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Detection of Deletion Mutants in Rice Via Overgo Hybridization Onto Membrane Spotted Arrays

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Abstract

Overlapping nucleotides or overgos were designed and used to detect deletions in rice mutants that had been generated by treatment with a chemical (diepoxybutane, DB) or irradiation (gamma ray, GR). As a proof of concept, DNA of three spotted leaf 11 (spl11) deletion mutants, GR5612, GR5717 and DB2487, and the wild-type DNA (IR64) were spotted onto nylon membranes in different concentrations and probed with radiolabeled overgos designed from different regions within the Spl11 gene. At least 3 μg per spot of DNA was required to show unambiguous and visible signals. Overgos designed from regions putatively deleted in GR5612 consistently did not hybridize to the GR5612 genomic DNA, whereas DNA from wild-type IR64 and from mutants in other regions consistently hybridized to the overgos. DNA of mutant DB2487 hybridized to all the overgos tested, suggesting the deletion is small and undetectable by the overgos. Overall, this study demonstrates that overgos can be employed to detect, verify, and characterize deletions, particularly in single copy genes, in mutant genomes. However, the technique requires careful adjustment of DNA concentration before spotting and the spotting of relatively high DNA concentrations onto the membranes.

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Abbreviations

Overgos:

overlapping nucleotides

Spl11 :

spotted leaf 11

DB:

diepoxybutane

GR:

gamma ray

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Acknowledgments

MGQ Diaz is a recipient of a Postdoctoral Grant from the University of the Philippines System Faculty Development Program. The authors would like to thank Dr. Guo-Liang Wang for providing the spl11 mutant seeds and the sequences of the presumed deleted segments. This work is supported by the Generation Challenges Program, USDA-CSREES-NRI, and the USAID-IRRI linkage program.

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Authors and Affiliations

  1. Institute of Biological Sciences, University of the Philippines Los Baños College, Laguna, 4031, Philippines

    M. G. Q. Diaz

  2. Department of Bioagricultural Sciences and Pest Management and Program in Plant Molecular Biology, Colorado State University, Fort Collins, 80523-1177, CO, USA

    M. G. Q. Diaz & J. E. Leach

  3. Department of Plant Pathology, Kansas State University, Manhattan, KS, USA

    M. Ryba

  4. Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute, Los Baños, Philippines

    H. Leung

  5. Cornell University, Ithaca, NY, USA

    R. Nelson

Authors
  1. M. G. Q. Diaz
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  2. M. Ryba
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  3. H. Leung
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  4. R. Nelson
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  5. J. E. Leach
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Correspondence to J. E. Leach.

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Diaz, M.G.Q., Ryba, M., Leung, H. et al. Detection of Deletion Mutants in Rice Via Overgo Hybridization Onto Membrane Spotted Arrays. Plant Mol Biol Rep 25, 17–26 (2007). https://doi.org/10.1007/s11105-007-0002-7

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  • DOI: https://doi.org/10.1007/s11105-007-0002-7

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