Plant Molecular Biology Report pages 253-275 Volume 8(4) Protocol 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, and Michael McClelland (BWSS, MM) California Institute of Biological Research, 11099 North Torrey Pines Road, Suite 300, La Jolla, CA 92037, USA (ph: 1-619-535-5485; fax: 1- 619-535-5472); (RJH, AGA) Department of Genetics, Iowa State University, Ames, IA 50011, USA Key words: Pulsed-field gel elec~'ophoresis, rice, Oryza sativa, physical mapping, genome analysis Abstract:. The Oryza sativa (rice) genome is small (600 to 900 megabasepairs) 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 of Arabidopsis 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. Abbreviations: BSA, bovine serum albumin; pcv, packed cell volume; kb, kilobase pairs; Mb, megabase s pairs; meC, 5-methylcytosine; PFE, pulsed-field el electrophoresis; RE, restriction endonuclease; RFLP, restriction fragment ngth polymorphism; TAFE, transverse alternating field electrophoresis; ESP, KGB, LET, PIM, PWS, TBEM, THE, and SCE are defined inMaterials andMethods. 253