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| Characterizing intrapopulational genetic variation of Norway spruce and quantifying root-associated micro-organisms by newly developed DNA markers |
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R. Schubert, R. Riegel and G. Müller-Starck
Technische Universität München, Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt, Fachgebiet für Forstgenetik, Am Hochanger 13, 85354-Freising;
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Polymerase chain reaction (PCR) represents the most innovative technology for the screening of genetic variation. Moreover, this technology is the most sophisticated tool to detect plant pathogens and symbiotic fungi.
After sequence analysis of elicitor-induced cDNA clones, oligonucleotide primer pairs were designed for amplifying 6 polymorphic Mendelian-inherited loci in Norway spruce [Picea abies (L.) Karst.], an ecologycally and economically important European conifer. Analyzing the Kranzberg test population (location near Freising, Bavaria), these new DNA markers revealed a trend to more even frequency distributions and larger intrapopulational variation in contrast to 18 codominant and polymorphic isoenzyme gene markers (ref. 3). Since the mentioned DNA markes are targeted to stress defense genes, genotypic selective effects distinguishing between tolerant and sensitive subsets of Picea abies were confirmed for some of the proved loci by analyzing sulfur dioxide tolerant clones in the Ore mountains (Saxony) as well as by investigating a sodium chloride-polluted spruce stand in Bavaria (ref. 2). These pilot studies indicate that the new multiallelic DNA markers are beneficial to population genetics and ecological genetics of spruce.
Based on the sequence data of the internal transcribed spacer regions ITS1/ITS2 of the ribosomal gene cluster, specific primers and dual-labelled fluorogenic probes were designed for the PCR-based detection of both pathogen and mycorrhizal DNA. This real time PCR-technology monitors the amplification of the diagnostic marker fragment directly during thermocycling and therefore no post-PCR sample handling is required for product quantification. In cooperation with W. Oßwald (Freising) and G. Bahnweg (Neuherberg), we quantified Phytophthora citricola DNA (ref. 1), causing root-rot, in seedling roots of European beech (F. sylvatica) and pendunculate oak (Q. robur). Furthermore, DNA of the ectomycorrhizal fungus Piloderma croceum, normally colonizing spruce roots, was extracted from agar-cultivated cells and quantified by real-time PCR (cooperation with S. Raidl, Munich). These is the first report on the successfull application of novel real-time PCR techniques in the fields of plant pathology and mycorrhiza research.
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References:
(1) Böhm J., Hahn A., Schubert R., Bahnweg G., Adler N., Nechwatal J., Oehlmann R. and Oßwald W. (1999): Real-time quantitative PCR: DNA determination in isolated spores of the mycorrhizal fungus Glomus mosseae and monitoring of Phytophthora infestans and Phytophthora citricola in their respective host plants. J. Phytopathology 147, 409-416.
(2) Schubert R. (2000): Genomanalyse und DNA-Markerentwicklung als molekulare Werkzeuge zur Züchtung stresstoleranter Kulturpflanzen und Waldbäume inklusive Pathogendiagnostik. Habilitationsschrift zur Erlangung des akademischen Grades Dr. rer. nat. habil. Für das Fachgebiet Molekulargenetik und Pflanzenzüchtung, vorgelegt dem Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München.
(3) Schubert R., Müller-Starck G. and Riegel R. (2001): Development of EST-PCR markers and monitoring their intrapopulational genetic variation in Picea abies (L.) Karst. Theor. Appl. Genet. 102, in press.
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