Molecular Breeding

, Volume 15, Issue 4, pp 349–366 | Cite as

Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR

  • Didier Merdinoglu
  • Giséle Butterlin
  • Lucie Bevilacqua
  • Vincent Chiquet
  • Anne-Françoise Adam-Blondon
  • Stéphane Decroocq


Despite their numerous advantages, the use of microsatellites as genetic markers could be limited because of the low number of loci that can be simultaneously analysed per experiment. To increase the information per simple sequence repeat (SSR) assay in the grapevine, we developed a large set of new markers suitable for multiplexing and multi-loading. We produced microsatellite motif-enriched genomic libraries containing preferentially large size inserts which allowed us to design primers generating a wide range of allele sizes in a very standard and unique PCR condition. Three hundred and fifty clones were sequenced and 190 of them (54%) contained microsatellite motifs with suitable flanking regions for primer design. We developed 169 new SSR markers giving suitable signal with fluorescent-based DNA detection. The total number of alleles detected varied from 1 to 8 per locus with an average of 3.5 and the mean expected heterozygosity was 0.544 (range: 0 0.86). Sixty-eight loci (40%) were perfect types, 73 (43%) were imperfect and 28 (17%) were compound or imperfect-compound. The number of alleles generated by perfect and imperfect type loci was positively correlated to the length of the microsatellite motif. Forty-six multiplex sets based on 125 selected loci were developed. Considering their allele size range, up to four PCR multiplex were pooled together for multi-loading. The 169 SSR loci developed in this study represent a new and informative set of markers easy to combine for multiplexing and multi-loading according to the needs of any user and suitable for large scale genetic analyses in grapevine.


Grapevine Microsatellite Multiplex PCR Polymorphism Simple sequence repeats 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Adam-Blondon, A-F., Roux, C., Claux, D., Butterlin, G., Merdinoglu, D., This, P. 2004Mapping 245 SSR markers on the Vitis vinifera genome: a tool for grape geneticsTheor. Appl. Genet.10910171027CrossRefPubMedGoogle Scholar
  2. Aceto, S., Sica, M., Gamba, G., Montieri, S., Farina, A., Gaudio, L. 2003Isolation and characterization of microsatellite loci from Asparagus acutifolius (Liliaceae)Mol. Ecol. Notes3242243CrossRefGoogle Scholar
  3. Algharim, H.J., Almirall, J.R. 2003Development of micro satellite markers in Cannabis sativa For DNA typing and genetic relatedness analysesAnal. Bioanal. Chem.37612251233CrossRefPubMedGoogle Scholar
  4. Bowers, J.E., Dangl, G.S., Meredith, C.P. 1999Development and characterization of additional microsatellite DNA markers for grapeAm. J. Enol. Vit.50243246Google Scholar
  5. Bowers, J.E., Dangl, G.S., Vignani, R., Meredith, C.P. 1996Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.)Genome39628633Google Scholar
  6. Bowers, J.E., Meredith, C.P. 1997The parentage of a classical wine grapeCabernet SauvignonNat. Genet.168487CrossRefPubMedGoogle Scholar
  7. Butcher, P.A., Decroocq, S., Gray, Y., Moran, G.F. 2000Developmentinheritance and cross-species amplification of microsatellite markers from Acacia mangiumTheor. Appl. Genet.10112821290CrossRefGoogle Scholar
  8. Cipriani, G., Freza, G., Peterlunger, E., Tectolin, R. 1994Grapevine Fingerprint using microsatellite repeats.Vitis33211215Google Scholar
  9. Dalbo, M.A., Ye, G.N., Weeden, N.F., Steinkellner, H., Sefc, K.M., Reish, B.I. 2000Gene controlling sex in grapevines placed on a molecular marker-based mapGenome43333340CrossRefPubMedGoogle Scholar
  10. Decroocq, V., Favé, M.G., Hagen, L., Bordenave, L., Decroocq, S. 2003Development and transferability of apricot and grape EST microsatellite markers across taxaTheor. Appl. Genet.106912922PubMedGoogle Scholar
  11. Di Gasparo, G., Peterlunger, E., Testolin, R., Edwards, K.J., Cipriani, G. 2000Conservation of microsatellite loci within the genus VitisTheor. Appl. Genet.101301308CrossRefGoogle Scholar
  12. Edwards, K.J., Barker, J.H.A., Daly, A., Jones, C., Karp, A. 1996Microsatellite libraries enriched for several microsatellite sequences in plantsBiotechniques20758760PubMedGoogle Scholar
  13. Fischer, B., Salakhutdinov, I., Akkurt, M., Eibach, R., Edwards, K.J., Töpfer, R., Zyprian, E.M. 2003Quantitative trait locus analysis of fungal disease resistance factor on a molecular map of grapevineTheor. Appl. Genet.108501515CrossRefPubMedGoogle Scholar
  14. Grando, M.S., Bellin, D., Edwards, K.J., Pozzi, C., Stefanini, M., Velasco, R. 2003Molecular linkage maps of Vitis vinifera L. and Vitis riparia MchxTheor. Appl. Genet.10612131224PubMedGoogle Scholar
  15. Jakse, J., Kindlhofer, K., Javornik, B. 2001Assessment of genetic variation and differentiation of hop genotypes by microsatellite and AFLP markersGenome44773782CrossRefPubMedGoogle Scholar
  16. Juillet, N., Freymond, H., Degen, L., Goudet, J. 2003Isolation and characterization of highly polymorphic microsatellite loci in the bladder campion, Silene vulgaris (Caryophyllaceae)Mol. Ecol. Notes3358359CrossRefGoogle Scholar
  17. Lagercrantz, U., Ellegren, H., Anderson, L. 1993The abundance of various polymorphic microsatellite motifs differ between plants and animalsNucl. Acid Res.2111111115Google Scholar
  18. F. Lefort, P. Roubelakis-Angelakis 2000 Nuclear SSR primers of the laboratory of plant physiology and biotechnology.The Greek Vitis Databasehttp://www.biology.uch. gr/gvd/contents/general-info/01.htm.Google Scholar
  19. Lemes, M.R., Brondani, R.P.V., Grattapaglia, D. 2002Multiplexed systems of microsatellite markers for genetic analysis of mahogany, Swietenia macrophylla king (Meliacelae), a threatened neotropical timber speciesJ. Hered.93287291CrossRefPubMedGoogle Scholar
  20. Maguire, T.L., Edwards, K.J., Saenger, P., Henry, R. 2000Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae)Theor. Appl. Genet.101279285CrossRefGoogle Scholar
  21. Marinoni, D., Akkak, A., Bounous, G., Edwards, K.J., Botta, R. 2003Development and characterization of microsatellite markers in Castanea sativa (Mill.)Mol. Breeding11127136CrossRefGoogle Scholar
  22. Masi, P., Spagnoletti Zeuli, P.L., Donini, P. 2003Development and analysis of multiplex microsatellite markers sets in common bean (Phaseolus vulgaris L.)Mol. Breeding11303313CrossRefGoogle Scholar
  23. Mitchell, S.E., Kresovich, S., Jester, C.A., Hernandez, C.J., Szewc-McFadden, A.K. 1997Application of multiplex-PCR and fluorescence-basedsemi-automated allele sizing technology for genotyping plant genetic resourcesCrop. Sci.37617624Google Scholar
  24. Morgante, M., Olivieri, A.M. 1993PCR-amplified microsatellites as markers in plant geneticsPlant J.3175182CrossRefPubMedGoogle Scholar
  25. Nei, M. 1973Analysis of gene diversity in subdivided populationsProc. Natl. Acad. Sci. USA7033213323PubMedGoogle Scholar
  26. Powell, W., Gordon Marchay, C., Provan, J. 1996aPolymorphism revealed by simple sequence repeatsTrends Plant Sci.1215222Google Scholar
  27. Powell, W., Morgante, M., Andre, C., Hanafey, M., Vogel, J., Tingey, S., Rafalski, A. 1996bThe comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysisMol. Breeding2225238CrossRefGoogle Scholar
  28. Riaz, S., Dangl, G.S., Edwards, K.J., Meredith, C.P. 2004A microsatellite marker based framework linkage map of Vitis vinifera LTheor. Appl. Genet.108864872CrossRefPubMedGoogle Scholar
  29. Sanchez-Escribano, E.M., Martin, J.P., Carreno, J., Cenis, J.L. 1999Use of sequence-tagged microsatellite site markers for characterizing table grape cultivarsGenome428793CrossRefGoogle Scholar
  30. Sargent, D.J., Hadonou, A.M., Simpson, D.W. 2003and characterization of polymorphic microsatellite markers from Fragaria viridisa wild diploid strawberryMol. Ecol. Notes3550552CrossRefGoogle Scholar
  31. Scott, K.D., Eggler, P., Seaton, G., Rossetto, M., Ablett, E.M., Lee, L.S., Henry, R.J. 2000Analysis of SSRs derived from grape ESTsTheor. Appl. Genet.100723726CrossRefGoogle Scholar
  32. Sefc, K.M., Guggenberger, S., Regner, F., Lexer, C., Glössl, J., Steinkellner, H. 1998aGenetic analysis of grape berries and raisins using microsatellite markersVitis37123125Google Scholar
  33. Sefc, K.M., Lopes, M.S., Lefort, F., Botta, R., Roubelakis-Angelakis, K.A., Ibáñez, J., Pejić, I., Wagner, H.W., Glössl, J., Steinkellner, H. 2000Microsatellite variability in grapevine cultivars from different European regions and evaluation of assignment testing to assess the geographic origin of cultivarsTheor. Appl. Genet.100498505CrossRefGoogle Scholar
  34. Sefc, K.M., Regner, F., Glössl, J., Steinkellner, H. 1998bGenotyping of grapevine and rootstock cultivars using microsatellite markersVitis371520Google Scholar
  35. Sefc, K.M., Regner, F., Tureschek, E., Glössl, J., Steinkellner, H. 1999Identification of microsatellite sequences in Vitis ripariatheir application for genotyping of different Vitis speciesGenome42367373CrossRefPubMedGoogle Scholar
  36. Sefc, K.M., Steinkellner, H., Glössl, J., Kampfer, S., Regner, F. 1998cReconstruction of a grapevine pedigree by microsatellite analysisTheor. Appl. Genet.100498505CrossRefGoogle Scholar
  37. Sefc, K.M., Steinkellner, H., Wagner, H.W., Glössl, J., Regner, F. 1997Application of microsatellite markers to parentage studies in grapevinesVitis36179183Google Scholar
  38. Stamati, K., Blackies, S., Brown, J.W.S., Russell, J. 2003A set of polymorphic SSR loci for subarctic willow (Salix lanataS. lapponumS. herbacea)Mol. Ecol. Notes3280282CrossRefGoogle Scholar
  39. Thomas, M.R., Cain, P., Scott, N.S. 1994DNA typing of grapevines: A universal methodology and database for describing cultivars and evaluating genetic relatednessPlant. Mol. Biol.25939949CrossRefPubMedGoogle Scholar
  40. Thomas, M.R., Scott, N.S. 1993Microsatellite repeats in grapevine reveal DNA polymorphisms when analyzed as sequence-tagged sites (STSs)Theor. Appl. Genet.86985990Google Scholar
  41. Tommasini, L., Batley, J., Arnold, G.M., Cooke, R.J., Donini, P., Lee, D., Law, J.R., Lowe, C., Moule, C., Trick, M., Edwards, K.J. 2003The development of multiplex simple sequence repeat (SSR) markers to complement distinctness, uniformity and stability testing of rape (Brassica napus L.) varietiesTheor. Appl. Genet.10610911101PubMedGoogle Scholar
  42. Toth, G., Gaspari, Z., Jurka, J. 2000Microsatellites in different eukaryotic genomes: survey and analysisGen. Res.10967981CrossRefGoogle Scholar
  43. Wang, Z., Weber, J.L., Zhong, G., Tanksley, S.D. 1994Survey ofplant short tandem DNA repeatsTheor. Appl. Genet.8816Google Scholar
  44. Zhang, L.S., Becquet, V., Li, S.H., Zhang, D. 2003Optimization of multiplex PCR and multiplex gel electrophoresis in sunflower SSR analysis using infrared fluorescence and tailed primersActa Bot. Sin.4513121318Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Didier Merdinoglu
    • 1
  • Giséle Butterlin
    • 1
  • Lucie Bevilacqua
    • 1
  • Vincent Chiquet
    • 1
  • Anne-Françoise Adam-Blondon
    • 2
  • Stéphane Decroocq
    • 3
  1. 1.Unité Mixte de Recherche 1131, Institut National de la Recherche AgronomiqueUniversité Louis PasteurColmar cedexFrance
  2. 2.Unité Mixte de Recherche en Génomique VégétaleInstitut National de la Recherche AgronomiqueEvry cedexFrance
  3. 3.Unité de Recherche sur les Espéces Fruitiéres et la VigneInstitut National de la Recherche AgronomiqueVillenave d’Ornon cedexFrance

Personalised recommendations