Molecular and cytogenetic evidence for an allotetraploid origin of Trifolium dubium (Leguminosae)

Suckling clover, Trifolium dubium Sibth., is a European grassland legume that has spread to many parts of the world. The present work shows that it is an allotetraploid (2n=4x=30) combining the genomes of T. campestre Schreb. (2n=2x=14) and T. micranthum Viv. (2n=2x=16), two diploid species of similar geographic distribution. T. dubium has two nuclear ITS sequences that closely match those of T. campestre and T. micranthum. Genomic in situ hybridisation using genomic DNA of T. campestre and T. micranthum as probes has differentiated the ancestral sets of chromosomes in T. dubium cells. Comparative fluorescence in situ hybridisation analyses of 5S and 18S-26S rDNA loci were also consistent with an allotetraploid structure of the T. dubium genome. A marked preponderance of ITS repeats from T. campestre over those from T. micranthum indicated that concerted evolution has resulted in partial homogenisation of these sequences by depletion of the T. micranthum-derived 18S-26S rDNA repeats. In parallel with this, the epigenetic phenomenon of nucleolar dominance has been observed in T. dubium such that the chromatin associated with the 18S-26S rDNA loci derived from T. campestre is decondensed (transcriptionally active), whilst that from T. micranthum remains highly condensed throughout the cell cycle. T. dubium, therefore, appears to have arisen by way of hybridisation between forms of the diploid species T. campestre and T. micranthum accompanied by chromosome doubling. The observed genomic changes in rDNA resulting from interspecific hybridisation provide evidence for the process of genome diploidisation in T. dubium.