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Fingerprinting and Genetic Stability of Rubus Using Molecular Markers

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Fingerprinting and Genetic Stability of Rubus Using Molecular Markers AN ABSTRACT OF THE THESIS OF Nina Rosa F. Castillo for the degree of Master of Science in Plant Physiology presented on September 13, 2006. Title: Fingerprinting and Genetic Stability of Rubus Using Molecular Markers Abstract approved: _____________________________________________________ Barbara M. Reed Two studies used DNA markers to assess their usefulness in germplasm identification and evaluation of genetic stability in four cryopreserved Rubus accessions that were stored for over 10 years in liquid nitrogen. In the first study, 12 robust Simple Sequence Repeat (SSR) markers were developed from two microsatellite-enriched libraries of ‘Meeker’ raspberry and ‘Marion’ blackberry. Of the 70 and 78 SSR-containing sequences identified, four SSR markers were obtained from the ‘Meeker’ library and eight from that of ‘Marion’. These twelve genomic SSRs and one Expressed Sequence Tag- (EST)-SSR designed from an (AT)6- containing R. idaeus sequence (AF292369) from Genbank were used for fingerprinting 48 raspberry and 48 blackberry cultivars stored at the Repository. The SSR markers developed in Rubus were highly polymorphic. Twelve SSRs generated 96 alleles in raspberries and 177 in blackberries. These markers distinguished between the raspberries and blackberries except in ‘Logan’ and ‘Boysen’ clones. Neighbor Joining cluster analysis based on the proportion of shared allele distance using 13 SSRs separated the cultivars into two main groups: the raspberries and the blackberries. Hybrid berries and cultivars with uncommon ancestry grouped separately from the two major groups. The raspberry and blackberry groups were further divided according to their pedigrees. In the second study, two types of markers, SSRs and Amplified Fragment Length Polymorphisms (AFLP) were used to evaluate genetic fidelity of regrown cryopreserved Rubus shoot tips stored for 12 years in liquid nitrogen. Analyses were done on two groups of plants separated based on the length of time they were subcultured after storage. Group one plants were analyzed after subculturing for seven months using Platinum Taq polymerase in the PCR reactions while Group two plants were analyzed immediately after recovery from cryopreservation using AmpliTaq Gold polymerase in the PCR reactions. No polymorphism was detected in either group of plants based on SSR analysis using 10 loci. Ten AFLP primer pairs amplified 547 fragments in R. grabowskii, 400 in ‘Mandarin’, 530 in ‘Silvan’, and 521 in ‘Hillemeyer’ Group one plants. An appreciably lower number of PCR products were amplified in Group two plants: 331 fragments in ‘Hillemeyer’, and 379 in ‘Silvan’. Differences in number of AFLP markers between Groups one and two were caused by use of different polymerases during the analysis. AFLP markers, with a high marker index, revealed polymorphism in three of four Rubus genotypes in Group one. However, no polymorphism was detected in Group two plants based on AFLP analysis. Recovery of plants from cryopreservation was low in the three accessions that exhibited AFLP polymorphisms (R. grabowskii, ‘Silvan’ and ‘Mandarin’). ‘Hillemeyer’ regrowth was 80% while R. grabowskii was 40%, ‘Silvan’, 20% and ‘Mandarin’, 10%, indicating less than ideal regrowth for the three genotypes. Such polymorphism might have been generated through somaclonal variants regenerated from callus tissue. Genotypic influence on stability may explain why those three genotypes were prone to variation while ‘Hillemeyer’ remained genetically stable despite long culture periods. High recovery rates and careful treatment and monitoring of regrown plants should therefore be employed to ensure maintenance of genetic fidelity of cryopreserved plants. The variation detected may also be transient and requires further morphological and molecular analysis of adult regrown cryopreserved plants that were transplanted and are growing in the greenhouse. ©Copyright by Nina Rosa F. Castillo September 13, 2006 All Rights Reserved Fingerprinting and Genetic Stability of Rubus Using Molecular Markers by Nina Rosa F. Castillo A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented September 13, 2006 Commencement June 2007 Master of Science thesis of Nina Rosa F. Castillo presented on September 13, 2006. APPROVED: Major Professor, representing Plant Physiology Head of the Department of Horticulture Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Nina Rosa F. Castillo, Author ACKNOWLEDGEMENTS My sincere appreciation goes to my major professor, Dr. Barbara Reed for accepting me as her advisee and giving me the opportunity to pursue the study. The support and encouragement had been valuable as I finish my degree. My sincere appreciation also goes to Dr. Nahla Bassil for accommodating me in her laboratory and for funding the research. The technical guidance, knowledge and skills imparted and unending patience are greatly appreciated. Thank you also to Dr. Chad Finn for sharing the resources that I need especially during the preparation of the manuscript; Dr. Kim Hummer for sharing valuable information especially during the final stages of the research; Dr. Valerian Dolja for reviewing the manuscript; members of the molecular genetics lab of the repository: especially to Barb Gilmore for the assistance throughout the experiment, Wambui Njuguna and Adrienne Oda for always lending a hand; members of the tissue culture lab, Jeanine DeNoma and Janine de Paz for helping me in media preparation and micropropagation; Brian Knaus and Peter Boches for helping me during the data analysis; my friends Vidyasagar, Sathuvalli and Jack Peters for all the support and help extended. Special thanks are due to my family, my dad, mom, sisters, Vicky and Joy, brothers-in-law, Greg and Rigor, my nephew, Jethro for all the emotional support and encouragement and most especially to my husband, Neil, and son, Nikki, for the patience and love always. CONTRIBUTION OF AUTHORS Dr. Nahla Bassil and Dr. Barbara Reed assisted with the experimental design, analysis, and writing of all chapters. Dr. Chad Finn provided all the information for the raspberries and blackberries used in fingerprinting in Chapter 2. TABLE OF CONTENTS Page CHAPTER 1. INTRODUCTION .................................................................................. 1 The Genus Rubus ...................................................................................................... 2 Raspberries........................................................................................................ 4 Blackberries ...................................................................................................... 5 Plant Genetic Resources of Rubus ............................................................................ 6 Rubus Conservation .................................................................................................. 8 Seed Storage...................................................................................................... 9 Field Genebanks.............................................................................................. 10 In Vitro Methods ............................................................................................. 11 Genetic Variation .................................................................................................... 12 Cryopreservation..................................................................................................... 14 Markers and Their Uses in Rubus........................................................................... 17 Molecular Marker Use in Assessing Genetic Fidelity ............................................ 27 Research Objectives................................................................................................ 29 Literature Cited ....................................................................................................... 30 CHAPTER 2. MICROSATELLITES FOR CULTIVAR IDENTIFICATION IN RASPBERRIES AND BLACKBERRIES.......................................................... 44 Abstract ................................................................................................................... 45 Introduction............................................................................................................. 46 Materials and Methods............................................................................................ 49 Results..................................................................................................................... 60 Discussion ............................................................................................................... 63 TABLE OF CONTENTS (Continued) Page Literature Cited ....................................................................................................... 71 CHAPTER 3. EVALUATION OF GENETIC STABILITY OF CRYOPRESERVED Rubus MERISTEMS USING SSR AND AFLP MARKERS............................. 77 Abstract ................................................................................................................... 78 Introduction............................................................................................................. 79 Materials and Methods...........................................................................................
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