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.     

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.     

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.     

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.     

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.     

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.     

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.     

Development of high yielding IR64 × <Emphasis Type="Italic">Oryza rufipogon</Emphasis> (Griff.) introgression lines and identification of introgressed alien chromosome segments using SSR markers

Modern rice varieties that ushered in the green revolution brought about dramatic increase in rice production worldwide but at the cost of genetic diversity at the farmers’ fields. The wild species germplasm can be used for broadening the genetic base and improving productivity. Mining of alleles at productivity QTL from related wild species under simultaneous backcrossing and evaluation, accompanied by molecular marker analysis has emerged as an effective plant breeding strategy for utilization of wild species germplasm. In the present study, a limited backcross strategy was used to introgress QTL associated with yield and yield components from Oryza rufipogon (acc. IRGC 105491) to cultivated rice, O. sativa cv IR64. A set of 12 BC₂F₆ progenies, selected from among more than 100 BC₂F₅ progenies were evaluated for yield and yield components. For plant height, days to 50% flowering and tillers/plant, the introgression lines did not show any significant change compared to the recurrent parent IR64. For yield, 9 of the 12 introgression lines showed significantly higher yield (19–38%) than the recurrent parent IR64. Four of these lines originating from a common lineage showed higher yield due to increase in grain weight and another three also from a common lineage showed yield increase due to increase in grain number per panicle. For analyzing the introgression at molecular level all the 12 lines were analyzed for 259 polymorphic SSR markers. Of the total 259 SSR markers analyzed, only 18 (7.0%) showed introgression from O. rufipogon for chromosomes 1, 2, 3, 5, 6 and 11. Graphical genotypes have been prepared for each line and association between the introgression regions and the traits that increased yield is reported. Based on marker trait association it appears that some of the QTL are stable across the environments and genetic backgrounds and can be exploited universally.     

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.     

Genetic variation within and between two cultivars of red clover (Trifolium pratense L.): Comparisons of morphological,isozyme, and RAPD markers

Summary Morphological, isozyme and random amplified polymorphic DNA (RAPD) markers were used to estimate genetic variation within and between cultivars of red clover (Trifolium pratense L.), an important temperate forage legume. Two cultivars of red clover, Essi from Europe and Ottawa from Canada, were evaluated. Six monogenic morphological characters were observed for 80 plants from each of these two cultivars. All six morphological loci were polymorphic in the cultivar Essi whereas only four loci were polymorphic in the cultivar Ottawa. Forty plants from each cultivar were assayed for isozyme markers. A total of 21 enzyme-coding loci with 43 alleles was detected using twelve enzyme systems. Thirteen and nine of these loci were polymorphic in Essi and Ottawa, respectively. The mean number of alleles per locus was 1.81 in Essi and 1.67 in Ottawa. Seventeen random 10-mer primers were screened for RAPD markers. Nine primers which gave clear and consistent amplified products were used to assay 20 individuals from each cultivar. Each primer gave from 7 to 20 amplified bands with an average of 14.8 bands per primer. One hundred and eight of 116 putative loci were polymorphic in Essi and 90 of 98 loci were polymorphic in Ottawa. High within-cultivar variation was observed in both cultivars using both isozyme and RAPD markers. This high polymorphism makes these markers useful for germplasm characterization and genetic studies in red clover.     

A single dominant gene for downy mildew resistance in broccoli

Downy mildew, incited by Peronospora parasitica (Pers.: Fr.) Fr., is a destructive disease of broccoli (Brassica oleraceaL., Italica Group). Resistant cultivars represent a desirable control method to provide a practical, environmentally benign, and long-term means of limiting damage from this disease. Doubled-haploid (DH) lines developed by us exhibit a high level of downy mildew resistance at the cotyledon stage. To determine the mode of inheritance for this resistance, a resistant DH line was crossed to a susceptible DH line to make an F₁, from which F₂ and backcross (BC) populations were developed. All populations were evaluated for response to artificial inoculation with P. parasitica at the cotyledon stage. All F₁ plants (including reciprocals) were as resistant as the resistant parent, indicating no maternal effect for this trait. F₂ populations segregated approximately 3resistant to 1 susceptible, BC populations using the resistant parent as the recurrent parent contained all resistant plants, and the BC to the susceptible parent segregated 1 resistant to 1 susceptible. These results indicate that resistance is controlled by a single dominant gene. This gene should be easily incorporated into F₁ hybrids and used commercially to prevent downy mildew at the cotyledon stage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Influence of recurrent selection for flowering on flowering and yields in kura clover

Summary These investigations were conducted to determine the effectiveness of first- and second-season selection programs for increasing forage and seed yield in kura clover (Trifolium ambiguum Bieb.).Rhizo kura clover, the base population, was grown as spaced plants in two programs for six generations of selection for flowering in the seedling and second seasons. Remnant seed for each generation of selection was used to establish spaced plant and broadcast evaluation trials.The first-season selection program was effective in increasing flowering not only in the first season, but for two subsequent seasons. The second-season selection for flowering had little or no effect. However, vigor, forage and seed yields were not increased by either program, and the first-season program progressively reduced vigor and forage yields as generations of selection increased. The cause of this is unknown but may be associated with inbreeding depression or detrimental physiological effects of first-season flowering. Reselection to eliminate possible inbreeding effects is being conducted to further examine the possibility of increasing seedling and aftermath vigor in kura clover.     

Effects of Soil Moisture Deficit on Forage Quality, Digestibility, and Protein Fractionation of Kura Clover

Kura clover (Trifolium ambiguum M.B.) is a persistent rhizomatous forage legume with great potential for permanent pastures. The effects of a soil moisture deficit on forage quality, digestibility and protein fractionation of two cultivars of Kura clover (Endura and Rhizo) were investigated in this study for 1 year. The responses of alfalfa (Medicago sativa L.) and red clover (Trifolium pratense L.) were also characterized. Stands of each species were field‐grown and submitted to two soil water regimes promoting soil moisture deficits and well‐watered (i.e. control) conditions. There were no interactions between species and soil water regimes. Soil moisture deficit increased acid detergent fibre (ADF) but reduced acid detergent lignin (ADL) content and consequently increased forage digestibility. It had only minor effects on protein content and fractionation. Species varied for most parameters measured. Kura clover generally had the lowest neutral detergent fibre (NDF), ADF and ADL contents, and consequently the greatest digestibility (83.9 %). Species also differed in their crude protein fractionation. Kura clover and red clover had a lower proportion of non‐protein nitrogen (NPN; A fraction) and a greater proportion of true protein (B fraction) (30.9 and 64.5 %, respectively) than alfalfa (36.4 and 57.4 %, respectively). Kura clover also had the lowest proportion (i.e. 4.7 %) of acid detergent insoluble protein (ADIP; C fraction) of all species tested. Endura Kura clover often had a higher forage quality than Rhizo. This study confirms that Kura clover produces high‐quality forage and provides the first estimates of protein fractionation in this species.     

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.     

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     

Growth and reproductive characteristics in backcross hybrids derived from Trifolium repens L. × T. nigrescens Viv. interspecific crosses

Interspecific hybridisation with the close relative, Trifolium nigrescens (Ball clover) is a possible strategy to achieve increased reproductive potential of white clover (Trifolium repens). Fertile F₁ plants have been used as the basis for two generations of backcrossing to T. repens as the recurrent parent. F₁ and backcrossed plants were assessed in both glasshouse and field for a range of morphological traits, including inflorescence production, and the level of water soluble carbohydrates in the stolons. Plants resulting from two generations of backcrossing had an increased allocation of dry matter to inflorescence production in comparison with T. repens. Variation within these plants for agronomic traits (e.g. stolon length, dry weights etc.) suggests that selection for these traits is feasible and in combination with increased inflorescence production offers a potentially valuable approach to germplasm improvement in white clover. This revised version was published online in August 2006 with corrections to the Cover Date.     

AFLP analysis of introgression in coffee cultivars (Coffea arabica L.) derived from a natural interspecific hybrid

S.288 an offspring of a putative spontaneous interspecific hybrid between tetraploid Coffea Arabica (2n = 4x = 44) and diploid C. liberica(2n = 2x = 22) and 17 arabica coffee introgression lines (representing F₂ and F₄) derived from the cross S.288 x Kent arabica were evaluated for introgression of C. liberica genetic material. In all, 36 AFLP primer combinations were used in the analysis. The AFLP profiles of introgressed lines were compared to five accessions each of C. arabica and C. liberica. A total of 137 polymorphic bands were scored among the 29 accessions analysed. The introgressed genotypes exhibited 102 marker bands consisting of 65 additional bands and 37missing bands associated with introgression of C. liberica genetic material. C. liberica accessions of EA group (C. liberica var liberica of Guinean origin) seemed to be the likely progenitor in the origin of natural hybrid. Analysis of genetic relationships in the introgressed lines suggested that introgression was limited to few fragments. Segregation and wide variation in number of marker fragments in the F₂ and F₄progenies were attributed to chromosome recombinations. The differences in the level of introgression between introgressed parent, F₂ and F₄ groups was not pronounced. Therefore the alien genetic material appeared to be fixed and there was no elimination or counter-selection over generations, from introgressed parent to F₄.In C. arabica accessions, only 35 polymorphic bands were seen confirming the low genetic diversity. On the contrary, although representing a small amount of alien genome introgression, the Liberica-introgressed genotypes provided notable genetic diversity. Considering the fact that the diploid species of Coffea constitute a valuable source of genetic diversity, the potential implications of variability generated by Liberica-introgressed genotypes in C. arabica breeding are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Classification of a world collection of white clover cultivars

Summary Morphology, flowering, cyanogenesis and leaf markings of 109 white clover (Trifolium repens L.) cultivars, grown as spaced plants and in small plots, were measured. Principal component analysis and cluster analysis were used to compare cultivars. Cultivars were classified into four broad groups. The most important criteria for distinguishing between groups were leaf size, cyanogenesis and combinations of these. Group I, termed small, included small-leaved, prostrate cultivars; Group II, termed intermediate, included the majority of the cultivars which were characterised by medium sized leaves and relatively low cyanogenesis levels; Group III, termed large, included the large-leaved highly cyanogenic cultivars; and Group IV, termed ladino, included large-leaved acyanogenic cultivars.     

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 among elite red clover ( Trifolium pratense L.) parents used in Chile as revealed by RAPD markers

Red clover (Trifolium pratense L.) is one of the main forage species of temperate regions. Cultivars of red clover are heterogeneous which makes their genetic analysis difficult. We applied RAPDs (Random Amplifed Polymorphic DNA) in order to assess the genetic relationship and levels of genetic variability existing among a group of 16 elite red clover parents organised in four subsets of 4 parents each. Out of 55 primers 21 provided reproducible results. A total of 135 reliable and polymorphic RAPD bands were detected which were used to estimate genetic distances among pair-wise combinations of elite parents. Nei and Li's similarity values ranged from 0.60 to 0.77, with a mean of 0.66, which reflects a rather high genetic variability among the genotypes evaluated. Lower levels of genetic variability, as detected by polymorphic loci and mean heterogeneity values, were detected in a subset of parents selected for resistance to the stem nematode. Cluster analyses resolved the different sets of parents in a manner consistent with what is known from their breeding origins. An Analysis of Molecular Variance detected substantial levels of variation within subsets of parents. RAPDs represent a valuable source of genetic information for red clover breeding programmes. This revised version was published online in August 2006 with corrections to the Cover Date.