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


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.

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Correspondence to Dragan Perovic.

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Perovic Jelena and Silvar Cristina contributed equally to this work.

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

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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).

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