RESUMO
KEY MESSAGE: The widely divergent species 4xTrifolium ambiguum and 2xT.occidentale are inter-fertile long after speciation (including polyploidisation) has occurred. Tri-species hybrids (T. repens × T. ambiguum × T. occidentale) have the potential to achieve introgression of stress resistant traits from both wild species into white clover. Trifolium ambiguum and T. occidentale are geographically, adaptionally and phenotypically contrasting species in the white clover section (Trifoliastrum) of the genus. T. ambiguum occurs as a high-altitude polyploid series (2x, 4x, 6x) in W Asia and NE Europe. T. occidentale is a diploid coastal species, occurring at sea level in W Europe. This study investigated hybridisation between 4xT. ambiguum and 2xT. occidentale and considered the significance of the hybrids for introgression breeding of white clover. Partially fertile F1 hybrids between 4xT. ambiguum and 2x and 4xT. occidentale were generated by embryo rescue. Hybrid plant morphology and fertility varied widely and hybrids generally expressed traits from both species. Advanced generation (F2-F5) 4x hybrids were highly fertile and constitute a new synthetic allotetraploid species. FISH analyses of 4x hybrids showed multivalent chromosome configurations with homoeologous associations between T. ambiguum and T. occidentale chromosomes. Crosses of the hybrids with T. repens produced fertile tri-species progeny. These very divergent species remain inter-fertile long after speciation (including polyploidisation) has occurred. Tri-species hybrids have the potential to achieve introgression of stress resistance traits from both wild species into white clover.