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Sequence-tagged-site-facilitated PCR for barley genome mapping.

S Tragoonrung1, V Kanazin, P M Hayes

  • 1Plant and Soil Science Department, Montana State University, 59717, Bozeman, MT, USA.

TAG. Theoretical and Applied Genetics. Theoretische Und Angewandte Genetik
|November 9, 2013
PubMed
Summary

Sequence-tagged-site (STS) primers derived from known sequences enable efficient PCR amplification for barley genome mapping. This method detects insertion/deletion and point mutation polymorphisms, facilitating map conversion and analysis.

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Area of Science:

  • Genetics
  • Molecular Biology
  • Plant Science

Background:

  • Genome mapping projects increasingly favor polymerase chain reaction (PCR) over Southern blot analysis for speed, efficiency, and safety.
  • A key limitation in PCR-based mapping is the availability of informative primer sequences.
  • Sequence-tagged-site (STS) approaches offer an alternative to random amplified polymorphism detection (RAPD).

Purpose of the Study:

  • To investigate the utility of sequence-tagged-site (STS) primers derived from known sequences for PCR-based barley genome mapping.
  • To assess the conversion of existing barley genetic maps to utilize PCR technology.
  • To screen for polymorphisms in barley progeny using STS primers.

Main Methods:

  • Obtained eight pairs of primer sequences: four from published sequences and four from sequencing DNA clones from the North American Barley Genome Mapping Project (NABGMP).
  • Screened barley progeny from winter x spring and spring x spring crosses for polymorphisms using these primers.
  • Distinguished insertion/deletion polymorphisms via agarose gel electrophoresis and point mutation polymorphisms using polyacrylamide gel electrophoresis after restriction endonuclease digestion.

Main Results:

  • Developed and utilized STS primers for PCR amplification in barley.
  • Successfully identified both insertion/deletion and point mutation polymorphisms.
  • Initiated chromosomal assignments and co-segregation analysis to validate allelism of PCR-detected polymorphisms.

Conclusions:

  • Sequence-tagged-site (STS) primers derived from known sequences are effective for PCR-based polymorphism detection in barley.
  • This approach facilitates the conversion of existing barley genome maps to a PCR-based system.
  • The method allows for direct detection of insertion/deletion events and indirect detection of point mutations, enhancing mapping capabilities.