Aplikace metod založených na amplifikaci DNA při studiu genomu rodu Prunus

Angl. resum

Genetic analysis of the genus Diospyros ssp. using RAPD and i-PBS methods

Molecular techniques (RAPD and i-PBS) were used to study genetic diversity within persimmon collection at Horticulture Faculty of Mendel University in Lednice. The aim of the work was to distinguish 14 known and 6 of unknown origin persimmon cultivars. The basic screening of 20 OPT primers was applied to 4 cultivars differring in the place of origin. Within the group of screened primers there were chosen those, which gave polymorphic repeatable strong and middle strong bands. Selected primers were used for the RAPD reactions within the whole persimmon collection. Three OPA primers previously described in the literature were also used for the RAPD reactions within the whole persimmon collection. Additional 16 i-PBS primers previously described in the literature were also used for i-PBS analysis of the whole group of cultivars. Amplification was successful with 12 i-PBS primers. The FreeTree software package was used to gen­erate a similarity matrix and then to produce a dendrogram using UPGMA analyses. The similarity dendrograms of all persimmon cultivars were created based on both approaches and also on combination of both analyses by program Tree View. All the dendrograms clearly separated the assessed cultivars into 4 clusters. There are cluster of American persimmons – Meader’ (1), ’Garretson’ (2) and ’Early Golden’ (3). They are representatives of D. virginiana. Further part of dendrogram includes single D. lotus (5), which is also clearly separated from other cultivars of the genus Diospyros. The third cluster includes interspecific hybrids ’Rossiyanka’ (10) and ’Nikitskaiya Bordovaiya’ (13), which arised from crosses of D. virginiana and D. kaki. The last cluster is formed by cultivars of Japanese persimmon – ’Mikatani Gosho’, ’Zenjimaru’, ’Tone Wase’, ’Hiratanenashi’, ’Fuyu’, Chinese cultivar – ’Sansi’ and two Italian cultivars ’Vaniglia’ and ’Tipo’. They are clustered without significant distinction. The similarities and the differences revealed among incorporation of cultivars into groups were compared with the literature findings

Comparison of DNA methylation landscape between Czech and Armenian vineyards show their unique character and increased diversity

Grapevine is a worldwide crop and it is also subject to global trade in wine, berries and grape vine plants. Various countries, including the countries of the European Union, emphasize the role of product origin designation and suitable methods are sought, able to capture distinct origins. One of the biological matrices that can theoretically be driven by individual vineyards’ conditions represents DNA methylation. Despite this interesting hypothesis, there is a lack of respective information. The aim of this work is to examine whether DNA methylation can be used to relate a sample to a given vineyard and to access a relationship between a DNA methylation pattern and different geographical origin of analysed samples. For this purpose, DNA methylation landscapes of samples from completely different climatic conditions presented by the Czech Republic (Central Europe) and Armenia (Southern Caucasus) were com-pared. Results of the Methylation Sensitive Amplified Polymorphism method confirm uniqueness of DNA methylation landscape for individual vineyards. Factually, DNA methylation diversity within vineyards of Merlot and Pinot Noir cultivars represent only 16% and 14% of the overall diversity registered for individual cultivars. On the contrary, different geographical location of the Czech and Armenian vineyards was identified as the strongest factor affecting diversity in DNA methylation landscapes (79.9% and 70.7% for Merlot and Pinot Noir plants, respectively).Supported by the Ministry of Education, Youth and Sports of the Czech Republic, Project LTC18009, Program INTER-COS

The group of variants analysed by MSAP.

<p>The system used to identify individual variants in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126638#pone.0126638.t001" target="_blank">Table 1</a> is as follows: the cultivar abbreviation (MT = Müller Thurgau or R = Riesling) is followed by identification number of the cutting. Meaning of the suffixes: M-NI = maternal, non-infected plants; M-I = maternal, GFLV-infected plants; IV-NI = <i>in vitro</i> cultivated, non-infected plants; IV-I = <i>in vitro</i> cultivated, infected plants; TIV-NI = <i>in vitro</i> thermotherapy, non-infected plants; and TIV-HAI = <i>in vitro</i> thermotherapy, plants healed after GFLV infection. The notes “(1 year)” and “(8 weeks)” indicate the time interval elapsed since the transfer of plants from <i>in vitro</i> to non-sterile conditions.</p><p>The group of variants analysed by MSAP.</p

Comparison of average mutual epigenetic similarities between groups represented by individuals with the same attributes (time elapsed since exposure to stress and the nature of conditions during <i>in vitro</i> cultivation).

<p>The first column clearly shows a decreasing tendency of similarity of DNA methylation landscape, when compared with state in maternal plants. It is also noticeable that the most similar DNA methylation states for each group of variants were usually recorded when individuals from the same group were compared (i.e., values on the diagonal of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126638#pone.0126638.t002" target="_blank">Table 2</a>).</p><p>Comparison of average mutual epigenetic similarities between groups represented by individuals with the same attributes (time elapsed since exposure to stress and the nature of conditions during <i>in vitro</i> cultivation).</p

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

<div><p>The appearance of somaclonal variability induced by <i>in vitro</i> 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 <i>in vitro</i> 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.</p></div

Whole experimental pipeline of newly introduced approach combining MSAP and NGS techniques

<p>Whole experimental pipeline of newly introduced approach combining MSAP and NGS techniques</p

Extraction of contigs representing amplicons differentially appeared across the standard cultivars and their derived somaclones repeatedly for both pairs.

<p>Extraction of contigs representing amplicons differentially appeared across the standard cultivars and their derived somaclones repeatedly for both pairs.</p