BMC Genomics 2006, 7:12 http://www.biomedcentral.com/1471-2164/7/12 TFhigeuCreYP375 clan of plant cyt P450s The CYP75 clan of plant cyt P450s. The branch containing the CYP75 family is focused on a Neighbor-Joining tree con- structed using 670 plant cytochrome P450 monooxygenases. Flavonoid 3'-hydroxylases VvF3'H-1a to -1d and flavonoid 3',5'- hydroxylases VvF3'5'H-1a to -2b that were identified in grapevine are in bold. They split into two lower hierarchical branches that identified the CYP75B (flavonoid 3'-hydroxylases) and CYP75A (flavonoid 3',5'-hydroxylases) sub-families, respectively. Vitis 'breeding line 20/3'. The two genes segregated inde- pendently and were mapped to linkage groups LG17 and LG6, respectively (Figure 4). The linkage groups were annotated according to [22] and included also reference microsatellite markers and SSCP markers generated from BAC end-sequences (BES) of a 'Cabernet Sauvignon' BAC library. The locus containing the gene that codes for flavo- noid 3'-hydroxylase was identified by linkage analysis fol- lowing the segregation of the SSCP of two co-segregating bands amplified from the parent 'Cabernet Sauvignon' by means of the primer pair F3int (Table 2). The VvF3'H locus mapped to the middle of LG17 and co-segregated with the BES marker 5A23-FM and the microsatellite marker VMC9g4. The locus containing the genes that code for flavonoid 3',5'-hydroxylase was mapped by means of a polymorphic SSCP band amplified from the parent 'Cabernet Sauvignon' using the primer pair F35-1int. This marker mapped to the middle of LG6 and co-segregated with microsatellite marker VrZag30 and BES markers 9C17-FM and 17K4-FM (Figure 4). Other SSCP bands that were co-amplified by the same primer pair F35-1int could not be scored in the progeny due to faintness or homozy- gosis. SSCP marker F35-2int could not be mapped in the 'Cabernet Sauvignon' parental map, but co-segregated with VrZag30 and SSCP marker F35-1int in the male par- ent of the same cross (data not shown). Physical mapping and anchoring of BAC contigs to the genetic map A total of 34 BAC clones containing the genes VvF3'H and VvF3'5'H were identified by screening a set of 18,432 BACs corresponding to a 6X genome coverage [23]. The primer pair F3, specific for grapevine flavonoid 3'-hydrox- ylase, detected 1.75 gene copies per haploid genome equivalent. Two bands of 196 bp and 173 bp, correspond- ing to the VvF3'H-1 and the VvF3'H-2 gene fragments, respectively, were co-amplified from all but two of the BACs, while the 196 bp band alone was amplified from the other two BAC clones. The genes coding for flavonoid 3',5'-hydroxylases were searched in the BAC library using the primer pairs F35-1 and F35-2 (Table 2). The primer pair F35-1, specific for the genes VvF3'5'H-1, identified five BAC clones. The primer pair F35-2, specific for VvF3'5'H-2, identified two different BAC clones. Twelve Page 6 of 17 (page number not for citation purposes)