Molecular Breeding

, Volume 34, Issue 2, pp 421–429 | Cite as

A DNA marker assay based on high-resolution melting curve analysis for distinguishing species of the FestucaLolium complex

  • Martina Birrer
  • Roland Kölliker
  • Chloe Manzanares
  • Torben Asp
  • Bruno Studer


The grass breeding industry is interested in a fast and cheap method of identifying contamination in seeds of Italian and perennial ryegrass (Lolium perenne L. and L. multiflorum Lam., respectively). This study shows that high-resolution melting curve analysis in combination with an unlabelled probe assay is an effective method of detecting single nucleotide polymorphisms (SNPs) in diverse Italian and perennial ryegrass backgrounds. This method proved efficient in differentiating ryegrass species and reducing the effect of additional DNA sequence polymorphisms close to the target SNP on the melting curve profiles. For the identification of contamination in Italian and perennial ryegrass seed production, high-resolution melting curve analysis shows great potential, as it is a single closed-tube PCR reaction with an easy workflow, providing results in <2 h after DNA extraction.


High-resolution melting curve analysis Italian ryegrass (Loliummultiflorum Lam.) Perennial ryegrass (Lolium perenne L.) Single nucleotide polymorphism Unlabelled probe assay 



The authors would like to acknowledge Sonja Reinhard from Agroscope, Institute for Sustainability Sciences, ISS and Marianne Wettstein from the Institute of Agricultural Sciences, ETH Zurich, as well as Dr. Oliver Schultz and Dr. Robert Akkers from BIOKÉ for excellent technical support. We sincerely thank Prof. Dr. Achim Walter for hosting the Forage Crop Genetics group at ETH Zurich. This study was partly funded by the SNSF Professorship (Grant no: PP00P2_138988).

Supplementary material

11032_2014_44_MOESM1_ESM.docx (614 kb)
Supplementary material 1 (DOCX 614 kb)
11032_2014_44_MOESM2_ESM.docx (46 kb)
Supplementary material 2 (DOCX 45 kb)


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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Martina Birrer
    • 1
  • Roland Kölliker
    • 2
  • Chloe Manzanares
    • 1
  • Torben Asp
    • 3
  • Bruno Studer
    • 1
  1. 1.Forage Crop Genetics, Institute of Agricultural SciencesETH ZurichZurichSwitzerland
  2. 2.Institute for Sustainability Sciences, ISSAgroscopeZurichSwitzerland
  3. 3.Department of Molecular Biology and Genetics, Faculty of Science and TechnologyAarhus UniversitySlagelseDenmark

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