0.922 (VVMD 7). The lengths of the alleles were compared with results from other laboratories; lengths were either the same or different by 1-3 bp. We also tested a new approach to microsatellite data comparison using a coding system based on reference alleles as a size standard (THIS et al., 2004). The obtained profiles were mostly in accordance with profiles of reference cultivars (THIS et al., 2004); we discuss the possible reasons for observed discrepancies.</p><p style="text-align: justify;">A third allele in the microsatellite spectrum was observed in five samples where template DNA originated from leaves. Additional analyses with DNA isolated from phloem-enriched samples (L2 meristem layer) were performed and typical diallelic profiles obtained. The reason for the detected triallelism is assumed to be a periclinal chimerism.</p>