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.     

Morphological characteristics of hybrids between white clover, Trifolium repens L., and Caucasian clover, Trifolium ambiguum M. Bieb

Hybrids between the stoloniferous Trifolium repens (2n = 4x = 32) and the rhizomatous Trifolium ambiguum (2n = 4x = 32) have been produced using T. repens as the recurrent parent. Morphological characteristics of the parent species and the F₁, BC1, BC2 and BC3 hybrids were examined in a glasshouse experiment. Leaflet ratios and general plant shape indicated that the BC3 hybrid was similar to T. repens. Separation of plants into components of above- and below-ground growth showed that T. repens had a greater total plant dry weight than T. ambiguum but 24% of its dry weight was in roots compared with 22% in roots and 44% in rhizomes in T. ambiguum. The BC3 generation contained plants that were predominantly T. repens-like with stolons, but also had a small proportion of their total dry weight (3%) as rhizome, confirming the potential to produce plants that combine stoloniferous and rhizomatous growth. The proportions of rhizome and stolon varied within the BC3 generation, enabling farther selection and plant breeding. The BC3 hybrids produced similar numbers of inflorescences to T. repens however, fertility was lower, although there was some variation within the BC3 for both characters.     

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.     

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.     

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.     

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.     

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.     

Occurrence of numerically unreduced (2n) gametes in Alstroemeria interspecific hybrids and their significance for sexual polyploidisation

The F1 hybrids of seven diploid Alstroemeria species (2n=2x=16) were investigated for the production of numerically unreduced (2n) gametes and their mode of origin. Based on a survey of 17 interspecific hybrid combinations,consisting of 119 genotypes, it was found that the F1 hybrids of Chilean-Brazilian species mostly produced first division restitution (FDR) 2n gametes. These F1 hybrids were self-pollinated in order to obtain F2 seeds, which was an indication that the F1 plants also produced 2neggs simultaneously. All the F2 progeny plants were typical allotetraploids, most of which formed 16 bivalents and a small proportion formed multivalents during metaphase I stages of meiosis. Through genomic in situ hybridisation (GISH) it was proved that multivalent formation in F2plants, derived from A. inodora ×A. pelegrina hybrid, was due to homoeologous recombination but not from reciprocal translocations. In order to test the segregation pattern of the recombinant chromosomes, an F3 population from one genotype, P6C49-6, was investigated. The recombinant chromosomes assorted independently from each other supporting the hypothesis that the segregation of chromosomes in ring quadrivalents did not behave like those in translocation heterozygotes. It was concluded that in allopolyploids of Alstroemeria,bilateral sexual polyploidisation could accomplish genetic recombination by both homoeologous crossing-over as well as through the assortment of chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. II. Morphology,fertility and cytology of the F1 hybrid

Summary F₁ hybrids of triazine resistant Brassica napus and triazine susceptible B. oleracea were morphologically intermediate to the parent species. Of 49 hybrids examined, 44 had 28 chromosomes, two had 37, one had 38 and two had 56. The 38-chromosome plant was thought to be a matromorph, the others, A₁C₁C (28), A₁C₁CC (37) or A₁A₁C₁C₁CC (56) type hybrids. Pollen stainability averaged 9.0% in the sesquidiploid, 32.0% in the tetraploids and 89.5% in the hexaploids. All the interspecific hybrids were resistant to 1.0×10^-4 mol L^-1 atrazine. The sesquidiploid hybrids produced gametes with chromosome numbers ranging from 9 to 17 and the tetraploid hybrid gametes had chromosome numbers from 15 to 22. Most hybrids produced self-seed. The partial fertility of these hybrids may permit their backcrossing to one or both parents.     

Inheritance of downy mildew resistance at cotyledon and adult-plant stages in ‘Couve Algarvia’ (Brassica oleracea var. tronchuda)

Parasitic nematodes damage white clover (Trifolium repens) roots, negatively impacting forage yield and persistence. No single gene resistance to nematodes has been identified in white clover. Trifolium semipilosum (2n = 2x = 16) genotypes exhibiting either complete resistance or susceptibility to infection by the clover root-knot nematode (CRKN), Meloidogyne trifoliophila, were identified. F₁ progeny (n = 92) of a pair-cross between ‘TsR’, a plant heterozygous for the resistance phenotype and ‘TsS’, a plant homozygous for the susceptible phenotype, were challenged with infective CRKN juveniles and evaluated subsequently for root galling. Segregation analysis indicated the resistance phenotype may be conferred by a single dominant allele at a locus (designated TRKR, Trifolium Root-knot Resistance) subject to segregation distortion. TsR, TsS, and bulked resistant and bulked susceptible F₁ progeny (n = 12/bulk) were screened using T. repens microsatellite (SSR) markers. Three SSRs revealed polymorphism in TsR and the resistant bulk, of which prs090 and prs247 map to loci on T. repens linkage group D2. Progeny were genotyped with these three SSRs and 23 additional SSRs from T. repens groups D1 and D2. Linkage analysis in both TsR and TsS demonstrated macro-synteny between T. repens group D homoeologues and the T. semipilosum linkage group (designated D_Ts) containing the TRKR locus. Significant segregation distortion was detected in TsR, and recombination in the central region of the T. semipilosum linkage group was suppressed relative to T. repens in both parents. These data demonstrate the opportunities and challenges for comparative mapping in Trifolium and characterisation of loci conferring resistance to plant-parasitic nematodes.     

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.     

Contribution of White Clover Varieties in High-Productivity Systems under Grazing and Cutting

Two medium leaf size varieties of white clover (Trifolium repens L.) were grown in mixture with a range of grass species and varieties in a field experiment conducted over 4 years. The clovers were grown singly or as a blend in swards with a tetraploid perennial ryegrass (Lolium perenne L.), a diploid Italian ryegrass (Lolium multiflorum L.) or tetraploid hybrids between these two species (Lolium x boucheanum Kunth). These grasses were also grown in monoculture. Swards were managed in a regime involving two periods of sheep grazing, two periods of cattle grazing and a silage cut in the same year for 4 years. The objectives of this experiment were 3‐fold: (1) to examine the yield and persistency of white clover in highly productive systems with modern perennial, hybrid and Italian ryegrasses; (2) to compare the performance of white clover varieties grown singly in mixture with grasses with their performance together as a blend, and (3) to examine the persistency of new Italian ryegrass varieties, exemplified by AberComo. The clover content of the sward remained high throughout the experiment, with a mean of 31 % overall. Differences in clover yield between plots were largely attributable to the effects of companion grasses. Total yields were very similar in plots containing different clover varieties and in plots where these varieties had been grown singly or as a blend. Grass yields in mixture were greater than those in monoculture but showed similar relative rankings. The diploid Italian ryegrass variety AberComo showed an unexpectedly high yield in the 4th year of the experiment. The implications of these results for strategies for productive, sustainable agriculture using modern white clover and ryegrass varieties are discussed.     

Nitrogen:sulphur ratio alters competition between Trifolium repens and Lolium perenne under cutting: Production and competitive abilities

Grasslands species, like others crops, no longer benefit from high atmospheric sulphur deposition. This may cause sulphur limitation and in turn may induce a shift in plant communities by altering species performance and competitive abilities. To test this hypothesis, a greenhouse pot experiment was designed to investigate the interacting effects of sulphur (S) and nitrogen (N) availability on production, morphology and competitive abilities of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.). Plants were grown in monocultures and mixtures during 4 months. They were supplied with combinations of three levels of S and three levels of N and subjected to three defoliation events. Both S and N gradients altered plants performance and modulated competitive interactions. In L. perenne, above- and below-ground dry matter production, leaf and tiller number and contribution to mixture DM production were little affected by S, but highly by N. For T. repens, these traits were significantly increased by S, but only slightly by N. At establishment, T. repens was subjected to a significant interspecific competition at low N–low S and high N–high S. But at regrowth (cuts 2 and 3), T. repens was much more affected by intra- than interspecific competition. L. perenne was only sensitive to competition (intraspecific) when N supply was combined with no or moderate S supply. We conclude that N:S ratio of soil appears to drive species production, morphology and competitive abilities, which in turn affect leguminous–gramineous species ratio and grassland plant community structure. Since the growth of T. repens was significantly reduced in low S treatments, our results suggest that the drastic fall in atmospheric sulphur deposition could restrict leguminous species in high N soil conditions.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. III. First backcross to parental species

Summary Atrazine resistant Brassica napus × B. oleracea F₁ hybrids were backcrossed to both parental species. The backcrosses to B. napus produced seeds in both directions but results were much better when the F₁ hybrid was the pollen parent. Backcrosses to B. oleracea failed completely but BC₁s were rescued by embryo culture both from a tetraploid hybrid (2n = 4x = 37; A₁C₁CC) and sesquidiploid hybrids (2n = 3x = 8; A₁C₁C). Progeny of crosses between the tetraploid hybrid and B. oleracea had between 25 and 28 chromosomes. That of crosses between the sesquidiploid hybrid and B. oleracea had between 21 and 27. A few plants that had chromosome counts outside the expected range may have originated from either diploid parthenogenesis, unreduced gametes or spontaneous chromosome doubling during in vitro culture. Pollen stainability of the BC₁s ranged from 0% to 91.5%. All the BC₁s to B. oleracea were resistant to atrazine.     

Spontaneous wheat/rye translocations from female meiotic products of hybrids between octoploid triticale and wheat

Summary Heptaploid hybrids between octoploid triticale and wheat were backcrossed as female parents with wheat to examine the rye chromosome distribution in the resultant progenies using genomic in situ hybridization (GISH). One hundred and one backcross (BC) seeds were examined and whole rye chromosome additions and substitutions, wheat/rye centric and noncentric translocations and rye telocentric chromosomes were detected. Dicentric wheat/rye translocated chromosomes were also observed. Comparisons were made with previous results on the rye chromosome distribution from male gametes of the same cross and differences were found, where in the female derived population a deficit of plants with more than two rye chromosomes was apparent relative to the anther derived population.     

Bulked AFLP analysis for the assessment of genetic diversity in white clover (Trifolium repens L.)

The use of bulked leaf samples from individual plants for amplified fragment length polymorphism (AFLP) analysis was evaluated as a tool for assessment of genetic diversity in white clover (Trifolium repens L.). Bulking of leaf samples produced slightly simpler AFLP profiles compared to the combined profiles of individual plants from the same cultivar. Approximately 90% of bands which were present in individual plants were present in bulked samples of the same cultivar. The majority of those absent were rare bands, shared by less than 25% of individual plants. Replicate bulk samples gave almost identical banding patterns, demonstrating the robustness of the bulked AFLP technique. Cluster analysis of AFLP data derived from individual plants resulted in a phenogram similar to that produced from data derived from bulked samples of the same plants. AFLP analysis of bulked samples detected significant amounts of genetic variability among 52 cultivars and accessions with genetic similarity values ranging from 0.42 to 0.92. However, cluster analysis of AFLP data only partially reflected the geographic origin of cultivars and accessions and was not congruent with cluster analysis based on variation for morphophysiological characters. Bulked AFLP analysis provides a powerful tool for rapid assessment of genetic variability in white clover and may also be used for cultivar identification. This revised version was published online in August 2006 with corrections to the Cover Date.     

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 .     

Independent segregation of two isozyme markers and inter-plant differences in nuclear DNA content in the interspecific backcross (Allium fistulosum L. × A. cepa L.) × A. cepa L.

Summary From backcrosses of three interspecific hybrids (A. fistulosum x A. cepa) with a. cepa 14 diploid and 2 triploid plants were recovered.In this BC1 population introgression of A. fistulosum genetic material into the A. cepa nuclear genome was studied using two isozyme markers: Got-1 and Got-2. Both loci carried two alleles. A. cepa was monomorphic for both markers. A. fistulosum was polymorphic for Got-2. Based on their Got isozyme pattern seven out of the 14 diploid BC1 plants had a recombinant genotype. The loci appeared to be unlinked. Differences were observed in nuclear DNA contents between the diploid BC1 plants, indicating that gametes produced by the interspecific hybrids contained different combinations of chromosomal material from A. cepa and A. fistulosum.Abbreviations Adh alcohol dehydrogenase - Got glutamate oxaloacetate transaminase - Idh isocitrate dehydrogenase - Pgi phosphoglucoisomerase - Pgm phosphoglucomutase