DUPLICATIONS THAT SUPPRESS AND DELETIONS THAT RESTORE EXPRESSION FROMA CHALCONE SYNTHASE MULTIGENE FAMILY

Seed coat color in soybean is determined by four alleles of the classi cally defined I (inhibitor) locus that controls the presence or absenc e as well as the spatial distribution of anthocyanin pigments in the s eed coat. By analyzing spontaneous mutations of the I locus, we demons trated that the I locus is a region of chalcone synthase (CHS) gene du plications. Paradoxically, deletions of CHS gene sequences allow highe r levels of CHS mRNAs and restore pigmentation to the seed coat. The u nusual nature of the I locus suggests that its dominant alleles may re present naturally occurring examples of homology-dependent gene silenc ing and that the spontaneous deletions erase the gene-silencing phenom ena. Specifically, mutations from the dominant i(l) allele (yellow see d coats with pigmented hila) to the recessive i allele (fully pigmente d) can be associated with the absence of a 2.3-kb HindIII fragment tha t carries CHS4, a member of the multigene CHS family. Seven independen t mutations exhibit deletions in the CHS4 promoter region. The dominan t I allele (yellow seed coats) exhibits an extra 12.1-kb HindIII fragm ent that hybridizes with both the CHS coding region and CHS1 promoter- specific probes. Mutations of the dominant I allele to the recessive i allele (pigmented seed coats) give rise to 10.4- or 9.6-kb HindIII CH S fragments that have lost the duplicated CHS1 promoter. Finally, gene expression analysis demonstrated that heterozygous plants (I/i) with yellow seed coats have reduced mRNA levels, indicating that the 12.1-k b HindIII CHS fragment associated with the dominant I allele inhibits pigmentation in a trans-dominant manner. Moreover, CHS gene-specific e xpression in seed coats shows that multiple CHS genes are expressed in seed coats.