title: Forward Genetics by Sequencing EMS Variation-Induced Inbred Lines.
abstract: 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 ethyl methanesulfonate (EMS)-mutagenizedSorghum 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 sequences in wild-type siblings and heterozygous in segregating families. This region contained a single nonsynonymous change that cosegregated with dwarfism in a validation population and caused a premature stop codon in theSorghumortholog encoding the gibberellic acid (GA) biosynthetic enzymeent-kaurene oxidase. Application of exogenous GA 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 cosegregation with the trait. Mutagenized lines lacking obvious phenotypic alterations are available for an extension of this approach: mapping with a known marker set in a line that is phenotypically identical to starting material for EMS mutant generation.
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Charles
authors.identifier: 0000-0003-0598-5980
authors.initials: C
authors.lastname: Addo-Quaye
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Elizabeth
authors.identifier: 0000-0002-1540-8928
authors.initials: E
authors.lastname: Buescher
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Norman
authors.initials: N
authors.lastname: Best
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Vijay
authors.initials: V
authors.lastname: Chaikam
authors.affiliation: United States Department of Agriculture-Agricultural Research Service, Donald Danforth Plant Science Center, St. Louis, Missouri 63132.
authors.forename: Ivan
authors.identifier: 0000-0001-6680-1722
authors.initials: I
authors.lastname: Baxter
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907 bdilkes@purdue.edu.
authors.forename: Brian P
authors.identifier: 0000-0003-2799-954X
authors.initials: BP
authors.lastname: Dilkes
mesh_terms: D000483:Alleles; D001483:Base Sequence / Q000235:genetics; D002874:Chromosome Mapping; D018389:Codon, Nonsense / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D018745:Genome, Plant / Q000235:genetics; D005875:Gibberellins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D016296:Mutagenesis / Q000187:drug effects / Q000235:genetics*; D009154:Mutation; D011995:Recombination, Genetic; D045868:Sorghum / Q000187:drug effects / Q000235:genetics*
chemical_list: D018389:Codon, Nonsense; D005875:Gibberellins; D003577:Cytochrome P-450 Enzyme System; D005020:Ethyl Methanesulfonate; C007842:gibberellic acid; C066398:ent-kaurene oxidase
keywords: SNP; Sorghum; gibberellin; mapping; mutant