Forward genetics by sequencing EMS variation induced inbred lines.

In order to leverage novel sequencing techniques for cloning genes in eukaryotic organisms with complex genomes, the false positive rate of variant discovery must be controlled for by experimental design and informatics. We sequenced five lines from three pedigrees of EMS mutagenized Sorghum bicolor, including a pedigree segregating a recessive dwarf mutant. Comparing the sequences of the lines, we were able to identify and eliminate error prone positions. One genomic region contained EMS mutant alleles in dwarfs that were homozygous reference sequence in wild-type siblings and heterozygous in segregating families. This region contained a single non-synonymous change that cosegregated with dwarfism in a validation population and caused a premature stop codon in the sorghum ortholog encoding the giberellic acid biosynthetic enzyme ent-kaurene oxidase. Application of exogenous giberillic acid rescued the mutant phenotype. Our method for mapping did not require outcrossing and introduced no segregation variance. This enables work when line crossing is complicated by life history, permitting gene discovery outside of genetic models.This inverts the historical approach of first using recombination to define a locus and then sequencing genes. Our formally identical approach first sequences all the genes and then seeks co-segregation with the trait. Mutagenized lines lacking obvious phenotypic alterations are available for an extention of this approach: mapping with a known marker set in a line that is phenotypically identical to starting material for EMS mutant generation.