http://www.loc.gov/mods/v3 http://www.loc.gov/standards/mods/v3/mods-3-2.xsd
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 RNA 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 sBAC115Jl4, 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
Issued also on microfiche from University Microfilms Inc.
Includes bibliographical references: pages 179-201.