Visual markers for tomato derived from the anthocyanin biosynthetic pathway

Abstract

Genes along the anthocyanin biosynthetic pathway have been some of the most useful genetic markers for both molecular and classical genetic studies. In order to exploit such markers for molecular studies in tomato, we have cloned and characterized genes encoding two steps in the pathway, chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR). We show that CHS is comprised of a multigene family with at least two unlinked genes being actively expressed in hypocotyls. These genetic results offer an explanation for the biochemical finding that none of the characterized anthocyanin-deficient tomato mutants completely lacks CHS activity. In contrast, a number of lines of evidence support the conclusion that DFR is encoded by a single gene in tomato: 1) only one class of cDNA transcript has been recovered from multiple isolations, 2) this transcript maps to a single genetic locus, 3) the intensity and banding pattern on Southern blots using this cDNA as a probe is consistent with there being only one DFR sequence at this locus, and 4) a single mutation, anthocyanin without (aw), completely abolishes DFR activity. A genomic clone of DFR was introduced into a line homozygous for the aw mutation by Agrobacterium transformation. While the aw mutation results in the absence of anthocyanin pigmentation, transformants containing a DFR transgene were fully pigmented. Hybridization analysis of progeny segregating for the presence or absence of anthocyanin indicated that pigmenation resulted from complementation by the DFR transgene. In addition to showing that the aw locus encodes the structural gene for DFR, these experiments demonstrate the applicability of DFR as a non-destructive, easily scored, visual marker for tomato.