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High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley

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Abstract

An original method has been established for the identification of novel alleles of eukaryotic translation initiation factor 4E (eIF4E) gene, which is required for resistance to agronomically important bymoviruses, in barley germplasm. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which so far has only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allowed significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The method described here involved an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover the coding sequence. This strategy further reduced the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in eIF4E approached 100%. Results of the current study are promising and suggest that this method could also potentially be applied to the discovery of superior alleles controlling other important traits in barley as well in other model and crop plant species.

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Acknowledgments

The authors wish to thank Chris Bass and Thomas Baldwin for providing barley RNA samples and sequences of novel barley eIF4E alleles, Andrew L. Phillips, Antonio Hernández López and Carlos Bayon for technical advice on the HRM technology and the LightScanner® System, Richard Parkinson and other greenhouse staff for the production of excellent plant material, Harold E. Bockelman (National Small Grains Collection, USDA-ARS, Aberdeen, Idaho, USA) for providing numerous barley genotypes, and John A. Lucas for critical reading of the manuscript. This study was supported by the grant BBE0071981 from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom (“Innovation in Crop Science - Exploitation of Genetics for Sustainability” initiative). The LightScanner® System (Idaho Technology Inc.) utilising Hi-Res Melting™ was purchased from the BBSRC’s Tools and Resources Development Fund (TRDF) grant BBE0251611. Rothamsted Research receives grant-aided support from the BBSRC.

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Authors and Affiliations

  1. Department of Plant Pathology and Microbiology, Centre for Sustainable Pest and Disease Management, Rothamsted Research, Harpenden, AL5 2JQ, UK

    Bernhard J. Hofinger, Hai-Chun Jing, Kim E. Hammond-Kosack & Kostya Kanyuka

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  1. Bernhard J. Hofinger
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  2. Hai-Chun Jing
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  3. Kim E. Hammond-Kosack
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  4. Kostya Kanyuka
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Correspondence to Kostya Kanyuka.

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Communicated by A. Schulman.

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Hofinger, B.J., Jing, HC., Hammond-Kosack, K.E. et al. High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley. Theor Appl Genet 119, 851–865 (2009). https://doi.org/10.1007/s00122-009-1094-2

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  • DOI: https://doi.org/10.1007/s00122-009-1094-2

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