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

Abstract

BackgroundWhite clover (Trifolium repens) is a ubiquitous weed of the temperate world that through use of improved cultivars has also become the most important legume of grazed pastures world-wide. It has long been suspected to be allotetraploid, but the diploid ancestral species have remained elusive. Putative diploid ancestors were indicated by DNA sequence phylogeny to be T. pallescens and T. occidentale. Here, we use further DNA evidence as well as a combination of molecular cytogenetics (FISH and GISH) and experimental hybridization to test the hypothesis that white clover originated as a hybrid between T. pallescens and T. occidentale.ResultsT. 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.ConclusionsEvidence 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.

Achievements and Challenges in Improving Temperate Perennial Forage Legumes

“…Utilization of the secondary and tertiary gene pool may also offer morphological and other adaptations to drought tolerance (Marshall et al, 2001). Recent evidence on the putative progenitor species and related germplasm that can be introgressed Williams et al, 2012) suggests there are opportunities for adaptive introgression of drought tolerance into white clover.…”

“…A systematic assessment of phylogeny in the Trifolium genus (Ellison et al, 2006) has enabled further utilization of wide hybrids and provides experimental evidence for identifying the progenitor species of allotetraploid white clover (Williams et al, 2012). This has implications for introgression breeding influencing the agronomic adaptation of white clover to a range of ecophysiological and pasture management conditions.…”

“…Partially fertile backcrosses to T. ambiguum were also reported with different ploidy levels . More recently, interspecific hybridization between white clover's putative diploid progenitor species has been used to create distinct new populations of value to plant breeding (Williams et al, 2012).…”

Legume Crops Phylogeny and Genetic Diversity for Science and Breeding

“…There are some exceptions: allotetraploid white clover originated as a hybrid between T. pallescens Schreb. and T. occidentale D. E. Coombe (Williams et al, 2012). …”

Forage Plant Ecophysiology: A Discipline Come of Age

“…White clover is now known to be an allotetraploid (2n = 4x = 32) with progenitor species Trifolium occidentale Coombe and Trifolium pallescens Schreb [9]. Both because of expected increase in incidence of drought through changing climatic conditions, as well as from an interest in extending the range of white clover, techniques have been developed for the introgression of genes into white clover from the more strongly rooted and drought resistant T. ambiguum (Caucasian clover), through interspecific hybridisation and backcrossing.…”