Forward genetics by sequencing EMS variation-induced inbred lines

Charles Addo-Quaye, Elizabeth Buescher, Norman Best, Vijay Chaikam, Ivan Baxter, Brian P. Dilkes

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

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)- 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 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 the Sorghum ortholog encoding the gibberellic acid (GA) biosynthetic enzyme ent-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.

Original languageEnglish
Pages (from-to)413-425
Number of pages13
JournalG3: Genes, Genomes, Genetics
Volume7
Issue number2
DOIs
StatePublished - 2017

Keywords

  • Gibberellin
  • Mapping
  • Mutant
  • SNP
  • Sorghum

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