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.

Asynapsis and unreduced gamete formation in a Trifolium interspecific hybrid.

BACKGROUND: Unreduced gametes, a driving force in the widespread polyploidization and speciation of flowering plants, occur relatively frequently in interspecific or intergeneric hybrids. Studies of the mechanisms leading to 2n gamete formation, mainly in the wheat tribe Triticeae have shown that unreductional meiosis is often associated with chromosome asynapsis during the first meiotic division. The present study explored the mechanisms of meiotic nonreduction leading to functional unreduced gametes in an interspecific Trifolium (clover) hybrid with three sub-genomes from T. ambiguum and one sub-genome from T. occidentale. RESULTS: Unreductional meiosis leading to 2n gametes occurred when there was a high frequency of asynapsis during the first meiotic division. In this hybrid, approximately 39% of chromosomes were unpaired at metaphase I. Within the same cell at anaphase I, sister chromatids of univalents underwent precocious separation and formed laggard chromatids whereas paired chromosomes segregated without separation of sister chromatids as in normal meiosis. This asynchrony was frequently accompanied by incomplete or no movement of chromosomes toward the poles and restitution leading to unreduced chromosome constitutions. Reductional meiosis was restored in progeny where asynapsis frequencies were low. Two progeny plants with approximately 5 and 7% of unpaired chromosomes at metaphase I showed full restoration of reductional meiosis. CONCLUSIONS: The study revealed that formation of 2n gametes occurred when asynapsis (univalent) frequency at meiosis I was high, and that normal gamete production was restored in the next generation when asynapsis frequencies were low. Asynapsis-dependent 2n gamete formation, previously supported by evidence largely from wheat and its relatives and grasshopper, is also applicable to hybrids from the dicotyledonous plant genus Trifolium. The present results align well with those from these widely divergent organisms and strongly suggest common molecular mechanisms involved in unreduced gamete formation.

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 with 6x T. ambiguum 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. Inter-fertility of the hybrids with allotetraploid T. repens showed that T. occidentale can provide a genetic bridge between 6x T. ambiguum and white clover to produce plants with new phenotypes combining the traits of all three species. Use of this information should enable potentially valuable stress tolerance traits from 6x T. ambiguum to be used in white clover breeding for the first time.

Fluorescence chromosome banding and FISH mapping in perennial ryegrass, Lolium perenne L.

BACKGROUND: The unambiguous identification of individual chromosomes is a key part of the genomic characterization of any species. In this respect, the development and application of chromosome banding techniques has revolutionised mammalian and especially, human genomics. However, partly because of the traditional use of chromosome squash preparations, consistent fluorescence banding has rarely been achieved in plants. Here, successful fluorescence chromosome banding has been achieved for the first time in perennial ryegrass (Lolium perenne), a forage and turf grass with a large genome and a symmetrical karyotype with chromosomes that are difficult to distinguish. RESULTS: Based on flame-dried chromosome preparations instead of squashes, a simple fluorescence Q-banding technique using quinacrine mustard, unambiguously identified each chromosome and enabled the development of a banded karyotype and ideogram of the species. This Q-banding technique was also shown to be compatible with sequential FISH mapping enabling labelled genes and molecular markers to be precisely assigned to specific cytogenetic bands. A technique for DAPI-banding, which gave a similar pattern to Q-banding, was also introduced. This was compatible with FISH mapping and was used to anchor a single copy gene from an earlier mapped linkage group of L. perenne, thus providing a step towards integration of the genetic and cytogenetic maps. CONCLUSIONS: By enabling the allocation of genes mapped by other methods to physically identified chromosome positions, this work will contribute to a better understanding of genomic structures and functions in grasses.

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

BACKGROUND: White 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. RESULTS: T. 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. CONCLUSIONS: 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. 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.

Eco-geographically divergent diploids, Caucasian clover (Trifolium ambiguum) and western clover (T. occidentale), retain most requirements for hybridization.

BACKGROUND AND AIMS: DNA sequence similarities and hybridization patterns in Trifolium (clovers) section Trifoliastrum suggest that rapid radiation from a common ancestral source led to this complex of diverse species distributed across Europe, western Asia and North Africa. Two of the most geographically and ecologically divergent of these species are the rhizomatous T. ambiguum from high altitudes in eastern Europe and western Asia and the stoloniferous T. occidentale from sea level in western Europe. Attempts were made to hybridize these species to ascertain whether, despite this separation, gene flow could be achieved, indicating the retention of the genetic factors necessary for hybridization. METHODS: Three F(1) hybrids formed after embryo rescue were described, characterized by conventional and molecular cytogenetics, subjected to fertility tests and progeny generations were developed. RESULTS AND CONCLUSIONS: Partially fertile hybrids between Trifolium ambiguum and T. occidentale were obtained for the first time. The F(1) hybrids produced seeds after open-pollination, and also produced triploid progeny in backcrosses to T. occidentale from the functioning of unreduced gametes in the hybrids. These plants were fertile and produced progeny with T. occidentale and with T. repens. Meiotic chromosome pairing in the F(1) showed six to eight bivalents per pollen mother cell, indicating pairing between the parental genomes. A chromosome-doubled form of one hybrid, produced using colchicine, showed some multivalents, indicative of interspecific chromosome pairing. The hybrid plants were robust and combined phenotypic characteristics of both species, having stolons, thick roots and a few rhizomes. Results show that despite separation by the entire breadth of Europe, the speciation process is incomplete, and these taxa have partially retained most of the genetic compatibilities needed for hybridization (possibly except for endosperm development, which was not tested). The fertile progeny populations could lead to new clover breeding strategies based on new hybrid forms.

Epichloë endophytes grow by intercalary hyphal extension in elongating grass leaves.

A fundamental hallmark of fungal growth is that vegetative hyphae grow exclusively by extension at the hyphal tip. However, this model of apical growth is incompatible with endophyte colonization of grasses by the symbiotic Neotyphodium and Epichloë species. These fungi are transmitted through host seed, and colonize aerial tissues that develop from infected shoot apical meristems of the seedling and tillers. We present evidence that vegetative hyphae of Neotyphodium and Epichloë species infect grass leaves via a novel mechanism of growth, intercalary division and extension. Hyphae are attached to enlarging host cells, and cumulative growth along the length of the filament enables the fungus to extend at the same rate as the host. This is the first evidence of intercalary growth in fungi and directly challenges the centuries-old model that fungi grow exclusively at hyphal tips. A new model describing the colonization of grasses by clavicipitaceous endophytes is described.

Molecular and cytogenetic evidence for an allotetraploid origin of Trifolium dubium (Leguminosae).

Suckling clover, Trifolium dubium Sibth., is a European grassland legume that has spread to many parts of the world. The present work shows that it is an allotetraploid (2n = 4x = 30) combining the genomes of T. campestre Schreb. (2n = 2x = 14) and T. micranthum Viv. (2n = 2x = 16), two diploid species of similar geographic distribution. T. dubium has two nuclear ITS sequences that closely match those of T. campestre and T. micranthum. Genomic in situ hybridisation using genomic DNA of T. campestre and T. micranthum as probes has differentiated the ancestral sets of chromosomes in T. dubium cells. Comparative fluorescence in situ hybridisation analyses of 5S and 18S-26S rDNA loci were also consistent with an allotetraploid structure of the T. dubium genome. A marked preponderance of ITS repeats from T. campestre over those from T. micranthum indicated that concerted evolution has resulted in partial homogenisation of these sequences by depletion of the T. micranthum-derived 18S-26S rDNA repeats. In parallel with this, the epigenetic phenomenon of nucleolar dominance has been observed in T. dubium such that the chromatin associated with the 18S-26S rDNA loci derived from T. campestre is decondensed (transcriptionally active), whilst that from T. micranthum remains highly condensed throughout the cell cycle. T. dubium, therefore, appears to have arisen by way of hybridisation between forms of the diploid species T. campestre and T. micranthum accompanied by chromosome doubling. The observed genomic changes in rDNA resulting from interspecific hybridisation provide evidence for the process of genome diploidisation in T. dubium.

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.