Bulked segregant AFLP analysis to identify markers for the introduction of the rhizomatous habit from Trifolium ambiguum into T. repens (white clover)

Bulked segregant analysis (BSA) using the technique of amplified fragment length polymorphism (AFLP^TM) was carried out on backcross hybrids between the important pasture species white clover and its relative Trifolium ambiguum. The introgression of the rhizomatous trait fromTrifolium ambiguum, previously shown to be associated with enhanced drought tolerance but which takes eighteen months to manifest itself, was analysed in a series of third generation backcross families with white clover as the recurrent parent. Eleven families differed in the proportions of plants classified according to rhizome content. Three of these families were selected for BSA-AFLP based on the creation of separate DNA pools from plants with large amounts of rhizomes or no rhizomes. Following an initial screen with119 primer combinations, 53 combinations were selected on the basis of the number of clear, reproducible polymorphisms they produced. Polymorphic bands were found that distinguished between rhizomatous and non-rhizomatous bulks in the three families. However, only one polymorphic band was found that unambiguously reflected the presence of the rhizomatous trait in individual plants in these families. This polymorphic band was also shown to be present in the BC2 parent, the BC1 and the original T. ambiguum parent. Given the aneuploid nature of the BC2 plants this band would appear to be a chromosome-specific marker tightly linked to the locus controlling rhizome development. The identification of a consistent marker for the rhizomatous trait opens up the possibility of applying marker assisted introgression and thereby facilitating the rapid incorporation of this trait into diverse germplasm. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nitrogen Fixation in Hybrids of White Clover (Trifolium repens L.) and Caucasian Clover (Trifolium ambiguum M. Bieb)

Hybrids between white clover (Trifolium repens L.) and Caucasian clover (or Kura clover, Trifolium ambiguum M. Bieb) are a potential route for the improvement of drought tolerance and persistence in white clover. However, to be agronomically viable they must show no significant reduction in their potential for nitrogen fixation relative to white clover. A comparative study of growth rate and nitrogen fixation was carried out in flowing solution culture without a supply of mineral nitrogen to the plants. The two parental species and two generations of backcross hybrids, with white clover as the recurrent parent, were assessed. The growth rate and N content of T. ambiguum were significantly lower than those of the other lines. However, dry matter production, nodule biomass per plant and rates of fixation were similar in second‐generation backcross plants and white clover. The results suggest that the agronomic potential of this novel germplasm is not compromised by limitations with respect to nitrogen fixation.     

Rhizobium specificity of Trifolium ambiguum M. Bieb × T. repens L. hybrid clovers

Growth of full-sib families of an F₁ interspecific hybrid between Trifolium ambiguum M.Bieb and T. repens L, and two generations of backcross hybrids (BC₁F₂ and BC₂F₁) with T. repens as the recurrent parent, were compared to their parental species. Plants were grown in a N-free medium and inoculated with Rhizobium leguminosarum biovar trifolii rhizobia effective on T. ambiguum or T. repens. Hybridisation produced progeny that nodulated with rhizobia from either T. ambiguum or T. repens, but plant growth varied. Mean weights of T. repens and hybrids, particularly F₁ and BC₁F₂, were higher when inoculated with a mixture of strains isolated from field grown T. repens than with the New Zealand inoculant strain for T. repens. When inoculated with the mix of rhizobia from T. repens, mean weights were 242, 189, 132, 125, and 100 mg/plant for T. repens, BC₂F₁, BC₁F₂, F₁ and T. ambiguum, respectively. However, although the mean weight of BC₂F₁ lines was significantly less than T. repens, there was considerable variation in individual full-sib families indicating the potential to select within BC₂F₁ hybrids for high plant growth/symbiotic nitrogen fixation. The weight of T. ambiguumplants inoculated with the New Zealand inoculant strain for hexaploid T. ambiguum was similar to T. repens inoculated with the mix of rhizobia from T. repens (253 and 242 mg/plant, respectively). Mean fresh weights of F₁ hybrid plants were similar when inoculated with rhizobia for T. ambiguum or T. repens (125 and 130 mg/plant, respectively). However, weight of T. repens, BC₁F₂ and BC₂F₁ hybrids inoculated with rhizobia for T. ambiguum were all less than 90 mg/plant. There was a significant relationship between plant fresh weight and ethylene production. The results indicate that measuring weights of inoculated plants growing in N-free media is a rapid initial method of screening a range of plant germplasm for plant growth/symbiotic nitrogen fixation rates. This revised version was published online in August 2006 with corrections to the Cover Date.     

Trifolium ambiguum as a source of variation in the breeding of white clover

Summary Hybrids between T. ambiguum and T. repens were successfully established after ovule culture. The hybrids were male sterile but one of the hybrids produced a single seed when backcrossed to T. repens, while the other produced a significantly greater number of seeds. The majority of the backcross hybrids from the latter had 48 chromosomes, being produced from the fertilisation of unreduced gametes of the F1 hybrid by T. repens pollen. Regular meiosis in the BC1 hybrid resulted in the production of balanced gametes containing the full complement of T. repens chromosomes and the polyhaploid set of T. ambiguum chromosomes. There was no difficulty in establishing a substantial population of BC2 hybrids. The meiotic behaviour of the BC1 hybrid indicated that it should be possible to transfer genes from T. ambiguum into white clover T. repens. The significance of the results in terms of the use of alien variation in the improvement of white clover is discussed.     

Variation for reproductive and agronomic traits among T. repens × T. nigrescens third generation backcross hybrids in the field

Interspecific hybridisation with Trifolium nigrescens Viv. is a possible strategy to improve the reproductive potential of white clover (Trifolium repens L.). Following the development of a fertile F1 hybrid, three generations of backcrossing have been carried out usingT. repens as the recurrent parent. Vegetative characteristics, stolon growth and seed yield components of the backcross (BC) 2 and 3 generations, as well as the parental species were measured on spaced plants grown in the field. Leaf size and plant spread of the BC2 and BC3 generations were less than T. repens but there was no difference in plant fresh weight. Numbers of inflorescences per plant and florets per inflorescence of the backcrosses were greater than T. repens however this was not reflected indifferences in seed yield per plant asT. repens had more seeds per floret and per plant than the backcrosses. Differences in stolon length, the proportion of flowering nodes and the pattern of axillary bud development were observed between T. repens and the backcrosses. Significant variation among the BC 3 generation for vegetative and reproductive traits was observed. Individual plants among the BC 3 generation were identified that combine high forage yield, substantial inflorescence production and good fertility, and these will form the basis of further selection. This revised version was published online in August 2006 with corrections to the Cover Date.     

Quality characteristics of backcross hybrids between Trifolium repens and Trifolium ambiguum

Two generations of novel backcross hybrids between white clover (Trifolium repens L.) and the related species Trifolium ambiguum,with white clover as the recurrent parent, were analysed for forage quality characteristics. Water soluble carbohydrate (WSC) levels, nitrogen concentrations (%N), and dry matter digestibility (DMD) were recorded for the hybrids and parental species grown in deep soil bins with and without a companion grass. Half the experimental material was subjected to moisture stress by being left unwatered through the four-week period when measurements were made. Four such four -week cycles of drought were imposed. The first and second generation backcrosses had the highest values for WSC, but were lowest for N%. There was no difference between the different legume lines (parents and backcrosses) for DMD. No effects of either moisture stress or presence of a grass companion on quality traits were observed. The WSC and %N of the grass component differed significantly depending on which legume line it was grown with. The importance of these results for the potential use of these hybrids in agriculture is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparing mating designs to restore seed production of interspecific hybrids between Trifolium repens (white clover) and Trifolium uniflorum

Interspecific hybrids between Trifolium uniflorum and cultivated white clover (Trifolium repens) have highly useful characteristics for temperate pastoral systems derived from both parent species. However, the early hybrids (F₁ and BC₁) also have unacceptably poor seed production for commerce. This study analysed the basis for the poor seed production and investigated breeding strategies for overcoming the problem. The BC₁F₁ generation produced lower‐than‐expected numbers of heads per plant and seeds per floret. Backcrossing of selected hybrids to white clover corrected these deficiencies and created new variation. Seed numbers were also returned to near target levels by recurrent selection within the BC₁ generation. Thus, it was possible to retain a theoretical average of 25% of T. uniflorum genome and still achieve high seed production per plant. The BC₁F₂ and BC₂F₁ generations produced high seed numbers per plant, along with reasonable variation. Both of these second‐generation hybrid forms have high reproductive potential and should be the focus for the selection of the desired combinations of agronomic and seed production traits.     

Introgression as a Route to Improved Drought Tolerance in White Clover (Trifolium repens L.)

Backcross hybrids between the important forage legume white clover Trifolium repens , which is stoloniferous, and the related rhizomatous species T. ambiguum have been produced using T. repens as the recurrent parent. The drought tolerance of parents and two generations of backcross plants, possessing both stolons and rhizomes, was studied in deep soil bins in a glasshouse both as monocultures and in mixtures with perennial ryegrass. Soil moisture content, leaf relative water content, and leaf water potential were measured on plants subjected to drought cycles of 4 weeks duration and those watered normally and maintained at field capacity. Six cycles of drought, carried out over 2 years, showed that T. ambiguum and the first and second generation backcross hybrids maintained a greater leaf relative water content and higher leaf water potential than T. repens at comparable levels of soil moisture. The dry matter yield of T. repens was significantly reduced over each drought cycle compared to the watered treatments, while the yield of T. ambiguum increased over the 4-week cycle in both treatments. The yield of the backcross generations was intermediate between those of the two parental species. The implications of these results for white clover germplasm improvement programmes are discussed.     

Effects of inbreeding on nodal root system morphology and architecture of white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.)

Plants of white clover (Trifolium repens L.) cultivar Crau, a self-fertile Crau genotype, and nine generations of inbred progeny were raised in sand culture in a glasshouse experiment. Digital images of the root systems were made and root morphological characteristics were determined on all the plants. Root architectural parameters were measured on the Crau parent and the S₁, S₄, S₆, and S₉ inbred lines. The clover roots became shorter and thicker with inbreeding but the number of root tips per plant was unchanged. Root architecture (branching pattern) was largely unaffected by inbreeding. It is concluded that inbreeding white clover will lead to shorter, thicker roots, and reduced nutrient uptake efficiency compared with the parent clover. The degree to which these deleterious traits are overcome during the development of F₁ hybrids needs to be determined.     

Development and characterization of interspecific hybrids of Trifolium alexandrinum×T. apertum using embryo rescue

This is the first report on the development of interspecific hybrids between Trifolium alexandrinum and T. apertum using embryo rescue and characterization of F₁ plants. T. apertum was used as the male parent and T. alexandrinum as the female parent. Development of interspecific hybrids under natural conditions is not successful and so embryo rescue was attempted. Of the several combinations tried, pollination 2 days after emasculation and embryos rescued 11 days after pollination was found to be the best. For embryo culture, EC3 medium consisting of MS basal supplemented with 2.3 μM kinetin and 3% sucrose was used. Germinated embryos were transferred to LSP3 medium 25 days after inoculation wherein most of the cultures showed multiple shoots that were split and subcultured on RL1 medium for rooting. After hardening, about 75% of hybrids were successfully transferred to the field. The hybrids, in general, showed morphological traits intermediate between the two parents; however, a few hybrids showed better growth than either parent. Some F₁ plants were almost 3 weeks later in flowering than the female parent. Pollen fertility among these plants ranged from 78 to approximately 100%. Chromosomal associations at diakinesis and isozyme banding patterns for acid phosphatase (ACP), superoxide dismutase (SOD) and peroxidase also confirmed the hybrid nature of the plants.     

Development of breeding populations from interspecific hybrids between Trifolium repens L. and T. occidentale Coombe

Trifolium occidentale is a diploid wild relative of T. repens (white clover) with adaptation to dry, saline coastal habitats. Transfer of drought and salt‐tolerant adaptive traits to white clover could be potentially valuable if interspecific hybridization can be achieved efficiently and leads to fertile hybrid populations. To achieve hybridization, 4x plants of T. occidentale were generated. Efficient techniques for generation of 4x plants and their identification using dry pollen shape are described. Interspecific 4x F₁ plants were achieved without embryo rescue. F₂ populations and first backcross hybrids to white clover were also efficiently achieved. Although male and female fertility were lower than in white clover, they were adequate to produce large amounts of seed from small numbers of inflorescences. Thus, early generation interspecific hybrid prebreeding populations can be readily developed, opening the way for transfer of traits from T. occidentale to white clover.     

Intra-varietal variation for in vitro plant regeneration in the genus Trifolium

Summary Seedlings of Trifolium repens showed considerable variation with regard to the morphology and growth of their calli, and their ability for in vitro differentiation of shoots. One of the lines selected for regeneration in primary callus cultures also showed shoot formation from protoplasts. Somatic embryogenesis in callus cultures of T. pratense and T. arvense occurred only in selected seedling lines. This paper highlights the importance of screening a large number of plants within a cultivar of outbreeding species to achieve reproducible plant regeneration from tissue culture.     

Plant Regeneration from Protoplasts of Trifolium repens and Lotus corniculatus

Protoplasts were isolated from leaves of Lotus corniculatus and Trifolium repens and from cotyledons of L. corniculatus. These protoplasts divided and produced colonies. The plating efficiency of protoplasts of both species was improved when agarose was used as a supporting medium. Plants were regenerated more regularly and in larger numbers from colonies of L. carniculatus than T. repens. The use of a culture line of T. repens that had been selected for its response in culture markedly increased the proportion of protoplast-derived cultures which regenerated shoots. One regenerant of T. repens (P6) was analysed for morphological and cytalogical variation. This plant was abnormal and highly aneuploid with a wide range of chromosome numbers.     

The use of endosperm balance number for predicting gamete selection in complex polyploid interspecific Trifolium repens × T. nigrescens crosses

Endosperm balance number (EBN) was used to predict the outcome of crosses among a series of complex polyploid interspecific hybrids between Trifolium repens and T. nigrescens. Normal endosperm development, and hence seed‐set, requires a 2 : 1 maternal : paternal EBN ratio in endosperm. Based on their crossability, T. repens (2n = 4x = 32) and T. nigrescens (2n = 2x = 16) were assigned the same EBN of 4. This number was used to predict the relative success of different backcrosses and intercrosses. The method was further developed to successfully predict which gametes would effect fertilization when complex polyploid hybrids with uneven genomic constitutions (5x, 7x) produced mixtures of euploid and aneuploid gametes. This was achieved by allocating EBNs to all potential gametes of complex polyploids. Allocation of gamete EBNs enabled not only prediction of the success or failure of a cross, but also which gametes would be filtered to effect fertilization in complex crosses. This enhanced ability to understand the potential outcomes of complex crosses will increase the efficiency of plant breeding programmes that integrate secondary gene‐pools into economic species by interspecific hybridization.     

Nitrogen Fixation by Hybrids of White Clover (Trifolium repens L.) and Trifolium nigrescens

Development of hybrids between white clover ( Trifolium repens L.) and Trifolium nigrescens provides a novel route for genetically improving the reproductive capacity of white clover, provided the hybrids are agronomically viable, particularly with respect to N₂ fixation. A comparative study of growth and rates of N₂ fixation over 21 days was conducted with the parental species, F ₁ hybrids and backcross hybrids, in flowing solution culture, without a supply of mineral N to the plants. T. nigrescens was unable to fix N₂ in association with the strains of Rhizobium leguminosarum biovar. trifolii selected for inoculation. Rates of N₂ fixation per plant increased in the order T. nigrescens < F ₁ hybrid < T. repens < backcross 1. Specific rates of N₂ fixation (days 0–21) increased in the order T. nigrescens < F ₁ hybrid < backcross 1 <  T. repens . Dry matter production and nodule biomass per plant increased at a higher rate in backcross 1 hybrids than in T. repens. The results suggest that the potential for N₂ fixation by backcross 1 hybrids is at least as great as that by T. repens .     

Genetic variability in herbage and seed yield in selected half-sib families of berseem clover, Trifolium alexandrinum L.

Berseem clover Trifolium alexandrinum L. is an annual forage legume commonly grown in pure stands and in grass mixtures in the Medi-terranean basin. Six populations were naturally cross-pollinated in 1990 and 1991 by a half-sib breeding method. In 1992 and 1993. 54 half-sib maternal plants, six original populations, and six advanced populations were field evaluated for forage (short cycle, harvest made at seven or eight inlernodes: long cycle, harvested at flowering) and seed yield (no forage harvest) in experiments at the Forage Crop Institute at Foggia, Italy (typical Mediterranean location). Genetic variability, narrow-sense herilability, genetic and phenotypic correlations among forage and seed yield component trails were investigated. The genetic variance in dry matter among maternal half-sib populations in short cycle was 51% greater than in long cycle. Narrow-sense heritabilily was 35% higher in short cycle than long cycle for dry matter and 26% higher for seed weight than seed yield. The magnitude of the genetic variance components and genetic correlations suggested that selection among plants of maternal half-sib populations would be more effective for improving dry matter in short than in long cycle harvests. The selection applied in the study was not effective for increasing seed yield per se however, the trait may be increased by selecting indirectly for seed weight.     

Self-compatibility and heterosis in white clover (Trifolium repens L.)

Inbred lines of white clover (Trifolium repens L.) have been produced utilising the rare self-fertility (Sf) allele. Twenty-two lines of four distinct groups have been maintained through five generations of selfing by single seed descent. Fourteen lines were used in crosses to produce F¹s. Both parents and hybrids were analysed for a range of morphological characters. Positive heterosis for dry matter production was observed in half the hybrids. No other trait showed significant heterosis. The degree of heterosis appears to be related to the extent of variation in morphological characters between the parental lines, some combinations of lines yielding heterotic hybrids in all cases, others in none. The majority of the F¹ hybrids are superior in terms of dry matter production to eight control varieties. This revised version was published online in July 2006 with corrections to the Cover Date.     

Responses to divergent selection for adventitious root growth in red clover (Trifolium pratense L.)

Summary Red clover (Trifolium pratense L.) persistence is improved by the growth of adventitious roots from the crown. The effect of one cycle of divergent phenotypic selection for adventitious roots in red clover was evaluated and root and shoot traits including degree of flowering habit correlated. Three classes of single-cross progenies were produced by crossing plants within and between the high and low root selections. Parents and progeny were grown as spaced plants. Adventitious root score and volume were significantly correlated (p<0.05); however they generally had low or nonsignificant correlations with other variables. A significant correlation (r=-0.36, p<0.01) was found between adventitious root score and flowering score (growth type) in 1988 but not in 1989. Significant variation was found among the three classes of single-cross progeny for adventitious root score and crown diameter. The effect of progeny class on score of adventitious roots was not linear, suggesting dominance effects for the absence of adventitious roots may have been present. A narrow-sense heritability estimate of 0.30 was found by midparent offspring regression of the adventitious root score. This low heritability value implies that successful selection would require progeny testing.     

Characterization of novel inbred lines of white clover (Trifolium repens L.). I. Dynamics of plant growth and nodule development in flowing solution culture

Growth, dry matter partitioning between shoots and roots, and extent of nodulation were characterised in four novel self-fertile highly inbred lines (referred to as A, B, C and D) of white clover (Trifolium repens L.) thought to differ fundamentally in their nitrogen relations and therefore regarded as candidate material for genetic mapping. Plants were inoculated with a mixture of three strains of Rhizobium and grown in flowing nutrient solutions without N for 18 d. Half the plants were then supplied with 20 μM NO₃ ^- during a 36 d treatment period, the remaining plants acting as ‘controls’ solely dependent upon N₂ fixation for acquisition of N. Total dry matter production and shoot:root ratios were similar in all lines supplied with NO₃ ^- and in control plants of lines B and C. Growth was severely reduced in control plants of line D and to a lesser extent in control line A. These effects were attributed to a failure to develop effective N₂ fixation capacity after apparently normal infection. Mean nodule size, nodule numbers and nodule d.w. per plant were extremely low in line D compared with the other lines, irrespective of whether NO₃ ^- was supplied. Lines A, B and C differed in the severity with which NO₃ ^- decreased mean d.w. per nodule and total nodule d.w. per plant relative to corresponding control plants, with line C being least sensitive. Nitrate also decreased the total number of nodules per plant relative to control plants after day 14 in all lines except C, and most severely in line B. This revised version was published online in August 2006 with corrections to the Cover Date.     

The characteristics of European,Mediterranean and other populations of white clover (Trifolium repens L.)

Indigenous populations of white clover (Trifolium repens L) collected from Europe, the Mediterranean area, as well as other parts of the world have been grown as spaced plants at Aberystwyth. Data were collected on various morphological characters, green weight and leaf marks.Leaflet length, width, area, plant height and green weight were all strongly correlated, but not the flowering date or leaflet shape (= ratio length/width).The mean leaflet size of Mediterranean populations (the supposed centre of origin) was very variable, and decreased northwards. All British populations were uniformly small leaved.Most of the populations were much less vigorous than the bred control varieties and only a few equalled them. Nevertheless some of the introductions exhibited seasonal growth patterns, especially growth into the autumn, and large leaves that could be usefully incorporated in improved varieties. Populations originating one step back towards the centre of origin will probably be the most useful when immediate adaptation is the aim.