A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.)

David G. Caldwell, Nicola McCallum, Paul Shaw, Gary J. Muehlbauer, David F. Marshall, Robbie Waugh (Lead / Corresponding author)

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Abstract

Two large-scale ethylmethanesulfonate (EMS) mutant populations from barley (Hordeum vulgare L.) cv. Optic have been developed to promote both forward and reverse genetics in this crop. Leaf material and seed from approximately 20 000 M2 plants were individually harvested, freeze-dried and archived. DNA was isolated from 9216 plants from the 20 and 30 mM EMS treatments and assembled into 1152 eight-plant pools. To facilitate PCR-based mutation scanning an approach has been employed that combines cleavage of heteroduplexes using the Cel nuclease (Cel I), post-cleavage intercalating dye labeling and the subsequent detection of cleaved products on a Transgenomic WAVE-HS. The populations were evaluated by screening for induced mutations in two genes of interest and the induced mutations were validated by sequence analysis. To enhance the screening process, 12-16 M3 progeny from each of the M2 plants were assessed for visible phenotypes and the data entered into a web accessible database (http://bioinf.scri.sari.ac.uk/distilling/ distilling.html).

Original languageEnglish
Pages (from-to)143-150
Number of pages8
JournalPlant Journal
Volume40
Issue number1
Early online date6 Aug 2004
DOIs
Publication statusPublished - 1 Oct 2004

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Keywords

  • Barley
  • Cel I
  • Ethylmethanesulfonate
  • Mutagenesis
  • Reverse genetics

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