Isolation, molecular characterisation and chromosomal location of repetitive DNA sequences in Brassica

Abstract

Plant genomes contain high amounts of repetitive DNA sequences, of which several are present in many species within a taxonomic family, whereas others can exhibit species- or genome-specificity. Genome-specific repetitive sequences can for example be used as chromosome landmarkers for the discrimination of genomes in natural or artificial hybrids at a very early stage of development.Rapeseed, B. napus (2n=38, AACC), is a natural hybrid derived from the closely related species B. rapa (2n=20, AA), turnip rape, and B. oleracea (2n=18, CC), cabbage. The discovery of repetitive sequences specific to the C genomes of Brassica, besides providing information on chromosome and genome evolution in this group, might allow the identification of some or all C genome chromosomes from B. napus. The aim of this work was to find repetitive sequences able to differentiate at the chromosomal level the highly homeologous A (B. rapa) and C (B. oleracea) genomes of Brassica in order to assist in the physical identification of B. napus chromosomes. For this purpose, C genome specific repetitive sequences were searched in genomic DNA libraries from B. oleracea.Phage and plasmid libraries were screened using either B. oleracea or B. rapa total genomic DNA as probe in plaque and dot-blot hybridisations of replica filters. In the screening of more than 100,000 plaques from the phage libraries only 0.45%-2.40% of the clones exhibited strong hybridisation signals, indicating the presence of repetitive DNA in their inserts. Since only a very small amount of the clones could be identified as containing repetitive DNA, phage libraries seem not to be suitable for the identification of species-specific repetitive sequences from B. oleracea, when total genomic DNA is used as probe.In the plasmid libraries 1164 clones were screened by dot-blot hybridisation of replica filters. In these libraries ca. 21% of the clones exhibited strong hybridisation signals with B. oleracea genomic DNA and could be identified as containing repetitive sequences. Fifteen clones, which showed stronger hybridisation signals with the C- than with the A- genome DNA, were selected for further Southern blot analyses, to verify the dot-blot results. Three clones (pBo1.6, pBo1.27 and pBo2.157) were confirmed as more frequent in the C genome ("C genome enriched clones"). These clones, as well as two highly repetitive clones (pBo2.94 und pBo1.173), which showed very strong hybridisation signals with both the A and the C genome DNAs in the dot blot experiments, were sequenced, compared with DNA sequence databases and localised through FISH on the chromosomes of B. oleracea, B. rapa and B. napus. The highly repetitive clones were also tested in B. carinata (Ethiopian mustard). In addition, the ribosomal RNA loci of the A and C genomes were localised by FISH on Brassica chromosomes using different rDNA probes.In accordance with the literature, three, six and nine chromosome pairs were identified as containing 5S and/or 45S rDNA loci in B. oleracea, B. rapa and B. napus, respectively. The highly repetitive clones, pBo2.94 (238 bp) und pBo1.173 (158 bp), were identified as homologous to already described centromeric sequences from Brassica (up to 97% sequence identity). Accordingly, these clones were localised in the centromeric regions of 12, 12-14, 16 and 28-30 chromosomes from B. carinata, B. oleracea, B. rapa and B. napus, respectively.Sequence analyses of the C genome enriched clones showed that two of them, pBo1.27 (182 bp) and pBo2.157 (205 bp), have high similarity with En/Spm- transposon-like sequences (87% and 98% sequence identity, respectively). The chromosomal localisation of these sequences in Brassica by FISH showed a dispersed distribution of these elements in the A and the C genome. The higher frequency of these sequences in the C genome was, however, more evident in the Southern blot hybridisations than in the FISH, probably because of the high degree of amplification necessary for the visualisation of the hybridisation sites in FISH.The third clone, pBo1.6 (203 bp), displayed in a segment varying between 116 bp and 132 bp up to 89% sequence identity with telomere-like DNA from many plant species. This sequence was localised through FISH at telomeric/subtelomeric and interstitial regions of all chromosomes from B. oleracea, whereas in B. rapa no signal was detected in most of the cells. In B. napus chromosomes with and without hybridisation signals were found. Frequently more than 18 chromosomes hybridised with pBo1.6 in this polyploid, suggesting that the sequence may have either spread to A genome chromosomes and amplified after the formation of this hybrid or that sequences present in a small amount in the A chromosomes have been amplified in the polyploid genome.The discovery of a sequence highly enriched in the C genome of Brassica opens the opportunity for detailed studies regarding the subsequent evolution of DNA sequences in polyploid genomes. Moreover, pBo1.6 may be useful in applied genetics for the determination of the chromosomal location of transgene DNA in genetically modified oilseed rape.