Warren M. Williams

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.

Nutrient responses and macronutrient composition of some Trifolium repens×Trifolium uniflorum interspecific hybrids

Abstract. Interspecific hybridisation is being utilised in white clover (Trifolium repens L.) breeding programs to overcome factors currently restricting productivity and persistence. Valuable new traits that may be introduced from the wild relative T. uniflorum include root characteristics and other adaptations to its natural, Mediterranean habitat. This study examined the effect of hybridisation on growth and macronutrient composition of white clover compared with T. uniflorum and T. repens × T. uniflorum backcross 1 (BC1) hybrids in two glasshouse sand culture experiments. Shoot and root dry weights of BC1 hybrids were greater than of white clover in low-concentration nutrient treatments but not in a more concentrated treatment. Decreases in dry weight with decreasing nutrient treatment strength were also smaller for some BC1 hybrids compared with white clover and other hybrid families. Most foliar macronutrient levels were adequate for white clover growth, but mean shoot or leaf phosphorus (P) concentrations were below published critical levels. Higher dry matter production under these low internal P concentrations suggests that some T. repens × T. uniflorum BC1 hybrids may be more tolerant of lower soil P levels than white clover. Such adaptations are likely to have been inherited from T. uniflorum. However, transgressive segregation may also be occurring, as T. uniflorum was larger than white clover in some, but not all, cases of low nutrient supply.

Drought resistance of Trifolium repens × Trifolium uniflorum interspecific hybrids

Abstract. White clover (Trifolium repens L.) is a widely used and highly valued temperate legume; however, its productivity and survival are restricted under dryland and drought conditions. This study investigated whether drought resistance of white clover could be improved by interspecific hybridisation with Trifolium uniflorum L. After almost 4 months without irrigation in a rain-shelter facility, shoot dry weight (DW) decreased significantly less in first-generation backcross (BC1) hybrids (–47%) than second-generation backcross (BC2) hybrids (–68%) and white clover (–69%). Stolon morphological parameters such as internode length and leaf lamina area also decreased less under water stress in the BC1 hybrids than in BC2 and white clover. There was also lower senescence in BC1 under water stress than in the other clover types. Genotypes with smaller changes in leaf lamina area, internode length, senescence and lateral spread had smaller changes in shoot DW, and there were significant correlations between constitutive levels of some characteristics and the effect of water stress on shoot DW. Under water stress, the growth form of the BC1 hybrids was compact, dense and prostrate, whereas white clover was more spreading and open. Increased allocation of dry matter to roots under drought, and greater root diameter, may also have influenced the ability of BC1 hybrids to maintain water uptake and key physiological processes. Overall, the data confirm that the drought resistance of white clover can be improved through hybridisation with T. uniflorum.

Epichloë fungal endophytes and the formation of synthetic symbioses in Hordeeae (=Triticeae) grasses

This review examines two classes of organism that live in symbiosis; grasses, and fungi. Specifically it deals with grasses of the tribe Hordeeae (formerly Triticeae) of the subfamily Poöideae and the Epichloë fungi of family Clavicipitaceae. Epichloë endophytes, particularly asexual forms, have important roles in pastoral agricultural systems in the Americas, Australia, and New Zealand. Selected strains add value to some grass‐based forage systems by providing both biotic and abiotic stress resistance. The importance of cereal grasses such as wheat, barley, rye, and oats to human and animal nutrition and indeed to the foundation and maintenance of human civilization is well documented. Both organism classes, Epichloë endophytes and cereal grasses, are of great importance in their own contexts. Here, we seek to review these two classes of organism and examine the possibility of bringing them together in symbiosis with the ultimate goal of improving cereal production systems.

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.

Effect of hybridisation with Trifolium uniflorum on tap root survival in white clover

A field study was conducted to determine whether tap root survival of white clover could be improved by hybridisation with Trifolium uniflorum. Tap root fragmentation and percentage of surviving tap roots were measured in 13, 16 and 19–20 month old plants. There were no intact healthy tap roots in white clover or second backcross (BC2) hybrids (12.5% T. uniflorum genes) post 13 months, but these were still present in T. uniflorum and first backcross (BC1) hybrids (25% T. uniflorum genes). Survival of T. uniflorum tap roots was higher than BC1, BC2 and white clover—30% of plants had intact, healthy tap roots at 19–20 months. The BC1 generation (31%) also had higher tap root survival than BC2 (13%) and white clover (11%) at 13 months. Although improved survival was not expressed as strongly in older BC1 plants, tap root deterioration was slower than in white clover and BC2. There is potential for targeted selection of specific genotypes and traits to further increase tap root lifespan in BC1 hybrids as there has been no previous selection for root traits in this material. The relationships between root diameter, leaf size and persistence in T. uniflorum and hybrids may differ from those expected for white clover cultivars. Characteristics of nodal rooting would also be expected to play a part in longer-term productivity and persistence.

The mechanism of seed coat-imposed dormancy revealed by oxygen uptake in Chatham Island forget-me-not Myosotidium hortensia (Decne.) Baill.

ABSTRACT Chatham Island forget-me-not Myosotidium hortensia (Boraginaceae) is a monotypic genus endemic to the Chatham Islands of New Zealand. Myosotidium hortensia is threatened in its natural environment by introduced grazing animals and weeds. Further to this, the conservation and reintroduction efforts of M. hortensia are hindered by their dormant seeds. The dormancy is imposed by the seed coat but the underlying dormancy mechanisms are not well understood. Understanding the dormancy mechanisms in this species will help in revegetation efforts. This work aims to elucidate mechanisms by which the seed coat imposes dormancy in M. hortensia by determining the germination after manipulation of the seed coat and measuring changes in oxygen uptake in response to this manipulation. The structure of the seed coat was assessed using scanning electron microscopy and the presence or absence of lignin in the seed coat determined. As with other Boraginaceae, the seed coat of M. hortensia does not restrict water penetration into the seed. Oxygen uptake was 4.5 fold higher in decoated than in intact seed. There was no significant increase in oxygen uptake for the seeds when their seed coat had been pierced or pierced and resealed with vaseline. However, the germination rate, as measured by radicle emergence, was faster in these seeds. These findings, in addition to the structural analysis of the seed coat which revealed that the seeds have secondary thickening but no lignin, confirmed that mechanical constraint of embryo growth was preventing germination.

Seed production trait associations and inheritance in interspecific hybrids between Trifolium repens (white clover) and Trifolium uniflorum

Abstract. Trifolium repens L. (white clover) is an important component of temperate pastures, but its root morphology makes it vulnerable to drought and pest attack. T. uniflorum is a wild species, adapted to dry environments, with deep woody roots but poor vegetative growth and only 1–3 florets per inflorescence (head). Interspecific hybridisation to incorporate the drought tolerance and root characteristics of T. uniflorum into white clover led to primary hybrids (F1 and BC1) with poor seed production. Advanced-generation hybrids expressed high variation for almost all seed-production traits, and seed production responded to selection. To inform future breeding programs, trait associations and heritabilities were analysed. Numbers of heads per plant, florets per head and seeds per floret were important factors with moderate–high heritabilities. The derived traits, numbers of seeds per head, florets per plant and seeds per plant, expressed low–moderate heritabilities. No negative associations between seed production and root traits were found in the hybrids, nor were there any negative associations among head production, persistence and foliage production. Selection for improved seed-production traits should be effective without adversely affecting vegetative traits.