Study on molecular breeding by the structural analysis of rice genome.

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Techniques for the isolation and fractionation of intact chromosomal DNA have been developed. The isolation of high-molecular-weight (HMW) DNA is performed in agarose blocks and it is then digested for fractionation by pulsed-field gel electrophoresis (PFGE), which can be used to resolve DNAs that are several mega-base pairs in length.Rice is an excellent model system, among crop plants for genome analysis, because of the low degree of complexity of its genome. Well-developed maps of restriction fragment length polymorphisms are available for rice, but methods for handling large pieces of DNA have not been developed that would allow analysis of its genome by PFGE.We have isolated high-molecular-weight DNA of over 5.7 Mb in length from isolated rice germ nuclei. The high-molecular-weight DNA was digested with a number of restriction endonucleases. An ethidium-stained gel after pulsed-field electrophoresis revealed that few of these endonucleases have the ability to cleave rice chromosomal DNA to large fragments of over 200 kb in length, perhaps because of the lower level of methylation of nucleotides in the rice genome than in other genomes. Some restriction endonucleases that do not cut rDNA repeats produced fragments with arrays of rDNA repeats of more than 1.1 Mb, as estimated by Southern analysis. Therefore, it appears that the rice rDNA cluster is constructed with a "physical" succession of more than 130 copies of 8-kb repeating units of rDNA. Genes for alpha-amylase were also detected by Southern method. It indicates our procedure is suitable for analysis of low-copy-number genes.