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

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Abstract

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.

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Authors and Affiliations

  1. Unité Mixte de Recherche 1131, Institut National de la Recherche Agronomique, Université Louis Pasteur, 28 rue de Herrlisheim, 68021, Colmar cedex, France

    Didier Merdinoglu, Giséle Butterlin, Lucie Bevilacqua & Vincent Chiquet

  2. Unité Mixte de Recherche en Génomique Végétale, Institut National de la Recherche Agronomique, 2 rue Gaston Crémieux, CP 5708, 91057, Evry cedex, France

    Anne-Françoise Adam-Blondon

  3. Unité de Recherche sur les Espéces Fruitiéres et la Vigne, Institut National de la Recherche Agronomique, 71 avenue Edouard-Bourleaux, 33 883, Villenave d’Ornon cedex, France

    Stéphane Decroocq

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  1. Didier Merdinoglu
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  2. Giséle Butterlin
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  3. Lucie Bevilacqua
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  4. Vincent Chiquet
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  5. Anne-Françoise Adam-Blondon
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  6. Stéphane Decroocq
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Correspondence to Didier Merdinoglu.

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Merdinoglu, D., Butterlin, G., Bevilacqua, L. et al. Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR. Mol Breeding 15, 349–366 (2005). https://doi.org/10.1007/s11032-004-7651-0

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