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Molecular Breeding

, Volume 32, Issue 1, pp 61–69 | Cite as

A versatile fluorescence-based multiplexing assay for CAPS genotyping on capillary electrophoresis systems

  • Jelena Perovic
  • Cristina Silvar
  • Janine Koenig
  • Nils Stein
  • Dragan PerovicEmail author
  • Frank Ordon
Article

Abstract

Recent advances in next-generation sequencing techniques and the development of genomics resources for crop plants with large genomes allow the detection of a large number of single nucleotide polymorphisms (SNPs) and their use in a high-throughput manner. However, such large numbers of SNPs are on the one hand not needed in some plant breeding projects and on the other hand not affordable in some cases, raising the need for fast and low-cost innovative techniques for marker detection. In marker selection in plant breeding programs, cleaved amplified polymorphic sequence (CAPS) markers still play a significant role as a complement to other high-throughput methods for SNP genotyping. New methods focusing on the acceleration of CAPS-based genotyping are therefore highly desirable. The combination of the classical CAPS method and a M13-tailed primer multiplexing assay was used to develop an agarose-gel-free protocol for the analysis of SNPs via restriction enzyme digestion. PCR products were fluorescence-labeled with a universal M13 primer and subsequently digested with the appropriate restriction endonuclease. After mixing differently labeled products, they were detected in a capillary electrophoresis system. This method allowed the cost-effective genotyping of several SNPs in barley in a multiplexed manner at an overall low cost in a short period of time. This new method was efficiently combined with the simultaneous detection of simple sequence repeats in the same electrophoresis run, resulting in a procedure well suited for marker-based selection procedures, genotyping of mapping populations and the assay of genetic diversity.

Keywords

CAPS SNP Fluorescence-based multiplexing M13 tail Marker-assisted selection Barley 

Supplementary material

11032_2013_9852_MOESM1_ESM.xls (34 kb)
Supplementary Table 1. SNP and SSR markers used in this study. (XLS 34 kb)

References

  1. Boutin-Ganache I, Raposo M, Raymond M, Deschepper CF (2001) M13-tailed primers improve the readability and usability of microsatellite analyses performed with two different allele sizing methods. Biotechniques 31:24–28PubMedGoogle Scholar
  2. Chagné D, Batley J, Edwards D, Forster JW (2007) Single nucleotide polymorphisms genotyping in plants. In: Oraguzie N, Rikkerink E, Gardiner S, De Silva H (eds) Association mapping in plants. Springer, New York, pp 77–94Google Scholar
  3. Close TJ, Prasanna RB, Lonardi LS, Wu Y, Rostoks N, Ramsay L, Druka A, Stein N, Svensson JT, Wanamaker S, Bozdag S, Roose ML, Moscou ML, Chao S, Varshney RK, Szűcs P, Sato K, Hayes PM, Matthews DE, Kleinhofs A, Muehlbauer GJ, DeYoung J, Marshall DF, Madishetty K, Fenton KD, Condamine P, Graner A, Waugh R (2009) Development and implementation of high-throughput SNP genotyping in barley. BMC Genomics 10:582–594PubMedCrossRefGoogle Scholar
  4. Ha BK, Boerma HR (2008) High-throughput SNP genotyping by melting curve analysis for resistance to southern root-knot nematode and frogeye leaf spot in soybean. J Crop Sci Biotech 11:91–100Google Scholar
  5. Hirotsu N, Murakami N, Kashiwagi T, Ujiie K, Ishimaru K (2010) Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species. Plant Methods 6:12PubMedCrossRefGoogle Scholar
  6. Koenig J, Kopahnke D, Steffenson BJ, Przulj N, Romeis T, Roeder MS, Ordon F, Perovic D (2012) Genetic mapping of a leaf rust resistance gene in former Yugoslavian barley landrace MBR1012. Mol Breed. doi: 10.1007/s11032-012-9712-0
  7. Konieczny A, Ausubel FM (1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J 4:403–410PubMedCrossRefGoogle Scholar
  8. Kosambi DD (1944) The estimation of map distances from recombination values. Ann Eugen 12:172–175Google Scholar
  9. Kota R, Wolf M, Michalek W, Graner A (2001) Application of denaturing high-performance liquid chromatography for mapping of single nucleotide polymorphisms in barley (Hordeum vulgare L.). Genome 44:523–528PubMedGoogle Scholar
  10. Kota R, Varshney RK, Prasad M, Zhang H, Stein N, Graner A (2008) EST-derived single nucleotide polymorphism markers for assembling genetic and physical maps of the barley genome. Funct Integr Genomics 8:223–233PubMedCrossRefGoogle Scholar
  11. Miedaner T, Korzun V (2012) Marker-assisted selection for disease resistance in wheat and barley breeding. Phytopathology 102:560PubMedCrossRefGoogle Scholar
  12. Mondini L, Nachit MM, Porceddu E, Pagnotta MA (2011) HRM technology for the identification and characterization of INDEL and SNP mutations in genes involved in drought and salt tolerance of durum wheat. Plant Genet Resour 9:166–169CrossRefGoogle Scholar
  13. Oetting WS, Lee HK, Flanders DJ, Wiesner GL, Sellers TA, King RA (1995) Linkage analysis with multiplexed short tandem repeat polymorphisms using infrared fluorescence and M13 tailed primers. Genomics 30:450–458PubMedCrossRefGoogle Scholar
  14. Pavy N, Parsons L, Paule C, Mackay J, Bousquet J (2006) Automated SNP detection from a large collection of white spruce expressed sequences: contributing factors and approaches for the categorization of SNPs. BMC Genomics 7:174PubMedCrossRefGoogle Scholar
  15. Pellio B, Streng S, Bauer E, Stein N, Perovic D, Schiemann A, Friedt W, Ordon F, Graner A (2005) High-resolution mapping of the Rym4/Rym5 locus conferring resistance to the barley yellow mosaic virus complex (BaMMV, BaYMV, BaYMV-2) in barley (Hordeum vulgare ssp. vulgare L.). Theor Appl Genet 110:283–293PubMedCrossRefGoogle Scholar
  16. Poland JA, Brown PJ, Sorells ME, Jannik J (2012) Development of high density genetic maps for barley and wheat by using a novel two enzyme genotyping by sequencing approach. PLoS ONE 7(2):e32253PubMedCrossRefGoogle Scholar
  17. Ramsay L, Macaulay M, Degli Ivanissevich S, McLean K, Cardle L, Fuller J, Edwards KJ, Tuvesson S, Morgante M, Massari A, Maestri E, Marmiroli N, Sjakste T, Ganal M, Powell W, Waugh R (2000) A simple sequence repeat-based linkage map of barley. Genetics 156:1997–2005PubMedGoogle Scholar
  18. Riedel C, Habekuß A, Schliephake E, Niks R, Broer I, Ordon F (2011) Pyramiding of Ryd2 and Ryd3 conferring tolerance to a German isolate of Barley yellow dwarf virus-PAV (BYDV-PAV-ASL-1) leads to quantitative resistance against this isolate. Theor Appl Genet 123:69–76PubMedCrossRefGoogle Scholar
  19. Schuelke M (2000) An economic method for the fluorescent labelling of PCR fragments. Nat Biotechnol 18:233–234PubMedCrossRefGoogle Scholar
  20. Silvar C, Perovic D, Casas AM, Igartua E, Ordon F (2011) Development of a cost-effective pyrosequencing approach for SNP genotyping in barley. Plant Breed 130:394–397CrossRefGoogle Scholar
  21. Stein N, Herren G, Keller B (2001) A new DNA extraction method for high–throughput marker in a large–genome species such as Triticum aestivum. Plant Breed 120:354–356CrossRefGoogle Scholar
  22. Stein N, Prasad M, Scholz U, Thiel T, Zhang H, Wolf M, Kota R, Varshney K, Perovic D, Grosse I, Graner A (2007) A 1,000-loci transcript map of the barley genome: new anchoring points for integrative grass genomics. Theor Appl Genet 114:823–839PubMedCrossRefGoogle Scholar
  23. Struss D, Plieske J (1998) The use of microsatellite markers for detection of genetic diversity in barley populations. Theor Appl Genet 97:308–315. doi: 10.1007/s001220050900 CrossRefGoogle Scholar
  24. Thiel T, Michalek W, Varshney RK, Graner A (2003) Exploiting EST databases for the development and characterization of gene derived SSR markers in barley. Theor Appl Genet 103:411–422. doi: 10.1007/s00122-002-1031-0 Google Scholar
  25. Van Ooijen JW (2006) Join Map®4.0 software for the calculation of genetic linkage maps in experimental populations. Kyazma BV, WageningenGoogle Scholar
  26. Varshney RK, Graner A, Sorrells ME (2005) Genic microsatellite markers in plants: features and applications. Trends Biotechnol 23:48–55PubMedCrossRefGoogle Scholar
  27. Varshney RK, Marcel TC, Ramsay L, Russell J, Röder MS, Stein N, Waugh R, Langridge P, Niks RE, Graner A (2007) A high density barley microsatellite consensus map with 775 SSR loci. Theor Appl Genet 114:1091–1103. doi: 10.1007/s00122-007-0503-7 PubMedCrossRefGoogle Scholar
  28. Werner K, Friedt W, Ordon F (2005) Strategies for pyramiding resistance genes against the barley yellow mosaic virus complex (BaMMV, BaYMV, BaYMV-2). Mol Breed 16:45–55CrossRefGoogle Scholar
  29. Xu Y, Lu Y, Xie C, Gao S, Wan J, Prasanna BM (2012) Whole-genome strategies for marker-assisted plant breeding. Mol Breed 29:833–854CrossRefGoogle Scholar
  30. Yang TJW, Lin WD, Schmidt W (2010) Transcriptional profiling of the Arabidopsis iron deficiency response reveals conserved transition metal homeostasis networks. Plant Physiol 152:2130–2141PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Jelena Perovic
    • 1
  • Cristina Silvar
    • 2
    • 3
  • Janine Koenig
    • 2
  • Nils Stein
    • 1
  • Dragan Perovic
    • 2
    Email author
  • Frank Ordon
    • 2
  1. 1.Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK)GaterslebenGermany
  2. 2.Julius Kühn-Institute (JKI), Federal Research Institute for Cultivated Plants, Institute for Resistance Research and Stress ToleranceQuedlinburgGermany
  3. 3.Dpto. de Bioloxía Animal, Bioloxía Vexetal e EcoloxíaUniversidade da CoruñaCoruñaSpain

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