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

分离和分离完整染色体DNA的技术已经发展起来。高分子量（HMW） DNA的分离是在琼脂糖块中进行的，然后通过脉冲场凝胶电泳（PFGE）进行消化分离，PFGE可用于分离长度为几个兆碱基对的DNA。由于水稻基因组的复杂性较低，因此在作物基因组分析中是一个很好的模式系统。水稻的限制性片段长度多态性图谱已经开发得很好，但是处理大片段DNA的方法还没有开发出来，无法通过PFGE分析其基因组。我们从分离的水稻胚核中分离到长度超过5.7 Mb的高分子量DNA。高分子量的DNA被一些限制性内切酶消化。脉冲场电泳后的乙二胺染色凝胶显示，这些内切酶很少有能力将水稻染色体DNA切割成长度超过200 kb的大片段，这可能是因为水稻基因组中核苷酸的甲基化水平低于其他基因组。据Southern分析估计，一些不切割rDNA重复序列的限制性内切酶产生的片段的rDNA重复序列超过1.1 Mb。因此，水稻rDNA集群似乎是由超过130个8kb重复rDNA单位的“物理”序列构建而成的。用Southern法检测α -淀粉酶基因。结果表明，该方法适用于低拷贝数基因的分析。

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.