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Evaluation of Genetic Diversity and Stability of Colocasia Cultivars Using ISSR Seukmin Hong Mekime09@Gmail.Com Stephen F. Austin State University SFA ScholarWorks Electronic Theses and Dissertations Fall 12-15-2017 Evaluation of Genetic Diversity and Stability of Colocasia Cultivars Using ISSR seukmin hong [email protected] Follow this and additional works at: https://scholarworks.sfasu.edu/etds Part of the Agriculture Commons Tell us how this article helped you. Repository Citation hong, seukmin, "Evaluation of Genetic Diversity and Stability of Colocasia Cultivars Using ISSR" (2017). Electronic Theses and Dissertations. 129. https://scholarworks.sfasu.edu/etds/129 This Thesis is brought to you for free and open access by SFA ScholarWorks. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of SFA ScholarWorks. For more information, please contact [email protected]. Evaluation of Genetic Diversity and Stability of Colocasia Cultivars Using ISSR Creative Commons License This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License. This thesis is available at SFA ScholarWorks: https://scholarworks.sfasu.edu/etds/129 Evaluation of Genetic Diversity and Stability of Colocasia Cultivars Using ISSR By Seukmin Hong, B.S. Biotechnology Presented to the faculty of the graduate school of Stephen F. Austin State University In Partial Fulfillment Of the Requirements For the degree of Masters of science in General Agriculture STEPHEN F. AUSTIN STATE UNIVERSITY December 2017 Evaluation of Genetic Diversity and Stability of Colocasia Cultivars Using ISSR By Seukmin Hong, Biotechnology BS APPROVED: _____________________________________ Jared Barnes, Ph. D., Thesis Director _____________________________________ Bea Clack, Ph. D., Committee Member _____________________________________ Yuhui Weng, Ph. D., Committee Member _________________________ Richard Berry, D.M.A Dean of the Graduate School ABSTRACT Using 44 ornamental Colocasia cultivars planted at Stephen F. Austin State University (Nacogdoches, TX), cultivar assessment, genetic diversity, stability, and relationship were examined with banding patterns produced from Inter-simple sequence repeat (ISSR) markers. Comparing banding patterns of vegetatively propagated clones of each cultivar, genetic stability was examined. Unweighted pair group method with arithmetic mean (UPGMA) and principal coordinate analysis (PCO) were performed to examine genetic relationship that can explain a need for the new classification Colocasia gigantea and recent movement of re-classifying Colocasia antiquorum. Average Shannon’s diversity index for all loci was found to quantify genetic diversity. The genetic stability analysis confirmed all 44 cultivars commonly found in market have identical genetic characteristics. The genetic relationship analysis supports the new classification of Colocasia antiquorum but does not strongly support the need for re-classification of Colocasia gigantea based on our results. Shannon’s diversity index suggested that 44 ornamental Colocasia cultivars have high gene pool. i TABLE OF CONTENTS ABSTRACT ................................................................................................. i TABLE OF CONTENTS ............................................................................. ii LIST OF FIGURES .................................................................................... iv LIST OF TABLES ....................................................................................... v CHAPTER ONE ..........................................................................................1 Plant Breeding and Cultivar Assessment Using Biotechnology ...............1 General Description of Colocasia ............................................................3 Colocasia Breeding .................................................................................4 Genetic Diversity of Colocasia ................................................................7 Project Introduction ............................................................................... 10 CHAPTER 2 ............................................................................................. 14 Introduction ........................................................................................... 14 DNA isolation from Colocasia leaf tissue ........................................... 14 Marker Choices .................................................................................. 16 Genetic Stability Analysis ................................................................... 18 Genetic Diversity Analysis ................................................................. 18 Materials and Methods .......................................................................... 23 Sample collection and preparation ..................................................... 23 DNA Isolation ..................................................................................... 25 Quantification of DNA ........................................................................ 26 Polymerase Chain Reaction Based Inter Simple Sequence .............. 26 Agarose Gel Electrophoresis ............................................................. 28 Banding Patter Scoring ...................................................................... 28 Statistical Analysis and Genetic Quantification .................................. 29 Results and Discussion ......................................................................... 29 DNA Isolation and Quantification ....................................................... 29 Banding Pattern Scoring .................................................................... 30 Genetic Stability Analysis ................................................................... 34 UPGMA .............................................................................................. 35 ii Principal Coordinates Analysis........................................................... 40 Genetic Diversity Quantification ......................................................... 44 Conclusion ................................................................................................ 49 Literature Cited ......................................................................................... 50 Appendix A ............................................................................................... 59 Appendix B ............................................................................................... 61 Appendix C ............................................................................................... 64 Appendix D ............................................................................................... 69 Appendix E ............................................................................................... 73 Appendix F ............................................................................................... 83 VITA ......................................................................................................... 99 iii LIST OF FIGURES Figure 1. Representation of Colocasia trial garden five beds (Bed A to E).. ............................................................................................................ 24 Figure 2. Dendrogram of 44 Colocasia cultivars produced using UPGMA with Jaccard’s coefficient.. .......................................................... 39 Figure 3. PCO scatter graph of 44 Colocasia cultivars using PC1 as X- axis and PC2 as Y-axis............................................................................. 43 iv LIST OF TABLES Table 1. List of Colocasia cultivars with breeder information and number of clones sampled. ........................................................................... 13 Table 2. List of primers with sequence and annealing temperature. .............. 27 Table 3. Thermocycler setting for PCR-ISSR. ................................................ 27 Table 4. Observed bands produced from each primer. .................................. 32 Table 5. Number of bands produced after PCR-ISSR. .................................. 33 Table 6. Percent of bands shared by all cultivars and unique bands for each cultivar in all 44 cultivars ....................................................................... 34 Table 7. Seven most similar Colocasia cultivars where the similarity index value was higher than overall similarity index average. ....................... 40 Table 8. Comparable Shannon’s diversity index of other Colocasia cultivars and ornamental genera. .................................................................. 44 v CHAPTER ONE Plant Breeding and Cultivar Assessment Using Biotechnology People have exploited plant genetics to benefit humankind for more than ten thousand years (Ross-Ibarra et al., 2007). A tremendous diversity of plant cultivars have been created, found, and selected by man, and the discovery of new traits that can benefit horticulture continues today. Breeding began with domestication as plants with desirable characteristics such as bigger fruit, higher yield, reduced branching, taller height, the loss or reduction of seed dispersal, the loss of seed dormancy, changes in photoperiod sensitivity, and the loss or reduction of toxic compounds were selected (Gepts, 2004; Hammer, 1984). Many benefits that arose from selection such as obtaining food from a farm, minimized travel to find a food source. However, society now faces new obstacles like the need for feeding a dramatically increasing population
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