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

Warren M Williams,S Hussain,Nicholas W Ellison,Helal A Ansari,Isabelle M Verry

doi:10.1186/1471-2229-12-55

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.

Highlights

White clover (Trifolium repens) is a ubiquitous weed of the temperate world that through use of improved cultivars has become the most important legume of grazed pastures world-wide
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
DNA sequences In the present work, comparisons were made of the chloroplast trnL intron DNA (cpDNA) sequences of white clover and five geographically divergent T. pallescens accessions, all verified by their distinctive ITS DNA sequence

Summary

Introduction

White clover (Trifolium repens) is a ubiquitous weed of the temperate world that through use of improved cultivars has 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. T. occidentale is a predominantly self-fertile, strictly maritime species with a very narrow adaptation, occurring only very close to the sea in confined habitats on the gulfstream coasts of Europe [9] (Figure 1a). T. pallescens is a predominantly cross-pollinating but self-fertile alpine clover, presently occurring only above 1,800 meters in Europe (Figure 1b). It has a narrow adaptation within the alpine zone [10].

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