The disertation thesis was focused on the analysis of variability of two nepoviruses genomes, Grapevine fanleaf virus -- GFLV and Arabis mosaic virus -- ArMV. The thesis described single isolates of these nepoviruses and their until recently non-explored genome portions coded by RNA1 strand, the coding regions for 1BHel and 1EPol proteins precisely. The main topic of this thesis was the identification of infected plants that were infected by nepoviruses GFLV and ArMV. Based on design of specific primers there was established sequence homology of coding sequences. Another topic was the development and optimalization of diagnostic system for the efficient GFLV and ArMV detection. This work was based on hypothesis of very high level GFLV and ArMV genomes variability. The results were recieved mainly from sequencing analyses performed by capillary automatic sequencer ABI-PRISM 310 (Applied Biosystems, Carlsbad, USA). Obtained results were assessed with CLC Main Workbench 5 software (CLC Bio, Aarhus, Denmark). Real-time PCR TaqMan multiplex was also designed using this software. This real-time tool is able to detect and quantify both nepoviruses within one PCR reaction simultaneously. There were sequenced and analysed the genetic codes at the nucleotide level for MP 2B , 2CCP, 1BHel and 1EPol proteins in this work. The first one was assessed because of the development of real-time PCR TaqMan multiplex. In this case were sequenced 290 bp from 14 GFLV and ArMV isolates, the variability level was established in frame of nucleotides 71.7-97.6%. The partial genetic code for 2CCP was assessed in frame of 6 GFLV isolates and the variability was established in 935 bp fragment to 83-86% at the nucleotide level and 81-91% at the amino acid level. The partial genetic code for 1BHel was sequenced in frame of 6 isolates GFLV and the variability was established in 379 bp in frame of all accessible GFLV and ArMV isolates, the variability was established 86.4-98.9% at the nucleotide level and 96-100% at the aminoacid level. The partial genetic code for 1EPol protein was sequenced in 6 GFLV isolates in frame of 365 bp and the variability was established for all accessible sequences in GenBank/NCBI. The variability was established at the nucleotide level 79.5-100% and at the aminoacid level 91.4-100%. All received sequences longer than 300 bp were submitted to GenBank/NCBI. The obtained partial genetic codes for 1BHel a 1EPol proteins coded in RNA1 were unique at the time of publication. This work provided the important results which were used subsequently like source information for construction of vectors intended for the preparation of transgenic plants. Then the incurred grapevine would show resistance against GFLV. Moreover, based on results of this work there was created certified methodology of the real-time PCR TaqMan multiplex method which can be standartly used as an efficient diagnostic tool for simultaneous detection of GFLV and ArMV. This real-time system was succesfully compared with ELISA what would be interesting mainly for diagnostic laboratories engaged in the certification processes of propagation material.
| Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:249287 |
| Date | January 2013 |
| Creators | Eichmeier, Aleš |
| Source Sets | Czech ETDs |
| Language | Czech |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis |
| Rights | info:eu-repo/semantics/restrictedAccess |
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