Experimental evidence for the ancestry of allotetraploid Trifolium repens and creation of synthetic forms with value for plant breeding

T. pallescens plants were identified with chloroplast trnL intron DNA sequences identical to those of white clover. Similarly, T. occidentale plants with nuclear ITS sequences identical to white clover were also identified. Reciprocal GISH experiments, alternately using labeled genomic DNA probes from each of the putative ancestral species on the same white clover cells, showed that half of the chromosomes hybridized with each probe. F1 hybrids were generated by embryo rescue and these showed strong interspecific chromosome pairing and produced a significant frequency of unreduced gametes, indicating the likely mode of polyploidization. The F1 hybrids are inter-fertile with white clover and function as synthetic white clovers, a valuable new resource for the re-incorporation of ancestral genomes into modern white clover for future plant breeding.Evidence from DNA sequence analyses, molecular cytogenetics, interspecific hybridization and breeding experiments supports the hypothesis that a diploid alpine species (T. pallescens) hybridized with a diploid coastal species (T. occidentale) to generate tetraploid T. repens. The coming together of these two narrowly adapted species (one alpine and the other maritime), along with allotetraploidy, has led to a transgressive hybrid with a broad adaptive range.White clover, an allotetraploid (2n=4x=32) stoloniferous herb, is naturally distributed through the grasslands of Europe, W Asia and N Africa, from low to high latitudes and altitudes and, because of its broad adaptation, has become the most extensively used legume of grazed pasture world-wide. Its origin has been long debated [1-4]. The identity of the ancestors has remained elusive and, despite many attempts [e.g. [3,5-7] there has been no successful re-synthesis. A phylogenetic analysis of Trifolium based on the nuclear internal transcribed spacer region of 18 S–26S rDNA (ITS) and chloroplast trnL intron DNA (cpDNA) sequences [8] suggested that the closest extant diplo