Characterisation of repeated sequences from microdissected B chromosomes of Crepis capillaris.
The B chromosome of Crepis capillaris was isolated from the standard chromosomes by microdissection, and the chromosomal DNA amplified using the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The PCR product was cloned and a B-specific library created and characterised. Southern and in situ hybridisation analyses of the DOP-PCR product from microdissected B chromosomes confirmed that the B chromosome is composed mainly of sequences also present in the A chromosomes but lacks the main repeated DNA families located in the A-chromosomal heterochromatin. From 100 clones analysed, 12% of the generated B-chromosomal library was shown to be composed of dispersed repeats located in both the A and B chromosomes. No B-specific repeated sequence was detected. One of the most abundant repeated DNAs within the library, the family B134, was further characterised. Repeating units show a sequence similarity range from 69% to 90% and are characterised by their richness in (CA)n repeats. In situ hybridisation revealed that members of this family are dispersed throughout the A and B chromosomes but are more concentrated in the pericentromeric heterochromatin of the B, indicating that the molecular organization of B heterochromatin is different from that of the A chromosomes. Compared with the A chromosomes, the Bs contain about 20,000 copies per micron more of the B134 sequence. This indicates that B134 was amplified on the B chromosome after its origin. The B134 sequences in the B chromosomes have also diverged from those on the A chromosomes. Although the DNA composition of A and B chromosomes is similar, Bs are evolving separately from A chromosomes at the molecular level.
Jamilena
M
M
Department of Botany, School of Plant Sciences, University of Reading, Reading RG6 2AS, UK.
Garrido-Ramos
M
M
Ruiz Rejón
M
M
Ruiz Rejón
C
C
Parker
J S
JS
D001483:Base Sequence; D015139:Blotting, Southern; D002875:Chromosomes / Q000737:chemistry*; D003001:Cloning, Molecular; D017931:DNA Primers; D018744:DNA, Plant / Q000737:chemistry; D006570:Heterochromatin; D017404:In Situ Hybridization, Fluorescence; D008846:Micromanipulation; D008969:Molecular Sequence Data; D010944:Plants / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D018744:DNA, Plant; D006570:Heterochromatin