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pathway may have multiple phenotypic effects. The pleiotropic role of the flavonoid pathway is a complication in the effort to link phenotype to molecular changes, but, in addition, many of the genes of the flavonoid pathway are now known to consist of small multigene families (Table 1), so it is essential to associate a mutation in a particular gene with a phenotype of interest. That is, one must identify the gene family member responsible for the observed phenotype.

MOLECULAR CHARACTERIZATION OF THE GENES OF FLAVONOID BIOSYNTHESIS IN IPOMOEA

The first committed step in the flavonoid biosynthetic pathway is encoded by the enzyme chalcone synthase (CHS), which catalyzes the formation of naringenin chalcone from three molecules of malonyl-CoA and one molecule of p-coumaroyl-CoA (Kreuzaler and Hahlbrock, 1975). Several lines of evidence suggested that the A/a locus might encode a CHS gene. First, the albino phenotype is epistatic to all other flower color variants, suggesting an early point of action in the pathway, and, second, the albino phenotype is consistent with a blockage at CHS. In an attempt to show that the A/a locus encodes a chalcone synthase enzyme, we initiated efforts to clone chalcone synthase genes from a genomic library of I. purpurea. Multiple screenings of the library by using a heterologous probe from parsley (Reinhold et al., 1983) were unsuccessful. Subsequent screening of the library with a tomato CHS clone (O'Neill et al., 1990) resulted in the cloning of four genes (CHS-A, -B, -C, and a pseudogene) initially identified as CHS on the basis of nucleotide sequence similarity to other published CHS gene sequences (Durbin et al., 1995).

To provide a comparative context for Ipomoea CHS gene family evolution, one can look at the Petunia CHS gene family. Petunia is important because both Ipomoea and Petunia are in the same flowering plant order (Solanales), and the Petunia CHS gene family had been extensively characterized, with as many as 12 genes provisionally identified (Koes et al., 1987). The four I. purpurea genes appear to share a common line of descent with an unusual Petunia gene (CHS-B) that is relatively distant in nucleotide sequence from other Petunia CHS genes.

Subsequently, Fukada-Tanaka et al. (1997) cloned and characterized two more CHS genes from Ipomoea (CHS-D and -E) by using differential display and AFLP-based mRNA fingerprinting (Habu et al., 1997). These genes proved to be more closely related in nucleotide sequence to the majority of CHS genes characterized in Petunia. Biochemical analysis of Ipomoea CHS genes A, B, D, and E revealed that only CHS-D and -E are capable of catalyzing the condensation reaction that results in naringenin chalcone (Shiokawa et al., 2000). The CHS-A and -B genes appear to en-



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