Molecular Genetics and Genomics

, Volume 272, Issue 2, pp 181–193

Mapping Ds insertions in barley using a sequence-based approach

  • L. D. Cooper
  • L. Marquez-Cedillo
  • J. Singh
  • A. K. Sturbaum
  • S. Zhang
  • V. Edwards
  • K. Johnson
  • A. Kleinhofs
  • S. Rangel
  • V. Carollo
  • P. Bregitzer
  • P. G. Lemaux
  • P. M. Hayes
Original Paper

DOI: 10.1007/s00438-004-1035-3

Cite this article as:
Cooper, L.D., Marquez-Cedillo, L., Singh, J. et al. Mol Genet Genomics (2004) 272: 181. doi:10.1007/s00438-004-1035-3
  • 104 Views

Abstract

A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley (Hordeum vulgare subsp. vulgare). The long-term objective of this project is to identify a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics. AcTPase and Ds-bar elements were introduced into immature embryos of Golden Promise by biolistic transformation. Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in new lines, each containing a stabilized Ds element in a new location. The sequence of the genomic DNA flanking the Ds elements was obtained by inverse PCR and TAIL-PCR. Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 19 independent lines using primarily restriction digest-based assays of PCR-amplified single nucleotide polymorphisms and PCR-based assays of insertions or deletions.The proncipal strategy was to identify and map sequence polymorphisms in the regions corresponding to the flanking DNA using the Oregon Wolfe Barley mapping population. The mapping results obtained by the sequence-based approach were confirmed by RFLP analyses in four of the lines. In addition, cloned DNA sequences corresponding to the flanking DNA were used to assign map locations to Morex-derived genomic BAC library inserts, thus integrating genetic and physical maps of barley. BLAST search results indicate that the majority of the transposed Ds elements are found within predicted or known coding sequences. Transposon tagging in barley using Ac/Ds thus promises to provide a useful tool for studies on the functional genomics of the Triticeae.

Keywords

Transposon taggingBarleyReverse genetics Ac/DsMapping

Supplementary material

supp.pdf (439 kb)
(PDF 11 KB)

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • L. D. Cooper
    • 1
  • L. Marquez-Cedillo
    • 1
  • J. Singh
    • 2
  • A. K. Sturbaum
    • 3
  • S. Zhang
    • 2
  • V. Edwards
    • 3
  • K. Johnson
    • 4
  • A. Kleinhofs
    • 4
  • S. Rangel
    • 2
  • V. Carollo
    • 5
  • P. Bregitzer
    • 3
  • P. G. Lemaux
    • 2
  • P. M. Hayes
    • 1
  1. 1.Department of Crop and Soil ScienceOregon State UniversityCorvallisUSA
  2. 2.Department of Plant and Microbial BiologyUniversity of CaliforniaBerkeleyUSA
  3. 3.National Small Grains Germplasm Research FacilityUSDA-ARSAberdeenUSA
  4. 4.Department of Crop and Soil Sciences, School of Molecular BiosciencesWashington State UniversityPullmanUSA
  5. 5.Western Regional Research CenterUSDA-ARSAlbanyUSA