Abstract
Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This 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 coverage based on an estimated genome size of 8.7 Mb. The library contains 4608 clones with an average insert size of 146 kb. To generate a physical map, the entire library was fingerprinted with
Hin
dIII, and the fingerprinted clones were assembled into contigs using the
Fingerprint Contig
software (
FPC
; Sanger Centre, UK). The
FPC
analysis placed 3410 clones in six large contigs. The ends of 1152 BAC inserts were sequenced to generate a sequence-tagged connector (STC) framework. To join and orient the contigs, high-density BAC colony filters were probed with 41 known gene probes and 17 end sequences from contig boundaries. STC sequences were searched against the public databases using
FASTA
and
BLASTX
algorithms. Query results allowed the identification of 113 high probability matches with putative functional identities that were placed on the physical map. Combined with the hybridization data, a high-resolution physical map with 194 positioned markers represented 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 function to produce a functionally categorized database of relevant
B. japonicum
STC sequences that can also be traced to specific locations in the physical map.