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Abstract Background Trifolium ambiguum occurs as a 2x, 4x, 6x polyploid series in W Asia. The 6x form is the most agronomically desirable, having strong rhizomatous spread and drought tolerance. These traits would be potentially very valuable if they could be transferred to white clover (T. repens) which is the most important agronomic clover species. However, to-date, no fertile interspecific hybrids with 6x T. ambiguum are available. Previously, 2x T. occidentale from W Europe has produced synthetic fertile hybrids with both 2x and 4x T. ambiguum and these were inter-fertile with white clover. Here we ask whether 2x T. occidentale can form fertile hybrids with 6x T. ambiguum and act as a genetic bridge to white clover and bring these species together as part of a common gene pool. Results Ten verified F1 (6x T. ambiguum x 2x T. occidentale) hybrids were produced by embryo rescue and seven were studied further. All four investigated for chromosome number were 2n = 4x = 32 and FISH confirmed the expected 21 T. ambiguum and 8 T. occidentale chromosomes. Hybrid fertility was extremely low but 2n female gametes functioned with white clover pollen to produce seeds. Derived plants were confirmed using FISH and were successfully backcrossed to white clover to produce partially fertile breeding populations. Conclusions Although T. occidentale and 6x T. ambiguum are widely separated by geography and ecological adaptation they have maintained enough genomic affinity to produce partially fertile hybrids. Inter-fertility of the hybrids with allotetraploid T. repens showed that T. occidentale can provide a genetic bridge between 6x T. ambiguum and white clover to produce plants with new phenotypes combining the traits of all three species. Use of this information should enable potentially valuable stress tolerance traits from 6x T. ambiguum to be used in white clover breeding for the first time.

Key message: The widely divergent species 4xTrifolium ambiguum and 2xT.occidentale are inter-fertile long after speciation (including polyploidisation) has occurred. Tri-species hybrids (T. repens × T. ambiguum × T. occidentale) have the potential to achieve introgression of stress resistant traits from both wild species into white clover. Trifolium ambiguum and T. occidentale are geographically, adaptionally and phenotypically contrasting species in the white clover section (Trifoliastrum) of the genus. T. ambiguum occurs as a high-altitude polyploid series (2x, 4x, 6x) in W Asia and NE Europe. T. occidentale is a diploid coastal species, occurring at sea level in W Europe. This study investigated hybridisation between 4xT. ambiguum and 2xT. occidentale and considered the significance of the hybrids for introgression breeding of white clover. Partially fertile F1 hybrids between 4xT. ambiguum and 2x and 4xT. occidentale were generated by embryo rescue. Hybrid plant morphology and fertility varied widely and hybrids generally expressed traits from both species. Advanced generation (F2–F5) 4x hybrids were highly fertile and constitute a new synthetic allotetraploid species. FISH analyses of 4x hybrids showed multivalent chromosome configurations with homoeologous associations between T. ambiguum and T. occidentale chromosomes. Crosses of the hybrids with T. repens produced fertile tri-species progeny. These very divergent species remain inter-fertile long after speciation (including polyploidisation) has occurred. Tri-species hybrids have the potential to achieve introgression of stress resistance traits from both wild species into white clover. [ABSTRACT FROM AUTHOR]

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Six dryland pastures were established at Lincoln University, Canterbury, New Zealand, in February 2002. Production and persistence of cocksfoot pastures established with subterranean, balansa, white or Caucasian clovers, and a perennial ryegrass-white clover control and a lucerne monoculture were monitored for nine years. Total annual dry-matter (10.0-18·5 t DM ha−1) and sown legume yields from the lucerne monoculture exceeded those from the grass-based pastures in all but one year. The lowest lucerne yield (10 t ha−1 yr−1) occurred in Year 4, when spring snow caused ungrazed lucerne to lodge and senesce. Cocksfoot with subterranean clover was the most productive grass-based pasture. Yields were 8·7-13·0 t DM ha−1 annually. Subterranean clover yields were 2·4-3·7 t ha−1 in six of the nine years which represented 26-32% of total annual production. In all cocksfoot-based pastures, the contribution of sown pasture components decreased at a rate equivalent to 3·3 ± 0·05% per year ( R2 = 0·83) and sown components accounted for 65% of total yield in Year 9. In contrast, sown components represented only 13% of total yield in the ryegrass-white clover pastures in Year 9, and their contribution declined at 10·1 ± 0·9% per year ( R2 = 0·94). By Year 9, 79% of the 6.6 t ha−1 produced from the ryegrass-white clover pasture was from unsown species and 7% was dead material. For maximum production and persistence, dryland farmers on 450-780 mm yr−1 rainfall should grow lucerne or cocksfoot-subterranean clover pastures in preference to ryegrass and white clover. Inclusion of white clover as a secondary legume component to sub clover would offer opportunities to respond to unpredictable summer rainfall after sub clover has set seed. [ABSTRACT FROM AUTHOR]

Liveweight production was quantified from six dryland pastures at Lincoln University, Canterbury, New Zealand, over an 8 year period. The experiment was a randomised complete block with six replicates. The pastures were established in autumn 2002 with cocksfoot (CF) and subterranean (CF/Sub), balansa (CF/Bal), white (CF/Wc) or Caucasian (CF/Cc) clovers, a perennial ryegrass/white clover (RG/Wc) control and a lucerne (Luc) monoculture. Liveweight (LWt) production from Years 2 (2003–04) to 9 (2010–11) was 7940 kg LWt/ha from the lucerne monoculture, 7270 kg LWt/ha from the CF/Sub pasture and 5680 ± 171 kg LWt/ha from CF/Bal, CF/Wc, CF/Cc and RG/Wc pastures. Annually, LWt production ranged from 443 kg/ha (CF/Wc pastures with hoggets grazing in spring in 2006–07) to 1285 kg/ha from CF/Sub pastures in Year 9 when ewes with twin lambs at foot grazed in spring. On average, grass-based pastures produced 64%–70% of their total annual LWt production in spring compared with 57% from lucerne monocultures. In spring, relationships between LWt and total spring DM yield showed that 7.3 ± 0.68 kg LWt/ha was produced per 100 kg total DM (R2= 0.87) grown in 5 of the 6 years under the hogget/weaned lamb grazing system. Relationships between LWt and metabolisable energy or N yield failed to improve this relationship, which suggests that DM yield from persistent improved pasture components was the major factor responsible for differences in LWt production from these optimally grazed pastures. [ABSTRACT FROM PUBLISHER]

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 F1 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 F1 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 F1 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. [ABSTRACT FROM AUTHOR]

Background and Aims: Using two parental clones of outcrossing Trifolium ambiguum as a potential model system, we examined how during seed development the maternal parent, number of seeds per pod, seed position within the pod, and pod position within the inflorescence influenced individual seed fresh weight, dry weight, water content, germinability, desiccation tolerance, hardseededness, and subsequent longevity of individual seeds. [ABSTRACT FROM PUBLISHER]

A single 8× F1 hybrid between Caucasian clover (T. ambiguum) and white clover (T. repens) was backcrossed to seven white clover plants to generate fertile 6× BC1 families. To determine the most useful combination of desired traits of both parental species, and to develop a breeding strategy for this population, subsequent generations were produced in various ways. Self‐pollination and intercrossing among BC1F1 families produced F2, and later, F3 and F4 generations. Second, third and fourth backcrosses to white clover produced BC2, BC3 and BC4 generations. All generations were compared with the parental species in a single experiment grown in sand. Expression of the desired below‐ground traits of Caucasian clover was best in the BC1 generation. By contrast, the best expression of the desired above‐ground white clover traits was shown by the BC2F1 generation. However, the BC2F1 was pentaploid and did not stably transmit its desirable features to subsequent generations. On the other hand, advancement in the BC1 from F1 to F4 revealed significant positive variation in most of the important traits and suggested that selection would be successful. A breeding strategy was developed based on the genetically stable BC1 generation, advancing to BC1F6 by selecting for increases in shoot dry matter (DM), stolon numbers, stolon growth, improved nodal rooting radius, larger numbers of flowers and high fertility and seed‐set. [ABSTRACT FROM AUTHOR]

The high nutritive value and persistence under a wide range of climatic and soil fertility conditions make Caucasian clover a potentially useful forage legume but there is little information about the performance of livestock grazing Caucasian clover/grass swards. This study compared liveweight gains of lambs grazing Caucasian clover/perennial ryegrass and white clover/perennial ryegrass swards on high fertility (Olsen P 20 mg L−1, SO4-S 12 mg kg−1) and low fertility (Olsen P 11 mg L−1, SO4-S 7 mg kg−1) soils from 1998 to 2001 in the South Island of New Zealand. Mean annual liveweight gains were 1178 kg ha−1 for Caucasian clover/perennial ryegrass and 1069 kg ha−1 for white clover/perennial ryegrass swards at high fertility compared with 1094 kg ha−1 and 1015 kg ha−1, respectively, at low fertility. There was a higher mean proportion of clover in Caucasian clover/perennial ryegrass (0·19) than white clover/perennial ryegrass (0·11) swards, but there were no differences in total herbage production between the two clover/perennial ryegrass swards. The mean concentration of crude protein in the herbage of Caucasian clover (302 g kg DM−1) was higher than that in white clover (287 g kg DM−1) and grass herbage (227 g kg DM−1). Estimated mean metabolizable energy concentrations in the herbage were 12·5 MJ kg DM−1 for the two clovers and 11·6 MJ kg DM−1 for grass herbage. The difference in liveweight gain between swards on soils of high and low fertility was associated with an increase in total herbage production of similar composition and nutritive value, giving a greater number of grazing days for the swards on soils of high than low fertility. [ABSTRACT FROM AUTHOR]

The effect of sowing date (SD) and sowing rate of perennial ryegrass (PRG) on the establishment of Caucasian and white clovers in New Zealand was assessed. Clovers were sown in spring on 24 September (SD1) and 9 November (SD2) 1999, and in autumn on 4 February (SD3) and 31 March (SD4) 2000. On each date, clovers were sown with 0, 3, 6 or 12 kg ha−1 of PRG. Total herbage dry matter (DM) production up to 6 November 2000 was 13–16 t DM ha−1 for SD1 and SD2 when sown with 3–12 kg ha−1 of PRG, and 7–10 t DM ha−1 for sown clover monocultures. For SD3 and SD4, total herbage production was 6–9 t DM ha−1 with PRG, while total herbage production of clover monocultures was 5·4 t DM ha−1 for SD3 and 2·6 t DM ha−1 for SD4. By 6 November 2000, white clover contributed proportionately more than 0·15 of herbage mass when sown with 3–12 kg ha−1 of PRG on SD1, SD2 or SD3, but less than 0·09 when sown on SD4. The proportion of Caucasian clover never exceeded 0·09 of herbage mass in any of the swards. White clover was successfully established in spring and in autumn with 3–12 kg ha−1 of PRG provided the 15-mm soil temperature was above 14 °C. None of the combinations of Caucasian clover and PRG provided an adequate proportion of legumes during the establishment year. This unsuccessful establishment of Caucasian clover with PRG was attributed to its inability to compete for available light as a seedling due to slow leaf area expansion from secondary shoot development and a high root:shoot ratio. Alternative establishment strategies for Caucasian clover may include the use of slow establishing grasses, cover crops and temporal species separation. [ABSTRACT FROM AUTHOR]

Seedling competition for resources during establishment affects the potential success of individual species within a pasture. Germination, emergence and leaf expansion are key characteristics that contribute to the competitive ability of species. In this study, development and growth characteristics of Caucasian clover, white clover and perennial ryegrass (PRG) seedlings were quantified. A base temperature of <4°C and an optimum temperature of ∼27°C were found for development in each species. Thermal time (Tt) requirements for 75% of final germination were lower for Caucasian clover (46°C d) and white clover (40°C d) than for PRG (76°C d), but Tt requirements for 50% of final emergence were similar (∼110°C d). The phyllochron (°C d leaf−1) for primary stem leaves was slower for Caucasian clover (109°C d) than for white clover (94°C d) and PRG (101°C d). Appearance of the first PRG tiller, which indicates the initiation of secondary leaf development, occurred after 373°C d, compared with 532°C d for the first white clover stolon. Caucasian clover crown shoots did not develop until >1180°C d. Consequently, white clover and PRG had more leaves (∼15 plant−1) and faster shoot relative growth rates (∼0·062 mg mg−1 d−1) than Caucasian clover (5 leaves plant−1, 0·049 mg mg−1 d−1). [ABSTRACT FROM AUTHOR]

Bulked segregant analysis (BSA) using the technique of amplified fragment length polymorphism (AFLPTM) was carried out on backcross hybrids between the important pasture species white clover and its relative Trifolium ambiguum. The introgression of the rhizomatous trait from Trifolium 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 with 119 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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. [ABSTRACT FROM AUTHOR]

Trifolium ambiguum M. Bieb and T. repens L. are taxonomically related but very difficult to cross. The rare hybrids so far reported between these two species were obtained only by embryo culture. This difficulty has been overcome in the present research by the creation of a “fertile bridge” between T. ambiguum and T. repens. Characters of interest can now be transferred from T. ambiguum to T. repens by using this “fertile bridge” without the use of sophisticated techniques. An array of backcross progenies was generated from crosses between a T. ambiguum× T. repens F1 hybrid (8 x H-435) and its parental species. The 8 x hybrid was cross-fertile only with T. repens and resulted in 145 seeds from 1578 reciprocal crosses. Eleven of nineteen initially grown BC1F1 plants were all hexaploid with an average pollen stainability of 41.6%. A high frequency of multivalents at metaphase-I indicated that both autosyndetic and allosyndetic pairing occurred. Backcrosses of 6 x BC1F1 plants to T. repens resulted in 5 x BC2F1 plants with an average pollen stainability of 59.3%. On the other hand, 6 x BC1F1×6 x T. ambiguum crosses did not produce any seed and only two pentaploid plants were obtained from 6 x BC1F1×4 x T. ambiguum crosses. The difficulty encountered in generating 6 x backcross progeny with 6 x T. ambiguum was overcome by intercrossing the 6 x BC1F1 plants and producing 6 x BC1F2 plants with an average pollen stainability of 65.8%. One of these 6 x BC1F2 plants was cross-compatible as a female with 6 x T. ambiguum and resulted in CBC2 plants that were all cross-compatible with 6 x T. ambiguum. The 6 x BC1F2 plants are likely to be superior to 6 x BC1F1 progeny, as they have exhibited better expression of the combined rhizomatous and stoloniferous growth habit, improved fertility, more frequent nodal rooting and heavier nodulation. Consequently, the 6 x BC1F2 plants can either be used directly in the selection programme or as a “fertile bridge” between the two parental species. The present work has resulted in the development of a series of fertile hybrids by the manipulation of chromosome numbers, combining the agronomic characteristics of the parent species in varying genome balances and at a range of ploidy levels. It is concluded that the initial sterility of the primary interspecific hybrids need not be a barrier to successful inter-breeding. [ABSTRACT FROM AUTHOR]