4xTrifolium ambiguum and 2xT. occidentale hybridise despite wide geographic separation and polyploidisation: implications for clover breeding

Key message: Barriers to hybridisation between 4x T. ambiguum and T. occidentale are incomplete long after speciation (including polyploidisation) has occurred. Tri-species hybrids (T. repens x T. ambiguum x T. occidentale) have the potential to achieve introgression of both T. ambiguum and T. occidentale traits into white clover. Abstract: Trifolium ambiguum and T. occidentale are geographically isolated species in the white clover section (Trifoliastrum) of the clover genus. T. ambiguum occurs as a polyploid series (2x, 4x, 6x) at high altitudes, mainly in W Asia and NE Europe. T. occidentale is a diploid coastal species, occurring only at sea level on the Gulf Stream coasts of W Europe. Here we examine the hypothesis that divergence of polyploid (4x) T. ambiguum and 2x T. occidentale has occurred in the absence of genetic barriers to hybridisation and investigate the significance of this for introgression breeding of white clover. Partially fertile F1 hybrids between 4x T. ambiguum and 2x and 4x T. 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) hybrids were highly fertile. FISH and meiotic analyses of 4x hybrids revealed multivalent chromosome configurations showing homoeologous associations between T. ambiguum and T. occidentale chromosomes. Crosses of the hybrids with T. repens produced fertile tri-species progeny. We conclude that barriers to interspecific hybridisation are still incomplete long after speciation (including polyploidisation) has occurred. Tri-species hybrids have the potential to achieve introgression of both T. ambiguum and T. occidentale traits into white clover.