Efficient Identification of Causal Mutations through Sequencing of Bulked F from Two Allelic Bloomless Mutants of .

Sorghum ( Moench, L.) plant accumulates copious layers of epi-cuticular wax (EW) on its aerial surfaces, to a greater extent than most other crops. EW provides a vapor barrier that reduces water loss, and is therefore considered to be a major determinant of sorghum's drought tolerance. However, little is known about the genes responsible for wax accumulation in sorghum. We isolated two allelic mutants, () and , from a mutant library constructed from ethyl methane sulfonate (EMS) treated seeds of an inbred, BTx623. Both mutants were nearly devoid of the EW layer. Each mutant was crossed to the un-mutated BTx623 to generated F populations that segregated for the phenotype. Genomic DNA from 20 F plants from each population was bulked for whole genome sequencing. A single gene, Sobic.001G228100, encoding a GDSL-like lipase/acylhydrolase, had unique homozygous mutations in each bulked F population. Mutant harbored a missense mutation in the gene, whereas had a splice donor site mutation. Our findings thus provide strong evidence that mutation in this GDSL-like lipase gene causes the phenotype, and further demonstrate that this approach of sequencing two independent allelic mutant populations is an efficient method for identifying causal mutations. Combined with allelic mutants, MutMap provides powerful method to identify all causal genes for the large collection of mutants in sorghum, which will provide insight into how sorghum plants accumulate such abundant EW on their aerial surface. This knowledge may facilitate the development of tools for engineering drought-tolerant crops with reduced water loss.