Ovule abortion in white clover (Trifolium repens L.)

Plants of white clover ( Trifolium repens L.) cv. Olwen were grown in an open glasshouse maintained at a mean temperature of 20^oC and ovule growth and seed production measured. Differences in the rate of growth of ovules within ovaries were observed as early as 2 days after pollination. Ovules reached a maximum size after 8 days with the smallest only half the size of the largest. After 8 days, the smallest ovules became flaccid and shrivelled. Ovule position within the ovary had little effect on the frequency of seed set and although there was an apparently higher probability that central ovules produced a seed than those nearer the peduncle or style this was not statistically significant. Inflorescence position and floret position on the inflorescence had a significant effect on the number of seeds per floret and seed weight; the first formed inflorescences and the first florets to be pollinated on each inflorescence had more seeds per floret and heavier seeds and fewer florets with no seed than later pollinated florets. There were also differences between florets within the same whorl. The role of a number of factors which may influence floret site utilisation are discussed.     

On the origin of the cyanogenic polymorphism in Trifolium repens L.

Trifolium repens L. and Trifolium nigrescens Viv. are two of the approximately six cyanogenic species known in the genus Trifolium. The two species are closely related: T. nigrescens is considered to be one of the diploid ancestors of the amphidiploid T. repens . We studied morphology, meiosis and the cyanogenic system in T. repens (amphidiploid), T. nigrescens (diploid) and their reciprocal hybrids. A comparison of the enzyme linamarase in the species and hybrids shows that there is a general resemblance between their linamarases. Immunological studies indicate that the linamarases must have a somewhat different three-dimensional structure. These facts are consistent with the view that T. nigrescens (or an ancestral form of this species) has donated the Li gene of T. repens . The other putative parent, T. occidentale has probably not donated an active Li gene. The hypothesis of the origin of the Li gene does not explain its polymorphism in European populations of T. repens , as T. nigrescens is monomorphic for cyanogenesis and amphidiploids do not segregate for genes which are homozygous dominant in one of the parents. Segregation for Li could be caused by a gene mutation or a small exchange between homeologous chromosomes. The latter event is more probable. A nigrescens-repens exchange would give rise to a chromosomal region with reduced homology to both parental chromosomes. The genes in the region of exchange will be tightly linked due to diminished cross-over frequency. It has been known for years that Li has effects on the vegetative and reproductive characters in T. repens and we have recently shown that these effects must be the result of genes linked to Li . As the associated characters influence the fitness of the cyanotypes, not only the origin but also the maintenance of the cyanogenic polymorphism is closely related to the evolutionary history of T. repens .     

Influence of boron on the reproductive growth of white clover (Trifolium repens L.) cultivars

The effect of boron on the reproductive growth of four contrasting white clover cultivars was examined in a controlled environment and glasshouse experiment. Plants grown under a nutrient regime excluding boron and those receiving 0.5 mg/litre boron had fewer stolons, reproductive stolons and inflorescences than those receiving 1, 2 or 3 mg/litre. Inflorescences were also smaller and had fewer seeds per floret. A boron concentration of 1 mg/litre gave optimum reproductive growth, higher levels of boron giving no further increase. Peduncle length and nectar quantity, but not nectar quality, were also influenced by boron indicating a possible beneficial effect of this element on pollinator activity. Cultivars differed in both vegetative and reproductive growth. Cv. Gwenda, a small leaved cultivar, had more stolons but fewer inflorescences than cv. Menna and smaller inflorescences than cv. Olwen, a large leaved cultivar. Generally, these cultivars responded similarly to boron. The implications of these results for the reproductive growth of white clover are discussed.     

Detection of selection in populations of white clover (Trifolium repens L.)

Seed populations of white clover polymorphic for the presence/absence of both ovariogenic glucosides and the hydrolysing enzyme linamarase, were introduced into three natural populations. Over the first six months of life a significant increase in the frequency of linamarase containing individuals occurred. Estimated selection coefficients against plants lacking linamarase were in the region of 0.3. This result may have been due to selection at the enzyme locus alone, or to selection favouring cyanogenic individuals which possess both cyanogenic glucosides and enzyme.     

Rapid evolution of an adaptive cyanogenesis cline in introduced North American white clover (Trifolium repens L.)

White clover is polymorphic for cyanogenesis (HCN production after tissue damage), and this herbivore defence polymorphism has served as a classic model for studying adaptive variation. The cyanogenic phenotype requires two interacting biochemical components; the presence/absence of each component is controlled by a simple Mendelian gene (Ac/ac and Li/li). Climate-associated cyanogenesis clines occur in both native (Eurasian) and introduced populations worldwide, with cyanogenic plants predominating in warmer locations. Moreover, previous studies have suggested that epistatic selection may act within populations to maintain cyanogenic (AcLi) plants and acyanogenic plants that lack both components (acli plants) at the expense of plants possessing a single component (Acli and acLi plants). Here, we examine the roles of selection, gene flow and demography in the evolution of a latitudinal cyanogenesis cline in introduced North American populations. Using 1145 plants sampled across a 1650 km transect, we determine the distribution of cyanogenesis variation across the central United States and investigate whether clinal variation is adaptive or an artefact of population introduction history. We also test for the evidence of epistatic selection. We detect a clear latitudinal cline, with cyanogenesis frequencies increasing from 11% to 86% across the transect. Population structure analysis using nine microsatellite loci indicates that the cline is adaptive and not a by-product of demographic history. However, we find no evidence for epistatic selection within populations. Our results provide strong evidence for rapid adaptive evolution in these introduced populations, and they further suggest that the mechanisms maintaining adaptive variation may vary among populations of a species.     

A comparison of ovule abortion in self-incompatible and self-fertile lines of white clover (Trifolium repens L.)

Plants of white clover (Trifolium repens L.) cv. Olwen and three genotypes of each of three self-fertile inbred lines of white clover were grown from seed in a bee-proof glasshouse. When in flower, a series of crosses within and between the self-incompatible (SI) and self-fertile (SF) lines was made and ovule growth and seed production measured 7 and 28 days after pollination (DAP) respectively. At harvest, the SF plants had a lower proportion of ovules aborting and more seeds per floret than the SI plants, though the mean seed weight of the SI plants was greater. Ovule abortion was observed in both the SF and SI plants though the number of aborted ovules was greater in the SI plants. The stage when abortion occurred also differed. All of the ovule abortion in the SI plants occurred within a 7-day period after pollination. In the SF plants, some abortion was observed in this period but also between 7 and 28 DAP, when the seed was harvested. This suggested that abortion, up to 7 DAP, is due primarily to genetic factors whilst subsequent abortion, between 7 and 28 DAP, is due to non-genetic factors.     

Effect of pollen competition and stigmatic receptivity on seed set in white clover (Trifolium repens L.)

The effect of competition between incompatible and compatible pollen grains on the seed production of white clover was studied. Stigmatic receptivity was also studied. A selection line of white clover that has red leaves was used as a pollen donor and as a genetic marker to allow determination of the pollen donor responsible for ovule fertilisation. Results show that incompatible pollen did not inhibit compatible pollen grains from fertilising ovules and producing seeds, although it did slightly impair seed yield. At temperatures of 20/10°C (day/night) stigmas of white clover remained receptive to additional pollination up to 32 h after an initial compatible pollination and 40 h after an incompatible pollination. The results imply that factors other than inadequate pollination and the self-incompatibility system are responsible for failure of white clover to attain its potential seed yield. Received: 31 August 1999 / Revision accepted: 28 February 2000     

Studies on the feeding of Sitona lineatus L. (Coleoptera: Curculionidae) on white clover (Trifolium repens L.) seedlings

Measurements of feeding damage by sitona weevil (Sitona lineatus L.) adults on differing numbers of seedlings of white clover (Trifolium repens L.) at the first and fourth trifoliate leaf stage were made in the glasshouse at 20°C. S. lineatus consumed more of the trifoliate component of the seedling. Sitona adults caused significant yield reduction at all levels of plant population. Total clover consumption increased with increasing size of sitona population, but consumption per adult weevil was reduced.     

Inheritance of phosphorus response in white clover (Trifolium repens L.)

Genotypes of white clover that exhibited divergent responses to P were identified in a glasshouse pot trial. Six high P-responding genotypes were selected from previously identified high P-responding cultivars and 5 low P-responding genotypes were selected from previously identified low P-responding cultivars. These were crossed in a full diallel design without selfing and reciprocals were kept separate. The P-response of progeny lines was compared with parents. High P-response was dominant over low P-response with progeny from crosses between high and low P-response genotypes being similar to the high P-response parent. Reciprocal effects were not significant. The general combining abilities of high P-response genotypes were generally greater than that of the low P-response genotypes, although there were significant specific combining abilities. Narrow sense heritabilities for P response were moderate, 0.46 based on the linear coefficient and 0.33 based on the quadratic coefficient of the fitted response curves.The mode of inheritance, feasibility of manipulating differences in P response by breeding and future directions of this work are discussed.Deceased.Deceased.     

Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs), can greatly accelerate breeding programs. The aim of this study was to develop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative trait loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA) _n repeats yielded 793 clones containing SSR loci. The majority of SSRs consisted of perfect dinucleotide repeats, only 7% being trinucleotide repeats. After exclusion of redundant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR loci and PCR products in the expected size range were amplified from 101 loci. These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four primer pairs were tested in an F₂ population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amplification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provide valuable tools for molecular breeding of white clover but may also have applications in related taxa. Received: 3 April 2000 / Accepted: 12 May 2000     

Response to soil aluminium of two white clover (Trifolium repens L.) genotypes

An aluminium (Al) tolerant genotype of white clover was compared with an Al susceptible genotype in artificial soil profiles in which exchangeable Al increased with depth. The tolerant genotype had a greater proportion of its root mass deeper in the soil than the susceptible genotype. Nitrogenase activity showed a similar pattern. Shoot Al concentration did not vary between the genotypes but root Al in the susceptible line was twice that in the tolerant genotype. Plant potassium content in the susceptible line was relatively less, probably in response to higher aluminium content.     

Effects of temperature and photoperiod on vegetative growth of white clover (Trifolium repens) ecotypes

Effects of photoperiod and temperature on vegetative growth of seedling populations and clones of white clover ( Trifolium repens L.) originating from various latitudes (58°48'–69°54'N) and altitudes (up to 1100 m above sea level) were studied in a phytotron. Dry matter production, stolon elongation, petiole elongation and leaf lamina size were enhanced by long photoperiod. The requirement for long photoperiod increased with decreasing temperature. At 6°C the maximum growth was recorded under 24-h daylength. At 18°C already an increase in photoperiod from 12 to 15 h significantly enhanced growth, and maximum growth was obtained at 21-h photoperiod. The studied populations responded similarly to daylength, and the results did not indicate photoperiodic ecotypes in the material. The southernmost clones and populations generally had the highest dry matter production at all temperature treatments (6–18°C). Variation between clones within one location was, however, significant, and rapidly growing clones were found also in high-latitude locations. Dry matter production was poorly correlated with the morphological characters observed, but in some cases significant correlation with leaf lamina size was found.     

Competition between cyanogenic and acyanogenic morphs of Trifolium repens

Summary To investigate the cost of the dimorphic cyanogenic system in Trifolium repens L., genotypes of the cyanogenic (T_c) and acyanogenic (T_a) morphs were grown in mixtures over a range of ratios and in pure stands at two levels of total density (low in a first experiment, high in a second experiment). The principles of the competition analysis employed were those related to the inverse linear model response. The morphs were compared using two interaction indices, the substitution rate and the relative resource total (RRT). The relative fitness of the two morphs, i.e. biomass and number of flowers per plant, suggests that the T_a morph has a competitive advantage over the T_c morph.     

Translocation of phosphorus from nodal roots in two contrasting genotypes of white clover (Trifolium repens)

Patterns of translocation of recently-assimilated phosphorus (P) exported from'young' source roots (located 3–4 nodes from the stolon apex) and 'old' source roots (located near the base of the stolon) on the primary stolon of clonal plants of the forage legume white clover ( Trifolium repens L.) were determined using ³²P. Plants of a small-leaved genotype and of a large-leaved genotype were grown in sand culture at two notionally limiting or near-limiting rates of P supply and one non-limiting rate of supply. The small-leaved genotype showed little response in growth rate to the full range of P treatments whereas growth of the large-leaved genotype at the non-limiting rate of P supply was 2. 4 times greater than at the two low rates of P supply. Source roots of both genotypes exported only 26–30% of the P they acquired to the shoot within 24 h when P supply was limited whereas at the high-P rate 54% of recently-assimilated P was exported. Patterns of translocation of exported P to specific sinks differed little between the genotypes and the P treatments; branches were the main sink, accounting for nearly 80% of the estimated amounts of P (μg day⁻¹) exported from young and old roots combined. Translocation patterns from individual roots were determined largely by the modular structure of plants and by the location of the root relative to the major sinks, and were therefore consistent with the same source-sink principles which govern carbohydrate translocation in clonally-growing species. There were strong suggestions that storage of P in stolons and roots played a much greater role in the growth of the small-leaved plants than of the large-leaved plants.     

An analysis of the costs and benefits of the cyanogenic system in Trifolium repens L.

Summary The effect of the cyanogenic glucosides linamarin and lotaustralin and their hydrolyzing enzyme linamarase was studied in a B₂ generation segregating for the genes Ac and Li. Plants containing the glucosides are protected against grazing by snails both in the seedling stage and as adult plants. In seedlings, however, there is a direct effect on survival, whereas in adult plants the leaf area of plants containing linamarin/lotaustralin is less reduced under intense grazing. Linamarase has no effect on grazing by snails, possibly as a result of the presence of -glucosidase activity in the gut of these animals. The genes Ac and Li, or genes tightly linked to them, have other effects as well: plants possessing one dominant Ac allele produce fewer flowers than homozygous ac plants. I compared this difference in flower production to the metabolic cost of producing the cyanogenic glucosides. The energy content of the difference in flower head production far exceeded the metabolic cost of cyanoglucoside production in Acac plants. It is possible that the cost of maintaining a certain level of cyanoglucosides is much more important for the plant than the initial cost of biosynthesis. The importance of the effects of Ac and Li in the maintenance of cyanogenic polymorphism in white clover is discussed.     

Nitrogen relations of a low nitrate uptake inbred line of white clover (Trifolium repens L.)

The nitrogen relations of an inbred line of white clover (Trifolium repens L.) thought to exhibit an abnormally low capacity for NO₃ ^– uptake (line LNU) were compared with a line regarded as normal with respect to NO₃ ^– uptake (line NNU). Growth, nodulation, N₂ fixation and NO₃ ^– uptake were measured over 7 weeks in flowing solution culture (Experiment 1) by plants dependent for N acquisition on either (i) NO₃ ^– uptake, (ii) NO₃ ^– uptake +N₂ fixation, or (iii) N₂ fixation only. Effects of plant N status on the short-term uptake and translocation of ¹⁵NH₄ ⁺ and ¹⁵NO₃ ^– were also investigated (Experiment 2). Nitrate uptake per plant by –fix/+NO₃ ^– line LNU was 50% of uptake by line NNU over 35 days, and there were significant differences in specific uptake rates of NO₃ ^– between the lines over the first 24 days. The `low NO<sub>3</sub> <sup>–</sup> uptake' phenotype was indistinct under +fix/+NO<sub>3</sub> <sup>–</sup> treatment. Nitrate lowered specific rates of nitrogen fixation by line NNU but had no effect on line LNU. Only low N status line LNU plants had lower short-term rates of NH<sub>4</sub> <sup>+</sup> and NO<sub>3</sub> <sup>–</sup> uptake than line NNU. It is concluded that the `low NO₃ ^– uptake' phenotype of line LNU is inconsistently expressed. Circumstantial evidence points to increased NO₃ ^– efflux and decreased xylem translocation of NO₃ ^– as possible explanations for the lower NO₃ ^– uptake by line LNU.