Construction of a bacterial artificial chromosome (BAC) library and identification of overlapping BAC clones with chromosome 4-specific RFLP markers in rice

To facilitate construction of physical map of the rice genome, a bacterial artificial chromosome (BAC) library of IR64 genomic DNA was constructed. It consists of 18432 clones and contains 3.28 rice genomic equivalents. The insert size ranged from 37 to 364kb with an average of 107 kb. We used 31 RFLP markers on chromosome 4 to screen the library by colony hybridization. Sixty eight positive clones were identified with 2.2 positive clones per RFLP marker. The positive clones were analyzed to generate 29 contigs whose sizes ranged from 50 to 384 kb with an average of 145.6 kb. Chromosome walking was initiated for ten contigs linked to resistance genes. Thirty eight BAC clones were obtained and two contigs were integrated. Altogether, they covered 5.65 Mb (15.1%) of chromosome 4. These contigs may be used as landmarks for physical mapping of chromosome 4, and as starting points for chromosome walking towards the map-based cloning of disease resistance genes which were located nearby.