Plant Molecular Biology Reporter

, Volume 8, Issue 4, pp 253–275 | Cite as

Analysis of rice (Oryza sativa L.) genome using pulsed-field gel electrophoresis and rare-cutting restriction endonucleases

  • Bruno W. S. Sobral
  • Rhonda J. Honeycutt
  • Alan G. Atherly
  • Michael McClelland
Protocol

Abstract

TheOryza sativa (rice) genome is small (600 to 900 megabase pairs) when compared to that of other monocotyledonous plants. Rice was the first of the major cereals to be successfully transformed and regenerated. An RFLP map with approximately 300 markers is readily available, and the DNA content per map unit is only two to three times that ofArabidopsis thaliana. Rice is also the main staple food for the majority of peoples in the world. We developed techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice, used statistical methods to determine which restriction endonucleases are rare-cutting, and used pulsed-field gel electrophoresis (PFE) to separate large fragments of rice DNA. Southern hybridization to blotted rice PFE gels was used to show that the digests were complete. The long-term goal of our work is to generate an integrated genetic/physical map for the rice genome, as well as helping to establish rice as a model for map-based gene cloning and genome analysis.

Key words

Pulsed-field gel electrophoresis rice Oryza sativa physical mapping genome analysis 

Abbreviations

BSA

bovine serum albumin

pcv

packed cell volume

kb

kilobase pairs

Mb

megabase pairs

5meC

5-methylcytosine

PFE

pulsed-field gel electrophoresis

RE

restriction endonuclease

RFLP

restriction fragment length polymorphism

TAFE

transverse alternating field electrophoresis

ESP, KGB, LET, PIM, PWS, TBEM, THE, and SCE

are defined inMaterials and Methods

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Copyright information

© Kluwer Academic Publishers 1990

Authors and Affiliations

  • Bruno W. S. Sobral
    • 1
  • Rhonda J. Honeycutt
    • 2
  • Alan G. Atherly
    • 2
  • Michael McClelland
    • 1
  1. 1.California Institute of Biological ResearchLa JollaUSA
  2. 2.Department of GeneticsIowa State UniversityAmesUSA

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