Andrew G. Griffiths

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.

An integrated genetic linkage map for white clover (Trifolium repens L.) with alignment to Medicago

BackgroundWhite clover (Trifolium repens L.) is a temperate forage legume with an allotetraploid genome (2n=4×=32) estimated at 1093 Mb. Several linkage maps of various sizes, marker sources and completeness are available, however, no integrated map and marker set has explored consistency of linkage analysis among unrelated mapping populations. Such integrative analysis requires tools for homoeologue matching among populations. Development of these tools provides for a consistent framework map of the white clover genome, and facilitates in silico alignment with the model forage legume, Medicago truncatula.ResultsThis is the first report of integration of independent linkage maps in white clover, and adds to the literature on methyl filtered GeneThresher®-derived microsatellite (simple sequence repeat; SSR) markers for linkage mapping. Gene-targeted SSR markers were discovered in a GeneThresher® (TrGT) methyl-filtered database of 364,539 sequences, which yielded 15,647 SSR arrays. Primers were designed for 4,038 arrays and of these, 465 TrGT-SSR markers were used for parental consensus genetic linkage analysis in an F1 mapping population (MP2). This was merged with an EST-SSR consensus genetic map of an independent population (MP1), using markers to match homoeologues and develop a multi-population integrated map of the white clover genome. This integrated map (IM) includes 1109 loci based on 804 SSRs over 1274 cM, covering 97% of the genome at a moderate density of one locus per 1.2 cM. Eighteen candidate genes and one morphological marker were also placed on the IM. Despite being derived from disparate populations and marker sources, the component maps and the derived IM had consistent representations of the white clover genome for marker order and genetic length. In silico analysis at an E-value threshold of 1e-20 revealed substantial co-linearity with the Medicago truncatula genome, and indicates a translocation between T. repens groups 2 and 6 relative to M. truncatula.ConclusionsThis integrated genetic linkage analysis provides a consistent and comprehensive linkage analysis of the white clover genome, with alignment to a model forage legume. Associated marker locus information, particularly the homoeologue-specific markers, offers a new resource for forage legume research to enable genetic analysis and improvement of this forage and grassland species.

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.

Marker-Trait Associations for Flavonoids and Biomass in White Clover (Trifolium repens L.)

White clover, a primary forage legume in temperate permanent pasture, is limited by poor adaptation to abiotic stress factors such as water scarcity. Flavonoids contribute to abiotic stress tolerance in plants. Genetic analysis of flavonoid accumulation may help in understanding its relationship to plant growth and morphology in white clover. The objective of this research was to discover marker-trait associations for biochemical and morphological traits previously identified as associated with drought tolerance, using clonally replicated white clover plants. Parents and 131 progeny of a bi-parental cross between the cultivar ‘Grasslands Kopu II’ (K2) and the ecotype ‘Tienshan’ (T) were genotyped with 104 microsatellite (SSR) markers and a Diversity Array Technology (DArT) assay, revealing 320 polymorphisms segregating from parent K2 and 208 from parent T. Markers on linkage group (LG) 1–2 were significantly (p < 0.005) associated with concentrations of the flavonols quercetin (Q) and kaempferol (K) and the Q:K ratio (QKR). A cluster of linked markers including prs406 accounted for 21 %, 167 % and 53 % change in Q, K, and QKR trait values, respectively. Polymorphic loci on LGs 6–1 and 7–1 in parent K2 influenced shoot and root dry matter. Loci on LGs 7–2 and 8–1 influenced root dry matter but not shoot dry matter. Root to shoot ratio was influenced by loci associated with markers from parent T on LGs 4–1 and 8–1. These results support a hypothesis that flavonoid metabolism is under close genetic control and largely independent of genetic factors influencing growth, suggesting that it might be possible to improve both abiotic stress tolerance and growth potential in white clover. These marker-trait associations revealed deleterious alleles in an elite cultivar, and indicate the potential value of diversity from wild germplasm for white clover improvement.