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A simple method for detection of Czech bolting garlics (Allium sativum L.) using microsatellite analysis: methodology for practice
Ovesná, Jaroslava ; Mitrová, Katarína ; Kučera, Ladislav
The aim of the methodology was to verify and compile a set of microsatellite markers for the purpose of verification to be used for the undisputed identification of selected types of Czech bolting garlics. The object of submitted methodology is to simplify procedure that allow to determine Czech bolting garlic using microsatellite analysis (SSR- Single Sequence Repeats), which is applicable in testing laboratories. The described method allows to identify the variety and breeding material of bolting garlic of Czech origin using DNA markers (microsatellite alleles) that are present only for this group of varieties and breeding lines. By the amplification of the genome containing the microsatellite locus by polymerase chain reaction (PCR) using specific, fluorescently labeled primers, and subsequent analysis of the lengths of PCR products. Methodology newly brings a unique set of diagnostic SSR markers, describe the evaluation and interpretation of the results.
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Methodology for detecting the presence of cranberries and lingonberries in processed products using molecular SSR markers: methodology for practice
Kučera, Ladislav ; Ovesná, Jaroslava
The subject of the methodology is a procedure for diagnosing the presence of lingonberries (Vaccinium vitis-idaea L.) and cranberries (Vaccinum macrocarpon L.) in processed products such as tea or fruit ingredients of many food products (jams, jelly, purree, tea). The methodology exploit DNA sequences that are typical for each plant species. Thus, molecular DNA markers that characterize the plant species by SSRs (single sequence repeats) are used. The methodology describes the extraction of DNA from the assumed matrix, the use of selected SSR markers and the analytical data evaluation procedure. The method can be used in any solid equipped molecular-genetic laboratory. The method is applicable for the characterization of DNA isolated from any part of the plant, fruit, and derived food or food supplements. Method describes the procedure and necessary equipment to perform the analysis. Protocol recommends how to evaluate the results.
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Detection of five garlic viruses using real- time PCR analysis: methodology for practice
Mitrová, Katarína ; Svobodová, Leona ; Ovesná, Jaroslava
The objective of the assay is detection of five viruses in garlic using real- time PCR analysis. . This assay allows to identify five of the viruses in the varieties of garlic - Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Garlic common latent virus (GCLV), Shallot latent virus (SLV) and Garlic mite-borne filamentous virus (GMbFV) based on the detection of gene sequences for the coat protein by SYBR Green real-time PCR. This method was shown in our work to be a suitable tool for the detection of highly variable pathogens, such as garlic viruses. The method brings a new procedure for detection of these viruses.
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The genetic basis of Czech garlic "paličák" (A. sativum L. ssp. ophioscorodon) focusing on the biosynthetic pathways of the secondary metabolites
Čermák, Vladimír ; Ovesná, Jaroslava (advisor) ; Demnerová, Kateřina (referee)
Czech bolting garlic (Allium sativum L.) is the name for Czech varieties, which are categorized into subspecies sativum ssp. An inflorescence production and specific onion morphology is typical for this type of garlic. The genetic basis, that has been described by the analysis of microsatellites, are discussed in this thesis. Transcription analysis has revealed a large number of unigenes that have been assigned by genetic ontology to individual functions in the organism. Therefore, genetic differences from other varieties were confirmed. The impact of the environment and cultivation practices, including large- scale production, is being explored. The results can be used for further research or breeding. Consumers and experts have rated the Czech bolting garlic as a variety with a distinctly pungent taste and aroma. Substances causing this strong characteristic taste are secondary metabolites, alk(en)ylcysteine-S-oxides (ACSO), especially alliin and methiin. Chemical analysis did not confirm the exceptionally high value of these taste precursors or other substances that should distinguish Czech bolting garlic. Secondary metabolism includes other important substances that are used mainly in pharmacy and healthcare, where garlic has been used for thousands of years due to antibacterial effects,...
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Study of factors influencing efficiency of Canabis sativa transformation
Širl, Marek ; Ovesná, Jaroslava (advisor) ; Vondráková, Zuzana (referee)
Hemp (Cannabis sativa L.) is a multi-use crop, able to provide fibre celulose a hurds for industrial treatment seeds for oil preparation biomass for energy conversion and produces secondary metabolites useful for pharmaceutical application. For its resistence to stress ability to accumulate high concentration of heavy metals and low cultivations demands, it can also be used for phytoextractions. Current research is focused on establishment of cultivation protocol, which allows transformation of callus cultures, and their regeneration with high efficiency. In this thesis, several varieties of hemp were transferred to in vitro conditions and were tested for their ability to form callus. The best results were achieved using the hypocotyl segments in a nutrient medium supplemented with 1 mg/L of naphtylacetic acid and one of these two synthetic cytokinins 0,5 mg/L of thidiazuron or 5 mg/L of 6-benzylaminopurine. No significant difference in the use of these two cytokinins were observed. None of the explants on four different test media for regeneration of shoots were able to succesfully regenerate. Transformation of hemp was tested using two different methods. Transformed protoplasts from hemp leafs after agroinfiltration were isolated. This method turn out to be unsuitable for use with hemp due to its...
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A transcriptomic-based comparison of barley cultivars differing with respect to their low temperature acclimation capacity
Janská, Anna ; Ovesná, Jaroslava (advisor) ; Vaňková, Radomíra (referee) ; Honys, David (referee)
The PhD thesis is focused on a transcriptomics-based comparison of barley cultivars differing with respect to their low temperature acclimation capacity, with a particular focus on genes transcribed in the leaf and crown. The crown was of interest because of its importance for the winter survival of the plant. To involve both the first and the second phase of hardening, the test plants were exposed first to +3řC for 21 days, followed by - 3řC for one day. Freezing damage was assessed by measuring electrolyte leakage (Papers 2 and 3), using a modified version of a protocol developed by Prášil and Zámečník (1998). The same protocol was adapted to evaluate crown regrowth (Paper 2); for this purpose, the plants were cooled, then replanted and cut above the crown, and their survival rate calculated over the following week. Each RNA sample was queried by hybridization to an Affymetrix 22 K Barley1 GeneChip Genome Array (Close et al. 2004). The data were statistically analysed with the help of the software packages R, MAS 5.0 (Ihaka & Gentleman 1996) (Papers 2 and 3), Gene Spring GX 7.3 (Agilent Technologies, Santa Clara CA) and MapMan (Thimm et al. 2004; Usadel et al. 2005) (Paper 2), the "Self-Organizing Maps" algorithm (Kohonen et al. 1996) (Paper 3) and MIPS FunCat (Ruepp et al. 2004) (Paper 2). Paper...
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