Although the analysis of length polymorphism at STR loci has become a method of choice for grape cultivar identification, the standardization of methods for this purpose lags behind that of methods for DNA profiling in human and animal forensic genetics. The aim of this study was thus to design and validate a grapevine STR protocol with a practically useful level of multiplexing. Using free bioinformatics tools, published primer sequences, and nucleotide databases, we constructed and optimized a primer set for the simultaneous analysis of six STR loci (VVIi51, scu08vv, scu05vv, VVMD17, VrZAG47, and VrZAG83) by multiplex PCR and CE with laser-induced fluorescence, and tested it on 90 grape cultivars. The new protocol requires subnanogram quantities of the DNA template and enables automated, high-throughput genetic analysis with reasonable discriminatory power. As such, it represents a step toward further standardization of grape DNA profiling.
- MeSH
- algoritmy MeSH
- DNA rostlinná analýza genetika MeSH
- genetické markery genetika MeSH
- mikrosatelitní repetice genetika MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- reprodukovatelnost výsledků MeSH
- víno MeSH
- Vitis klasifikace genetika MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
The first isolated cytokinin, 6-furfurylaminopurine (kinetin or Kin), was identified almost 55years ago. Its biological effects on plant cells and tissues include influences on such processes as gene expression, cell cycle, chloroplast development, chlorophyll biosynthesis, stimulation of vascular development, delay of senescence, and mobilization of nutrients. In the present study we prepared a series of eight N9-substituted Kin derivatives, and characterized them with available physicochemical methods such as CI+ mass spectrometry and (1)H NMR spectroscopy. All compounds were tested in three classical cytokinin bioassays: a tobacco callus assay, an Amaranthus assay, and a senescence assay with excised wheat leaves. The ability of the compounds to interact with Arabidopsis cytokinin receptors CRE1/AHK4 and AHK3 was tested in a bacterial receptor assay. Prepared derivatives with certain substitutions of the N9-atom of the purine moiety enhanced the cytokinin activity of the parent compound in the bioassays to a remarkable degree but negatively affected its perception by CRE1/AHK4 and AHK3. The ability of compounds to delay the senescence of excised wheat leaves in both dark and light conditions, was highly correlated with their ability to influence membrane lipid peroxidation, which is a typical symptom of senescence. Our results were corroborated by gene expression profiling of those genes involved in cytokinin metabolism and perception, plant senescence, and the stress response, and suggest that prepared kinetin derivatives might be used as potent anti-senescence agents.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- kinetin chemická syntéza chemie farmakologie MeSH
- molekulární struktura MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- proteiny huseníčku metabolismus MeSH
- pšenice účinky léků metabolismus MeSH
- puriny chemická syntéza chemie MeSH
- stárnutí účinky léků MeSH
- tabák účinky léků metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
