Transposon insertion in a cinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize

Maize brown midrib1 (bm1) mutant plants have reduced lignin content and offer significant advantages when used in silage and biofuel applications. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the conversion of hydroxycinnamyl aldehydes to monolignols, a key step in lignin biosynthesis. Maize CAD2 has been implicated as the underlying gene for bm1 phenotypes since bm1 plants have reduced CAD activity and lower CAD2 transcript level. Here, we describe a Dow AgroSciences maize bm1 mutant (bm1-das1) that contains a 3,444-bp transposon insertion in the first intron of CAD2 gene. As a result of chimeric RNA splicing, cad2 mRNA from bm1-das1 contains a 409-bp insert between its 1st and 2nd exons. This insertion creates a premature stop codon and is predicted to result in a truncated protein of 48 amino acids (AA), compared to 367 AA for the wild type (WT) CAD2. We have also sequenced cad2 from the reference allele bm1-ref in 515D bm1 stock and showed that it contains a two-nucleotide (AC) insertion in the 3rd exon, which is predicted to result in a truncated protein of 147 AA. The levels of cad2 mRNA in the midribs of bm1-das1 and bm1-ref are reduced by 91 and 86 % respectively, leading to reductions in total lignin contents by 24 and 30 %. Taken together, our data show that mutations in maize CAD2 are responsible for maize bm1 phenotypes. Based on specific changes in bm1-das1 and bm1-ref, high throughput TaqMan and KBioscience's allele specific PCR assays capable of differentiating mutant and WT alleles have been developed to accelerate bm1 molecular breeding.