BMC Genomics 2006, 7:12 http://www.biomedcentral.com/1471-2164/7/12 FiGniceglnuuedrtieincg4mthaep VovfF'C3'a5b'Herannedt SVavuF3vi'gHnolonc'iLG 6 (a) and LG 17 (b) Genetic map of 'Cabernet Sauvignon' LG 6 (a) and LG 17 (b) including the VvF3'5'H and VvF3'H loci. VvF3'H and VvF3'5'H loci (in bold) were mapped with the use of the SSCP markers F3H and F35-1int, respectively. BES markers (underlined) were generated from the end-sequences of BAC clones positive for VvF3'H and VvF3'5'H and mapped using the SSCP technique. VMC, VVI, UDV, VVMD, VVS, and VrZag are microsatellite markers. Numbers on the left indi- cate the genetic distance between the markers expressed in centiMorgan (cM). additional BAC clones were identified by both primer pairs. The number of positive BAC clones per haploid genome equivalent was on average 2.4 for the gene VvF3'5'H-1 and 2.75 for the gene VvF3'5'H-2. Local phys- ical maps around the VvF3'H and VvF3'5'H loci were con- structed using all positive BAC clones identified. Those BAC clones were used to query 2,035 BAC contigs result- ing from a whole genome physical map. This map was assembled using FPC software [24] at a 10-50 cut-off from 30,151 fingerprints of BAC clones derived from the same library and corresponding to an approximate coverage of 10X [S. Paillard, unpublished data]. Two flavonoid 3'-hydroxylase genes, VvF3'H-1 and VvF3'H-2, were tightly linked in contig ctg253 that con- sisted of 28 BAC clones and approximately covered 700 kb (Figure 5). Six BAC clones contained both isogenes, whilst two BAC clones (6A08 and 5A23) contained only VvF3'H-1. BAC clone 5A23 was initially not included in the ctg253 following computational contig assembly because it did not meet the cut-off due to a low number of fingerprint bands. However, it was later placed in the con- tig upon PCR anchorage to other BAC clones using the BES-derived primers (BES5A23-FM). The same marker BES5A23-FM was used to anchor contig ctg253 to the 'Cabernet Sauvignon' genetic map. The marker based on the SSCP of the VvF3'H gene fragments and the BES- derived marker co-segregated at the level of resolution of the genetic map. Flavonoid 3',5'-hydroxylase genes were present in four contigs (ctg313, ctg871, ctg1320, and ctg2373) that cov- ered approximately 450, 600, 280 and 140 kb, respec- tively. Each one of the contigs ctg313, ctg1320 and ctg2373 contained both VvF3'5'H-1 and VvF3'5'H-2 gene fragments. Contig ctg871 contained only the VvF3'5'H-2 gene fragment. Contigs ctg313, ctg871, ctg1320 and ctg2373 included 26, 29, 10 and 3 BAC clones, respec- tively, of which 14, 10, 10 and 3, respectively, contained either one or both isoforms of flavonoid 3',5'-hydroxy- lases as shown in Figure 6. VvF3'5'H-1 and VvF3'5'H-2 fragments were amplified from each individual positive BAC clone of contigs ctg313, ctg871, ctg1320 and ctg2373 using the primers pairs F35-1int and F35-2int, respec- tively, and analyzed by the SSCP technique. Based on BAC order and the number of SSCP haplotypes, at a first approximation we hypothesize the occurrence of four loci containing VvF3'5'H-1-type sequences and seven loci containing VvF3'5'H-2-type sequences over the four contigs (Figure 6). Two paralogous copies of gene VvF3'5'H-1 were identified in ctg313. The first paralog had two allelic variants, that could be detected by SSCP analy- sis, one present in three BAC clones 6H20, 9C17, 31P20 (corresponding to the SSCP band genetically mapped from 'Cabernet Sauvignon') and the other in the remain- ing five clones (data not shown). Two BAC clones (28B15 and 99I17) spanned the region that included both para- logs. A third potential paralogous copy of the gene VvF3'5'H-1 was identified in contig ctg1320 whose BAC clones contained one of the two SSCP haplotypes present in the BACs from contig ctg313. An additional different haplotype of the gene VvF3'5'H-1 was identified in contig ctg2373 that included just three BAC clones. We could detect a potential of seven paralogous copies of the gene VvF3'5'H-2 in contigs ctg313, ctg871, ctg1320 and ctg2373. The primers pair F35-2int identified three types of gene fragments distinguishable by size, hereafter referred to as short, medium and long (Figure 6). All three gene fragments were found in ctg1320 in the close vicinity of the gene copy of VvF3'5'H-1. Three BAC clones con- tained all three forms at the same time, confirming the paralogous nature of these VvF3'5'H-2 gene copies. The medium sized gene fragment VvF3'5'H-2 was also ampli- fied in ctg313 and ctg2373 where it showed a SSCP hap- lotype different from that found in ctg1320 (data not shown). A different locus carrying the longer fragment of Page 7 of 17 (page number not for citation purposes)