Rapid Identification of Causal Gene Mutations through Sequencing Bulked F2 Derived from Independent Alleles.

Sorghum (Sorghum bicolor Moench, L.) plant accumulates copious layers of extra-cuticular waxes on its aerial surfaces, more spectacular than most other crops. It provides a vapor barrier to reduce water loss and is considered as a major determinant of its superior drought tolerance. However, little is known about the genes responsible for wax accumulation in sorghum. We isolated two allelic mutants completely void of extra-cuticular wax layer, named bloomless1 (bm1-1) and bm1-2, from two independent mutant lines. Each bm mutant was crossed to BTx623, the parent for the sorghum pedigreed mutant library, to generated F2 populations segregating for the bm phenotype. Genomic DNA from 20 bm F2 plants from each population were bulked and sequenced to 15x coverage of the whole genome on Illumina x10. A gene encoding a GDSL-like Lipase/Acylhydrolase was found to harbor a mis-sense mutation in bm1-1 and a splice donor site mutation in bm1-2. Thus, we provided strong evidence that this GDSL-like Lipase gene is the causal gene. The function of the gene in extra-cuticular wax accumulation will be discussed.