Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genic male-sterile rice 5460S

In order to develop a detailed physical map of the thermo-sensitive genic m ale-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene , a high-quality rice bacterial artificial chromosome (BAC) library from TG MS rice 5460S was constructed. The method of constructing BAC library was e xamined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice hapl oid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolat ed and appeared stable after 100 generations of serial growth. Hybridizatio n of BAC clones with mitochondrial and chloroplastic genes as probes demons trated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tms1 gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of posit ive clones were successfully isolated using thermal asymmetric interlaced P CR (TAIL PCR) technique.