Microsatellite Markers for Grapevine: Tools for Cultivar Identification & Pedigree Reconstruction

Abstract

Microsatellites have become a favorite type of DNA marker for identification of grapevine cultivars, and their properties enable a wide range of applications from cultivar identification based on various parts of the grapevine plant to pedigree reconstruction and genome mapping (Sefc et al. 2001). Genetic markers have the advantage that the DNA of a certain plant is identical in all cells of any tissue at any stage of development and is not influenced by environmental or sanitary conditions of the plants. DNA can be obtained from every kind of plant tissue available, e.g. wood, leaves or berries, and analyses can therefore be carried out at any time of the year. Thanks to the PCR (Polymerase Chain Reaction) technology, minute amounts of DNA can be analyzed, which extends the usefulness of the method from plant tissue to grapevine products such as must and wine. The typical microsatellite sequence consists of five to about one hundred tandem repeats of short, simple sequence motives composed of 1 to 6 nucleotides (e.g. (GA)n, (GATA)n (Fig. 1). In eukaryotes, an estimated 104 to 105 microsatellite loci are scattered randomly throughout the genome. This abundance of microsatellite sequences in eukaryote genomes constitutes an almost unlimited source of polymorphic sites that may be exploited as genetic markers. Thomas et al. (1993) first investigated the use of microsatellite DNA for identifying grapevine cultivars. They showed that microsatellite sequences were abundant in grapevine and very informative for identifying V. vinifera cultivars. Moreover primer sequences were conserved across other Vitis species and Muscadinia (Thomas and Scott 1993). It was also demonstrated through pedigree analysis that the microsatellite alleles were inherited in a co-dominant Mendelian manner (Thomas and Scott 1993) confirming their suitability for genetic mapping and investigation of genetic relatedness (Thomas et al. 1994). As other groups throughout the world became interested in grapevine microsatellite genotyping, a large number of markers has been developed by individual groups (e.g. Bowers et al. 1996, 1999b, Sefc et al. 1999, Arroyo-Garcia et al. 2004, Adam-Blondon et al. 2004, Di Gaspero et al. 2005, Merdinoglu et al. 2005, Goto-Yamamoto et al. 2006) and in the framework of the Vitis Microsatellite Consortium (VMC, 1997-2004). The approach used for microsatellite marker development in these studies was the construction of a genomic library from a grapevine cultivar or rootstock, screening of the library with microsatellite probes, sequencing of microsatellite containing clones, design of PCR primers from the sequences flanking the microsatellite, optimization of PCR conditions and characterization of the microsatellite polymorphism. Furthermore, public databases as those compiled from cDNA and EST (Expressed Sequence Tag) type data were recognized as a source of microsatellite loci, and a large grape EST dataset (Ablett et al. 1998) was employed for the development of microsatellite markers (Scott et al. 2000b). Over 100 microsatellites were readily identified, and the further investigation of some of these loci showed enough polymorphism to allow the discrimination of several grapevine and rootstock cultivars, and transferability of the primers to species of the related genera Cissus and Cayratia. To date, many microsatellite markers have been developed and mapped in Vitis, and locus information including primer sequences and mapping position can be retrieved from the UniSTS database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=unists).