Molecular Evolution of the Gapc Gene Family In

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Molecular Evolution of the Gapc Gene Family In MOLECULAR EVOLUTION OF THE GAPC GENE FAMILY IN AMSINCKIA SPECTABILIS (BORAGINACEAE) Joëlle R. Pérusse Department ofBiology McGill University, Montreal November 2001 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment ofthe requirements ofthe degree ofMasters of Science Joëlle R. Pérusse © 2001 National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1 A ON4 canada canada You, file Votre réftl_ Ou, file Notre rélé,_ The author bas granted a non­ L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library ofCanada to Bibliothèque nationale du Canada de reproduce, loan, distnbute or sell reproduire, prêter, distribuer ou copies ofthis thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership ofthe L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son penmsslOn. autorisation. 0-612-78937-3 Canada PREFACE This thesis carries a credit weight of 39 credits, from a total of45 credits required for the Master's degree. Graduate credits are a measure ofthe time assigned to a given task in the graduate program. They are based on the consideration that a term offull-time graduate work is equivalent to 12 to 16 credits, depending on the intensity ofthe program. 2 AB8TRACT This thesis investigates the molecular evolution ofthe cytosolic glyceraldehyde 3­ phosphate dehydrogenase (GapC) gene family in two varieties ofAmsinckia spectabilis (Boraginaceae) that differ in mating system. Examination of sequence variation suggests that the gene family consists ofthree or four members. Compared with GapC in Arabidopsis thaliana, the GapC locus in A. spectabilis has at least two fewer introns and one intron that is double in length. Strong purifying selection was detected at each putative locus since the divergence ofthe Amsinckia spectabilis lineage from species in the aIlied family Solanaceae. Mean nuc1eotide diversity across aIl observedO loci is 0.0036 for the inbreeding variety spectabilis, and 0.0049 for the outbreeding variety microcarpa. Outbreeding populations were systematicaIly more diverse than inbreeding populations at GapC-IV These results are discussed in the context oftheory for the fate of duplicate genes, and the background selection hypothesis. 3 RESUME La présente thèse étudie l'évolution moléculaire de la famille des gènes de la glycéraldehyde 3-phosphate déshydrogénase cytosolique (GapC) chez deux variétés d'Amsinckia spectabilis qui diffèrent de système de reproduction. L'examen de la variation des séquences suggère que la famille des gènes GapC comprend trois ou quatre membres chez Amsinckia spectabilis. Les loci GapC chez Amsinckia spectabilis contiennent un intron deux fois plus long, et au moins deux introns de moins, comparé à GapC chez Arabidopsis thaliana. Une forte sélection purificatrice fut détectée à chaque locus GapC depuis la divergence de la lignée à laquelle appartient Amsinckia spectabilis et des espèces de la famille Solanaceae. La diversité génétique évaluée à partir des séquences d'ADN est de 0.0036 chez la variété auto-féconde spectabilis, et de 0.0049 chez la variété à fertilisation croisée microcarpa. Les populations à fertilisation croisée étaient systématiquement plus diverses au locus GapC-IV que celles auto-fécondes. Ces résultats sont discutés dans le contexte de la théorie sur le sort des gènes dupliqués et de l'hypothèse de "background selection", 4 ACKNOWLEDGEMENTS First and foremost, l would like to thank my thesis supervisor, Daniel J. Schoen, for his guidance and support throughout this degree, and acknowledge his editorial expertise and his valuable help in data analysis. Thanks also to the other members ofmy supervisory committee, Candace Waddell and Tom Bureau, for excellent advice and use oftheir facilities. l also want to thank my undergraduate thesis advisor, Terry A. Wheeler, for getting me involved in research. Thank you to Lily, Steve, Aaron, Aura, Hien and Nabil, who have helped me with laboratory techniques and troubleshooting. Thanks to Nikoleta for doing most of the sequencing. Thanks to Mark, Claire and Frank ofthe McGill Phytotron for excellent plant care, and to Isabelle for doing part ofthe planting. l have enjoyed while at McGill the company of, and discussions ofevolutionary topics with, Steve, Sara, Mattieu, Aura, Natalia, Denis, Gray, Malorie, Adrian, Kathy and others. Thanks to my sister Marie-Andrée, and my friends Vanessa, Malorie, Alex K., Alex S., Adrian, Lily, Andrea, Jade and Jessica, for their encouragements, for making me laugh and for keeping me entertained. 5 Last but not least 1 would like to thank my parents for teaching me the value of education and hard work, and for instilling in me the love of leaming. To my beloved family, for your support and for aIl the "little" things that you did for me while 1 was hard at work. This thesis is dedicated to you. This work was supported in part by a postgraduate scholarship from the Natural Sciences and Engineering Council of Canada (NSERC) to lR. Pérusse and by a NSERC operating grant to D.l Schoen. 6 TABLE OF CONTENT Preface 2 Abstract 3 Resume 4 Acknowledgments 5 Table of Content. 7 List of Tables 8 List of Figures 10 Introduction 12 Materials and Methods................................................................. 34 Results 45 Discussion 55 Conclusion 63 Literature cited 65 Tables 87 Figures 101 Appendix A 108 7 LIST OF TABLES Table 1. Amsinckia spectabilis varieties studied 87 Table 2. Primer pairs and corresponding annealing temperatures 88 Table 3. List of individuals with the 18 bp deletion in their GapC gene sequence ............................................................................................... 89 Table 4. Putative locus-defining substitutions in relation to the consensus Amsinckia GapC sequence shown in Figure 1 90 Table 5. Linkage disequilibrium between putative loci-defining sites 91 Table 6. Analysis of selective constraint in four putative GapC loci in Amsinckia spectabilis 93 Table 7. Analysis of selective constraint in four putative GapC loci in Amsinckia spectabilis (singletons removed) 94 8 Table 8. Analysis of selective constraint for each putative A. spectabilis GapC locus analyzed in combination with Solanaceous GapC coding sequence data ................................................................................................ 95 Table 9. Functional consequences of amino acid substitutions in haplotypes of Amsinckia spectabilis at GapC loci 96 Table 10. Nucleotide variation at four putative Gap C loci in Amsinckia spectabilis varieties that differ in mating system 97 Table 11. Nucleotide variation at four putative GapC loci in Amsinckia spectabilis varieties that differ in mating system (singletons removed) 98 Table 12. Nucleotide variation at GapC-I and GapC-IV loci within Amsinckia spectabilis populations that differ in mating system 99 Table 13. Nucleotide variation at GapC-I and GapC-IV loci within Amsinckia spectabilis populations that differ in mating system (singletons removed) 100 9 LIST OF FIGURES Figure 1. Alignment of the partial GapC gene sequence ofArabidopsis thaliana and Amsincka spectabilis. ............................................................. 101 Figure 2. Graphical representation ofintronJexon structure in a partial GapC gene sequence ofArabidopsis thaliana and Amsinckia spectabilis 102 Figure 3. Neighbor-joining tree of GapC haplotypes in Amsinckia spectabilis .............................. '" '" 103 Figure 4. Haplotypes produced by single individua1s in Amsinckia spectabilis populations .... 104 Figure 5. Representative result ofPCR-RFLP ana1ysis, depicted here for population 28. .......................................................................... .. 105 Figure 6. Results from a Southern hybridization between a GapC fragment and Amsinckia spectabilis genomic DNA digested with Hha l (lane 1) and Hind III (lane 2) 106 Figure 7. ML tree ofGapC sequences from Amsinckia spectabilis and Solanaceous species tobacco, tomato, petunia and potato 107 10 INTRODUCTION DNA sequences are a valuable source of infonnation on the evolutionary process. Many disciplines, from comparative biology to population genetics, profit from sequence data analyses. For example, studies involving phylogenetics and biogeography (Whelan et al. 200l; Pagel 1999) have reconstructed gene trees, primarily based on plastid sequences (Simon et al. 1994), which have successfully been compared to species trees based on morphological characters. The increased ease ofobtaining molecular genetic data has resulted in a greater resolution of historical demographic parameters for populations (Goldstein and Harvey 1999), and in more accurate measures ofpopulation divergence estimates (Edwards and Beerli 2000). Population geneticists have put much emphasis on the level and patterns ofevolution as revealed by DNA sequences, with a particular emphasis on natural selection, drift and recombination (Aquadro 1997). Methods have been developed
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