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

The objective of this project was to investigate the possibility of introgression of stress \nresistance traits from T. ambiguum (A) into T. repens (R) by interspecific hybridisation, \nusing two approaches. The first used T. occidentale Coombe (O) as a genetic bridge \nbecause this species has chromosome pairing homology to both the two other species. \nThe second approach attempted direct integration of genomes from the two species \nthrough ploidy manipulation. \nFor the first approach, four crossing strategies used T. occidentale as a genetic bridge. \nEach started with different multispecies hybrids with various genomic contributions \nfrom the three parental species. The second approach began with 5x and 7x T. ambiguum \nx T. repens hybrids (ARRRR and AAARRRR). All the initial hybrids were repeatedly \nselfed, inter-crossed and backcrossed with colour-marked white clover and advanced \nprogenies with reasonable levels of fertility were obtained in every strategy. Advanced \nhybrids were selected on the basis of flow cytometric ploidy estimation, phenotypes and \nsomatic chromosome counts and were characterised for chromosome pairing and \nintrogression events using both conventional and molecular cytogenetics. The advanced \nhybrid progenies were also grown in a sandpit to determine the relative expressions of \nthe parental traits. \nChromosome analyses showed evidence of chromosome elimination, chromosome \naddition/substitution, allosyndetic pairing involving A-derived chromosomes and interspecific \ngenomic recombination. GISH analysis revealed that the genetic bridge strategy \n1, which started with RRAO, gave a plant with four apparently large A-R and A-O \nchromosomal exchanges along with an A chromosome addition and a O-R substitution. \nNo apparent signs of introgression were detected by GISH in the other strategies but \nintrogression could not be ruled out because the sample was small and the morphology \nof the hybrids tested in the sandpit showed the expression of characters from both the \nparental species. These plants need to be characterised by using more genetic markers. \nThe advanced progeny in the strategy based on direct integration of AxR genomes \nshowed a low level of inter-specific chromosome pairing consistent with an absence of, \nor very low level of, introgression. The introgression revealed by GISH in the advanced \nprogeny of RRAO hybrids provides evidence that using T. occidentale as genetic bridge \nhas worked by disrupting the genomic integrity in T. repens. The material having \nintrogression has many applications from white clover improvement point of view. \nWhile direct hybrids with A & R genomes did not lead to any apparent introgression.