Development of a map-based cloning system in Sorghum bicolor: 1. Isolation of megabase-size DNA and construction of a bacterial artificial chromosome library and 2. Genetic and physical mapping of the 5S rDNA locus

A method was developed for the isolation of megabase-size DNA from Sorghum bicolor. The DNA prepared by this method was larger than 1 Mb in size and readily digestible with restriction enzymes. The DNA was shown to be suitable for physical mapping, and was successfully used for the construction of BAC and YAC libraries. The results demonstrate that this method will be useful for isolation of such DNA from sorghum and other closely related grasses such as sugarcane.;The BAC system was tested in plants by constructing an ordered 13,440 clone sorghum BAC library. The library has an average insert size of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations. No chimeric clones were detected as determined by fluorescence in situ hybridization. The library was screened with twelve sorghum and maize probes and all but one sorghum probe hybridized to at least one BAC clone. These results demonstrate that this BAC library will be useful for several physical mapping and map-based cloning applications not only in sorghum but other related cereal genomes.;The sorghum 5S rRNA gene was investigated to study the molecular organization of the 5S rRNA clusters using the method for isolation of megabase-size DNA from sorghum and the sorghum BAC library. Two size classes of 5S rDNA repeats were isolated from sBAC115J14, and one size class of 5S rDNA repeats was isolated from sBAC120A6. A large segment (118 bp) is tandemly duplicated in the long 5S rDNA repeats. The 5S rDNA locus and two BACs were co-localized by FISH at one major locus on a medium size chromosome. The subtelomeric location of the 5S rDNA locus was confirmed by RFLP mapping of sequences immediately adjacent to the 5S rDNA locus. The results from the analysis of two BACs, PFGE analysis of several enzyme digests, and two different ladder type hybridization patterns of 5S rDNA repeats in genomic DNA suggested that two arrays of repeats, having evidently different characters, co-exist in the same 5S rDNA locus of sorghum.