The varieties Aubin vert E-1, Bachet E-1, Franc noir E-1, Knipperlé E-1, Sacy 783 and Romorantin 466 were obtained from the Espiguette Estate of the Institut Français de la Vigne et du Vin (IFV, formerly ENTAV). Aligoté 01, Auxerrois 01, Chardonnay 102, Gamay noir 06, Pinot noir 74 and Pinot noir 102 were obtained from the Foundation Plant Services vineyard at UC Davis. Gouais blanc (Plant ID 39794 and 39798) was obtained from the Foundation Plant Services greenhouse.
Leaves were crushed onto Whatman FTA cards. Discs punched from the cards were washed with three aliquots of 800 µl of the elution buffer provided by the manufacturer, followed by two washes with 800 µl of 10 mM Tris-HCl pH 8.0, 0.1 mM EDTA. Triton X100 was added to the washes to 1% v/v, followed by overnight incubation at room temperature. A 400 µl sample of each of these washes was taken and 800 µl of 96 per cent ethanol and 120 µl of 4 M sodium acetate were added. Samples were then incubated at −80°C for 20 min followed by centrifugation at 13 000 rpm in an Eppendorf microcentrifuge. The supernatant was discarded and the pellets were washed with 500 µl of 70 per cent ethanol and then dissolved in 100 µl water. Whichever DNA preparation gave best yields in PCR from the five washes of a given card was used for subsequent analysis.
Primers for a SNP, identified within the region covered by the primers for the microsatellite locus cpSSR4, and designated cp4527, were synthesized as follows:
- Forward AGCAACCCGAATAAGATAAA.
- Reverse AGATGATTGAATAGACCCGC.
The polymorphic locus corresponded to position 4527 in the GenBank accession DQ424856.1. PCR was carried out using Taq polymerase from Bioline according to the manufacturer's instructions with 1 µl of DNA template solution prepared as described above. A FAM label was incorporated onto cpSSR PCR products for genotyping as described by Boutin-Ganache et al. (2001)
. For the cpSSR loci the cycling parameters were 3 min at 94°C, 33 cycles of (30 s at 94°C, 45 s at Tm, 1 min at 72°C), 10 cycles of (30 s at 94°C, 45 s at 53°C, 1 min at 72°C), 10 min at 72°C. Tm was set at 1°C below the predicted annealing temperature for the primers used. For cp4527 the cycling parameters were 2 min at 94°C, 35 cycles of (1 min at 94°C, 1 min at 51°C, 2 min at 72°C), 7 min at 72°C. Products were analysed in 2.5 per cent agarose gels, stained with ethidium bromide and visualized using UV-light. PCR products were purified when necessary using an Illustra GFX PCR Purification kit (GE Healthcare, Amersham, UK) according to the manufacturer's instructions. Genotyping was carried out on an ABI3730 genotyping machine at the National Institute of Agricultural Botany, Huntingdon Road, Cambridge, UK and DNA sequencing was carried out by Geneservice, Cowley Road, Cambridge, UK. PCR and sequencing of the cp4527 locus were performed in duplicate. No evidence of polymorphism within individual cultivars at any of the chloroplast loci was found, either within our data or when comparing our data with those of Arroyo-Garcia et al. (2006)
. This is consistent with the fact that cultivars are invariably propagated asexually and only very low levels of somaclonal variation are seen within cultivars, even with nuclear microsatellites (Bowers et al. 1999
; Riaz et al. 2002