Donald T M, Houben A, Leach C R, Timmis J N
Department of Genetics, University of Adelaide, Australia.
Genome. 1997 Oct;40(5):674-81. doi: 10.1139/g97-089.
Ribosomal RNA genes are present near the end of the short arm and, to a lesser extent, near the centromere of the B chromosomes of some populations of Brachycome dichromosomatica. The internal transcribed spacer (ITS2) was amplified by PCR from total leaf DNA using primers within the conserved regions encoding the 5.8S and 25S stable rRNA species. Comparison of PCR amplified ITS2 sequences from several individual plants without B chromosomes with corresponding sequences derived from microdissected B chromosomes revealed two consistent differences between the rDNA of A and B chromosomes. One of these differences produced an SfcI restriction site that was present only in the ITS2 of the B-chromosome rDNA. Amplification by PCR of ITS2 from total genomic DNA from plants with and without B chromosomes showed an additive relationship between the amount of PCR product containing the SfcI site and the number of B chromosomes present. Quantitative analysis indicated that the proportion of total nuclear rDNA present on a single B chromosome varied between 2 and 4% in different A chromosome backgrounds. Similar experiments, with appropriate positive and negative controls, using reverse transcriptase PCR of the equivalent region within the 40S precursor rRNA, suggested that the B-chromosome rDNA was not transcribed. Similarly, PCR of reverse transcribed total RNA from plants containing B chromosomes using primers specific for the B chromosome ITS2 was unable to detect a transcript from the B chromosome.
