Partner und Internationale Organisationen
(Englisch)
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A, B, CH, CZ, D, DK, E, F, FIN, GR, H, I, IRL, L, N, NL, P, PL, S, UK
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Abstract
(Englisch)
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Treatments against pathogens or pests are often very specific, and therefore require the ability to unambiguously recognize and correctly identify taxa as a fundamental first step. Taxa are traditionally recognized based upon morphological characters. However, not all species lend themselves to this approach because of insufficient phenotypic variation. The use of genetic markers represents a viable addition or alternative to traditional methods of species recognition, as the development of molecular techniques during the last two decades has provided a variety of simple, yet robust and reliable diagnostic tools. The aim of this project was to establish a molecular identification key for agronomically important thrips species. The development of such a diagnostic key involves three major steps: 1) Potential target DNA sequences and appropriate diagnostic molecular techniques have to be evaluated. We used PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) to analyze a 480 bp long fragment of the mitochondrial cytochrome oxidase I (COI) coding gene. Unlike other molecular methods this approach is relatively inexpensive and highly reproducible, a prerequisite when applied in different laboratories and on different equipment. The initial step of a PCR amplification of the target DNA is an ideal tool when only minimal amounts of template DNA are available, allowing to analyze all developmental stages of single thrips. 2) The method has to be adapted to accommodate all species to be diagnosed, and the diagnostic patterns have to be established. The target sequence was first PCR amplified using a published 'universal' primer. Direct sequencing of all PCR products confirmed the amplification of the homologous target sequence in all species. However, quality and amount of obtained PCR product varied considerably between different species due to differences in the primer recognition sequences. Subsequent modification of primers to better match the thrips sequences resulted in satisfactory PCR results for all species. Twelve different restriction enzymes have been screened and two (AluI, Sau3AI) -producing homologous bands (verified by direct sequencing)- were chosen for subsequent analyses. These enzymes produce restriction patterns that allow unambiguous identification of all thrips species analyzed so far. 3) The quality (i.e. the robustness) of the molecular key has to be assessed on two levels; within and among species polymorphism. Within species genetic polymorphism was assessed by analyzing several individuals (min. 10 - max. 132) per species collected from different populations or countries. A total of 249 individuals representing 10 species were analyzed. Restriction enzymes produced patterns that allow unambiguous identification of all species. Direct sequencing confirmed the low within and high among-species polymorphism and confirmed COI being an ideal region for identification purposes in thrips. Finally, statistical support for the quality of the key was given by applying a hierarchic cluster analysis on the entire data set (nucleotide sequences) and previously published sequences from additional 22 thrips species. In the resulting neighbour joining dendrogram all conspecific individuals clustered together with maximal (100%) bootstrap support.
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