forward genetics by sequencing ems variation-induced inbred lines

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ID: 207860
2017
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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.
Reference Key
addo-quaye2017g3:forward Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors ;Charles Addo-Quaye;Elizabeth Buescher;Norman Best;Vijay Chaikam;Ivan Baxter;Brian P. Dilkes
Journal separation and purification technology
Year 2017
DOI
10.1534/g3.116.029660
URL
http://g3journal.org/lookup/doi/10.1534/g3.116.029660
Keywords
mapping snp sorghum gibberellin

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