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.

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

Phenotypic (rp), genotypic (rg), genotype × location (rgl) and error (re)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 rg 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.

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

The study described in this thesis focuses on a quantitative understanding of the complex interactions in binary mixtures of perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) under cutting. The first part of the study describes the dynamics of growth, production and the structural characteristics of contrasting grass and clover cultivars under field conditions. This basic information is used in the second part to quantify light absorption, C0 2 assimilation, radiation use efficiency (RUE) and light competition of the species using a modelling approach. Both species showed a seasonal pattern in growth of the dry matter (DM) and leaf area index (LAI) during the season so that the grass-dominated swards during spring shifted to clover dominance in summer. Without N fertilisation (-N), this seasonality was mainly controlled by the weather conditions. However, in fertilised mixtures (+N) grass was the dominant component of the mixture during the whole season and clover growth was always limited by light. The competitive ability and persistence of clover were determined by the structural characteristics of the cultivars. In the -N swards, both large and small-leaved clover cultivars had a higher proportion of their leaf area at the top canopy layers than their companion grass. In the +N mixtures, this was observed only in largeleaved clover, whereas the small-leaved cultivar was strongly overtopped. The difference between cultivars; was mainly due to the pattern of allocation of the DM into the supporting tissues. Experimental results showed two sources of vertical heterogeneity within the mixed grass-clover canopies: different patterns of LAI distribution and leaf dispersion. The validity of the canopy light partitioning model was considerably improved by introducing these sources of variation into the model. Using this model the RUE of species was calculated for different regrowth periods. Grass and clover had a different RUE in mixture and monoculture, but overall RUE was higher in grass, particularly in spring. The variation in the DM yield of grass under different treatments was due to changes in RUE and absorbed radiation. However, in clover these differences were mainly due to the amount of absorbed radiation. Quantification of light competition showed that in spring grass was the strongest competitor. In summer, the competitive ability of clover was related to N level and clover cultivar. Without N, both large and small-leaved clover were better competitors than grass. However, in the +N swards only the large-leaved clover had the same competitive ability as its companion grass. In both species a leaf N profile developed during regrowth, parallel to the light profile within the canopy. The effect of the observed compared to a uniform leaf N profile on canopy C02 assimilation of species was low, but it was different between mixtures and monocultures. It was concluded that the effect of canopy structure on productivity of species was more important than their assimilatory characteristics. The persistence of white clover under cutting may be improved by choosing cultivars with a higher competitive ability, based on canopy structure.

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Modelling interactions in grass-clover mixtures

Information on the variation available for different plant attributes has enabled germplasm collections to be effectively utilised in plant breeding. A world sourced collection of white clover germplasm has been developed at the White Clover Resource Centre at Glen Innes, New South Wales. This collection of 439 accessions was characterised under field conditions as a preliminary study of the genotypic variation for morphological attributes; stolon density, stolon branching, number of nodes. number of rooted nodes, stolon thickness, internode length, leaf length, plant height and plant spread, together with seasonal herbage yield. Characterisation was conducted on different batches of germplasm (subsets of accessions taken from the complete collection) over a period of five years. Inclusion of two check cultivars, Haifa and Huia, in each batch enabled adjustment of the characterisation data for year effects and attribute-by-year interaction effects. The component of variance for seasonal herbage yield among batches was large relative to that for accessions. Accession-by-experiment and accession-by-season interactions for herbage yield were not detected. Accession mean repeatability for herbage yield across seasons was intermediate (0.453). The components of genotypic variance among accessions for all attributes, except plant height, were larger than their respective standard errors. The estimates of accession mean repeatability for the attributes ranged from low (0.277 for plant height) to intermediate (0.544 for internode length). Multivariate techniques of clustering and ordination were used to investigate the diversity present among the accessions in the collection. Both cluster analysis and principal component analysis suggested that seven groups of accessions existed. It was also proposed from the pattern analysis results that accessions from a group characterised by large leaves, tall plants and thick stolons could be crossed with accessions from a group that had above average stolon density and stolon branching. This material could produce breeding populations to be used in recurrent selection for the development of white clover cultivars for dryland summer moisture stress environments in Australia. The germplasm collection was also found to be deficient in genotypes with high stolon density, high number of branches high number of rooted nodes and large leaves. This warrants addition of new germplasm accessions possessing these characteristics to the present germplasm collection.

Pattern analysis of the diversity of morphological plant attributes and herbage yield in a world collection of white clover (Trifolium repens L.) germplasm characterised in a summer moisture stress environment of Australia

A random sample of 60 germplasm accessions from the Australian white clover germplasm collection was characterized in the field at Glen Innes, N.S.W. Genotypic variation for stolon and other morphological attributes was measured in one season. Herbage yield was evaluated over seven seasons to quantify the magnitude of accession-by-season interaction. There were significant (P < 0.01) differences among accessions, seasons, and accession-by-season interactions for herbage yield. Classification was used to group the accessions on their seasonal herbage yield. A summer active group, a winter active group and a group showing autumn activity for herbage yield were identified. There was significant (P < 0.01) variation among accessions for all plant attributes measured and their accession mean repeatability was moderate to high. There were strong genotypic correlations among the morphological attributes. Five of the morphological attributes (stolon density, stolon branching, plant spread, plant height, stolon thickness and leaf length) expressed a strong genotypic correlation with average herbage yield over seven seasons. The genotypic variation estimated for herbage production and plant attributes signifies the potential of the collection to be used as a source of variation for the genetic improvement of productivity and perenniality of white clover for Australian environments.

Genotypic variation for stolon and other morphological attributes of white clover (Trifolium repens L.). Populations and their influence on herbage yield in the summer rainfall region of New South Wales

L. A. Brien

Papers

Genotypic variation for stolon and other morphological attributes of white clover (Trifolium repens L.). Populations and their influence on herbage yield in the summer rainfall region of New South Wales