Genetic variation in a Norwegian germplasm collection of white clover (Trifolium repens L.)

Phenotypic (r_p), genotypic (r_g), genotype × location (r_gl) and error (r_e)correlations for important agronomic characters were estimated for eleven Norwegian populations of white clover (Trifolium repens L.), originating from between 58^°52′N and 69^°30′N latitude, and from altitudes of 10 to 450 m a.s.l. The populations were evaluated in monoculture clonal field plots for two years at two locations in Norway. All pairwise correlations between foliage height, leaflet length, dry matter yield (DMY) and general performance were positive and significant. Winter survival was positively correlated with general performance, spring growth and DMY, but negatively associated with internode length, while seed yield showed a small positive genotypic correlation with foliage height. Considerable differences were, however, revealed among populations with regard to sign and magnitude of the estimated correlations. Within-population estimates of r_g were consistently positive between DMY, general performance and most other characters; between foliage height on the one hand and leaflet length, winter survival and seed yield on the other hand, and between internode length and leaflet length, indicating pleiotropy. Correlations involving other character-combinations varied substantially among populations, and imply presence of linkage and/or coadaptation. Path coefficient analysis was used to partition the genotypic correlations between some of the characters into direct and indirect effects. Path coefficients revealed that foliage height had the highest positive direct effect on DMY, followed by winter survival and internode length. Leaflet length, which showed a positive, significant simple correlation with DMY, exhibited a negligible direct influence, counterbalanced nearly completely by the positive indirect effect on DMY via foliage height. Positive indirect effects on DMY via foliage height were also found for winter survival and internode length. The coastal populations from North and Middle Norway, and a southern highland population showed the largest expected responses to phenotypic selection for most of the characters. The results demonstrate that there is enough genetic variation in adapted local populations to develop new improved cultivars adapted to high-latitude environments that combine upright growth habit with sufficient winter-hardiness and persistency. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cultivar variation for seed development in white clover (Trifolium repens L.)

Summary A controlled environment study was undertaken to clarify the factors responsible for poor seed set and to study seed development, ovule degeneration and seed abortion, both morphologically and cytologically, in three Japanese cultivars of white clover. Although the mean number of ovules per floret was 4.2–5.1, the average number of seeds per floret was found to be only 2.3–2.7. Microscopic examination of carpels from 0 to 28 days following floret maturity and pollination showed that 26–33% and 8–17% of the total seeds lost occurred within the first three days and the third through fifth day following pollination, respectively. Beyond this period occasional seed abortion was observed at all stages of seed development, but this represented a very small proportion (2–7%) of the total seeds lost.A stain clearing technique was used to examine the cytoplasmic state of the embryo sac in intact, unfertilized, mature ovules and embryos of the ovules at 3 and 5-day periods following pollination. It was found that 20–22% of unfertilized and matured ovules were sterile, suggesting that ovule degeneration before fertilization was the major cause for the high percentage of seeds lost within a 0 to 3-day period following pollination. Cytological observations revealed that abortion of developing seed was due to a sudden arrest in embryo growth and that the early development of the embryo of such aborting seed was normal. Either nutrient shortage or meiotic irregularities may be the cause for high ovule sterility or post-fertilization abortion of developing seeds.     

Selection for root hair length in white clover (Trifolium repens L.)

Summary Results are given which show that the root hair length of a white clover population can be increased and decreased by selection. An increase of 50 m in root hair length over the mean of an unselected population of the cultivar Tamar was achieved resulting in an 11% calculated increase in the volume of soil explored by root hairs. The realised heritability for selection in the long root hairs was 0.33, and for the short root hairs 0.44.     

Selection of a highly-regenerative genotype of white clover (Trifolium repens L.) and plant regeneration from protoplasts derived from this genotype

Summary Callus cultures were induced from hypocotyl sections of 24 varieties of white clover (Trifolium repens L.). The calli did not show any significant difference of growth among the varieties. After the calli has been transferred to three regeneration media, green-spot formation was observed on calli derived from some seedlings. Remarkable intra- and intervarietal variations in the emergence of green spots and some trends between the origin of varieties and the frequency of green spots were observed. In most cases, the green spots turned brown without showing further differentiation, and only two genotypes formed shoots. A callus from a seedling of the Swedish variety Undrom has sustained high levels of plant regeneration throughout 24 months of culture. Protoplasts derived from this selected genotype were divided into cell colonies. 8P (Kao, 1977) medium containing 0.5 mg/1 2,4-D and 0.5 mg/1 kinetin was the most suitable medium for inducing divisions in protoplasts. When subcultured into solid B5 medium, the colonies produced calli, which when transferred to a regeneration medium, formed shoots. This genotype is expected to a useful subject for genetic engineering of white clover.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2ip 6-, -dimethylallylamino purine - NAA -naphthaleneacetic acid     

The inheritance of the cold induction response with respect to floral initiation in white clover (Trifolium repens L.)

Summary The inheritance of the cold induction response in the UK cultivar of white clover Menna, was assessed in controlled environments.Only 50% of the population flowered without prior cold induction. The cold period increased the number of stolons which became reproductive.All families from the crosses between induction and non-induction requiring genotypes produced progeny lacking a cold requirement for flowering.Heritability estimates for the number of flowers per plant, reproductive buds per stolon and stolon number were high.The necessity of eliminating the cold induction requirement in potentially valuable white clover cultivars in relation to seed production at lower latitudes is discussed.     

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.     

Recurrent selection programs for white clover (Trifolium repens L.) using self-compatible plants. I. Selection of self-compatible plants and inheritance of a self-compatibility factor

Summary When white clover plants were self-pollinated, 75% of a 143-plant population did not set seed. Plants which did set small numbers of seed probably did so as a result of pseudo-self-compatibility. One highly self-compatible plant was selected from the breeding materials. The S₂ progeny of this plant was autogamous and genetically fixed for morphology and isozyme patterns. Genetic analyses of self-compatibility were conducted as far as the F₃ progeny. It was concluded from these data that self-compatibility was inherited in a simple Mendelian way and that plants homozygous for self-compatibility were obtained. There was no correlation between presence or absence of self-compatibility and growth vigor. The possibility of application of self-compatible plants to recurrent selection procedures is outlined and the utilization of self-compatible plants in white clover breeding programs is discussed.     

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.     

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.     

Effects of divergent selection on correlations between morphological traits in white clover (Trifolium repens L.)

The effects of two generations of divergent selection for stolon diameter and stolon amount on the pattern of phenotypic correlations between important agronomic traits in white clover (Trifolium repens L.) were studied. The effects of selection were: the proportions of the variances of traits under direct selection were considerably lower within selection lines than overall and this was associated with a loss of correlation between traits. Unselected traits showed higher proportions of variances remaining within the selection lines and shifts from non-significant to significant correlations from the plants overall to selection lines. The significance of these results and their relevance to strategies of germplasm improvement is discussed. This revised version was published online in July 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.     

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.     

Variation within and among Ladino white clover ecotypes for agronomic traits

Summary One set of 285 genotypes from three landraces and one natural population and a second set of 165 genotypes from two landraces and one natural population were evaluated in separate trials. Variation among populations was mostly accounted for by the difference between landrace and natural population genotype groups, the former showing features related to higher seed yield and short-term aerial dry matter (DM) yield and to lower persistence, cold tolerance and protein content. Compared to variance among ecotypes, that within ecotypes was never lower and it was at least four-fold higher for important traits such as aerial and total (aerial plus stolon) DM yield, stolon density, production of seed heads and cold tolerance. Landraces and natural populations showed similar levels of intra-population variation for all traits except cold tolerance, for which landraces tended to somewhat greater variation. Trait associations were less definite at the genotype level than what previously found in terms of mean values of the ecotypes. This finding and the relatively high broad sense heritability which was observed for all components of DM and seed yield can support the breeding activity on this clover. The level of variation and of heritability found for protein content do not suggest the routine evaluation of this trait in breeding programmes.     

Genetic variation among cultivars of red clover (Trifolium pratense L.) detected by RAPD markers amplified from bulk genomic DNA

Summary The use of random amplified polymorphic DNA (RAPD) markers obtained from bulked samples was investigated for cultivar identification in red clover. Pooled samples were examined in order to minimize variation within cultivars. To determine the appropriate number of individuals to include in the bulked samples representing each cultivar, DNA samples from two, three, four, five, ten and twenty individuals were pooled. Twenty was found to be an appropriate number of red clover individuals per bulk in order to amplify only the DNA sequences shared among most individuals in each cultivar. Fourteen 10-mer primers were used to amplify genomic DNA from combined leaf samples of 15 red clover cultivars from European, Japanese and North American origins. A total of 79 amplified products, of which 55 were polymorphic, was obtained. Cultivar-specific bands were observed with 13 primers. The amplification patterns obtained from two primers could distinguish all 15 red clover cultivars. Rogers' genetic distances for all 105 pairwise comparisons were calculated to evaluate relationships among these cultivars. Cluster analysis based on these genetic distances separated these 15 cultivars into three groups, with two of the groups consisting of a single Japanese cultivar each, while the third group included cultivars from European, North American, and Japanese origins.     

Factors affecting seed yield in white clover

Summary Eighteen clones of white clover chosen at random were examined for their seed setting possibilities. Large differences in the size of the inflorescence and the rate of fertilization were found among the clones, so that breeding strains of white clover with better seed-setting seems possible. The number of ovules per floret seemed not very variable.     

A study of dominance at the locus controlling cyanoglucoside production in Trifolium repens L.

Summary The Ac allele for cyanoglucoside production in white clover has been shown to be incompletely dominant and a dosage effect is indicated at this locus. The recessive allele, ac, when homozygous gives rise to absence of cyanoglucosides in the leaf tissue, whereas in heterozygous plants it has been shown to reduce the level of cyanoglucoside, to a level less than half the value found in Ac Ac plants.     

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.     

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.     

Investigation of yield and persistence of white clover using cluster analyses

Summary A visual growth score method was used to monitor the growth performance of 24 populations of white clover (Trifolium repens) over a three year period. The mean growth scores for each year were used as population x year data for cluster analyses. Cluster analysis methods based on a regression model where genotypes were grouped for similarity of both intercepts and slopes (Method 1) or slopes only (Method 2), were able to differentiate the persistent populations from those that showed a yield decline over time. The deviations from regression mean square was used as an indication of stability. Method 1 was able to cluster the high yielding persistent populations into a separate cluster, thus facilitating the selection of desirable populations. Deviations from regression identified the stable populations from the cluster. In combination with the simple growth score system, this analytical method is a powerful tool for evaluating a large number of breeding families of perennial pasture clovers and grasses for yield and persistence.