Abstract
Sorghum (Sorghum bicolor 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, bloomless40-1 (bm40-1) and bm40-2, 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 bm mutant was crossed to the un-mutated BTx623 to generated F2 populations that segregated for the bm phenotype. Genomic DNA from 20 bm F2 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 F2 population. Mutant bm40-1 harbored a missense mutation in the gene, whereas bm40-2 had a splice donor site mutation. Our findings thus provide strong evidence that mutation in this GDSL-like lipase gene causes the bm 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 bm 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.
Highlights
Forward genetics is a powerful approach to identifying gene mutations that cause phenotypes of interest, and elucidating the biochemical and molecular mechanisms and signaling processes underlying plant development and adaptation
scanning electron microscopy (SEM) of the WT sheath revealed that the stomata on the sheath were completely covered with fibrous wax crystals, whereas the abaxial surface of leaf blade was only partially covered (Figures 1b,c)
We demonstrated that sequencing of two independent allelic mutants represents a powerful approach to identifying causal gene mutations in sorghum
Summary
Forward genetics is a powerful approach to identifying gene mutations that cause phenotypes of interest, and elucidating the biochemical and molecular mechanisms and signaling processes underlying plant development and adaptation. Linkage of the mutant phenotype with DNA markers is analyzed, using as many markers as possible, until the mutation is mapped to a very small region flanked by two markers. To confirm the identity of the gene, the wild-type gene is introduced into the mutant through transformation to determine whether it can complement the mutant phenotype This last step can be bypassed if two or more independent mutant alleles are identified that carry unique mutations in the same gene because the odd of finding two independent mutations by chance on the same gene that lead to the same phenotype is near zero (Jander et al, 2002)
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