Abstract:In this study, bm-like1, a natural brown midrib mutant, was found to be steadily inherited after continual self-pollination. Phenotype analysis of the F2 population composed of homozygous bm-like1 mutant and B73 inbred lines indicated that the brown midrib phenotype of bm-like1 was controlled by a single recessive gene. Using BSA-seq and fine mapping, the target region was mapped to the interval of 32-36 Mb on maize chromosome 4, where BM3 was located. BM3 gene encodes a caffeic acid o-methyltransferase, which is an important enzyme in lignin biosynthesis. We crossed bm-like1 mutant with bm3 mutant for allelism testing and found that the F1 plants showed brown midrib phenotype, indicating that BM3 was the causal gene for bm-like1 mutant. Genomic sequence analysis revealed that a 1439-bp insertion at 144 bp upstream of transcription start site of BM3 might cause the brown midrib phenotype of bm-like1, which is different from bm3 mutation of 5452-bp insertion in the second exon of BM3 gene. Real-time quantitative PCR and transcriptomic analysis indicated that the expression of BM3 gene in bm-like1 mutants was significantly decreased compared with the wild type plants, the differentially expressed genes between bm-like1 and wild type plants were mainly enriched in the secondary metabolites biosynthesis and metabolic pathway.