A candidate-gene approach to clone the sorghum Brown midrib gene encoding caffeic acid O-methyltransferase

Abstract

The brown midrib (bmr) mutants of sorghum have brown vascular tissue in the leaves and stem as a result of changes in lignin composition. The bmr mutants were generated via chemical mutagenesis with diethyl sulfate (DES) and resemble the brown midrib (bm) mutants of maize. The maize and sorghum brown midrib mutants are of particular value for the comparison of lignin biosynthesis across different, yet evolutionarily related, species. Although the sorghum brown midrib mutants were first described in 1978, none of the Brown midrib genes have been cloned. We have used a candidate-gene approach to clone the first Brown midrib gene from sorghum. Based on chemical analyses of the allelic mutants bmr12, bmr18 and bmr26, we hypothesized that these mutants had reduced activity of the lignin biosynthetic enzyme caffeic acid O-methyltransferase (COMT). After a northern analysis revealed strongly reduced expression of the COMT gene, the gene was cloned from the mutants and the corresponding wild types using PCR. In all three mutants, point mutations resulting in premature stop codons were identified: bmr12, bmr18 and bmr26 are therefore mutant alleles of the gene encoding COMT. RT-PCR indicated that all three mutants express the mutant allele, but at much lower levels relative to the wild-type controls. Molecular markers were developed for each of the three mutant alleles to facilitate the use of these mutant alleles in genetic studies and breeding programs.