Terry P. T. Michaelson-Yeates

An SSR and AFLP molecular marker-based genetic map of white clover (Trifolium repens L.)

A framework genetic map of white clover (Trifolium repens L.) has been constructed using an F2 progeny set derived from the intercross of fourth and fifth generation inbred genotypes carrying a self-fertile mutation (Sf). White clover SSR (TRSSR) and AFLP markers were used to derive a map with 135 markers (78 TRSSR loci and 57 AFLP loci) assigned to 18 linkage groups (LGs) with a total map length of 825 cm. Sixteen of these LGs are presumed to correspond to the 16 chromosomes of the white clover karyotype. A limited number of multiple loci were detected. Substantial segregation distortion was observed for both mapped and unmapped loci, with a bias towards heterozygous types and a preponderance of distorted markers on certain LGs. This observation, along with a high degree of residual heterozygosity within the inbred parental genotypes, suggests that reduced individual fitness due to loss of heterosis is a major effect for white clover and will limit the applicability of F2 or backcross mapping strategies for this species. A core set of map-assigned co-dominant, single locus SSR markers has been defined for whole genome scans of genetic variability in white clover.

Latent and Active Polyphenol Oxidase (PPO) in Red Clover (Trifolium pratense) and Use of a Low PPO Mutant To Study the Role of PPO in Proteolysis Reduction

Polyphenol oxidase (PPO) activity in leaf extracts of wild type (WT) red clover and a mutant line expressing greatly reduced levels of PPO (LP red clover) has been characterized. Both latent and active forms of PPO were present, with the latent being the predominant form. PPO enzyme and substrate (phaselic acid) levels fluctuated over a growing season and were not correlated. Protease activation of latent PPO was demonstrated; however, the rate was too low to have an immediate effect following extraction. A novel, more rapid PPO activation mechanism by the enzymes own substrate was identified. Rates of protein breakdown and amino acid release were significantly higher in LP red clover extracts compared with WT extracts, with 20 versus 6% breakdown of total protein and 1.9 versus 0.4 mg/g FW of free amino acids released over 24 h, respectively. Inclusion of ascorbic acid increased the extent of protein breakdown. Free phenol content decreased during a 24 h incubation of WT red clover extracts, whereas protein-bound phenol increased and high molecular weight protein species were formed. Inhibition of proteolysis occurred during wilting and ensilage of WT compared with LP forage (1.9 vs 5 and 17 vs 21 g/kg of DM free amino acids for 24 h wilted forage and 90 day silage, respectively). This study shows that whereas constitutive red clover PPO occurs predominantly in the latent form, this fraction can contribute to reducing protein breakdown in crude extracts and during ensilage.

Individual and multi-environment combined analyses identify QTLs for morphogenetic and reproductive development traits in white clover (Trifolium repens L.)

White clover (Trifolium repens L.) is a key component legume of temperate pasture agriculture and an important target for molecular marker-assisted plant breeding. A genetic map of white clover has been used to assess genetic control of agronomically important traits that vary in the F2(I.4R×I.5J) mapping family. Phenotypic analysis was performed for a range of vegetative morphogenesis traits (such as leaf area, internode length, plant height and plant spread) and reproductive morphogenesis and development traits (such as flowering date, floral intensity and seed yield), with both spatial and temporal replication. A multi-environment combined analysis (combined analysis) has been performed for traits assessed across multiple experimental datasets in order to identify consistent genetic effects. Quantitative trait locus (QTLs) were detected for the majority of traits, and the locations and magnitudes of QTL effects were compared between individual and combined analyses. This molecular genetic dissection of agronomic traits in white clover provides the basis for equivalent studies in more complex populations, design of marker-assisted selection strategies and comparative genetics with model legume species. Selection for QTLs derived from the combined analysis will permit robust improvement of phenotypic traits over different environments.

Relationships between genetic distance measured by RAPD-PCR and heterosis in inbred lines of white clover (Trifolium repens L.)

The RAPD-PCR technique has been used to generate DNA amplification profiles from a set of advanced generation inbred lines of white clover ( Trifolium repens L.). The genetic distances between lines have been estimated and have been compared with the extent of positive heterosis for dry matter yield in hybrid crosses. The analysis clearly separated the four major groups (H, J, R and S), with little within group variation. Heterosis, previously shown to be positively correlated with the extent of phenotypic variation between parental lines, was greatest between lines from groups showing the least genetic distance. The lack of correlation between heterosis and genetic divergence is discussed in the context of results from other systems. Additionally, the data allow a comparison of the extent of phenotypic variation for a trait under selective pressure with the degree of variation at the DNA level detected with the RAPD technique.

Effect of genotype of Trifolium repens on mycorrhizal symbiosis with Glomus mosseae

Eason, W. R., Webb, K. J., Michaelson-Yeates, T. P. T., Abberton, M. T., Griffith, G. W., Culshaw, C. M., Hooker, J. E., Dhanoa, M. S. (2001). Effect of genotype of Trifolium repens on mycorrhizal symbiosis with Glomus mosseae. Journal of Agricultural Science, 137 (1), 27-36. Sponsorship: BBSRC

Characterization of novel inbred lines of white clover (Trifolium repens L.). I. Dynamics of plant growth and nodule development in flowing solution culture

Growth, dry matter partitioning between shoots and roots, and extent of nodulation were characterised in four novel self-fertile highly inbred lines (referred to as A, B, C and D) of white clover (Trifolium repens L.) thought to differ fundamentally in their nitrogen relations and therefore regarded as candidate material for genetic mapping. Plants were inoculated with a mixture of three strains of Rhizobium and grown in flowing nutrient solutions without N for 18 d. Half the plants were then supplied with 20 μM NO3- during a 36 d treatment period, the remaining plants acting as ‘controls’ solely dependent upon N2 fixation for acquisition of N. Total dry matter production and shoot:root ratios were similar in all lines supplied with NO3- and in control plants of lines B and C. Growth was severely reduced in control plants of line D and to a lesser extent in control line A. These effects were attributed to a failure to develop effective N2 fixation capacity after apparently normal infection. Mean nodule size, nodule numbers and nodule d.w. per plant were extremely low in line D compared with the other lines, irrespective of whether NO3- was supplied. Lines A, B and C differed in the severity with which NO3- decreased mean d.w. per nodule and total nodule d.w. per plant relative to corresponding control plants, with line C being least sensitive. Nitrate also decreased the total number of nodules per plant relative to control plants after day 14 in all lines except C, and most severely in line B.

Trifolium ambiguum as a source of variation in the breeding of white clover

SummaryHybrids 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.

Effect of pollen competition and stigmatic receptivity on seed set in white clover (Trifolium repens L.)

Abstract The effect of competition between incompatible and compatible pollen grains on the seed production of white clover was studied. Stigmatic receptivity was also studied. A selection line of white clover that has red leaves was used as a pollen donor and as a genetic marker to allow determination of the pollen donor responsible for ovule fertilisation. Results show that incompatible pollen did not inhibit compatible pollen grains from fertilising ovules and producing seeds, although it did slightly impair seed yield. At temperatures of 20/10°C (day/night) stigmas of white clover remained receptive to additional pollination up to 32 h after an initial compatible pollination and 40 h after an incompatible pollination. The results imply that factors other than inadequate pollination and the self-incompatibility system are responsible for failure of white clover to attain its potential seed yield.

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.

Characterization of novel inbred lines of white clover (Trifolium repens L.). II. Variation in N2 fixation, NO3- uptake and their interactions

AbstractN2 fixation, nitrate uptake, and tissue N levels werecompared in four novel self-fertile inbred lines (referred to as A, B, C andD) of white clover (Trifolium repens L.) believed to differ markedly intheir nitrogen relations. Plants were inoculated with a mixture of Rhizobiumstrains and grown in flowing nutrient solutions without mineral N for 18 dprior to supplying half the plants with 20 µM