Skip to main content

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

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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • 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 31:24–28

    PubMed  CAS  Google Scholar 

  • 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 77–94

  • 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 10:582–594

    PubMed  Article  Google Scholar 

  • 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 11:91–100

    Google Scholar 

  • 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 6:12

    PubMed  Article  Google Scholar 

  • 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

  • Konieczny A, Ausubel FM (1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J 4:403–410

    PubMed  Article  CAS  Google Scholar 

  • Kosambi DD (1944) The estimation of map distances from recombination values. Ann Eugen 12:172–175

    Google Scholar 

  • 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 44:523–528

    PubMed  CAS  Google Scholar 

  • 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 8:223–233

    PubMed  Article  CAS  Google Scholar 

  • Miedaner T, Korzun V (2012) Marker-assisted selection for disease resistance in wheat and barley breeding. Phytopathology 102:560

    PubMed  Article  Google Scholar 

  • 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 9:166–169

    Article  CAS  Google Scholar 

  • 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 30:450–458

    PubMed  Article  CAS  Google Scholar 

  • 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 7:174

    PubMed  Article  Google Scholar 

  • 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 110:283–293

    PubMed  Article  CAS  Google Scholar 

  • 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):e32253

    PubMed  Article  CAS  Google Scholar 

  • 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 156:1997–2005

    PubMed  CAS  Google Scholar 

  • 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 123:69–76

    PubMed  Article  CAS  Google Scholar 

  • Schuelke M (2000) An economic method for the fluorescent labelling of PCR fragments. Nat Biotechnol 18:233–234

    PubMed  Article  CAS  Google Scholar 

  • 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 130:394–397

    Article  CAS  Google Scholar 

  • 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 120:354–356

    Article  CAS  Google Scholar 

  • 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 114:823–839

    PubMed  Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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 

  • Van Ooijen JW (2006) Join Map®4.0 software for the calculation of genetic linkage maps in experimental populations. Kyazma BV, Wageningen

  • Varshney RK, Graner A, Sorrells ME (2005) Genic microsatellite markers in plants: features and applications. Trends Biotechnol 23:48–55

    PubMed  Article  CAS  Google Scholar 

  • 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

    PubMed  Article  CAS  Google Scholar 

  • 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 16:45–55

    Article  CAS  Google Scholar 

  • Xu Y, Lu Y, Xie C, Gao S, Wan J, Prasanna BM (2012) Whole-genome strategies for marker-assisted plant breeding. Mol Breed 29:833–854

    Article  Google Scholar 

  • Yang TJW, Lin WD, Schmidt W (2010) Transcriptional profiling of the Arabidopsis iron deficiency response reveals conserved transition metal homeostasis networks. Plant Physiol 152:2130–2141

    PubMed  Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dragan Perovic.

Additional information

Perovic Jelena and Silvar Cristina contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Perovic, J., Silvar, C., Koenig, J. et al. A versatile fluorescence-based multiplexing assay for CAPS genotyping on capillary electrophoresis systems. Mol Breeding 32, 61–69 (2013). https://doi.org/10.1007/s11032-013-9852-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11032-013-9852-x

Keywords

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