Breaking Free: The Genomics of Allopolyploidy-Facilitated Niche Expansion in White Clover.

The merging of distinct genomes, allopolyploidization, is a widespread phenomenon in plants. It generates adaptive potential through increased genetic diversity, but examples demonstrating its exploitation remain scarce. White clover (Trifolium repens) is a ubiquitous temperate allotetraploid forage crop derived from two European diploid progenitors confined to extreme coastal or alpine habitats. We sequenced and assembled the genomes and transcriptomes of this species complex to gain insight into the genesis of white clover and the consequences of allopolyploidization. Based on these data, we estimate that white clover originated ∼15,000 to 28,000 years ago during the last glaciation when alpine and coastal progenitors were likely colocated in glacial refugia. We found evidence of progenitor diversity carryover through multiple hybridization events and show that the progenitor subgenomes have retained integrity and gene expression activity as they traveled within white clover from their original confined habitats to a global presence. At the transcriptional level, we observed remarkably stable subgenome expression ratios across tissues. Among the few genes that show tissue-specific switching between homeologous gene copies, we found flavonoid biosynthesis genes strongly overrepresented, suggesting an adaptive role of some allopolyploidy-associated transcriptional changes. Our results highlight white clover as an example of allopolyploidy-facilitated niche expansion, where two progenitor genomes, adapted and confined to disparate and highly specialized habitats, expanded to a ubiquitous global presence after glaciation-associated allopolyploidization.

Introgression of Trifolium ambiguum Into Allotetraploid White Clover (Trifolium repens) Using the Ancestral Parent Trifolium occidentale as a Bridging Species.

White clover (Trifolium repens) is an allotetraploid pasture legume widely used in moist temperate climates, but its vulnerability to drought, grazing pressure and pests has restricted its wider use. A related species, Caucasian clover (Trifolium ambiguum), is a potential source of resistances to drought, cold, grazing pressure and pests that could potentially be transferred to white clover by interspecific hybridization. Although direct hybridization has been achieved with difficulty, the hybrids have not been easy to backcross for introgression breeding and no interspecific chromosome recombination has been demonstrated. The present work shows that interspecific recombination can be achieved by using Trifolium occidentale, one of the ancestral parents of T. repens, as a bridging species and that large white clover breeding populations carrying recombinant chromosomes can be generated. A 4x hybrid between T. ambiguum and T. occidentale was crossed with T. repens and then backcrossed for two generations. Five backcross hybrid plants with phenotypes appearing to combine traits from the parent species were selected for FISH-GISH analyses. Recombinant chromosome segments from T. ambiguum were found in all five plants, suggesting that recombination frequencies were significant and sufficient for introgression breeding. Despite early chromosome imbalances, the backcross populations were fertile and produced large numbers of seeds. These hybrids represent a major new resource for the breeding of novel resilient forms of white clover.

Molecular phylogenetics of the clover genus (Trifolium--Leguminosae).

Trifolium, the clover genus, is one of the largest genera of the legume family. We conducted parsimony and Bayesian phylogenetic analyses based on nuclear ribosomal DNA internal transcribed spacer and chloroplast trnL intron sequences obtained from 218 of the ca. 255 species of Trifolium, representatives from 11 genera of the vicioid clade, and an outgroup Lotus. We confirm the monophyly of Trifolium, and propose a new infrageneric classification of the genus based on the phylogenetic results. Incongruence between the nrDNA and cpDNA results suggests five to six cases of apparent hybrid speciation, and identifies the putative progenitors of the allopolyploids T. dubium, a widespread weed, and T. repens, the most commonly cultivated clover species. Character state reconstructions confirm 2n=16 as the ancestral chromosome number in Trifolium, and infer a minimum of 19 instances of aneuploidy and 22 of polyploidy in the genus. The ancestral life history is hypothesized to be annual in subgenus Chronosemium and equivocal in subgenus Trifolium. Transitions between the annual and perennial habit are common. Our results are consistent with a Mediterranean origin of the genus, probably in the Early Miocene. A single origin of all North and South American species is hypothesized, while the species of sub-Saharan Africa may originate from three separate dispersal events.

A lineage-specific centromeric satellite sequence in the genus Trifolium.

We report the molecular structure, genomic organization, chromosomal distribution and evolutionary dynamics of TrR350, a satellite DNA isolated from the forage legume white clover (Trifolium repens L.; 2n = 4 x = 32). The basic repeating unit is an A+T rich 350 bp Hin dIII fragment with a complex dimeric structure consisting of an internal direct repeat of 156 bp packed between unrelated flanking sequences. Each 156 bp repeat has a conserved 24 bp motif repeating at two places. Most of the 24 bp short repeating units enclose a pentanucleotide CAAAA motif, presumed to be involved in breakage-reunion mechanism of tandemly repeating arrays. The dimers share high sequence homology among themselves while monomers within dimers show significant sequence divergence. Genomic Southern hybridization and/or fluorescence in situ hybridization (FISH) on 17 Trifolium species/subspecies revealed that it is a lineage-specific repeat confined to several species within the section Lotoidea originating in the Mediterranean region. The uniform length of the basic repeating unit and the centromeric localization in most of the species harbouring it reflects its extensive conservation in the lineage. However, the Hin dIII restriction profile in seven species also indicated independent evolution of this repeat.