Plant & Animal Genome IX Conference Plant & Animal Genome IX Conference
Poster: Sequencing & EST
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The legume family is one of the most important agricultural resources among plant species. In order to investigate the legume-specific biological processes such as symbiotic nitrogen fixation, we have started a large-scale genome analysis of a model legume, Lotus japonicus, by sequencing both cDNA and the genome. (Data on cDNA analysis will be presented by E. Asamizu at the poster session.) We have constructed a genomic library of L. japonicus in a transformation competent artificial chromosome (TAC) vector as a source of genome sequencing clones. The average insert sizes were 87kb, 96kb, 105 kb and 106kb for four independent preparation, a total of which is 7.7 haploid genome equivalent. The TAC libraries thus generated were arrayed in ninety-three 384 well microtiter plates, and 48 DNA pools each containing 384 clones were subjected to PCR screening. Seed clones for sequencing were selected by high throughput PCR screening using primer sets designed on the basis of ESTs and cDNA markers of L. japonicus and other legumes. The nucleotide sequence of each selected clone was determined according to the bridging shotgun method with an approximate redundancy of 5, followed by gene assignment by similarity search and computer prediction. Mapping of the seed clones on the basis of genome sequence information is also performed. The rate of the overall processes is estimated to be approximately 30 Mb per year. The latest status of the project will be presented.