Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2003 Agronomic and molecular characterization of Louisiana native Spartina alterniflora accessions Alicia Beatriz Ryan Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Accounting Commons Recommended Citation Ryan, Alicia Beatriz, "Agronomic and molecular characterization of Louisiana native Spartina alterniflora accessions" (2003). LSU Doctoral Dissertations. 2001. https://digitalcommons.lsu.edu/gradschool_dissertations/2001 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. AGRONOMIC AND MOLECULAR CHARACTERIZATION OF LOUISIANA NATIVE SPARTINA ALTERNIFLORA ACCESSIONS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy In The Department of Agronomy By Alicia Beatriz Ryan B.S., National University of Cordoba, Cordoba Argentina. 1992 M.S., International Center for Advanced Mediterranean Agronomic Studies Institute, Zaragoza Spain. 1997 December 2003 ACKNOWLEDGMENTS The author would like to express her heartfelt gratitude and sincere thanks to her major advisors, Drs. Bradley Venuto and Stephen Harrison, for their leadership, guidance, assistance, encouragement and patience throughout the completion of the work and the preparation of the manuscript. She feels very honored to have worked with them for the last five years. She is grateful to his graduate committee composed of Dr. Barry Moser, minor advisor, Department of Experimental Statistics, Dr. Charles Johnson, Department of Horticulture, and Dr. Irv Mendelssohn , Department of Oceanography and Coastal Science Wetland Biogeochemistry Institute for their time, thought, and critical review devoted to the preparation of the dissertation. My thanks goes to Dr. Prasanta Subudhi, Dr James Oard and Mary Bowen for the support and help in molecular genetic lab. My sincere thank goes to Michael Materne (USDA-NRCS) for their help during my study in the marsh project. This study could not be completed without the kind help, friendship and support of Alan Shadow, who provided excellent help in the field and green house. I would also like to thank the other people who made this project a success; Professor, student workers, “the wheat crew”, and everyone else who has contributed to make this project a success. She is also grateful to the Agronomy Department faculty and staff, especially Connie, Jere, Jodie, and Bobbie for their help, kindness and friendship. The author appreciates the friendship and valuable assistance of her ii colleagues in the Department of Agronomy, specially Jackie Prudente and Gabriel Aluko, and all other friends both in the U.S and Argentina. Most of all, I am grateful to my parents, sisters, nieces, nephews, brother in-laws, and relatives for their love, support and encouragement. Finally, a very heartfelt gratitude and appreciation goes to her dear husband, Hector Zapata, for his constant encouragement, patience great companion and moral support throughout her graduate program and for being there for her. Without them, the author could have not been through this stage of her life and career. To them, this piece of work is wholeheartedly dedicated. The author would like to praise God for the knowledge and wisdom that He showered upon her and for His infinite blessings. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS…………………………………………………………… ii LIST OF TABLES…………………………………………………………………... vii LIST OF FIGURES…………………………………………………………………. x ABSTRACT………………………………………………………………………….. xii CHAPTER 1 INTRODUCTION…………………………………………………… 1 1.1 Background of the Study………………………………………….…………… 1 1.2 Objectives of the Study………………………………………………………… 6 1.3 References……………………………………………………………………… 7 CHAPTER 2 LITERATURE REVIEW…………………...……………….………. 10 2.1 Coastal Wetlands………………………………………………………………. 10 2.1.1 Coastal Wetland Losses..….……………………………………… 11 2.1.2 Coastal Wetland Restoration……………………...…………….…. 11 2. 2 Biological Study, Spartina alterniflora……………………………………….. 13 2.2.1 Description.…………………………………………………………… 13 2.2.2 Genus Spartina.…………….………………………………………... 16 2.2.3 Biological Characteristics of Spartina alterniflora..………………. 18 2.2.3.1 Cytogenetics……………………………………………… 18 2.2.3.2 Physiological and Reproductive Characteristics……... 19 2.3 Utilization of Molecular Markers in Genetic Diversity Analysis and Germplasm Improvement……………………………………………………… 24 2.3.1 Molecular Marker Systems………………………………………….. 24 2.3.2 Types of DNA Markers………………………………………………. 25 2.3.2.1 Biochemical Markers………….…………………………. 26 2.3.2.2 DNA-based Markers………………………...…………… 26 2.3.2.2.1 Hybridization-based Techniques or the Non-PCR Techniques………………………… 26 2.3.2.2.2 Arbitrarily-primed PCR……………………… 27 2.3.3 Use of DNA Markers…………………………………………………. 28 2.3.3.1 Variety Identification or Fingerprinting…………………. 29 2.3.3.2 Genetic Diversity…………………………………………. 31 2.3.4 Use of RAPDs and AFLPs as a Tool for Analysis of Genetic Diversity in Spartina…………………………………………………... 34 2.3.5 Methods for Analyzing Molecular Variation at the Population Level…………………………………………………………………… 37 2.3.5.1 Cluster Analysis and Dendrogram Construction ….... 39 2.4 References……………………………………………………………………… 40 iv CHAPTER 3 FIELD EVALUATION AND SELECTION OF SUPERIOR SPARTINA ALTERNIFLORA GERMPLASM……………………. 49 3.1 Introduction……..……………………………………………………………….. 49 3.2 Materials and Methods………………………………………………………… 52 3.2.1 Evaluation and Selection……………………………………………. 52 3.2.1.1 Source Population...……………………………………….. 52 3.2.1.2 Cycle 1 Evaluation and Selection………………………... 53 3.2.1.3 Cycle 2 Evaluation and Selection………………………... 53 3.2.1.4 Cycle 3 Evaluation and Selection………………………... 57 3.2.2 Data Analysis…………………………………………………………. 60 3.3 Results and Discussion………………………………………………………... 62 3.3.1 Cycle 1 Evaluation and Selection…………………………………... 62 3.3.2 Cycle 2 Evaluation and Selection…………………………………... 70 3.3.3 Cycle 3 Evaluation and Selection…………………………………... 81 3.3.3.1 Performance of Selected Plants at Ben Hur Farm and Grand Terre Island …………………………………………. 81 3.3.3.2 Seed Production in Cycle 2..……………………………… 85 3.3.3.3 Correlation Among Reproductive Traits in Cycle 2 Selections……………………………………………………. 86 3.3.3.4 Genotypes Selected in Cycle 3…………………………… 89 3.4 References…………...…………………………………………………………. 93 CHAPTER 4 MOLECULAR CHARACTERIZATION OF SELECTED SPARTINA ALTERNIFLORA GERMPLASM …………………… 96 4.1 Introduction…………..………………………………………………………….. 96 4.2 Materials and Methods………………………………………………………… 101 4.2.1 Sample Collection and DNA Extraction………………………….. 101 4.2.2 RAPD Analysis…...…………………………………………………. 103 4.2.2.1 Optimization of RAPD Protocol…………………………… 103 4.2.2.2 RAPD Data Analysis………………………………………. 105 4.2.3 AFLP Analysis………………………………………………………. 106 4.2.3.1 Template Preparation of Genomic DNA .……………….. 106 4.2.3.2 Amplified Fragment Length Polymorphism Procedure... 107 4.2.3.2.1 Restriction Digestion of Genomic DNA and Ligation of Adaptors…………………………... 109 4.2.3.2.2 AFLP Pre-amplification……………………… 109 4.2.3.2.3 Selective AFLP Reaction Products………… 110 4.2.3.3 Gel Analysis of AFLP Reaction Products……………… 111 4.2.4 Genetic Diversity Analysis…………………………………………... 114 4.2.5 Comparison with Agronomic Data………………………………….. 114 4.3 Results and Discussion………………………………….…………………….. 115 4.3.1 RAPD Analysis……………………………………………………….. 115 4.3.1.1 Primer Screening and Variety Fingerprinting…………… 115 4.3.1.2 Cluster Analysis……………………………………………. 116 4.3.2 AFLP Analysis……………………………………… ……………….. 121 4.3.2.1 Primer Screening and Variety Fingerprinting…………… 121 v 4.3.2.2 Cluster Analysis……………………………………………. 122 4.3.3 Comparison Morphological and Molecular Data …………………. 129 4.4 References……………………………………………………………………… 130 CHAPTER 5. SUMMARY AND CONCLUSIONS………………………………. 135 APPENDIX: COLLECTION DATA AND SOLUTIONS FOR DNA ISOLATION 139 VITA…………………………………………………………………………………... 144 vi LIST OF TABLES 2.1 Species of the genus Spartina (Mobberley, 1956, Partridge 1987)……. 17 3.1 Geographical distribution of Spartina alterniflora accessions collected across South Louisiana during 1998………………………………………. 55 3.2 Spartina alterniflora accessions selected for field study in 1999 based on germination test and seedling vigor……………………………………. 57 3.3 Spartina alterniflora accessions selected during 2000 at Ben Hur Farm, Baton Rouge, LA……………………………..……………………… 58 3.4 Mean, standard deviation, and range for date of collection, seed weight and percent of germination among 126 native Spartina alterniflora accessions collected in South Louisiana during 1998……… 64 3.5 Pearson correlation coefficients for seed weight, germination rate, and collection date among 126 native Spartina alterniflora accessions collected in Louisiana during 1998…………...……………………………. 65 3.6 The 20 cycle 1 accessions of Spartina alterniflora identified for cycle 2 evaluation based on analysis of seed weight, percent germination, and collection date for 126 native accessions collected in South Louisiana during 1998………………………………………………………….……….. 70 3.7 Mean values for height, spread, vigor, rust, tiller density and days to first flowering of 20 cycle 2 Spartina alterniflora accessions grouped by the location of origin…………………………………………………………