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


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Article Info

DATE

2013-06

AUTHORS

Jelena Perovic, Cristina Silvar, Janine Koenig, Nils Stein, Dragan Perovic, Frank Ordon

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. More... »

PAGES

61-69

References to SciGraph publications

  • 2010-12. Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species in PLANT METHODS
  • 2007-04. A high density barley microsatellite consensus map with 775 SSR loci in THEORETICAL AND APPLIED GENETICS
  • 2006-12. Automated SNP detection from a large collection of white spruce expressed sequences: contributing factors and approaches for the categorization of SNPs in BMC GENOMICS
  • 2003-02. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.) in THEORETICAL AND APPLIED GENETICS
  • 2007-03. A 1,000-loci transcript map of the barley genome: new anchoring points for integrative grass genomics in THEORETICAL AND APPLIED GENETICS
  • 2007. Single Nucleotide Polymorphism Genotyping in Plants in ASSOCIATION MAPPING IN PLANTS
  • 2009-12. Development and implementation of high-throughput SNP genotyping in barley in BMC GENOMICS
  • 2005-08. Strategies for Pyramiding Resistance Genes Against the Barley Yellow Mosaic Virus Complex (BaMMV, BaYMV, BaYMV-2) in MOLECULAR BREEDING
  • 2000-02. An economic method for the fluorescent labeling of PCR fragments in NATURE BIOTECHNOLOGY
  • 2005-01. 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.) in THEORETICAL AND APPLIED GENETICS
  • 2008-08. EST-derived single nucleotide polymorphism markers for assembling genetic and physical maps of the barley genome in FUNCTIONAL & INTEGRATIVE GENOMICS
  • 2011-06. 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 in THEORETICAL AND APPLIED GENETICS
  • 2012-10. Genetic mapping of a leaf rust resistance gene in the former Yugoslavian barley landrace MBR1012 in MOLECULAR BREEDING
  • 2012-04. Whole-genome strategies for marker-assisted plant breeding in MOLECULAR BREEDING
  • 1998-07. The use of microsatellite markers for detection of genetic diversity in barley populations in THEORETICAL AND APPLIED GENETICS
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