A Eurasia-wide polyploid species complex involving 6x Trifolium ambiguum, 2x T. occidentale and 4x T. repens produces interspecific hybrids with significance for clover breeding

Background: Trifolium ambiguum occurs as a 2x, 4x, 6x polyploid series in W Asia, The 6x form is the most agronomically desirable, having strong rhizomatous spread and drought tolerance. These traits would be potentially very valuable if they could be transferred to white clover (T. repens) which is the most important agronomic clover species. However, to-date, no fertile interspecific hybrids are available. Previously, 2x T. occidentale from W Europe has produced synthetic fertile hybrids with both 2x and 4x T. ambiguum and these were inter-fertile with white clover. Here we ask whether 2x T. occidentale can form fertile hybrids with 6x T. ambiguum and act as a genetic bridge to white clover and bring these species together as part of a common gene pool. Results: Ten verified F1 (6x T. ambiguum x 2x T. occidentale) hybrids were produced by embryo rescue and seven were studied further. All four investigated for chromosome number were 2n=4x=32 and FISH confirmed the expected 21 T. ambiguum and 8 T. occidentale chromosomes. Hybrid fertility was extremely low but 2n female gametes functioned with white clover pollen to produce seeds. Derived plants were confirmed using FISH and were successfully backcrossed to white clover to produce partially fertile breeding populations. Conclusions: Although T. occidentale and 6x T. ambiguum are widely separated by geography and ecological adaptation they have maintained enough genomic affinity to produce partially fertile hybrids. This is consistent with a Eurasia-wide radiation of speciation from a presumed 2x ancestor leading to marked morphological divergence and involving polyploid speciation. Inter-fertility with allotetraploid T. repens showed that this species is part of the same complex radiation that has also involved interspecific hybridization. Use of this information enables potentially valuable 6x T. ambiguum genomes to be used in white clover breeding for the first time.