Defontaine, A.

Hallet, J. N.

Jean-Jaques, I.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 32, 189-190 (1993) Research Note Characterization of Vitis vinifera cultivars by Random Amplified Polymorphic DNA markers I. JEAN-JAQUES1), A. DEFONTAINE')*) and J. N. liALLET2) During the past two years an alternative strategy based on PCR technology has been described (WILLIAMS et al. 1990)and carried out successfully to differentiate cultivars of several plant species (CARLSON et al. 1991; KLEIN-LANKHORS et al. 1991; PARRAN et al. 1991; WILDE et al. 1992). This procedure, named RAPD for Random Amplified Polymorphic DNA, used single decamer primers of arbitrary sequence to amplify DNA fragments of polymorphic length. We have tested this method for the intraspecific characterization of Vitis vinifera and we report here the results obtained on 8 cultivars. D N A e x t r a c t i o n s were performed from clean, young and fully expanded leaves according to DoYLE and DoYLE (1987) with 1 %of polyvinylpyrrolidone (PVP) in the extraction buffer. Extracted DNA was purified using glass-max DNA isolation spin cartridge system purchased by Gibco Brl. Yield of DNA were measured using the HOECHST dye assay method with a TKO 100 minifluorimeter (Hoefer Scientific Instrument)� Random primers were provided by Operon Technologies Inc. and were not further purified. For R A P  D a n  a l y s i s  the protocol reported by WILLIAMS et al. (1990) was followed with minor modifications. Amplification reactions were carried out in 25 f.ll buffer containing 10 mM Tris pH 8.3, 50 mM KC!, 4 mM MgCl2, 200 f!M each of dATP, dGTP, dCTP, dTTP, 0.2 f!M primer and 0.5 unit of Taq Polymerase (CETUS). Amplification was performed in a Perkin Elmer CETUS DNA thermocycler programmed for 45 cycles of 1 min at 94 OC for denaturing, 1 min at 36 T for annealing, 2 min at 72 T for extension. More than 50 decamers of arbitrary sequences have been screened as primers on total DNA of grape cultivars in way to generate polymorphic PCR products. Many of them allowed the amplification of 2-15 DNA fragments sized from 0.1 to 5 kb. Under described standard conditions the electrophoretic patterns were highly reproducible. Among a set of successful primers, some determined complex profiles with numerous and not discrete bands which were difficult to interprete. Others generated simple and readable patterns but only few of them were discriminant between cultivars. The results obtained with two primers OPA 01 (CAGGCCCTTC) and OPA 18 (AGGTGACCGT) are presented in Figure A and B. OPA 01 allowed to obtain five "phenotypes" among 8 tested cultivars. Two pairs, namely Cabernet Sauvignon and Cabernet franc (Figure A, lanes 3 and 4) and Gamay and Chenin (Figure A, lanes 7 and 8), could not be differentiated. The OPA 18 primer gave more numerous amplified products (Figure B). It yielded to the differentiation of the 8 cultivars. However, two of them, Syrah and Chenin (Figure B, lanes 2 and 8) differed only by a weak but constant band (arrow) of their electrophoretic patterns. In fact only cross comparisons between patterns obtained with the two primers OPA 01 and OPA 18 led to a reliable identification of each cultivar. OPA 01 and OPA 18 have also been tested for clones discrimination within the same variety (data not shown) but without success. Experiments with a wide range of decamer primers are in progress. They indicate that each grape variety and certainly some clones within the same cultivar could be characterized by RAPD markers. As already demonstrated for other culti­vated plants these markers could also be very useful for genetic mapping of Vitis vinifera. 1) Institut Technique de la Vigne et du Vin Nantes, France 2) Lab. de Biologie V egetale et Biotechnol., Uni versite deN antes 2, rue de la Houssiniere, F -44072 Nantes *) to whom reprints should be requested 190 I. JEAN-JAQUES, A. DEFONTAINE and J. N. IiALLET Figure: Amplification patterns of polymorphic DNA from 8 cultivars of Vitis vinifera using single arbitrary 10-mer oligonucleotide primers, OPA 01 (A) and OPA 18 (B). Lanes 1-8 correspond respectively to Pinot noir, Syrah, Cabernet Sauvignon, Cabernet franc, Chardonnay, Grenache, Gamay noir, Chenin. SM: Hindiii digested lambda DNA+Haeiii digested Phi x 174 DNA. Agarose gel concentration 1.2 %. On figure A note that electrophoretic profiles of the two Cabernet cultivars (lanes 3 and 4) are identical even if the smallest band is very weak in lane 4 (arrow). Acknowledgements This study has been supported by grants of ONIVIN (Office National lnterprofessionneldu V in) under the coordination of ITV (Institut Technique de la Vigne et du Vin, Nantes). We are indebted to Mr. BoURSIQumfrom ENSAM (Ecole N ationale des Sciences Agronomiques de Montpellier) for his ampelographic advices and to the ENTAV (Etablissement National Technique pour !'Amelioration de la Viticulture) for technical help and free use of its Vitis collection. We also thank the ATAV (Association Technique pour !'Amelioration de la Viticulture du Val de Loire) for the gift of clone 327. CARLSON,J. E.;TuLSIERAM, L. K.; GLAUBrrz,J. C.; LuK, V. W. K.; KAuFFELDT, C.;RuTLEDGE, R.; 1991: Segregation of random amplified DNA markers in F1 progeny of conifers. Theoret. Appl. Genet. 83, 194-200. DoYLE, J. J.; DoYLE, J. L.; 1987: A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19 (1), 11-15. KLEIN-LANKHORS, R. M.; VERMUNT, A.; WEIDE, R.; TiHARSKAT, T.; ZABEL, P.; 1991: Isolation of molecular markers for tomato (L. esculentum) using random amplified polymorphic DNA (RAPD). Theoret. A pp!. Genet. 83, 108-114. PARRAN, 1.; KEssELI, R.; MrcHELMORE, R.; 1991: Identification of restriction fragment length polymorphism and random amplified polymorphic DNA markers linked to downy mildew resistance genes in lettuce using near isogenic lines. Genome 34, 1021-1027. WILLIAMS, J. G. R.; RUBELIK, A. R.; LJVAK, K. J.; RAFALSKl, J. A.; TrNGEY, S. V.; 1990: DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Res. 18, 6531-6535. 

Characterization of Vitis vinifera cultivars by random amplified polymorphic DNA markers

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Characterization of Vitis vinifera cultivars by random amplified polymorphic DNA markers

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