Single nucleotide polymorphisms (SNPs) are the most abundant of all markers, both in animal and plant genomes. In crops and tree species considerable investment has been recently made on this genomic technology. While large-scale characterisation of SNPs by high-throughput techniques is possible, such high-throughput platforms are not available to all plant breeding laboratories. This report compares alternative multi-purpose and affordable methods for SNP assay in grapevine (Vitis spp.). In particular, the efficiency, sensitivity and reliability of single-strand conformation polymorphism (SSCP) on both non-denaturant gels and fluorescence-based capillary electrophoresis are compared with minisequencing (single nucleotide extension reaction). The results indicate that when multiplexing in combination with minisequencing is a mid-throughput, reliable and flexible technique for the detection of SNPs and can therefore be used effectively to improve marker assisted breeding in grapevine.

Background Most of the grapevine ( Vitis vinifera L.) cultivars grown today are those selected centuries ago, even though grapevine is one of the most important fruit crops in the world. Grapevine has therefore not benefited from the advances in modern plant breeding nor more recently from those in molecular genetics and genomics: genes controlling important agronomic traits are practically unknown. A physical map is essential to positionally clone such genes and instrumental in a genome sequencing project. Results We report on the first whole genome physical map of grapevine built using high information content fingerprinting of 49,104 BAC clones from the cultivar Pinot Noir. Pinot Noir, as most grape varieties, is highly heterozygous at the sequence level. This resulted in the two allelic haplotypes sometimes assembling into separate contigs that had to be accommodated in the map framework or in local expansions of contig maps. We performed computer simulations to assess the effects of increasing levels of sequence heterozygosity on BAC fingerprint assembly and showed that the experimental assembly results are in full agreement with the theoretical expectations, given the heterozygosity levels reported for grape. The map is anchored to a dense linkage map consisting of 994 markers. 436 contigs are anchored to the genetic map, covering 342 of the 475 Mb that make up the grape haploid genome. Conclusions We have developed a resource that makes it possible to access the grapevine genome, opening the way to a new era both in grape genetics and breeding and in wine making. The effects of heterozygosity on the assembly have been analyzed and characterized by using several complementary approaches which could be easily transferred to the study of other genomes which present the same features.

Grapevine molecular maps based on microsatellites, AFLP and RAPD markers are now available. SSRs are essential to allow cross-talks between maps, thus upgrading any growing grapevine maps. In this work, single nucleotide polymorphisms (SNPs) were developed from coding sequences and from unique BAC-end sequences, and nested in a SSR framework map of grapevine. Genes participating to flavonoids metabolism and defence, and signal transduction pathways related genes were also considered. Primer pairs for 351 loci were developed from ESTs present on public databases and screened for polymorphism in the "Merzling" (a complex genotype Freiburg 993-60 derived from multiple crosses also involving wild Vitis species) x Vitis vinifera (cv. Teroldego) cross population. In total 138 SNPs, 108 SSR markers and a phenotypic trait (berry colour) were mapped in 19 major linkage groups of the consensus map. In specific cases, ESTs with putatively related functions mapped near QTLs previously identified for resistance and berry ripening. Genes related to anthocyanin metabolism mapped in different linkage groups. A myb gene, which has been correlated with anthocyanin biosynthesis, cosegregated with berry colour on linkage group 2. The possibility of associating candidate genes to known position of QTL is discussed for this plant.

EST (expressed sequence tags) sequencing, SNP (single nucleotide polymorphisms) development and haplotype assessment are powerful tools for the support of marker-assisted selection. The grapevine genome is currently being scavenged in our laboratory using an EST-SNP approach. Nine parental genotypes, used to create five inter- or intra-specific hybrids, have been tested to evaluate the degree of polymorphism between Vitis vinifera, Vitis riparia and a further intraspecific hybrid, measuring their nucleotide diversity. The SNPs were analysed on cDNA sequences of 4 functional classes of genes based on homology with genes present in a public database: sugar metabolism, cell signalling, anthocyanin metabolism and defence related. Primer pairs were deduced and used to amplify corresponding genomic sequences. Almost 12,000 bp of DNA have been scanned revealing differences among genotypes of up to 247 SNPs, with the highest rate of one SNP occurring every 78 bp when clones of different Vitis species are compared. Re-sequencing allowed the definition of haplotypes in the nine genotypes studied and these were confirmed by analysing segregating populations. The efficiency of SSCP, in comparison with re-sequencing, was considered for 25 gene fragments of the same 9 genotypes.