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Assessment of Genetic Variablity of Spigelia Marilandica and S

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Assessment of Genetic Variablity of Spigelia Marilandica and S ASSESSMENT OF GENETIC VARIABLITY OF SPIGELIA MARILANDICA AND S. GENTIANOIDES USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) MARKERS AND CLONAL PROPAGATION OF STEM CUTTINGS OF S. MARILANDICA, S. GENTIANOIDES VAR. ALABAMENSIS, AND S. MARILANDICA × S. GENTIANOIDES VAR. ALABAMENSIS F2 AND F3 HYBRIDS by AMANDA JANNETTE HERSHBERGER (Under the Direction of Carol Robacker) ABSTRACT Despite the ecological and ornamental potential of southeastern U.S. native Spigelia, little is known about the intraspecific or the interpopulation genetic variation. Spigelia habitat is becoming fragmented due to human activity, making it imperative to gain an understanding of natural genetic variation among and within species and populations. This study used amplified fragment length polymorphism (AFLP) analysis to determine interspecific and intraspecific genetic variation and to evaluate gene flow. Thirteen populations of two species of native Spigelia, S. marilandica (L.) (SM), S. gentianoides Chapm. ex A. DC. var. alabamensis K. Gould (SGA), and S. gentianoides var. gentianoides (SGG), were analyzed. Based on analysis of molecular variance (AMOVA) and estimates of Nei’s coefficients of gene diversity (HS, HT, and GST), the majority of variation found in Spigelia occurs within populations. Among species and among population variation were low, likely the effect of common ancestry as well as relatively frequent introgression among individuals (and populations) of Spigelia. An unrooted UPGMA i phenogram delineated three clades. The significance of these results is discussed in relation to breeding in Spigelia. In addition to the lack of information concerning genetic variation in Spigelia, little is known concerning clonal propagation strategies of SM and SGA. The effects of cutting date, indole-3-butyric acid (IBA) level, and genotype on rooting percentage, root number, and root length were evaluated. Stem cuttings were obtained from five genotypes of SM, one genotype of SGA, three genotypes of SM× SGA F2 hybrids, and two genotypes of SM × SGA F3 hybrids. IBA level significantly affected rooting percentage and root number, but not root length. The SM × SGA hybrids successfully rooted through all months evaluated, while SM and SGA genotypes exhibited a decline in rooting in cuttings taken in Sept. Results suggest that SM and SGA may be successfully propagated by treating stem cuttings taken in May, June, July, or Aug. with 0.3% IBA. Cuttings of SM × SGA hybrids can be taken through Sept. These protocols provide a basis for rapid propagation of Spigelia. INDEX WORDS: AFLP markers, Spigelia spp., native plants, endangered species, among population diversity, within population diversity, AMOVA, clonal propagation, bottom heat, IBA ii ASSESSMENT OF GENETIC VARIABLITY OF SPIGELIA MARILANDICA AND S. GENTIANOIDES USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) MARKERS AND CLONAL PROPAGATION OF STEM CUTTINGS OF S. MARILANDICA, S. GENTIANOIDES VAR. ALABAMENSIS, AND S. MARILANDICA × S. GENTIANOIDES VAR. ALABAMENSIS F2 AND F3 HYBRIDS by AMANDA JANNETTE HERSHBERGER B.S., Purdue University, 2006 M.S., University of Georgia, 2008 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements of the Degree DOCTOR OF PHILOSOPHY ATHENS, GA 2012 iii ©2012 Amanda Jannette Hershberger All Rights Reserved iv ASSESSMENT OF CLONAL PROPAGATION AND GENETIC VARIABILITY OF SPIGELIA MARILANDICA AND S. GENTIANOIDES USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) MARKERS by AMANDA JANNETTE HERSHBERGER Major Professor: Carol Robacker Committee: James Affolter Allan Armitage Melanie Harrison-Dunn Tracie Jenkins Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2012 v DEDICATION Writing this part is always the most difficult. It’s tempting to allow this dedication to become either too personal or completely devoid of any human component. This is my attempt at a happy medium. I have attended the University of Georgia for six years for both my M.S. and Ph.D. degrees and have met a number of people along the way that have taught me a lot about myself, for better or for worse. Many people have helped me so much that I wouldn’t even have enough room to thank each and every one of them here. Thank you to Lee Ann Kelly for all of your help in the beginning. I didn’t know you for very long, but thank you. Thanks to Suzzanne Tate for your patience and all of your help with my first project. Thank you to Sherrod Baden and Allen Byous for maintaining all of the Griffin material. Thanks to Noelle Barkley, Zhenbang Chen, Tyler Eaton, and Tracie Jenkins for all of your assistance with the DNA portion of my research. Thank you to all my grad student comrades for helping with various projects and for being entertaining. Thanks to Jim Gegogeine for being there through it all. Thank you to my husband, Luke, for becoming the best friend I in no way deserve and being the calm element in my life. Without you I am absolutely sure I never would have finished. This wouldn’t be a dedication from me if I didn’t also thank my dogs for being alive. Gordita, my curmudgeon of a dog, and Professor Finworth Solomon Jones (Jonesy), the playful and sweet counterpart to Gordita, share best friend status with Luke. I was so happy to come home from campus to your wagging tails. iv ACKNOWLEDGEMENTS There were many parts of this research that were not limited to execution and understanding of project plans. One of those factors is writing, an important portion with which I have always struggled. Dr. Robacker spent a considerable amount of time working with me on this without showing too much irritation. I hope that her influence is evident in this work. Enjoy! v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS…………………………………………………………………..v LIST OF TABLES……………………………………………………...…………………….vii LIST OF FIGURES…………………………………………………………………………...x CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW………………………….1 Purpose of studies……………………………………………………..1 Botanical and geographical description of Spigelia…………………..2 Molecular studies in Spigelia species……………….………………...7 AFLP background and technology……………………………………9 Spigelia propagation………………………………………………….15 Literature cited………………………………………………………..18 2 ASSESSMENT OF GENETIC VARIABILITY OF SPIGELIA MARILANDICA AND S. GENTIANOIDES USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) MARKERS………….26 Abstract……………………………………………………………….27 Introduction…………………………………………………………...28 Materials and Methods……………………………….……………….32 Results and Discussion………………………………………………..36 Literature cited……….……………………………………………….45 vi 3 CLONAL PROPAGATION OF STEM CUTTINGS OF SPIGELIA MARILANDICA, S. GENTIANOIDES VAR. ALABAMENSIS, AND S. MARILANDICA × S. GENTIANOIDES VAR. ALABAMENSIS F2 AND F3 HYBRIDS…………………………………………………………...60 Abstract……………………………………………………...………..61 Introduction…………………………………………………………...61 Materials and Methods………………………………………………..64 Results and Discussion………………………………………………..65 Literature cited………..………………………………………………69 4 CONCLUSIONS……………………………………………………………...80 Literature cited………………………………………………………..84 vii LIST OF TABLES Page Table 2.1. Phenotypic differences between S. marilandica, S. gentianoides var. gentianoides, and S. gentianoides var. alabamensis.................................................................49 Table 2.2. Locations of populations collected, grouped by species. Each population is numbered in succession and includes four to 11 individual plant samples……….………50 Table 2.3. List of adaptors and primers screened and used in this study to characterize the amplification fragment length polymorphism (AFLP) band patterns in two Spigelia species…………………………………………………………………………………….51 Table 2.4. Percentage of polymorphic loci, average genetic diversity within populations (HS), average genetic diversity within species (HT), and proportion of species genetic diversity attributed to among population variation (GST) for species of Spigelia…………………………………………………………………………………....52 Table 2.5. Percentage of polymorphic loci in all populations of Spigelia evaluated in this study…………… ………………………………………………………………………...53 Table 2.6. Analysis of molecular variation (AMOVA) for three Spigelia spp./varieties included in this study………… ……………………………………….…………………54 Table 2.7. Nei's unbiased measures of genetic distance (Nei, 1978) below diagonal and geographic distance (km) above diagonal……………………..………………………….55 Table 3.1. Spigelia genotypes and numbers of cuttings taken throughout duration of project………………………………………………………………………………………...72 viii Table 3.2. Levels of significance of analysis of variance (ANOVA) effects for rooting percentage, root number, and root length for individual genotypes of Spigelia………….73 Table 3.3. Effect of IBA level on rooting percentage, root number, and root length of Spigelia with all genotypes analyzed separately……………………………………….…74 Table 3.4. Effect of IBA level and cutting date on rooting percentage, root number, and root length of Spigelia over all months…………………………………………………...75 Table 3.5. Levels of significance of analysis of variance (ANOVA) effects for rooting percentage, root number, and root length for grouped genotypes of Spigelia (SM, SGA, and SM × SGA hybrids within their respective categories)……………………….……...76 Table 3.6. Effect of grouped genotype and cutting date on rooting percentage, root number, and root length of Spigelia over all months…………………………………….………...77 ix LIST OF FIGURES Page Figure 2.1. Spigelia collection sites for current project……………………………………...56 Figure 2.2.
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