Design of grapevine (Vitis vinifera L.) cultivar-specific SCAR primers for PCR fingerprinting

Among 34 grapevine cultivars (Vitis vinifera L.). eight putative genotype-specific RAPD markers, from 'Albarifio','Caino blanco', 'Chardonnay','Folle blanche','Grenache blanc','Malvasia Sitges', 'Torrontes' and 'Treixadura' respectively, were selected to transform into SCAR markers. Of these, seven markers were cloned and then five which showed a positive specific hybridization signal were sequenced. For these five markers, 30 sequence-specific primers ranging from I4 to 29 bases were designed to amplify genomic DNA from 64 grapevine cultivars under more-stringent PCR conditions. Only, two primer pairs, OpA11(1175)p17R/ p17F and OpD10(800)p14R/p14F still produced a specific SCAR marker, the 'Folle blanche' ScA11(1175) and the 'Malvasia Sitges' ScD10(800) respectively. Moreover, the ScA11(1175) marker was amplified only in 'Folle blanche' among the 64 cultivars tested with a large annealing temperature range using either two different Taq DNA polymerases or two separate thermocyclers. In addition, we discuss the initial polymorphism originated by the RAPD technique and suggest a new design of SCAR primers to obtain reliable cultivar-specific SCAR markers from single PCR-based bands for identification purposes.