Conservation genetics of Sclerocactus brevihamatus subsp. tobuschii and an assessment of its genetic relationship with Sclerocactus brevihamatus subsp. brevihamatus By Niraj Rayamajhi, B.S. A Thesis In PLANT AND SOIL SCIENCE Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Approved Dr. Jyotsna Sharma Chair of Committee Dr. Mark Burow Dr. Matthew Olson Dr. Mark Sheridan Dean of the Graduate School August, 2015 © 2015 Niraj Rayamajhi All Rights Reserved Texas Tech University, Niraj Rayamajhi, August 2015 ACKNOWLEDGEMENTS I wish to express my sincere appreciation to my advisor Dr. Jyotsna Sharma, for her affection, constant encouragement, guidance, support, advice, motivation, and most of all her patience. Her guidance helped me all the time in my research and writing this thesis. I also express my sincere regards to other members of the advisory committee, Dr. Matthew Olson and Dr. Mark Burow for their helpful advice, assistance, constructive comments, and helping me out with data analysis. I wish to extend my heartfelt thanks to Dr. Shannon Fehlberg and Jessica Allen at the Desert Botanical Garden and Dr. Ratan Chopra at Texas Tech University for guiding me with the methods. I would also like to thank all my lab mates for their cooperation during my research work in the laboratory. I am also highly obliged to all my professors at Texas Tech University for proving me quality education and enhancing my skills. I would like to thank the U.S. Fish and Wildlife Service and the Lady Bird Johnson Wildflower Center (Dr. Karen Clary) for providing funding to Dr. Sharma for this investigation. I also want to thank my parents Mr. Tanka Rayamajhi and Mrs. Indira Rayamajhi, and sister Srijana Rayamajhi for their constant support and motivation throughout my entire life. I must acknowledge my girlfriend Pragya, who has assisted in editing the thesis, and whose affection and encouragement are the source of my inspiration. ii Texas Tech University, Niraj Rayamajhi, August 2015 TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ ii LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES ......................................................................................................... viii I. INTRODUCTION AND BACKGROUND ........................................................... 1 Introduction ....................................................................................................... 1 Biodiversity ................................................................................................... 1 Genetic diversity ........................................................................................... 3 Conservation genetics .................................................................................. 4 The genus Sclerocactus and study taxa ........................................................ 5 Why study these taxa? .................................................................................. 7 Microsatellite markers ................................................................................. 8 Background ....................................................................................................... 9 Objectives of the study.................................................................................... 15 Significance of the study ................................................................................. 15 Literature Cited ............................................................................................... 17 II. MICROSATELLITE DEVELOPMENT AND PRIMER SELECTION FOR GENETIC STUDY IN SCLEROCACTUS BREVIHAHAMATUS SUBSP. TOBUSCHII AND SCLEROCACTUS BREVIHAMATUS SUBSP. BREVIHAMATUS ................................................................................................. 24 Abstract ........................................................................................................... 24 Introduction ..................................................................................................... 25 Materials and methods .................................................................................... 27 iii Texas Tech University, Niraj Rayamajhi, August 2015 DNA extraction and microsatellite development ........................................ 27 Primer selection ......................................................................................... 28 Results ............................................................................................................. 29 Conclusions ..................................................................................................... 29 III. CONSERVATION GENETICS OF SCLEROCACTUS BREVIHAMATUS SUBSP. TOBUSCHII AND AN ASSESSMENT OF ITS GENETIC RELATIONSHIP WITH SCLEROCACTUS BREVIHAMATUS SUBSP. BREVIHAMATUS ................................................................................................. 38 Abstract ........................................................................................................... 38 Introduction ..................................................................................................... 40 Materials and Methods .................................................................................... 44 Sample collection ....................................................................................... 44 DNA extraction ........................................................................................... 45 Polymerase chain reaction (PCR) amplification and genotyping .............. 45 Microsatellite analyses ............................................................................... 47 Results ............................................................................................................. 50 Diversity of the seven microsatellite loci ................................................... 50 Genetic diversity within populations of Sclerocactus brevihamatus subsp. tobuschii ..................................................................................................... 51 Genetic diversity within a population of Sclerocactus brevihamatus subsp. brevihamatus .............................................................................................. 52 Genetic differentiation or strucuture of Sclerocactus brevihamatus .......... 53 Bottleneck test of all the ten populations of Sclerocactus brevihamatus .... 55 Discussion ....................................................................................................... 55 iv Texas Tech University, Niraj Rayamajhi, August 2015 Genetic diversity within populations .......................................................... 56 Heterozygosity of Sclerocactus brevihamatus subsp. tobuschii in comparison to long-lived perennials, and plants with similar mating type and geographical distributions based on microsatellite markers .............. 57 Heterozygosity of Sclerocactus brevihamatus subsp. tobuschii in comparison to other cacti ........................................................................... 58 Heterozygosity of Sclerocactus brevihamatus subsp. tobuschii in comparison to other species of Sclerocactus based on microsatellite analysis ....................................................................................................... 62 Inbreeding coefficient of Sclerocactus brevihamatus subsp. tobuschii in comparison to other cacti, congeners and conspecific taxa ...................... 63 Population genetic structure ...................................................................... 65 Literature Cited ............................................................................................... 69 IV. CONCLUSIONS................................................................................................... 97 Conservation implications .............................................................................. 98 Future directions ............................................................................................. 99 Literature Cited ............................................................................................. 100 APPENDIX ......................................................................................................... 101 v Texas Tech University, Niraj Rayamajhi, August 2015 LIST OF TABLES 2.1. Seven optimized polymorphic microsatellite loci for Sclerocactus brevihamatus subsp. tobuschii...............................................................................33 3.1. Location and population code of ten populations of Sclerocactus brevihamatus..........................................................................................................75 3.2. Primer sequences and genetic diversity statistics of seven nuclear microsatellite markers isolated from Sclerocactus brevihamatus subsp. tobuschii (SBT)......................................................................................................77 3.3. Genetic diversity measures of ten Sclerocactus brevihamatus populations in the United States estimated based on seven nuclear microsatellites.................78 3.4. Pairwise geographic distances (kilometers) and pairwise Fst for ten populations of Sclerocactus brevihamatus based on seven nuclear microsatellites.............................................................................79