Investigation of the possibility of introgression from Trifolium ambiguum M. Bieb. into T. repens L. : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Breeding and Genetics in the Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand

Abstract

The objective of this project was to investigate the possibility of introgression of stress resistance traits from T. ambiguum (A) into T. repens (R) by interspecific hybridisation, using two approaches. The first used T. occidentale Coombe (O) as a genetic bridge because this species has chromosome pairing homology to both the two other species. The second approach attempted direct integration of genomes from the two species through ploidy manipulation. For the first approach, four crossing strategies used T. occidentale as a genetic bridge. Each started with different multispecies hybrids with various genomic contributions from the three parental species. The second approach began with 5x and 7x T. ambiguum x T. repens hybrids (ARRRR and AAARRRR). All the initial hybrids were repeatedly selfed, inter-crossed and backcrossed with colour-marked white clover and advanced progenies with reasonable levels of fertility were obtained in every strategy. Advanced hybrids were selected on the basis of flow cytometric ploidy estimation, phenotypes and somatic chromosome counts and were characterised for chromosome pairing and introgression events using both conventional and molecular cytogenetics. The advanced hybrid progenies were also grown in a sandpit to determine the relative expressions of the parental traits. Chromosome analyses showed evidence of chromosome elimination, chromosome addition/substitution, allosyndetic pairing involving A-derived chromosomes and interspecific genomic recombination. GISH analysis revealed that the genetic bridge strategy 1, which started with RRAO, gave a plant with four apparently large A-R and A-O chromosomal exchanges along with an A chromosome addition and a O-R substitution. No apparent signs of introgression were detected by GISH in the other strategies but introgression could not be ruled out because the sample was small and the morphology of the hybrids tested in the sandpit showed the expression of characters from both the parental species. These plants need to be characterised by using more genetic markers. The advanced progeny in the strategy based on direct integration of AxR genomes showed a low level of inter-specific chromosome pairing consistent with an absence of, or very low level of, introgression. The introgression revealed by GISH in the advanced progeny of RRAO hybrids provides evidence that using T. occidentale as genetic bridge has worked by disrupting the genomic integrity in T. repens. The material having introgression has many applications from white clover improvement point of view. While direct hybrids with A & R genomes did not lead to any apparent introgression.