Two Regulatory Genes of the Maize Anthocyanin Pathway Are Homologous: Isolation of B Utilizing R Genomic Sequences

Genetic studies in maize have identified several regulatory genes that control the tissue-specific synthesis of the purple anthocyanin pigments during development. Two such genes, R and B, exhibit extensive allelic diversity with respect to the tissue specificity and developmental timing of anthocyanin synthesis. Previous genetic studies demonstrated that certain B alleles can substitute for R function, and in these cases only one functional allele at either locus is required for pigment synthesis in the aleurone. In addition, biochemical studies have shown that both genes act on the same biosynthetic pathway, suggesting that the genes are functionally duplicate. In this report we describe DNA hybridization experiments that demonstrate that the functionally duplicate nature of B and R is reflected in DNA sequence similarity between the two genes. We took advantage of this homology and used the R genomic sequences to clone B. Two different strategies were pursued and two genomic clones isolated, a 2.5-kilobase BgIII fragment linked to the b allele in W23 inbred stocks and a 1.0-kilobase HindIII fragment linked to the B allele in CM37 stocks. Examination of several independent transposable element insertion mutations in B and revertant derivatives demonstrated that our clones recognize the functional B gene. Genomic clones representing the entire B-Peru allele were isolated, and a detailed restriction map was prepared. Using these clones we have identified a 2.2-kilobase mRNA in husks from plants containing either B-I or B-Peru alleles, but no B mRNA was detected in plants containing a b allele. The transcript is at least 100 times more abundant in strongly pigmented B-I husks than in weakly pigmented B-Peru husk tissue. Expression of functional B alleles in husk tissue correlates with the coordinate increase in mRNA levels of two structural genes of the pathway, A1 and Bz1, consistent with the postulated role of B as a regulatory gene.