Lignins are universal components in plants that form cross-links with carbohydrates, such as hemicelluloses in the cell wall. Lignin polymers lower fiber digestion in ruminants, and the degree of lignifications may be inversely proportional to forage crop digestibility. Cherney et al. (1991) Adv. Agron. 46:157-98. Maize containing a brown midrib (BMR) mutation exhibit a reddish brown pigmentation of the leaf midrib that is associated with significantly reduced lignin content, altered lignin composition, and improved digestibility. At least four independent BMR mutations have been identified in maize. Kuc et al. (1968) Phytochemistry 7:1435-6. These mutations, termed “bm1, bm2, bm3, and bm4,” all exhibit decreased lignin content when compared to control corn. bm3 mutations include insertions (bm3-1), deletions (bm3-2), and insertion/deletions (bm3-3) within the caffeic acid O-methyltransferase (COMT, e.g., GenBank Accession No. M73235) gene. Morrow et al. (1997) Mol. Breeding. 3:351-7; Vignols et al. (1995) Plant Cell 7:407-16.
The COMT gene controls enzyme activities involved in lignin biosynthesis. COMT utilizes S-adenylsylmethionine to transmethylate caffeic acid, which results in the production of ferulic acid. Coniferyl alcohol and sinapyl alcohol are ultimately generated from the ferulic acid. The combination of coniferyl, ferulic, and sinapyl alcohols in the presence of free radicals results in lignin production. Bm3 mutations have been characterized, and are thought to inhibit the transmethylation of caffeic acid by gene inactivation. Guillet-Claude et al. (2004) Theor. Appl. Genet. 110:126-35; Piquemal et al. (2002) Plant Physiology 130:1-11; He et al. (2003) Crop Sci. 43:2240-51; Morrow et al. (1997) Mol. Breeding. 3:351-7; Vignols et al. (1995) Plant Cell 7:407-16. However, a rapid method for specifically detecting and testing the zygosity of a particular plant at the bm3 locus has not been developed.