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Canada Du Canada Acquisitions and Direction Des Acquisitions Et Bibliographie Services Branch Des Services Bibliographiques 395 Wellington Strcel 395 National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Direction des acquisitions et Bibliographie services Branch des services bibliographiques 395 wellington Strcel 395. rue Wellinglon Ottawa. Onlano onawa (On13OO) K1AON~ K1AQN4 ' ..... r.... \.>t',.,.. ,.~, .." .•• NOTICE AVIS The quality of this microform is La qualité de cette microforme heavily dependent upon the dépend grandement de la qualité quality of the original thesis de la thèse soumise au submitted for microfilming. microfilmage. Nous avons tout Every effort has been made to fait pour assurer une qualité ensure the highest quality of supérieure de reproduction. reproduction possible. If pages are missing, contact the S'il manque des pages, veuillez university which granted the communiquer avec l'université degree. qui a conféré le grade. Some pages May have indistinct La qualité d'impression de print especially if the original certaines pages peut laisser à pages were typed with a poor désirer, surtout si les pages typewriter ribbon or if the originales ont été university sent us an inferior dactylographiées à l'aide d'un photocopy. ruban usé ou si l'université nous a tait parvenir une photocopie de : qualité inférieure. Reproduction in full or in part of La reproduction, même partielle, = this microform is governed by de cette microforme est soumise the Canadian Copyright Act, à la Loi canadienne sur le droit R.S.C. 1970, c. C-30, and d'auteur, SRC 1970, c. e-30, et subsequent amendments. ses amendements subséquents. Canada • GENETIC STUDIES OF RED CLOVER (TRIFOLIUM PRATENSE 1..) USING MORPHOLOGICAL, ISOZVME AND RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKERS PRASERT KONGKlATNGAM Oepartment of Plant Science McGilI University Montreal July 1995 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the reqùirements of the degree of Ooetor of Philosophy ID Prasert Kongkiatngam, 1995 National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Direction des acquisitions et Bibliographie Services Branch des services bibliographiques 395 Welli~ton Street 395. rue Welhnglon Ottawa. Oritano Ottawa (On,.roo) K1A0N4 K1AON4 The author has granted an L'auteur a accordé une licence irrevocable non-exclusive licence irrévocable et non exclusive allowing the National Ubrary of permettant à la Bibliothèque Canada to reproduce, loan, nationale du Canada de distribute or sell copies of reproduire, prêter, distribuer ou his/her thesis by any means and vendre des copies de sa thèse in any form or format, making de quelque manière et sous this thesis available to interested quelque forme que ce soit pour persons. mettre des exemplaires de cette thèse à la disposition des personnes intéressées. The author retains ownership of L'auteur conserve la propriété du the copyright in his/her thesis. droit d'auteur qui protège sa Neither the thesis nor substantial thèse. Ni la thèse ni des extraits extracts from it may be printed or substantiels de celle-ci ne otherwise reproduced without doivent être imprimés ou hiS/11er permission. autrement reproduits sans son autorisation. ISBN 0-612-08123-0 Canada • Short Title: Genetic Studies of Red Clover (Trifolium pratelL~e 1..) Prasert Kongkiatngam • ABSTRACT Genctic variation within and between two cultivars of red clover (Tri/oliam pratense L), Essi from Europe and Ottawa from Canada was estimated using morphological, isozyme and randol11 amplified polymorphie DNA (RAPD) markers. A total of 21 enzyme-coding loci with 43 alleles was deteeted using twelve enzyme systems. The mean number of aileles per locus was 1.81 in Essi and 1.67 in Ottawa. Nine 10-mer primers were used to assay 20 individuals from eaeh cultivar for RAPD m:-:rkers. Eaeh primer gave from 7 to 20 amplified bands with an average of 14.8 bands per primer. High within-eultivar variation was observed in both cultivars using both isozyme and RAPD markers. The mode of inheritanee of seven isozyme loci: Aat-2, Amy-l, Est-4, Est-7, Pgd-1, Pgd-2 and Skd-1. in red clover was verified. The genetic basis of banding patterns for 16 other isozyme loci: Aat-3, Adlz-1, Dia-l, Dia­ 2, Dia-3, Est-l, Est-2, Gpi-2, ldlz-l, Mdlz-l, Mdlz-2, Mdlz-3, Mdlz-4, Me-l, Me-2 and Pgm-2, was also postulated, based on the segregation patterns observed within cultivars. Two pairs oflinked enzyme-coding loci, Est-4jEst-7 and Pgd-2/Skd-1, were found with joint segregation analysis. Estimates ofgenetic variability of lS red clover cultivars from three different origins indicated that within-cultivar variation was much higher than between-cultivar variation. Allele frquencies of these isozymes could discriminate the five North American cultivars assayed, but they could not differentiate cultivars from Europe and Japan. The use of RAPD markers obtained frum bulked samples was investigated for cultivar identification in red clover. Pooled samples were examined in order to minimize variation within cultivars. Twenty was found to be an appropriate number of red clover individuals per bulk for homogenizing genetic variation within cultivars. Fourteen 1D-mer primers were used to amplify genomic DNA from combined leafsamples of lS red clover cultivars from European, Japanese and North American origins. A total of 79 amplified bands, of which SS were polymorphie, was obtained. Cultivar-specifie bands were observed • with 13 primers. The amplification patterns obtained from two primers could distinguish ail 15 red c10ver cultivars. Cluster analysis b'lscd on Rogers' genctic • distances separated these 15 cultivars inw four groups. but each group cont.lins cultivars from more than one geogr;:phical origin. Six lllorpholugiC<ll traits 'lIld 2.' enzyme-coding loci were used to study the genetic changes of two groups of red c10ver populations that have undergone three cycles of natural selection. MCC 124 is a mixture of seed of 20 cultivars originating from North America and Europe and MCC 125 is a polycross ofFusarium resistant clones largely from the cultivar OU:<.va. Thus MCC 125 is a much narrower based population. The percentages ofplants \Vith spreading hairs on stems and petioles. and \Vith hairy internodes increased dramatically after the firs. cycle of natural selection in the MCC 124 populations. The percentage of plants with leaf marks did not change in either group of populations. The percentage of plants with flowering stems in the first year of growth increased after the first cycle of selection in the MCC 124 populations. However, the percentage of flowering plants decreased slowly in the MCC 125 populations. These changes in morphological characters related to environmental adaptation indicated that natural selection had occurred in these red c10ver populations. The allele frequencies of most polymorphie loci did not change after three cycles of selection. ln the MCC 124 populations, the estimates of aver.lge exj>ected heterozygosity decreased slightly but the percentages of polymorphie loci and the mean number of alleles per locus were almost the same after three cycles of selection. ln the MCC 125 populations, all of these estimates decreased slightly. • ii • RÉSUMÉ Les variations génétiques parmi et entre deux cultivars de trèfle rouge (Trifolium pratense L) ont eté étudiées. Les cultivars Essi provenant d'Europe et Ottaw-.t provenant du Canada ont été analysés aux niveaux morphologique, de l'étude des isozymes et de l'étude des marqueurs RAPD ("Random Amplified Polymorphie DNA"). Un total de 21 loci contenant 43 allèles codant pour les enzymcsont été détectés en utilisant douze différents systèmes d'enzymes. Le nombre moyen d'allèles par locus était de 1.81 pour le cultivar Essi et de 1.67 pour Ottawa. Neuf amorces de 10 nucléotides ont été utilisées sur 20 individus de chacun des deux cultivars afin d'identifier les marqueurs RAPD appropriés. Chacune des amorces permet de detecter de 7 à 20 bandes amplifiées, avec une moyenne de 14.8 bandes detectées par amorce. L'étude des isozymes et des marqueurs RAPD a permis de détecter une grande variation au sein du même cultivar et cela pour les 2 cultivars étudiés. La transmission génétique de sept loci d'isczymes: Aat-2, Amy-l, &t-4, &t-7, Pgd-1, Pgd-2 et Skd-1 du tréfle rouge a été étudiée. La base génétique des patror,s de bandes pour 16 autre loci d'isozymes:Aat-3, Adh-1, Dia-l, Dia-2, Dia­ 3, Est-l, &t-2, Gpi-2, Idh-1, Mdll-1, Mdll-2, Mdll-3, Mdh-4, Me-l, Me-2 et Pgm-2 a aussi été postulée, basé sur le schéma de ségrégation observé parmis les cultivars. Deux paires de loci codant pour des enzymes Est-4jEst-7 et Pgd-2jSkd-1 ont été découvert à l'aide d'une analyse de ségrégation. L'estimation de la variabilité génétique de 15 cultivars de trèfle rouge, de trois différentes origines, indique clairement que la variation génétique est beaucoup plus importante au sein du même cultivar qu'entre les différents cultivars. Dest possible d'utiliser la fréquence des allèles de ces isozymes pour distinguer les cinq cultivars américains testé. Par contre, il est impossible d'utiliser cette méthode pour différencier les cultivars en provenance d'Europe et du Japon. L'utilisation de marqueurs RAPD obtenus déchantillons combinés a été étudiée afin de pennetté l'identification des cultivars de trèfle rouge. Les échantillons ont été combinés afin de minimiser la variation parmis les cultivars. Vmgt individus semblait être un nombre approprié de plants de trèfles rouges par échantillon combiné, ceci afin d'avoir une variation génétique • iü parmis les cultivars la plus homogène possible. Quatorze amorces de 10 nucléotides • ont été utilisées afin d'amplifier rADN génomique des échantillons comhinés contenant les feuilles de 15 cultivars de trèfle rouge. Ces cultivars étaient d'origine européenne.
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