Nicholas W. Ellison

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 = 4x= 32). The basic repeating unit is an A+T rich 350 bp HindIII 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 HindIII restriction profile in seven species also indicated independent evolution of this repeat.

Mechanisms of glyphosate resistance in two perennial ryegrass (Lolium perenne) populations.

BACKGROUND Perennial ryegrass (Lolium perenne) has developed resistance to glyphosate within New Zealand vineyards following many years of herbicide application. The objectives of this work were to confirm resistance within two populations obtained from affected vineyards, and to determine the mechanism of resistance to glyphosate. RESULTS Population O was confirmed to have a 25-fold resistance to glyphosate, whereas population J had a sevenfold resistance. Results of genotyping assays demonstrated a single nucleotide substitution at codon 106 of 5-enolpyruvylshikimate-3-phosphate synthase in population O but not population J. Glyphosate-resistant and glyphosate-susceptible populations did not differ in glyphosate absorption. However, in both resistant populations, much more of the absorbed (14) C-glyphosate remained in the treated leaf than occurred in the susceptible population. Significantly more glyphosate was found in the pseudostem region of susceptible plants compared with resistant plants. CONCLUSION Both target-site and non-target-site mechanisms of glyphosate resistance were found in the perennial ryegrass population with 25-fold resistance, whereas only the non-target-site mechanism of resistance was found in the population with sevenfold resistance. This is the first study of the mechanism of glyphosate resistance in perennial ryegrass.

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

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.

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.

Development of Trifolium occidentale as a Plant Model System for Perennial Clonal Species

Trifolium occidentale D.E. Coombe is a diploid, clonal perennial clover that is very closely related to white clover (T. repens L.). It has been previously reported to be self-pollinating and lacking in genetic diversity. However, new collections, especially in Spain and Portugal, have revealed that cross-pollinating populations with substantial genetic diversity do exist. This has led to T. occidentale being investigated as a potential genetic model species to facilitate the application of genomic methods for the improvement of white clover. Investigations have shown that T. occidentale has many attributes that make it suitable as a genetic model for white clover. It forms hybrids with white clover and the chromosomes of the two species pair and recombine at meiosis. Phylogenetic research shows that it is a very close relative, and probably an ancestor, of white clover. A framework linkage map based on SSR markers has shown it to be highly syntenic with white clover. A protocol for efficient transformation has been developed. An effective EMS mutagenesis method has been demonstrated by the induction of a high frequency of condensed tannin negative mutants. The clonal nature of T. occidentale is not shared by other dicotyledonous model species. It may, therefore, be useful for the genomic characterisation of traits associated with clonal growth and perenniality in this wider class of plants.

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.

Genomic and Geographic Origins of Timothy ( Phleum sp.) Based on ITS and Chloroplast Sequences

The relationship among members of the subgenus Phleum was determined using nuclear ribosomal ITS and chloroplast trnL intron DNA sequences. This subgenus is derived from a progenitor of the diploid Phleum alpinum subsp. rhaeticum. The relationships provide evidence of migration, hybridization, polyploidy and speciation associated with historical glaciations.

A molecular phylogenetic framework for cocksfoot (Dactylis glomerata L.) improvement

Abstract. The recently completed molecular phylogenetic analysis of Dactylis germplasm has provided a clear evolutionary history of the diploid Dactylis from which modern tetraploid germplasm and cultivars have evolved. This framework will allow us to use fully a wider range of both diploid and tetraploid germplasm for a more systematic improvement of cocksfoot. Germplasm of many diploid and tetraploid forms is under serious threat from habitat degradation and climate change, and many forms are currently poorly represented in genebanks. It is critical that a wide range of these forms is collected for storage and conservation. It is also critical that core collections are developed and maintained, using molecular phylogenetic and genetic diversity information as the basic framework. In order to apply molecular resources in an effective and balanced manner, pragmatic field breeding programs need to be continued in all major regions. This is a major concern for cocksfoot, as it is a species with limited international breeding investment. Viable, large-scale, cocksfoot breeding programs must be maintained internationally to allow adequate cultivar development, ongoing germplasm collection, introgression from wild germplasm and application of molecular resources.

Remnant oak savanna acts as refugium for meadow fescue introduced during nineteenth century human migrations in the USA

In 1990, an unknown forage grass was discovered growing in the shade of a remnant oak savanna in southwestern Wisconsin. Over 12 years, the practice of feeding mature hay on winter pastures spread this grass onto over 500 ha via seedling recruitment. Analysis of amplified fragment length polymorphic (AFLP) markers on 561 plants, compared to a diverse sample of wild European collections of perennial ryegrass (Lolium perenne L.), Italian ryegrass (L. multiflorum Lam.), meadow fescue (Festuca pratensis Huds. = L. pratense (Huds.) Darbysh.), and tall fescue (F. arundinacea Schreb.), identified a highly diverse population that was more closely allied with F. pratensis than the other species, based on genetic distances. Genomic in situ hybridization (GISH), using both Lolium- and Festuca-specific probes, led to effective hybridizations by only the Festuca-specific probes and gave indications of close homology to the F. pratensis genome. Similarly, genetic distance analysis using PCR-based Lolium expressed sequence tag (EST) markers on a subset of genotypes, compared to the four control species, clearly identified F. pratensis as the closest relative. Sequence analysis of the trnL intron of cpDNA distinguished the unknown plants from F. arundinacea, but not from Lolium. Additional survey work has identified this grass on 12 other farms within an area of about 20,000 ha. Soil samples accompanying plant samples indicated no seed banks and most farm records indicate no commercially introduced seeds during the twentieth century. We hypothesize that seeds of meadow fescue may have arrived with some of the earliest European immigrants to Wisconsin and spread along the historic Military Ridge Trail, a network of frontier U.S. Army forts connected by a major thoroughfare.

A Molecular Phylogenetic Framework for Timothy (Phleum pratense L.) Improvement

The recent molecular phylogenetic analysis of Phleum germplasm has provided a clear evolutionary history of the genus from which modern hexaploid germplasm and their cultivars have evolved. This framework will allow us to effectively use the full set of germplasm from all ploidies (2x, 4x, 6x and 8x) for a more systematic improvement of Timothy. The many new molecular forms of Phleum now known offer a huge potential to expand the gene pool of commercial hexaploid Timothy. The opportunity exists to hybridize and incorporate many new forms of molecular diversity into Timothy. However, genebank samples of many of these new forms are absent and urgently require collection. In addition, many of the novel diploid, tetraploid, hexaploid and octoploid forms are under serious threat from habitat degradation and climate warming in situ. It is also critical that core collections are developed and maintained using molecular phylogenetic and genetic diversity information as a basic framework. In order to apply molecular resources in an effective and balanced manner, it is important to ensure pragmatic field breeding programmes are continued in all major regions. This is a concern for Timothy, as it is a species with limited international breeding investment.