CONSTRUCTION AND CHARACTERIZATION OF A BACTERIAL ARTIFICIAL CHROMOSOME LIBRARY OF SORGHUM-BICOLOR

The construction of representative large insert DNA libraries is criti cal for the analysis of complex genomes. The predominant vector system for such work is the yeast artificial chromosome (YAC) system. Despit e the success of YACs, many problems have been described including: ch imerism, tedious steps in library construction and low yields of YAC i nsert DNA. Recently a new E.coli based system has been developed, the bacterial artificial chromosome (BAC) system, which offers many potent ial advantages over YACs. We tested the BAC system in plants by constr ucting an ordered 13 440 clone sorghum BAC library. The library has a combined average insert size, from single and double size Selections, of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations in liquid media. No chim eric clones were detected as determined by fluorescence in situ hybrid ization of ten BAC clones to metaphase and interphase S.bicolor nuclei . The library was screened with six sorghum probes and three maize pro bes and all but one sorghum probe hybridized to at least one BAC clone in the library. To facilitate chromosome walking with the BAC system, methods were developed to isolate the proximal ends of restriction fr agments inserted into the BAC vector and used to isolate both the left and right ends of six randomly selected BAC clones. These results dem onstrate that the S.bicolor BAC library will be useful for several phy sical mapping and map-based cloning applications not only in sorghum b ut other related cereal genomes, such as maize. Furthermore, we conclu de that the BAC system is suitable for most large genome applications, is more 'user friendly' than the YAC system, and will likely lead to rapid progress in cloning biologically significant genes from plants.