A marker-dense physical map of the Bradyrhizobium japonicum genome

Bacterial artificial chromosome (BAC) clones are effective mapping and sequ encing reagents for use with a wide variety of small and large genomes. Thi s report describes, the development of a physical framework for the genome of Bradyrhizobium japonicum, the nitrogen-fixing symbiont of soybean. A BAC library for B. japonicum was constructed that provides a 77-fold genome co verage based oil an estimated genome size of 8.7 Mb. The library contains 4 608 clones with an average. insert size of 146 kb. To generate a physical m ap, the entire library was fingerprinted with HindIII, and the fingerprinte d clones were assembled into contigs using the Fingerprint Contig software (FPC Sanger Centre, UK). The FPC analysis placed 3410 clones in six large c ontigs. The ends of 1152 BAC inserts were sequenced to generate a sequence- tagged connector (STC) framework. To join and orient the contigs, hf-h-dens ity BAC colony filters were probed with 41 known gene probes and 17 end seq uences from contig boundaries. STC sequences were searched against the publ ic databases using FASTA and BLASTX algorithms. Query results allowed the i dentification of 113 high probability matches with putative functional iden tities that were placed on the physical map. Combined with the hybridizatio n data, a high-resolution physical map with 194 positioned markers represen ted in two large contigs was developed, providing a marker every 45 kb. Of these markers, 177 are known. or putative B. japonicum genes. Additionally, 1338 significant BLASTX results (E < 10(-4)) were manually sorted by funct ion to produce a functionally categorized database of relevant B. japonicum STC sequences that can also be traced to specific locations in the physica l map.