BMC Genomics 2006, 7:12 http://www.biomedcentral.com/1471-2164/7/12 ful for mapping in untranslated regions (Table 1 and 2). Flavonoid hydroxylase gene fragments containing the intron were amplified in a 10 µl volume containing 200 µM of dNTPs, 0.4 µM of each primer, 0.5 U of Taq polymerase, 2.5 mM MgCl2 and 20 ng of template DNA. The PCR profile was 35 cycles of 92°C for 50s, annealing temperature as reported in Table 2 for 50s, 65°C for 1 min. Gene fragments were separated on 4% glycerol and 0.5 × MDE polyacrylamide gel (BioWhittaker Molecular Applications) for SSCP analysis, run at room temperature for 16 hours at 6 W and stained with silver nitrate. The seg- regation of F3int, F35-1int, F35-2int and 335 SSR markers was followed in the 'Cabernet Sauvignon' × Vitis 'breeding line 20/3' full-sib family [G. Di Gaspero, unpublished data]. Linkage groups of markers segregating from the 'Cabernet Sauvignon' parent were identified using Carthagene with a LOD of 5.0 [31]. Marker order was cal- culated using the command Build 3. Local physical maps A 6X BAC library of 'Cabernet Sauvignon' was screened by PCR with the F3, F35-1 and F35-2 primer pairs (Table 2) using a 3D pooling strategy [23]. Then, positive individual clones were pre-cultured in 2X LB medium for 20 hours and 2 µL of cell suspension were inoculated into the same medium and grown for 16 hours. Cultured clones were directly used as PCR template. Positive BACs were con- firmed using the corresponding F3int, F35-1int and F35- 2int primer pairs (Table 2). PCR products were then run into a SSCP gel (as above) for identifying the haplotype of each BAC clone. A set of 30,151 BAC fingerprints of the 'Cabernet Sauvignon' BAC clones [S. Paillard, unpub- lished data] were assembled into contigs using FPC v8.0 [24]. The first assembly was made at a cut-off value of 10- 50 and with a tolerance of 0.4 bp. After several steps of dQing and merging, 2,035 BAC contigs were obtained [S. Paillard, unpublished data]. BAC clones that were positive at the PCR screening were localized over the contigs. Clone order was determined by running the consensus band (CB) algorithm on each individual positive contig. Contig size was estimated by converting CB units into physical length, under the assumption of a band mean size of 1,100 bp. Positive contigs were anchored to the genetic map by means of already mapped SSR markers that were present in the assembled BACs [[23], Adam- Blondon, unpublished data]. Further SSCP markers (38C24-RM, 17K14-FM, 9C17-FM, 40A22-FM and 5A23- FM, Table 1 and 2) were developed on the BAC-end sequences (BES) [A.F. Adam-Blondon and S. Paillard, unpublished data] of the positive clones and enabled new genetic markers to be identified that were closely linked to VvF3'H and VvF3'5'H loci (Table 1 and 2). BES-derived markers (Table 1 and 2) were also used to cross-reference the BAC clones within a contig, validate the clone order and identify the BAC haplotypes in regions where allelic BACs were incorporated into a given contig. Transcript profiling Total RNA was extracted from berry skin and flesh, leaves, flower bunches, rootlets and seeds following the proce- dure described in [31]. Total RNA was treated with 0.5 U/ µg RQ1 DNase (Promega) in presence of an RNAse inhib- itor (RNAguard, Amersham Biosciences). First strand cDNA was synthesised using 2 µg of RNA, 0.5 µM (dT)18 primer and 50 U of M-MLV Reverse Transcriptase (Promega). Quantitative real-time PCR was carried out on a DNA Engine Opticon2 (MJ Research) using SYBR Green Jump Start Taq ReadyMix (Sigma). The primer pairs F3-1, F35-1 and F35-2 (Table 2) were used for expression anal- ysis of VvF3'H-1, VvF3'5'H-1 and VvF3'5'H-2, respectively. Each cDNA sample was analysed at two different dilutions and with two replicates per dilution. Gene transcripts were quantified comparing the threshold cycle (CT) with that of the constitutive actin gene [32]. Semi-quantitative PCR was performed on different tissues upon cDNA nor- malisation based on the expression of the actin gene and visualised on agarose gel stained with ethidium bromide. Primers pairs for the genes of the flavonoid pathway were retrieved from the literature: primers for Chs1, Chs2, Chs3, DFR, LDOX, UFGT were from [33], primers for Fls1 were from [34]. Primers for F3H and BAN were newly designed on the original gene sequences [35,36] to tag 150–200 bp gene fragments. Metabolite profiling during berry development Total soluble solids were quantified with a refractometer (Analytical control) and expressed as °Brix. Titratable acidity was measured with an automated titrator (Crison) and expressed as tartaric acid equivalents. Skin phenols were extracted for 4 hours in 1/10 (w/v) skin/solvent sus- pension of 100% methanol and 1% HCl, then centrifuged at 5,000 rpm for 15 min and filtered with a 0.2 µm PTFE filter (Chemtek Analytica). Total phenols were deter- mined following the procedure of [37]. The calibration curve was obtained with (+)-gallic acid (Sigma). Total fla- van-3-ols (catechins) were quantified as reported in [38]. The calibration curve was constructed with (+)-catechin (Extrasynthase). Absorbance was measured by a UV/VIS LAMBDA 2 spectrometer (Perkin-Elmer). Anthocyanins for the HPLC analysis were extracted for 4 hours from 200 mg of berry skin with 2 mL of methanol, then centrifuged and filtered with a 0.2 µm PTFE filter (Chemtek analitica). Methanol was evaporated under nitrogen flux and anthocyanins were re-suspended with 100–400 µL of 27:73 methanol:perchloric acid 0.3% (v/v). Anthocyanins were separated in a high performance liquid chromato- graph (Perkin Elmer Series 4) using a C18 Purospher RP- 18 (5 µm, 250 mm × 4 mm) column (Merck) protected by a C18 guard column, according to the procedure reported Page 15 of 17 (page number not for citation purposes)