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Application of multiplex-ready PCR for fluorescence-based SSR genotyping in barley and wheat

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Abstract

Microsatellites (SSRs) are widely used in cereal research, and their use in marker assisted breeding has increased the speed and efficiency of germplasm improvement. Central to the application of SSRs for many purposes are methodologies enabling the low-cost acquisition of large quantities of genetic information for gene and genotype identification. In this study, multiplex-ready PCR was evaluated in barley and bread wheat as an approach for rapid and more automated SSR genotyping on a fluorescence-based DNA fragment analyzer. Multiplex-ready PCR is a method that allows SSR genotyping to be performed using a standardized protocol. The method enables flexible fluorescence labeling of SSRs, generates a relatively constant amount of PCR product for each marker, and has a high amenability to multiplex PCR (the simultaneous amplification of several SSRs in the same reaction). A high (92%) compatibility of published SSRs with multiplex-ready PCR is demonstrated, and the usefulness of the method for large scale genotyping is shown by its application for whole genome marker assisted breeding in barley. A database of more than 2,800 barley and wheat SSRs, and a suite of bio-informatic tools were developed to support the deployment of multiplex-ready PCR for various genetic applications, and are accessible at http://www.genica.net.au. Multiplex-ready PCR is broadly applicable to cereal genomics research and marker assisted breeding, and should be transferable to similar analyses of any animal or plant species.

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Acknowledgments

This research was supported by the Molecular Plant Breeding CRC and Grains Research and Development Corporation, Australia. The authors gratefully acknowledge Dr. J. Eglinton from the University of Adelaide barley breeding program for providing access to the F5 breeding material.

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Authors and Affiliations

  1. Molecular Plant Breeding CRC, PMB 1, Glen Osmond, SA, 5064, Australia

    M. J. Hayden, T. M. Nguyen, A. Waterman, G. L. McMichael & K. J. Chalmers

  2. School of Agriculture, Food and Wine, Adelaide University, Waite Campus, Urrbrae, SA, 5064, Australia

    M. J. Hayden, T. M. Nguyen, A. Waterman, G. L. McMichael & K. J. Chalmers

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  1. M. J. Hayden
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  2. T. M. Nguyen
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  3. A. Waterman
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  4. G. L. McMichael
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  5. K. J. Chalmers
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Correspondence to M. J. Hayden.

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Hayden, M.J., Nguyen, T.M., Waterman, A. et al. Application of multiplex-ready PCR for fluorescence-based SSR genotyping in barley and wheat. Mol Breeding 21, 271–281 (2008). https://doi.org/10.1007/s11032-007-9127-5

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  • DOI: https://doi.org/10.1007/s11032-007-9127-5

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