9307162 Wu The goal of this proposed research is to develop two new methods for cleaving chromosome-size DNA specifically into large fragments in order to construct a contiguous physical map of each chromosome. These fragments will be used as starting materials in producing a low-density physical map of each chromosome without gaps. The major advantage of this method is that it will allow for the cleavage and recovery of much larger DNA molecules than can be achieved by the three previously published methods. Four novel cleavage-specific plasmids have been designed and two have been constructed for this research. These plasmids will be used to transform Oryza sativa (rice), followed by regeneration of the resulting transgenic plants. Quantitative slot-blot hybridization and genomic blot analysis will be carried out to determine the copy number of the integrated copy of the cleavage-specific plasmid in each transgenic plant. A collection of over 100 transgenic plants will be obtained and characterized, each with several copies of the cleavage-specific plasmids integrated in a different chromosome. Next, the chromosomal DNA from different transgenic plants will be specifically cleaved using a novel enzyme-based strategy, followed by fractionation of the DNA using pulsed field gel electrophoresis and hybridization analysis. The final result of this proposed research is to produce a contiguous, low-density physical map, with between 2 to 7 megabases (mb) in between two markers, of at least 8 out of the 12 chromosomes in rice. ***
