DNA FINGERPRINTING TECHNOLOGY FOR PLANT BREEDER'S RIGHTS

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  • 1 Biotechnology Laboratory, Univ. of British Columbia, Vancouver, B.C., Canada
  • 2 Biotechnology Laboratory, Dept. of Plant Science, and Dept. of Forest Science, Univ. of British Columbia, Vancouver, B.C., Canada

DNA fingerprinting is a potentially powerful molecular genetic technique that can be used to distinguish subtle differences in genome structure among closely related genotypes, such as many horticultural varieties. A DNA fingerprinting project is currently in progress at the Univ. of British Columbia (UBC) Biotechnology Laboratory to produce a set of DNA markers and an easy, reliable, and legally recognized analysis protocol that will enable the UBC Botanical Garden Plant Introduction Scheme (PISBG) to unambiguously identify any of their released varieties, even in dormant or juvenile form, wherever it is being propagated or sold. High-quality genomic DNA was isolated from the leaf samples of six PISBG species (Anagallis monellii, Artemesia stelleriana, Clematis, Genista pilosa, Microbiota decussata, and Penstemon fruticosa) using a modified CTAB DNA isolation protocol, and further purified by cesium chloride/ethidium bromide gradient. Samples of these genomic DNA preparations (10 ng) were then amplified by a 45-cycle polymerase chain reaction (PCR) protocol using 1.5-μm 10-nucleotide primers of arbitrary nucleotide sequence that amplify a variety of sites distributed across the genome. Following the amplification, PCR products [random amplified polymorphic DNA (RAPD) markers] were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. More than 70% of the 51 primers tested so far generated distinctive banding patterns (2–11 bands) with DNA samples from each species. Subtle changes in the genome or differences between genotypes can be detected by screening a series of such primers against DNA samples from the genotypes in question. Once a RAPD primer has been identified that consistently generates a different banding pattern between genotypes, it can be used as an identification tool for discriminating between those genotypes at any time in the future.

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