Cloning of a COBL gene determining brittleness in diploid wheat using a MapRseq approach

Plant tissue brittleness is related to cellular structure and lodging. MED0031 is a mutant identified previously from ethyl methane sulfonate treatment of diploid wheat accession TA2726, showing brittleness in both stem and leaf. In microscopic and histological observations, the mutant was found to have less large vascular bundles per unit area, a thinner sclerenchyma cell wall, and a broader parenchyma, compared with the wild type. The mutated gene, TmBr1, was mapped to a 0.056 cM interval on chromosome 5A^m. This gene was cloned using a MapRseq approach that searched the candidate gene through combination of the prior target gene mapping information with SNP calling and discovery of differentially expressed genes from RNA_seq data of the wild type and a BC₃F₂ bulk showing the mutant phenotype. TmBr1 encodes a COBL protein and a nonsense mutation within the region coding for the conserved COBRA domain caused premature translation termination. Introduction of TmBr1 to Arabidopsis AtCOBL4 mutant rescued the phenotype, demonstrating their functional conservation. Apart from the effect on cellulose content, the TmBr1 mutation might modulate synthesis of noncellulosic polysaccharide pectin as well. Application of the MapRseq approach to isolation of genes present in recombination cold spots and complicated genomes was discussed.