Medicine, Faculty of
Medical Genetics, Department of
DSpace
UBCV
Sakakibara, Stacey M.
Master of Science - MSc
University of British Columbia
The genus Rhododendron is one of the largest among the angiosperms, and has been the subject of intense interest in the horticultural field for the many species which are of commercial value. Hybrid rhododendrons are also valued, and many thousands of named cultivars have been registered with the International Rhododendron Registry. Rhododendrons make up an important sector of British Columbia's nursery industry, as they thrive in the mild climate of the Lower Mainland and Vancouver Island. The RAPD marker technique was applied in rhododendrons, first for DNA fingerprinting of commercial cultivars, and secondly, to study relationships within the genus For DNA fingerprinting, samples of rhododendron hybrids were obtained from Clay's Nursery in Langley, B.C. Reproducibility of the RAPD technique was established, as was clonal stability of the banding patterns generated. Thirteen RAPD primers were selected and used to generate RAPD banding patterns for the hybrid cultivars. Keys to identification of these cultivars were created based on the presence or absence of certain bands. The reproducibility of RAPDs coupled with the high level of polymorphism generated and the simplicity of the protocol makes the technique a good candidate for use in industry as a tool for quality control and identification of individuals whose identity may be in question. RAPDs were also evaluated for use in parentage analysis in rhododendrons. However, the relatively high frequency of non-parental bands observed limits the usefulness of the technique to certain situations in which there are only a few putative parents. For the taxonomic study, samples were collected from species in subsection Fortunea, as well as from some species thought to be related, and from an outgroup species. Cluster analysis on RAPD data generated from these samples did not give any evidence for the postulated relationships, but did highlight some other relationships. Individuals of the same species were generally clustered together, and individuals of the outgroup species were clustered in a distinct group from the other species. No distinctions were observed between species from more closely related subsections, however, which gives rise to questions regarding the current classification of Rhododendron. Further work is required to clarify and define these relationships.
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D N A F I N G E R P R I N T I N G IN R H O D O D E N D R O N S U S I N G R A N D O M A M P L I F I E D P O L Y M O R P H I C D N A by S T A C E Y M . S A K A K I B A R A B. Sc., University of British Columbia, Vancouver, British Columbia, 1994 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E STUDIES (Genetics Graduate Program) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1997 Stacey M . Sakakibara, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of JLyw^^^ . & The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T The genus Rhododendron is one of the largest among the angiosperms, and has been the subject of intense interest in the horticultural field for the many species which are of commercial value. Hybrid rhododendrons are also valued, and many thousands of named cultivars have been registered with the International Rhododendron Registry. Rhododendrons make up an important sector of British Columbia's nursery industry, as they thrive in the mild climate of the Lower Mainland and Vancouver Island. The RAPD marker technique was applied in rhododendrons, first for D N A fingerprinting of commercial cultivars, and secondly, to study relationships within the genus For D N A fingerprinting, samples of rhododendron hybrids were obtained from Clay's Nursery in Langley, B.C. Reproducibility of the RAPD technique was established, as was clonal stability of the banding patterns generated. Thirteen RAPD primers were selected and used to generate RAPD banding patterns for the hybrid cultivars. Keys to identification of these cultivars were created based on the presence or absence of certain bands. The reproducibility of RAPDs coupled with the high level of polymorphism generated and the simplicity of the protocol makes the technique a good candidate for use in industry as a tool for quality control and identification of individuals whose identity may be in question. RAPDs were also evaluated for use in parentage analysis in rhododendrons. However, the relatively high frequency of non-parental bands observed limits the usefulness of the technique to certain situations in which there are only a few putative parents. For the taxonomic study, samples were collected from species in subsection Fortunea, as well as from some species thought to be related, and from an outgroup species. Cluster analysis on R A P D data generated from these samples did not give any evidence for the postulated relationships, but did highlight some other relationships. Individuals of the same species were generally clustered together, and individuals of the outgroup species were clustered in a distinct group from the other species. No distinctions were observed between i i species from more closely related subsections, however, which gives rise to questions regarding the current classification of Rhododendron. Further work is required to clarify and define these relationships. TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables vii List of Figures viii Acknowledgements ix Dedication x Chapter 1. Introduction 1.1 Rhododendron 1 1.2 PCR & Random Amplified Polymorphic D N A 2 1.3 UsesofRAPD 3 1.4 RAPDs and Rhododendrons 5 1.5 Rhododendron Taxonomy 6 1.6 Rhododendron Subsection Fortunea 8 1.7 Species Distribution and Descriptions 11 1.7.1 Subsection Fortunea Species 11 1.7'.2 Other Species 13 1.8 Objectives 15 Chapter 2. DNA Fingerprinting in Rhododendrons Using RAPDs 2.1 Introduction 18 2.2 Materials & Methods 19 iv 2.2.1 Sample Collection 19 2.2.2 DNA Isolation 25 2.2.3 DNA Amplification 26 2.2.4 Primer Screening 27 2.2.5 Reproducibility 28 2.2.6 DNA Fingerprinting Conditions 28 2.2.7 Parentage Analysis 29 2.2.8 Analysis of DNA Fingerprints 29 2.3 Results 30 2.3.1 DNA Isolation 30 2.3.2 Primer Screening 30 2.3.3. Reproducibility 31 2.3.4 Parentage Analysis 35 2.3.5 DNA Fingerprinting 35 2.4 Discussion 40 2.4.1 Reproducibility Testing 40 2.4.2 Parentage Analysis 42 Chapter 3. Taxonomic Study of Rhododendron Subsection Fortunea 3.1 Introduction 47 3.2 Materials & Methods 49 3.2.1 Southern Blotting 56 V 3.2.4 Antibody Binding & Detection 59 3.2.5 Data Analysis 60 3.3 Results 60 3.3.1 Southern Blots 60 3.3.2 Cluster Analysis 61 3.4. Discussion 67 3.4.1 Southern Blots 67 3.4.2 Cluster Analysis 69 Chapter 4. General Conclusions 76 Bibliography 79 Appendix 1 83 Appendix II 84 Appendix III... .Compact disk containing collated RAPD patterns for hybrid cultivars vi LIST OF T A B L E S T A B L E PAGE 1. Classification of species under study. Comparison of the Balfourian 9 system of Rhododendron classification to Chamberlain's revision of subgenus Hymenanthes (based on RHS Yearbook, 1980). 2. Rhododendron hybrids collected for the D N A fingerprinting study and 20 their sources. 3. Rhododendron cultivars sampled for use in parentage analysis. 24 4. Sequences of primers selected for D N A fingerprinting and sizes of bands 32 scored for the taxonomic study. 5. Hybrid rhododendron cultivars used to study clonal stability 36 6. Data from parentage analysis. 37 7. Similarity matrix based on Jaccard's coefficient of R. sutchuenense, 46 R. x geraldii, and R. praevernum. 8. Rhododendron species and sources used in the taxonomic study 50 9. Southern blots: samples and probes 57 10. Results of goodness of fit tests comparing dendrograms generated by 64 three different clustering methods to the original similarity matrix. vii LIST OF FIGURES FIGURE P A G E 1. Map of China (from Davidian, 1988). 12 2. Rhododendron leaf shapes (from Cox, 1979). 16 3. Rhododendron corolla shapes (from Chamberlain, 1982). 17 4. Reproducibility of RAPDs in rhododendrons. 33 5. Clonal stability of RAPD banding patterns. 34 6. RAPD banding patterns of rhododendron hybrids and their parents. 38 7. Examples of D N A fingerprint composites found on the attached CD. 39 8. Southern blots and the RAPD gels from which they were derived. 62 Blots 232 and 225. 8.1 Blot 416. 63 9. Dendrogram of unweighted pair-group method with arithmetic average 66 (UPGMA) cluster analysis of the similarity matrix based on Jaccard's coefficient. 10. Dendrogram generated by the complete linkage clustering method 74 (CLINK) on the similarity matrix based on Jaccard's coefficient. viii A C K N O W L E D G M E N T S I am indebted to many people who have been extremely generous in assisting me on the way to completion of this thesis. Thanks to my supervisor, Dr. J.E. Carlson, for providing the opportunity to work on this project; Dr. Brian Ellis, who was instrumental in originating the contact with Clay's Nursery; members of my committee, Drs. Brian Holl, Carl Douglas and Fred Ganders for their guidance and suggestions in the formation of this work, and for critical reading of this thesis. Special thanks to Fred Ganders for sharing his expertise in rhododendrons and suggestions on species to study. Thanks to Les Clay of Clay's Nursery, Carolyn Jones of Van Dusen Botanical Gardens, Wilf Nicholls of the U B C Botanical Garden, Steve Hootman of the Rhododendron Species Foundation, and Colin Varner of the U B C Physical Plant Nursery for their generosity in providing tissue samples and access to their accession systems. Fumi, Todd and Brodie Sakakibara, Chieko and Yuki Inouye, and Michelle Hasebe all volunteered many hours of their time to help me collect samples, for which I am very grateful. Finally, thanks to my husband Brendan and my family for their unwavering encouragement and support. ix D E D I C A T I O N the memory of my teacher, Jack Schratter. CHAPTER 1: INTRODUCTION Random amplified polymorphic D N A (RAPD) is a relatively new technique, with various applications in biology. Based on the polymerase chain reaction (PCR), the technique utilizes a single short primer of arbitrary sequence to detect polymorphisms in the genomes of different individuals (Williams et al., 1990a; Welsh & McLelland, 1990). Because of the simplicity and speed with which results can be obtained, RAPDs are commonly used in D N A fingerprinting (Wilde et al., 1992), population genetic studies (Ballinger-Crabtree et al., 1992), and taxonomy (Demeke et al., 1992). Since no prior knowledge of genome size or sequence identity is required, RAPDs are especially useful in species which have not previously been studied using molecular biology. For example, rhododendrons are a commercially important crop within the nursery industry, and while a great deal of research is devoted to culture, care and breeding of these plants, relatively little has been done at the D N A level. The first objective of this study was to apply RAPDs to rhododendron cultivars for D N A fingerprinting, a need for which had existed since the advent of tissue culture for propagation (Knuttel, 1990; Bent, 1994). In addition, the fact that data from RAPDs is often analyzed for taxonomic purposes gave rise to the second main objective of the study, that is, to gain information on some of the relationships within the Rhododendron subsection Fortunea. 1.1 Rhododendron The history of cultivated rhododendrons is a long and fascinating one. While deliberate hybridization of evergreen azaleas is recorded from 17th century Japan (Williams et al., 1990b), widespread commercial breeding of rhododendrons began early in the 19th century 1 in England with the introduction of species from North America and the Himalayas (Davidian, 1982). The primary aim of these early hybridizers was to produce hardier plants; however, amateur breeders soon began to raise hybrids acclaimed for the beauty of their flowers or foliage or both. The introduction of certain horticulturally valued characters are often the aim of these controlled crosses, and the quest for novel floral forms, aroma or color, cold hardiness, heat tolerance, disease resistance and improved foliage has resulted in hybrid rhododendron cultivars numbering in the thousands. The advent of tissue culture has allowed large scale clonal propagation of plant selections, and this technology is now widespread in nurseries specializing in rhododendrons. However, because exchange and mass propagation of tissue culture material takes place when the plants are at ajuvenile stage, problems in identification can occur (Iqbal et al., 1995). Propagation of mislabeled plants has resulted in significant economic losses to nurseries (Knuttel, 1990), as well as substantial confusion as to the identity of certain plants on the part of amateur hobbyists. Thus, there is a need for a method of identification that is independent of tissue type and age, as well as environmental conditions. Given that an organism's D N A is unvarying with time and between different tissues, D N A fingerprinting has the potential to be a reliable means of plant identification. 1.2 PCR & Random Amplified Polymorphic DNA Methods of D N A fingerprinting based on the polymerase chain reaction (PCR) have become widely used in recent years as they offer a more rapid and simpler approach to detecting genetic differences between individuals than hybridization based procedures. D N A amplification using PCR involves several components in a reaction mixture. Template D N A 2 is isolated from the individual under study and short oligonucleotide primers which flank the region to be amplified are added in excess, along with a thermostable D N A polymerase (e.g. Taq D N A polymerase). Also present in the reaction are free nucleotides, M g + 2 , and buffer. PCR proceeds in several steps. First, the template is denatured by bringing the reaction to a high temperature, usually between 94-96°C. The primers are then allowed to anneal to the single stranded template by bringing the temperature down to a specific temperature between 40-65°C. D N A synthesis by the D N A polymerase is performed at around 72°C. The denaturing, annealing and extension steps comprise one cycle of PCR and are repeated in sequence from 25-35 times. Products from earlier rounds of PCR are utilized as templates for succeeding cycles; thus amplification of D N A is exponential. The technique widely known as random amplified polymorphic D N A (RAPD) was developed virtually simultaneously by Welsh and McLelland (1990) and Williams et al. (1990a). Whereas conventional PCR uses primers specific to a particular region of interest within the genome, the RAPD technique utilizes a short (usually 10 nucleotide) primer of arbitrary sequence. The primer is able to anneal to multiple sites within the genome, and amplification of several unknown sequences occurs. As this method requires low stringency for primer annealing, the annealing temperature is generally reduced for RAPDs. 1.3 Uses of R A P D RAPDs present several advantages to other methods of D N A fingerprinting. As no prior knowledge of genome size or sequence identity is required, RAPDs are especially useful in species which have not previously been studied using molecular biology. In addition, the technique is simple and fast and has the potential to be automated. RAPDs have been found 3 to detect high levels of polymorphism in numerous organisms, ranging from humans (Williams et al., 1990b), to insects (Ballinger-Crabtree et al., 1992), to microorganisms (Van Reenan and Dicks, 1996); and have often been used in plant crops of agricultural importance such as coffee (Orozco-Castillo et al., 1994), blueberries (Aruna et a l , 1993), celery (Yang & Quiros, 1993), cocoa (Wilde et al., 1992), raspberries (Parent et al., 1993), rapeseed (Mailer et al., 1994), peppers (Prince et a l , 1995), sweet potato (Connolly et a l , 1994), rice (Yu & Nguyen, 1994), broccoli and cauliflower (Hu and Quiros, 1991). Among the ornamental plants that have been studied using RAPDs are chrysanthemum (Wolff & Peters-Van Rijn, 1993), lilacs (Marsolais et al, 1993), and irises (Arnold et al., 1991). In addition to D N A fingerprinting, RAPDs have been utilized in population genetic studies to identify populations (Ballinger-Crabtree et al., 1992), demonstrate introgression (Arnold et a l , 1993), and to evaluate levels of genetic variation (Yu and Nguyen, 1994; Chandrashekhar and Nguyen, 1993; Williams et al., 1993). RAPDs have also been used to elucidate pedigree relationships in plants. In data analysis based on band sharing, Marsolais et al. (1993) were able to determine the origin of interspecific lilac hybrids and predicted that RAPDs will be useful for assessing the origins of cultivars and hybrids in many plants. If found to be valid in rhododendrons, this application of R A P D will be of great use to plant breeders, for many of the popular cultivars are of unknown or dubious parentage. -Finally, taxonomic studies based on phenetic relationships derived from band sharing have been done in poplar (Castiglione et al., 1994), Brassica (Demeke et a l , 1992) and celery (Yang and Quiros, 1993), to name a few. A l l have found RAPDs to be useful in 4 classification, although non-homologous bands of similar size may be a problem when comparing R A P D profiles between different species (Thormann et al., 1994). 1.4 RAPDs and Rhododendrons During the course of this research, a series of papers have been published by one other research group in which RAPDs have been used with rhododendrons. Iqbal et al. (1994) first showed that RAPDs can differentiate between individuals of different species by applying the technique to a single representative of four species; R. arborescens, R. atlanticum, R. formosum var. inaequale, and R. kawakamii. In a later paper, the authors confirmed that RAPD profiles were stable among clones of one individual. Using nine or ten clones each of R. arborescens, R. atlanticum and R. yedoense var. poukhanense (Iqbal et al., 1995a), and 10 RAPD primers, no polymorphisms were found among the clones of R. atlanticum and R. arborescens, while the R A P D profile of one clone of R. yedoense showed a difference of one band with one primer. The most recent paper to be published utilizes cluster analysis to study genetic relationships between twelve rhododendron species and hybrids (Iqbal et al., 1995b). It was found that individuals of the same species clustered together, and that hybrids with common parents clustered together. Iqbal et al. conclude that cluster analysis of RAPD data may be used to study ancestral relationships. It is clear that RAPDs have potential for use in studying genetic relationships within the genus Rhododendron, and in light of the sometimes controversial debate over classification of certain species, it was thought that a study of this nature may clarify some of the grey areas that are known to exist in the classification of the genus. 5 1.5 Rhododendron Taxonomy The genus Rhododendron is one of the largest among angiosperms, comprised of between 800-900 species, and still growing, as new species are continuously being described (Williams et al., 1990b). The magnificent flowers and attractive foliage of many rhododendrons are particularly valued in horticulture, and controlled hybridization and selection has resulted in a vast array of cultivars, numbering over 5000. As a result, rhododendrons account for an important sector of the nursery industry. Rhododendron has not been amenable to classification. Indeed, Cox has stated that " i f a thousand perfectly competent taxonomists tackled the genus...they would each produce different results" (p.81, 1979). One reason for this difficulty may be the fact that hybridization occurs freely between species of different subsections in the wild. As well, taxonomic significance of the morphological differences on which classification is based is not always clear (Chamberlain, 1982). In addition, the majority of species merge into one another in the wild and variation within natural populations is high (Leach, 1962). It seems evident that much speciation is taking place in areas where the ranges of many different and merging species overlap (Cox, 1979). Classification of this genus is of interest not only to botanists and taxonomists, but also to horticulturists and hobbyists. In the past, this has made changes in taxonomy of Rhododendron difficult to implement, as the vast majority of individuals in the field had adopted the "Series" system originated by Sir Isaac Bayley Balfour, and were reluctant to accept changes (Cox, 1979). Balfour's system was intended to be a temporary method of dealing with the influx of hundreds of new species from botanical exploration in Asia during the early part of this 6 century. It consisted of 43 series, each built around a single, well known "typical" species (Royal Horticultural Society, 1980). Unfortunately, Balfour died before revisions could take place, and his system was adopted by the British Rhododendron Society and published in The Species of Rhododendron (Stevenson, 1930). Thus, the Balfourian system became firmly established. A more natural classification which involved Subgenera, Sections and Subsections was proposed by Sleumer in 1949, but did not gain popular acceptance. Over the years confusion and miscommunication increased as various authors modified the two classifications and used these in their own writings. A complete revision of the genus was undertaken by Cullen (1982) and Chamberlain (1982), based on Sleumer's work and with contributions from the Philipsons (1986). As it stands today, the genus Rhododendron is divided into eight subgenera. Subgenus Rhododendron encompasses the lepidote (scaly) species of Rhododendron, and is composed of three sections, Vireya (mainly subtropical), Rhododendron, and Pogonanthum. The elepidote, or non-scaly species make up the remaining three subgenera. The former "Azalea" series of the Balfourian system have been divided into six subgenera, the most important of these being Azaleastrum and Tsutsusi, and the remaining elepidotes comprise subgenus Hymenanthes. Hymenanthes is composed of a single section, Pontica, which is further divided into twenty-four subsections. This revision has been generally accepted as the framework for the taxonomic organization of Rhododendron. It is, however, recognized that much work is still required for elucidation of relationships at lower taxonomic levels. The classifications in place at present may necessarily change as new data are acquired (Luteyn & O'Brien 1980). 7 1.6 Rhododendron Subsection Fortunea Subsection Fortunea within subgenus Hymenanthes has been described by Chamberlain (1982) as "a heterogeneous group of species that are nevertheless more closely allied to one another ... than they are to any species in any other subsection." As the Fortunei series described by Tagg (Stevenson 1930) and based on the classification of Balfour, this group was divided into six subseries as follows. Subseries Species Calophytum R. calophytum, R. openshawianum Davidii R. davidii, R. sutchuenense, R. planetum, R. praevernum Fortunei R. decorum, R. diaprepes, R. serotinum, R. fortunei, R. hemsleyanum, R. glanduliferum, R. discolor, R. houlstonii, R. vernicosum Oreodoxa R. oreodoxa, R. praeteritum, R. fargesii, R. erubescens Griffithianum R. griffithianum Orbiculare R. orbiculare Although the series has become a subsection and the subseries are no longer recognized, Cullen's revision did not introduce any substantial changes to this classification. However, the minor changes are summarized in Table 1. The majority of these adjustments have caused several taxa which were species to be classified as subspecies or varieties. Members of this subsection, particularly R. griffithianum and R. fortunei, have been used extensively as parents in hybridizations. In addition, several authors have suggested that some of the species included in subsection Fortunea are closely related to species in other 8 6 O TO CJ- TO TO' rS CT *n s g CT CTQ ,TO S " I i ^ 5 § 2 2 2. =• ^ r c, pi 5 w 3 § CO o C/3 <v £> 3 < 5 §? 2 >n 2 g cr TO Tl i-t 1 3* TO 5 -s TO cr CT X p 3 ^ © ^ 3. g £ S- I ^3 ^3 r^ > X © r 5 TO 3 r? W § » » CT* o 3 r« ro g •>!•>!•>! CT TO TO TO t » X X X « r r r §. < < £ § C/5 W to ^ CQ CO (/) JO •a t3 >a w 5' >3 > -TO P S 3 to » • C O O" S CT 5' ^ B K !> 2 p ffi 2. CT 2 3 e. on V TO a TO CT P. CT co cr cr CT i-l B. CT O CS 1/1 & CT cs c X CT 3 I cr CT 01 > 3 H * g" Si B a S 1 g. c 2 3 2-pa «i EC: ce 65 B s* 3 c B • C 2 as 8 o D co 3 5 * I CD 3 CT an » g *5ft » 3 to as ft D" 65 B a* B' >3 >3 3-o S co o a 1 B S ° O B X o o a <: a 3 ft a" e O CO n cT a s O o x sp o 2. CD W < 8 a-o a Co O a >3 >3 & 1 3 S" ce O rt 5S « . o 3 3 o o ^ a a s O o 3 g3 II CD «> >-t O g. w S' en _ ? H s- crq 2 era ST e >a >> *0 S ^ S3 a " I? S c 2 S B ' 2 a a e 3 CfQ era co rt 2 05 03 on 65 p t3 t o a a CO rt 1 U> us G O B 3^ TO Co ce e a" wi rt rt ©' D w 9 "1 65 3 a n e. B >3 >3 >3 >3 >0 >3 Co o Co e a-a TO a Co hrj TO 2 B 3 o B tf o < TO >! a & o s f? a-s TO a TO a Co TO cr 3^ TO TO* X c 3 X o cr TO Co O TO 0 1 a o cr o cr C 3 CO » S' CO rt O 3 subsections. Chamberlain noted that several subsections, including Fortunea, "could and probably should be subdivided". A study of phenology at the D N A level may give systematists additional characteristics to work with, as well as providing information to hybridizers as to the relationships within this important subsection. 1.7 Species Distribution and Descriptions 1.7.1 Subsection Fortunea Species The distribution of Fortunea has been documented by Chamberlain (1982). Most species are found in the provinces of Eastern and Southern China, ranging as far west as central Sichuan (Figure 1), however three species, R. decorum, R. vernicosum, and R. oreodoxa extend further west into northern Yunnan, southwest Sichuan, and Burma, and R. diaprepes is restricted to that area of northeast Burma and west Yunnan. R. griffithianum is found in eastern Nepal, northeastern India, and Bhutan (Figure 1). Of the species outside Fortunea that were included in this study, R. auriculatum may be found in central-eastern China around eastern Sichuan, while R. rex is distributed more towards the west, again across Yunnan and southwest Sichuan to northeast Burma. R. praestans is distributed similarly, but is restricted to northwest Yunnan and southeast Tibet (Xizang). R. williamsianum is endemic to central Sichuan; R. thomsonii ssp. thomsonii is restricted, like R. griffithianum, to eastern Nepal, northern India and Bhutan, while ssp. lopsangianum is found in south Xizang (Tibet). R. yakushimanum is found only in Japan. The Fortunea rhododendrons are shrubs or trees, reaching heights up to 18m. By all accounts a heterogeneous group, the leaves of these species range from oblong lanceolate {R. calophytum) to orbicular (R. orbiculare). Generally, the leaves are hairless on the top surface 11 CHINA Scale 1" = 447 Miles 1" = 720 Km. 0 ISO 360 5 00 720 900 1080 Km. 1 ' i ' I ' I 224 447 671 Miles Figure 1. Map of China (from Davidian, 1988) 12 at maturity while the lower surface can have whitish indumentum on the midrib (Chamberlain, 1982). The flowers are often glandular with corollas of 5-7 lobes, small calyxes, and over ten stamens (Cox, 1979). The corollas within this subsection range from open-campanulate to funnel campanulate and are prevalently colored white, pink, or rose ~ never yellow or red. Inflorescences range from five to thirty flowers arranged as an umbellate raceme (Davidian, 1988). 1.7.2 Other Species R. auriculatum, of subsection Auriculata, is a small tree up to six metres tall, with oblong leaves that bear glands at the margins and a funnel shaped corolla with seven lobes. This species, one of two in the subsection, is distinguished from Fortunea by its glandular-setulose young shoots, the hairs and glands on the leaf undersurface and the long buds bearing pointed scales (Chamberlain, 1982). R. praestans (subsection Grandia) is a shrub or small tree ranging from three to ten metres tall. The leaves are very large, oblong obovate in shape, with plastered indumentum below. The flowers of this species are eight lobed, with sixteen stamens, and range in color from white to magenta-rose. Cox (1979) suggests a relationship with R. calophytum on the basis of its large leaves, long pedicel and ventricose corolla. R. rex (subsection Falconera) also grows as a large shrub or small tree, from three to ten metres tall. Another of the large leafed species, individuals of this species possess oblanceolate foliage which is dark and shiny on the top surface and covered with thick indumentum below. The flowers can range in color from white to pink to mauve with a 13 crimson blotch and spots. The corolla is long, tubular campanulate in shape. Typically, sixteen stamens are found. The inflorescence carries from twenty to thirty flowers. Subsection Falconera is considered by Chamberlain (1982) to be closely allied to Subsection Grandia, and distantly related to Fortunea. As such, R. rex was chosen as a representative species from Falconera which may help to clarify the R. calophytum - R. praestans relationship. R. williamsianum (Subsection Williamsiana) is a dwarf shrub with ovate orbicular to broadly oblong leaves; the lower leaf surface is glabrous at maturity. The corolla is five or usually six lobed and campanulate, and is pale pink to rose or occasionally white in colour. Although Chamberlain considers this a distinct species with no close relatives, R. williamsianum is similar in general appearance, both in foliage and flower, to R. orbiculare (F. Ganders, pers. comm.). Cox (1979) states that this species has some affinities with R. souliei in subsection Campylocarpa. R. thomsonii individuals are shrubs to small trees, with orbicular or ovate leaves that are leathery and glaucous. The inflorescence is loose and composed of six to twelve flowers that are deep red in color, campanulate in shape. The calyx is cupular and unlike the Fortunea species, is often colored. This species is said to hybridize with R. campylocarpum in the wild (Chamberlain, 1982), and the foliage is often thought to be similar to R. orbiculare (F. Ganders, pers. comm.). R. yakushimanum (Subsection Pontica) is a very popular horticultural species. It grows as a shrub, and the leaves are elliptic or linear-lanceolate, the upper surface often covered with a thin indumentum. The corolla is five lobed, funnel-campanulate, and five to ten 14 flowers grow in a loose truss. Not thought to be closely allied to any of the Fortunea species, R. yakushimanum was used as an outgroup of sorts, and was also utilized in a preliminary study to determine levels of intraspecific variation. Rhododendron leaf morphology may be seen in Figure 2, while corolla shapes are shown in Figure 3. More detailed species descriptions may be found in Chamberlain's Revision of Rhododendron subgenus Hymenanthes (1982) and Cox's The Larger Species of Rhododendron (19791 1.8 Objectives In applying RAPDs to rhododendrons the objectives of this thesis were twofold. The first aim was to develop D N A fingerprints of commercial rhododendron cultivars using RAPDs, and to produce a fingerprint-based key for identification. The second goal was to study relationships within the Rhododendron subsection Fortunea utilizing cluster analysis of RAPD data, and to determine whether certain species classified within the subsection were more closely related to species in other subsections. A secondary objective was to evaluate the RAPD technique for its usefulness in determining parentage of certain hybrid rhododendrons. 15 A . Rhombic B. Oblong lanceolate C. Oblong O. Obovate E. Lanceolate F. Orbicular G . Oblanceolate H. Elliptic I. Oval J . Ovate Figure 2. Rhododendron Leaf Shapes (from Cox, 1979) 16 Figure 3. Rhodendron Corolla Shapes: a, b, campanulate; c, open-campanulate; d, funnel-campanulate; e, f, tubular-campanulate; g, oblique-campanulate. (from Chamberlain, 1982) 17 CHAPTER 2: DNA FINGERPRINTING IN RHODODENDRONS USING RAPDS 2.1 Introduction Rhododendrons are a commercially important horticultural crop in British Columbia. Nurseries specializing in rhododendrons rely upon tissue culture for large scale propagation of plant material. While a single nursery can propagate cultivars numbering in the hundreds, this method of clonal cultivation often leads to tissue culture tubes numbering in the thousands being processed. As conventional methods for identification of rhododendrons rely heavily on morphological characteristics of mature plants - especially flowers; and rhododendron cultivars are often sold in a juvenile form, handling of such large numbers can lead to mislabelling, and a single mistake can lead to hundreds of plants being propagated and sold under the wrong name. This in turn leads to financial losses to nurseries when these mislabelled plants are uncovered upon reaching maturity. A method of identifying or confirming the identity of these cultivars that is not dependent on morphological characters would therefore be very useful to those in the industry as a means of quality control. D N A fingerprinting using random amplified polymorphic D N A (RAPD) has become widely used in many crops of agricultural importance in recent years (Orozco-Castillo et al., 1994, Aruna et a l , 1993, Yang & Quiros, 1993, Wilde et al., 1992, Parent et al., 1993, Y u & Nguyen, 1994, Prince et al., 1995). D N A fingerprinting in itself is advantageous in that D N A is a stable trait which may be examined at any time during an organism's life cycle, and in many different tissues, thus precluding the necessity of examining mature plants to determine their identity. Using RAPDs as the tool for D N A fingerprinting has several 18 benefits over other methods. These include the speed with which fingerprints can be obtained, the relative simplicity of the protocol, the comparatively low cost, and the potential for automation. A l l of these advantages lend themselves well to a method of identification for use in industry. The objective of this study was to evaluate the method of random amplified polymorphic D N A for D N A fingerprinting rhododendron cultivars, and if the method proved to be successful, to obtain D N A fingerprints from rhododendrons of horticultural interest and to develop a key for identification of these plants. In addition, a small study was performed to investigate the potential use of RAPDs in determining parentage of hybrid rhododendrons. 2.2 Materials & Methods 2.2.1 Sample Collection Leaf samples were collected from 114 rhododendron cultivars at Clay's Nursery in Langley, B.C. (Table 2). To enable testing of clonal stability, where possible, duplicate material from a second individual clone was obtained from Van Dusen Gardens in Vancouver (Table 2). Additionally, samples from twelve hybrid cultivars that were not available from Clay's at the time of collecting were obtained from Van Dusen Gardens (Table 2). For parentage analysis, samples were collected from "families" consisting of a clonal hybrid cultivar and the two individuals which were the pollen and seed parents (Table 3). Where possible, newly unfolded leaves or those in their first year of growth were selected. After collection, plant material was stored for 24 hours to one week at 4°C. Leaves were then frozen in liquid nitrogen and ground to a fine powder using a Braun coffee grinder or a 19 Table 2. Rhododendron hybrids used in the DNA fingerprinting study and their sources. Hybrid Name Abbreviation Identification Number Source America A7 A7 Clay's Clay's Clay's Clay's Anah Kruschke A8 A8 Angelo A33 ' A33 Anna A40 A40 A n 77487 V D Anna Rose Whitney A9 A9 Clay's A R W u V D Arthur Bedford A B 7215 V D Aunt Martha A3 8 A3 8 Clay's Bambino Bm 91190 V D Beautiful Day B80 B80 Clay's Clay's Clay's Clay's Black Magic B55 B55 Blue Boy B77 B77 Blue Ensign B18 B18 B E u V D Blue Pacific B21 B21 Clay's BP 77387 V D Bonito B22 B22 Clay's Clay's Britannia B l B l Br 781090 V D Bud Flanagan B20 B20 Clay's Clay's Clay's Clay's Burnaby Centennial BO BO Caractacus C4* C4 Catawbiense Album C38* C38 Circus C27 C27 Clay's Clay's Clay's Cosmopolitan C7 C7 Cotton Candy C6* C6 CC u V D Countess of Derby CD 781096 V D County of York (Catalode) C37* C37 Clay's Crest Cr u V D Cunningham's White CW u V D Cutie C13 C13 Clay's Clay's Clay's Clay's Clay's Clay's Clay's Clay's Clay's Clay's Daphnoides D8 D8 Doc D51 D51 Dormouse D21 D21 Ebony Pearl E8 E8 Edith Boulter E13 E13 Edwin 0. Weber E48 E48 Exotic E40 E40 Fabia F l F l Freeman R. Stephens F21* F21 continued... 20 Table 2. continued Hybrid Name Abbreviation Identification Number Source General Eisenhower GE 781094 V D Golden Torch G57 G57 Clay's Clay's Clay's Goldsworth Orange G8 G8 Gomer Waterer G3 G3 GWO u V D GW1 781085 V D Grace Seabrook G33 G33 Clay's GS 81603 V D Grand Slam G37 G37 Clay's Clay's Clay's Grumpy G49 G49 Halfdan Lem H16 H16 H L u V D Hallelujah H19 H19 Clay's Clay's Clay's Heatherside Beauty H68 H68 Hotei H5 H5 Ht 751594 V D Hurricane H l l H l l Clay's Clay's Clay's Idealist 16 16 Ignatius Sargent 14 14 Jan Dekens J2* J2 Clay's JD 781084 V D Janet Blair J7 J7 Clay's Clay's Jean Marie de Montague J l J l J M 781087 V D Jennie Dosser J19 J19 Clay's Clay's Clay's Clay's Clay's Labrador L68* L68 Lee's Dark Purple L4 L4 Lem's Monarch L28 L28 Leo L18 L18 Linda L20 L20 Clay's Clay's Loder's White L6* L6 L W 7248 V D Loderi King George L27 L27 Clay's LKGO u V D . LKG1 u V D L K G 2 94713 V D Loderi Pink Diamond LPD 89183 V D LPD1 89183 V D Loderi Pretty Polly L I L I Clay's Loderi Superlative LS 89185 V D Loderi Venus L50 L50 Clay's L V 89184 V D LV1 u V D continued... 21 Table 2. continued Hybrid Name Abbreviation Identification Number Source Loderi White Diamond L W D 81604 V D Lord Roberts L8 L8 Clay's Clay's Markeeta's Flame M66 M66 MaF 93615 V D Mars M l M l Clay's Clay's Mrs. Betty Robertson M7 M7 M B R u V D Mrs. Furnival M5 M5 Clay's MF u V D Midsummer M28 M28 Clay's Clay's Clay's Naomi Pink Beauty N8 N8 Nova Zembla N l N l N Z u V D Odee Wright 03 03 Clay's Clay's Olin 0 . Dobbs 016 016 Olympic Lady 08* 08 Clay's OL u V D Ostbo's Red Elizabeth 01 01 Clay's Clay's Clay's Oudjik's Favorite O i l * O i l Pepperpot P29 P29 Percy Wiseman P70 P70 Clay's Peter Behring P48 P48 Clay's Clay's Clay's Phyllis Korn P l l P l l Pink Pearl P2 P2 PP 94714 V D Point Defiance P30 P30 Clay's Clay's Polar Bear P14 P14 PB u V D Potlatch P83 P83 Clay's Clay's Clay's Clay's Clay's Clay's Praecox P8 P8 Pr P8 Pygmalion P66 P66 Queen Nefertiti Q6 Q6 Ramapo R8 R8 Rp u V D Red Eye R42 R42 Clay's Clay's Clay's Clay's Rocket R6 R6 Ro R6 Rosamundi R2 R2 Roseum Elegans R4 R4 Clay's RE 7211 V D Rosy Dream R31 R31 Clay's Clay's Sappho S4 S4 Scintillation Sc u V D continued... 22 Table 2. continued Hybrid Name Abbreviation Identification Number Source Stephanie S54 S54 Clay's Clay's Clay's Clay's Sumatra S14 S14 Sir Charles Lemon S20 S20 Souvenir of W.C. Slocock S13 S13 SWCS u V D Surrey Heath S105 S105 Clay's Susan Sn 7283 V D Swamp Beauty S51 S51 Clay's Sweet Simplicity S96 S96 Clay's Clay's Sweet Sixteen S36 S36 Taurus T16 T16 Clay's Clay's Temple Belle T3 T3 Tidbit Tb 77377 V D Titian Beauty T37 T37 Clay's Clay's Clay's Clay's Clay's Tottenham T4 T4 Travis L . T40 T40 Trewithen Orange T6 T6 Trilby T l T l Trude Webster TW 77522 V D Unique U2 U2 Clay's U 781089 V D Vincent Van Gogh V20 V20 Clay's Clay's Vulcan V2 V2 VI u V D Whisperingrose W17 W17 Clay's Clay's Clay's Wilbrit W3 W3 Whirlaway Wh Woody's Friggin Riggin W7 W7 Clay's Clay's Yellow Pippin Berg's Yakushimanum Y16 Y16 Y9 Y9 Clay's Sources: Clay's = Clay's Nursery, Langley B.C. V D = Van Dusen Gardens, Vancouver B.C. Identification Number: Clay's = Catalogue number; Van Dusen Gardens = accession number u = unknown - sample possessed a name tag, but no accession number 23 Table 3. Rhododendron cultivars sampled for use in parentage analysis. Seed Parent Pollen Parent Offspring Hotei Jean Marie de Montague Catawbiense Album Mrs. Furnival Britannia Loderi Venus Loderi Venus Idealist Nova Zembla Countess of Derby Crest Leo Loderi King George Anna Rose Whitney Trilby Anna Anna Mrs. Betty Robertson Mist Maiden* Countess of Derby Beautiful Day Black Magic County of York Hurricane Jennie Dosser Markeeta's Flame Queen Nefertiti Odee Wright Peter Behring Trude Webster *Mist Maiden is a clonal form of R. yakushimanum, and is listed in Table 8. 24 or a mortar and pestle. The frozen, powdered tissue was placed in an appropriately sized Falcon tube (15 or 50 mis) and stored at -70°C to -80°C until D N A extraction. 2.2.2 DNA Isolation The method of D N A isolation was based on the protocol of Doyle and Doyle (1990). 7.5 ml of extraction buffer (3% cetyltrimethyl ammonium bromide (CTAB), 1.4M NaCl, 20mM EDTA (pH 8.0), lOOmM Tris-HCl (pH 8.0), 1% polyvinylpyrrolidone (PVP), and .5% p-mercaptoethanol added just prior to use) was aliquoted to a sterile 30 ml centrifuge tube, and preheated to 60°C in a water bath. Approximately 1 g of frozen powderedtissue was placed in the hot extraction buffer and incubated at 60°C from one to three hours with periodic vortexing. RNase A was then added to a concentration of 10 mg/ml and the tube was incubated at 37°C for 30 to 90 minutes. D N A was extracted with one volume of chloroform:isoamyl alcohol (24:1) and the tube was inverted to mix. The sample was then centrifuged at 2500 g for 10 minutes in a Beckman centrifuge using a JA-20 rotor. The aqueous layer was removed to a new tube, and the choloroform:isoamyl alcohol extraction was repeated. After the second extraction, the aqueous layer was removed to a 15 ml Falcon tube, and 4 M NaCl was added to a final concentration of 2.5M. D N A was precipitated with 2/3 volume room temperature isopropanol, and the sample was allowed to sit at room temperature for several hours to overnight. The tube was then centrifuged in a Heraeus tabletop centrifuge at 500 g for 2-3 minutes and the supernatant removed. In some cases, it was necessary to centrifuge the sample more vigorously (1600 g for 5-7 minutes) to obtain a pellet. In these instances, the pellet obtained was quite flocculent and viscous. About 10ml 25 of wash buffer (76% (v/v) ethanol and lOmM ammonium acetate) was added to the pellet, and the tube mixed with gentle swirling. Washing was allowed to proceed for several hours to days at room temperature. After washing, the tube was centrifuged for 10 minutes at 1 600 g, the pellet was transferred in a small volume of wash buffer to a 1.5ml Eppendorf tube, and further centrifuged at 13 000 rpm for 10 minutes in a refrigerated tabletop microcentrifuge. Supernatant was aspirated, and the pellet was allowed to air dry in a laminar flow hood. The pellet was then resuspended in an appropriate volume of TE buffer (lOmM Tris, ImM EDTA; pH 8.0). As the size of the pellet varied significantly between samples, volumes of TE required for resuspension ranged from 400 to 1000 ul. D N A concentration was estimated by gel quantification. A n initial 1/25 dilution of each sample was made in TE buffer and 5-10 |ul run on a 1.5% agarose gel for 45 minutes at 120V with lambda D N A standards of known concentration ranging from 5 to 100 nanograms of D N A per lane. After the gel was ethidium bromide stained for 30 minutes and destained in water for 10 minutes, the gel was placed on an ultraviolet transilluminator and photographed. Comparison of staining intensity between samples and standards allowed an estimate of the sample concentration to be made. The D N A was diluted to 0.8ng/jj,l for use in RAPDs, and these diluted samples stored at 4°C. D N A stock solutions were stored at -20°C. 2.2.3 DNA Amplification The conditions for R A P D assays were based on those established by Tulsieram et al. (1992). PCR amplification was performed in a GeneAmp PCR system 9600 thermal cycler (Perkin Elmer Cetus). R A P D amplification conditions were as follows: initial denaturing for 26 two minutes at 94°C followed by 45 cycles of one minute at 94°C, one minute at 36°C, and two minutes at 72C; final extension was for 10 minutes at 72°C, after which the temperature was held at 4°C. Various template and enzyme concentrations were tested, and an optimum concentration was selected for both. Each RAPD reaction contained the following components: 0.16 ng/ul template DNA, 0.3 u M oligonucleotide primer, 0.8 m M dNTP mixture, 2.3 m M MgC12, I X PCR Buffer II (10 m M Tris HC1, pH 8.3; 50 m M KC1), and .025 U/ul Taq D N A polymerase (Boehringer Mannheim). A premix composed of all ingredients except the template DNA, primer, and Taq D N A polymerase was prepared in bulk and stored at -20°C. Following amplification, 10 ul of 2X loading buffer (6X = 0.25% bromophenol blue, 0.25% xylene cyanol FF, 40% (w/v) sucrose in water) were added to each tube, and 7.5 pi of this mixture was loaded on to either a 1.5% agarose gel (primer screening) or 0.5% Synergel and 1.5% agarose (DNA fingerprinting). Agarose gels were subjected to electrophoresis in I X T A E buffer (0.04M Tris-acetate, 0.001M EDTA), and those with Synergel added were run in 0.5X TPE buffer (450mM tris-phosphate, lOmM EDTA) for 3.5 hours at 100 V (-0.20 V/cm2), stained with ethidium bromide, destained in water, and photographed on an ultraviolet transilluminator. 2.2.4 Primer Screening 274 primers were screened against D N A from four to ten cultivars obtained from Clay's Nursery in June, 1994. 3.3pl of 0.8 ng/ul D N A solution was aliquoted into each tube along with 3.3 ul of the primer being tested. Premix was measured out in the total volume required 27 for all RAPD reactions being performed, to which Taq D N A polymerase was added in the appropriate amount. 10ul of the premix + Taq was then added to each tube to bring the total reaction volume to 16.6 (jl Thirteen primers which gave clear, reproducible banding patterns were chosen for further use in D N A fiingerprinting. 2.2.5 Reproducibility Reproducibility of the R A P D technique was tested in several ways. Repeated amplifications on the same thermal cycler, banding patterns generated using several different thermal cyclers, and R A P D patterns generated using D N A from the same individual but isolated on different occasions were all evaluated for consistency. The cultivars used in this study are as follows: Britannia, Exotic, Grace Seabrook, Rocket, Red Eye, Tony, Whirlaway, Wilbrit, Yellow Pippin and Berg's yakushimanum. As well, clonal stability was examined using D N A from two or more individuals of one clone. In most cases one of the individuals was a young plant at Clay's Nursery, and the other was an established plant at Van Dusen Gardens. 2.2.6 DNA Fingerprinting Conditions The thirteen primers selected from the primer screening were used to generate RAPD D N A fingerprints from the whole set of rhododendron cultivars collected. Once primers were selected for the large fingerprinting study, many RAPD reactions were performed using the same primer. This allowed the primer to be added directly to the premix along with the Taq, precluding the need to add the primer individually to each tube. Reaction volume was adjusted such that 10 ul of premix + primer + Taq could still be aliquoted to each tube, 28 however each reaction now contained 2.5 (il of template, and the total reaction volume became 12.5 ul. A l l components of the reaction mixture remained in the same proportions as those for primer screening. 2.2.7 Parentage Analysis Information on the parentage of rhododendron hybrids was obtained from Rhododendron Hybrids by Salley & Greer (1986). Comparisons of banding patterns were made between hybrid offspring and their parents to determine whether RAPDs may have the potential to determine parentage among cultivars whose parentage is not certain. Band sharing among the hybrids and parents was calculated using Jaccard's coefficient of similarity (Sneath & Sokal, 1973): J= nAB nA + nB Where nA is the number of bands in individual A , nB is the number of bands in individual B, and nAB is the number of bands shared by A and B. 2.2.8 Analysis of DNA Fingerprints The purpose of this study was to generate D N A fingerprints that would distinguish among rhododendron cultivars. Accordingly, a key for identification was made based on the RAPD patterns generated for each cultivar. The key was created based on presence or absence of specific R A P D bands. Band sizes were estimated visually by comparison with the lOObp ladder that was run as a standard on each gel. Bands selected for inclusion in the key were strongly staining and discrete; as well, they were useful in distinguishing a subset of cultivars in this study. 29 2.3 Results 2.3.1 DNA Isolation The D N A isolation procedure yielded from 3.0 to 250 pg of D N A in different rhododendrons. As each R A P D reaction required either 2.0 or 2.64 ng of DNA, the protocol provided more than enough D N A for over 1000 RAPD reactions. Consistency of the powdered tissue appeared to play an important role in the yield of DNA. In general, the more finely ground the tissue was, the higher the yield of DNA. The appearance of the pellet varied between cultivars, with color ranging from white to yellow to brownish, and consistency from compact to viscous and sticky. However, RAPD fingerprinting patterns were readily generated regardless of the quality of the D N A pellet prior to resuspension. In some cases, the D N A isolation protocol failed to yield any detectable DNA. Due to time constraints, no further attempt was made to obtain D N A from those samples, which are marked with a star in Table 2. A scaling down of the D N A extraction procedure was attempted with a starting tissue weight of lOOmg (for protocol see Appendix I). In general, this procedure yielded D N A that gave rise to clear R A P D banding patterns, however the success depended to a much larger degree on the consistency of the powdered tissue. It is recommended that when using this protocol on small amounts of tissue that the starting material be very thoroughly powdered. 2.3.2 Primer Screening Of the 274 primers screened in this study, 78 yielded polymorphic bands, 6 gave monomorphic profiles, 63 did not amplify at all, and 126 generated sub-optimal banding 30 patterns. The thirteen primers selected for further use gave rise to clear, reproducible D N A fingerprints, and were considered to be useful for identification of cultivars in light of the high level of polymorphism they generated. These are listed in Table 4, along with their D N A sequences. 2.3.3 Reproducibility Repeated amplifications on the same thermal cycler consistently gave the same banding patterns for the individuals studied, as long as the amplification conditions and reaction components were constant. Three thermal cyclers were used to test for consistency of RAPD patterns between PCR machines. RAPDs generated on a Perkin Elmer 9600, a Perkin Elmer 480, and Hybaid Omnigene thermal cycler using the same D N A and primers were compared, and in all cases concordant results were obtained. It should be noted that while all the major bands were the same between thermal cyclers, a difference was seen in the amount of background generated (Figure 4). Another test of reproducibility involved RAPD assays with D N A isolated on several different occasions from tissue of the same individuals. RAPD patterns generated by the same primer were the same between D N A preparations of the same tissue in all cases. For clonal stability, identical RAPD patterns were generated for most of the cultivars under study with the exceptions of Jean Marie de Montague, Roseum Elegans, and Ramapo (Figure 5). The two samples of Jean Marie de Montague gave rise to completely different RAPD patterns, whereas the two samples of Roseum Elegans and Ramapo generated similar 31 O N t O O t O N O O N O O O N W t O ^ t O > o o o > > O o o o > > o Q O Q > o o > n o o o o o o o > n n n o H o Q o 3 ^ > o H O > > > > > > > a o H H o H O > O o H O > O H O O tO tO n o > o H o > > a o a* S o a o 3. o Go S" a-oo 1—* M I—i 1—> 1—1 oo 1—1 N O 1—> 1—1 4^ V o oo t—* 00 1—» - J —] t o 4^ t o o o o o o © o o O o o o o " © o " - J oo © 1—» N O 1—» 1—» N O >—» o t o O N o - - J 1>J o © t o V ^ O o o O N t o o O " o o O N o oo o o o —J oo o o o o 1—» o o oo p IO o U) J o o o I—* o © 1 . © - J o N O o N O t o o o oo o 1—» oo 1—I 00 o o - J o - - J O N - J o O N , . N O o O N t o o 00 00 N O >—* 1—* 1—» © p O N o t o o t o o o 00 o o O N 1—* o o o o o o p N O p N O o p O N O O 4^ N O O 4^ O 4^ t o O O o p NO t o p NO o p oo to - J to U i ON p NO p ON O NO o 3 re 0 5 e a S z ce a s « i e a B C3 Si 3 as A C/3 CC « US' re re a H » cr re" re N O re 3 « re o >tj T3 B re re re" re re a S9 O • 3 CTQ re •a B* CTQ » a a <-»• » o 3 o B s a Testing Reproducibi l i ty M u l t i p l e D N A I s o l a t i o n s M A B C D E 1500 bp -600 bp -D i f f e r e n t T h e r m a l C y c l e r s Perkin-Elmer 480 M 1 2 3 4 Perkin-Elmer 9600 M 1 2 3 4 1500 bp -600 bp -MM • • MJ _ • 8 i : *= • I — -Figure 4. Reproducibility of R A P D s in rhododendrons. Top, R A P D s run on D N A samples isolated on different occasions from one sample using primer 456. A . Britannia; B . Exotic; C. Grace Seabrook; D. Pink Pearl; E. Tony. Bottom, R A P D s run on the same D N A using different thermal cyclers. 1. Brittania; 2. Grace Seabrook; 3. Red Eye; 4. Rocket. M - 100 base pair D N A markers. 33 A . M l 2 3 4 5 6 7 8 1500 bp -600 bp -M i — - — - — « — . . . » . „ — • « — * — mm _ M , , !- ^ 4MMMF — .. B - M 1 2 3 1500 bp -600 bp -FigureS. A. Clonal stability of RAPD patterns. 1. Unique; 2. Vulcan; 3. Loderi King George; 4. Loderi Venus; 5. Mrs. Furnival; 6. Hotei; 7. Markeeta's Flame; 8. Nova Zembla B. Samples for which clonal stability was not established. 1. Jean Marie de Montague; 2. Roseum Elegans; 3. Ramapo All RAPDs generated with primer 225. M -100 base pair DNA ladder 34 but not identical banding profiles from some primers. A summary of the clones for which clonal stability was established is given in Table 5. 2.3.4 Parentage Analysis Results of the parentage analysis are shown in Table 6. Analysis was not performed on the family involving Catawbiense Album, Loderi King George, and County of York as D N A was not obtained from Catawbiense Album nor County of York; nor on the family involving Mist Maiden, as the D N A obtained from this cultivar rarely gave a clear banding pattern. Similarities between parents ranged from 27% to 36%, and the percentage of unique bands inherited by the hybrid from a single parent ranged from 16% to 33%. The frequency of non-parental bands found in the hybrid ranged from 3.2% to 22%. A sample of the RAPD profiles generated from these families is shown in Figure 6. 2.3.5 DNA Fingerprinting RAPD patterns for all hybrids fingerprinted have been collated and printed to the attached compact disc as TIF files. These reference images can be viewed using any program that recognizes TIF files, such as Adobe Photoshop. A n example of the composites found on the CD is shown in Figure 7. Key to fingerprinting The keys in Appendix II are recommended as a guide to narrowing down the identity of a rhododendron cultivar whose identity is in question. The keys are independent of each other, and are not constructed to be used in any specific order. As each key was created based on one primer and not all bands generated by each primer were included, full comparison of 35 Table 5. List of hybrid rhododendron cultivars used to study clonal stability. Name Source Clonal Stability Established Anna Rose Whitney Clay's, V D yes Anna Clay's, V D yes Blue Ensign Clay's, V D yes Blue Pacific Clay's, V D yes Britannia Clay's, V D yes Cotton Candy Clay's, V D no1 Gomer Waterer Clay's, V D yes Grace Seabrook Clay's, V D yes Halfdan Lem Clay's, V D yes Hotei Clay's, V D yes Jan Dekens Clay's, V D yes Janet Blair Clay's, V D yes Jean Marie de Montague Clay's, V D no Loder's White Clay's, V D no1 Loderi King George Clay's, V D yes Loderi Venus Clay's, V D yes Markeeta's Flame Clay's, V D yes Mrs. Betty Robertson Clay's, V D yes Mrs. Furnival Clay's, V D yes Nova Zembla Clay's, V D yes Olympic Lady . Clay's, V D no1 Pink Pearl Clay's, V D no1 Polar Bear Clay's, V D yes Ramapo Clay's, V D no Roseum Elegans Clay's, V D no Souvenir ofW.C. Slocock Clay's, V D yes Unique Clay's, V D yes Vulcan Clay's, V D yes 'clonal stability not established because D N A was not obtained from one of the clones 36 LI ^—^—j^i i—K r H - * » to H -3 8 5 & o £3 $ o o CD co CO O •-+> O o> 3-v: co CD H 2 P . cr CO a O N (v< o x O N N O t O P-P r o o-CD >-l CO i - i . i-l o fD a Cf DO (D cr fD CO o 3 t o <V1 0 S -t O < fD g CO o C CD CD 3 2 f >-+> P CD |-|- fD P-fD O a P ffQ 3 a p 3 fD P-fD O a 2 3; P o P o p p o ' p o " u > U ) 0 0 N ° 0 s - v= 0 s -N O o x ' N 1o t o O O o o O N o o o o N O iv< <vi O N a 4>- 4 s-4 s-N O S O 0 s - o x t^ S 1o *~~* N O (T( fD O O 4 s-t o <V) O N o x UJ O N -O N O (v< o O N o x /—\ <Vl - J o o t o © x " t o O N to t o o x <V< o o o o o I—* O N to to to t o t o - O . N O s o N O O N 0 X —s '"~s 4 s- 4 s-© ^ 0 0 ' O N H - H - H - N O U ) i—i 0 0 t o to o o to 4>-t o (v< o O N O N O N to to 0 S -O N O O t o O N o o O N O to (VI o o ^ 4 t o to to »— O N © (V< s o v O v O 0 s - 0 s - o x W ^ ^ 0 0 to O N o - o t o (Vl 0 0 N O (v> o o t o N O 4 s-o o O N (V) O N 0 S -<1 to -o o x 1o O -o O N (V> X 0 «-+• 2. CO CD 1 a P I n I-I CD CO 00 ^ <Vl t o (VI o o to -o °^ o x 'Zri N O O N 4 s-N O 0 S -N O t O O N N O •-o CD B a* h3 fD* a fD 3 coc § 3. & ^ § ' co a coC p 3 P-' CO fD p cr 3 e WoP-co^ P o co h3 W fD W B P t?co i l /—s CO ^3r fD 2. (-»-* fD p-t*to II O CO 1—1 fD fD P-8£ 01 o 0 cr 13 1 o 0 cr 13 1 _ o o * l l S D s. ° —. — 4- u> -i -i i" i' -i -i 4- K> 0 'Ji 1 i > > 2 ° 2. £ F 3 CO = 2 S co a " J? 2 — ~ o ?f r a o <«" n Z3 3 c —. h» r 73 - 7 ^ 3 | S> 15 § o ^ ^ — 1 • 2-r > a ~ im 3 0 rt o. a » =T re 3 o 1 _ a re" = ft a a % » s 2. CO a » ST -. a — -. ft -> 2 & - tC — - 1- -"1 r c 00 II — — -. EL' o o cr 13 _ L _ II II I II I illl o 0 cr 13 1 o 0 cr •0 1 1 I-I I ui o 0 cr 13 1 II I Ul o o cr 13 Ul o 0 cr 13 1 00 ft i i i i i i . ; i n ii i t > 00 ft > 00 ft — ft — K -. ft = 1 I I 11 I U l .. I l l l l > 03 n 6e C f Q e -t re 2 an i a 100 X mpl so re 9 - Ifd V) o ft an —> o r re > a y 3 fin > 2! C f Q re m 3 ""I • a a 3 • i . a a - 5' re *"| rs o ET 3 o- -a re o 3 OTQ ites re &i ri C / 3 3" 3" O 5" 3 3 re Si efer t re > o the PDs ( O" ""! ime aim i a c re a UK) ON o o © o RAPD banding patterns generated by all thirteen primers should be used to give a greater degree of certainty as to the identity of the plant under scrutiny. In addition, very faint bands or those of very large (greater than 2000 bp) or very small size (less than 300 bp) may not always be reproducible, and this should be taken into account when using the keys. Using the key to primer 225, seventy cultivars may be distinguished. Adding information from the other four keys, an additional 35 cultivars can be identified. Individual rhododendrons that were not distinguished by these five primers were Anah Kruschke, Aunt Martha, Gomer Waterer and Lee's Dark Purple. In addition, Loderi Pink Diamond and Loderi Superlative could not be distinguished from each other, nor could the group of Loderi Pretty Polly, Loderi King George, Loderi Venus, and Loderi White Diamond. The Loderi hybrids are all offspring of the same cross, and therefore it is not surprising that they should exhibit similar RAPD patterns. 2.4 Discussion 2.4.1 Reproducibility Testing The results of the reproducibility tests indicate that the protocol established for RAPDs in rhododendrons generates reproducible banding patterns in repeated reactions using the same thermal cycler, between different thermal cyclers, and on amplification of D N A from one individual which was isolated on different occasions. "Reproducible" in this context refers to a fingerprinting pattern in which the strong, definite bands are consistent. Very faint bands, while reproducible in this study, may not be consistent with all users. In scoring bands it is recommended that only prominent bands be used. 40 The clonal analysis uncovered three inconsistencies in the twenty-eight paired samples from Van Dusen Gardens and Clay's Nursery. The first instance involves the hybrid clone Jean Marie de Montague. It must be noted that the accession number of the sample obtained from Van Dusen Gardens was not verified, as the plant was identified based merely on the presence of a garden sign. Therefore, it seems likely that this sample was in fact misidentified, and the fingerprinting patterns generated from the one taken from Clay's Nursery should be taken as authentic. Parentage analysis involving these samples gives some support to this theory (see below). Conversely, there is also a chance that the sample collected at Clay's was mislabelled, as this has been known to happen in nurseries (Les Clay, pers. comm.). Therefore, RAPD banding profiles for both samples are included on the CD attached, as the identity of the true clone Jean Marie de Montague still needs to be verified. The different banding profiles generated by the two samples of Roseum Elegans and Ramapo collected are quite similar (Figure 5). Misidentification of one of the samples is less likely in the case of Roseum Elegans because the sample taken from Van Dusen Gardens was identified by accession number. The differences seen here may be due to somaclonal mutation in tissue culture, as the material sampled from Clay's nursery was propagated in this way. Substantial morphological variation has been observed in plants generated by tissue culture in some labs (Knuttel, 1990), and this example may illustrate the potential of RAPDs for diagnosing these changes. In the case of Ramapo, either of the two above explanations may apply. Because the identity of the clone sampled from Van Dusen Gardens was not verified by accession number, there is a chance that this individual was not properly identified. The fact that most of the banding patterns generated were very similar, and in 41 some cases identical, (primers 252 and 456) does seem to support the hypothesis of mutation in tissue culture; however, misidentification cannot be ruled out at this time because individuals that are closely related have been observed by the author to give very similar banding patterns as well. 2.4.2 Parentage Analysis The relatively frequent occurrence of non-parental bands in the RAPD profiles of the rhododendron hybrids under study may indicate that RAPDs will be of limited use in determining parentage among rhododendron cultivars. Other studies have differed in their findings with respect to non-parental bands in parentage analyses using RAPDs. Scott et al. (1992) found very low frequencies of non-parental bands in burying beetles (1.7%) and strawberries (0.2%), while Riedy et al. (1992) reported a very high frequency of non-parental bands in baboons and humans (an average of 4.4 and 2.7 non-parental bands respectively, generated by only five primers). The occurrence of non-parental bands in rhododendrons falls between these two extremes. These bands may have been generated by mutation, however they occur at too high a frequency for this to be the only explanation. It is possible that the non-parental bands were generated from a contaminant present during D N A isolation - even though samples were cleaned prior to extraction, the thick indumentum of some leaves may have prevented the removal of all contaminants. PCR artifacts may also have contributed to the presence of non-parental bands. It is possible that bands in the parents were not amplified due to kinetics - competition for primer binding sites may have prevented amplification. In addition, polymerase slippage during replication and non-template directed 42 addition of nucleotides by Taq are thought to generate artifactual product bands (Riedy et al., 1992). It may be noted that two sets of data are presented for the family involving Jean Marie de Montague. This may be attributed to the fact that the clonal identity of the two individuals collected was not established. The data suggest that the true clone is the one collected from Clay's nursery, as the frequency of non-parental bands when this individual is taken as the parent is 6.7%, much lower than the 16% frequency when the individual from Van Dusen Gardens is used. An excessive number of non-parental bands are also seen in the data for the family involving Jennie Dosser (22%). In fact, the number of non-parental bands exceeds the number of unique bands inherited from each parent. This may indicate that the documented parentage of this hybrid does not reflect the true parentage. It is possible that a mistake was made by the hybridizer. Many rhododendron hybrids are registered by amateur breeders, and controlled crosses are not always possible. However, the frequency of bands inherited from each parent is not markedly reduced, as might be expected i f a completely unrelated cultivar was mistakenly listed as a parent. Firm conclusions may not be made here, due to the small sample size of families under scrutiny. Further work is needed before any conclusions may be made as to the frequency of non-parental bands at which the identity of the parents may become suspect. While Scott et al. (1992) argue that a high frequency of non-parental bands does not preclude the use of RAPDs for parentage analysis, a high frequency of non-parental bands may lead to false exclusion of a possible parent in analyses of rhododendrons given the vast 43 numbers of cultivars that are available. Thus, the RAPD technique is not recommended for parentage determination when large numbers of putative parents need to be screened. RAPDs may be of use, however, in certain instances where identity of one or two putative pollen parents are in question, and the identity of the seed parent is known. In these cases, RAPDs may enable researchers to determine the most likely pollen parent. For further guidelines on analyses of RAPDs in such a case, refer to Scott et al. (1992). The above discussion applies to rhododendron "families" in which the parents are clonal cultivars. Many hybrid rhododendron cultivars are derived from crosses of two individuals of different species, or from a cross between a species and a hybrid. In these cases, i f the identity of the specific individual of the species in question is known, and material is available for D N A isolation, then parentage analysis can proceed in the limited capacity described above for hybrid - hybrid crosses. If a hybrid is thought to have originated from a cross between two species, another approach may be taken. Marsolais et al. (1993) determined the origin of interspecific lilac hybrids using RAPDs. By performing RAPDs on several members of the putative parental species, known hybrids of the two parents, as well as the hybrid in question, they were able to ascertain that the individual of interest was of the same parentage as known hybrid offspring of the two species. Hybrids of two species appear to cluster closely with the parent species, as evidenced in data presented in Chapter 3, Figure 9. Rhododendron x geraldii (abbr. Sg) is an acknowledged hybrid of R. sutchuenense (Su) and R. praevernum (Pv) (Chamberlain, 1982, Cox, 1979). The similarity coefficients of these taxa are shown in Table 7. While R. x geraldii does appear to share more similarity with R. sutchuenense than with R. praevernum, this may be due to the small sample size of R. 44 x geraldii. Thus, even if the exact individuals thought to have been the parents are not known it may be possible to determine the likely parent species, as hybrids will be expected to be more similar to these than to other species. It is recommended to perform analysis of this type only in cases in which there is a definite candidate for a putative parent species. The identity of the alleged parent may then be confirmed or refuted. The large number of clonal rhododendron cultivars and the presence of non-parental bands in the RAPD profiles of hybrids of known parentage precludes the use of RAPDs for large scale screening of rhododendrons to determine parentage. However, the technique may be useful in specific circumstances such as those in which there is a definite candidate for parentage that is to be tested. 45 9P GA C/3 CfQ W C/3 crq t o Sgl C/3 s Su6 C/3 s (Ji C/3 C 4> C/3 C W Su2 Sul o o o o p o o O o o o to <1 OJ O N 0 0 0 0 O N N O 4^ u> O N U l O N t o U l O N t o o O N N O O N O N 0 0 O N O o p o O o o o o O O O t o U l o o^ O N t o N O 1—k O N t o OO U> O N t o o b o 4^ N O U l O N 4^ O o o p p o o o o O o O i o -o O N O N - O •o t>J O J N O o U l O N t o 0 0 O N O N - O O N N O O N 4^ 4^ t o -o O N 4^ O N p o o o o o o O O p O L J tO 4^ O N 0 0 N O ^ • J -o o o o o t_o t o O N o o O N UJ 0 0 b o 4^ t o O O p O p p p O p O p U) U l b o U l o b o O N N O O N O N UJ O N U l OO O N U I N O O N - 0 t o O u> O N -o 4^ p o o O O p o o o p O N O N O N O N O N U ) o o t o O N N O O N t o o^ ui O N -o O O o O o O O o o O o O t o U l O N t o b o o o ~o o o o o O N O N -J 0 0 t o N O O N t o U l N O U l © o o o o o p p o O O o t o O N b o u> -J b o ~o ^ - J U l O N o - 0 b o -o ui O N -J 4^ o o o p o o o O o o i—' O N O N - O N O O N - O O N - 0 4^ t o O N O J o o O O o O o o o O o k> - 0 o O N t o t o o N O U l O N 0 0 t o O N 0 0 o o o p p o o o •—* to ui N O U l O N U l I—* 0 0 o <] U l O o o o o o o o t o U l - J O N U l N O o U l - J t o o o o < < < < W K> I— o o o o •-» •o -o o o U l t o N O O O O N to O O O *0 p o p O N b o ^ U i O N N O O U l t o o o o O O i — O N ^ O U l U l O N O O O O O N O O O O O O O O O »-> O J O N ^ o U l N O 0 0 o >-> O N O J O P P r" w bo b o o o U l o 4 ^ O o t o o U l O N O o o o o < t o h3 Ul C / 3 s C/3 c t o C/3 s C/3 s C/3 C/3 e O N C/3 C •OJ C/3 t o C/3 o C H » o * ? » p ^ era f / 5 £ 3 — M a 2. a S , w n 69 S" § s. 3 P s s s 1 & 2 ^ C Q . s 5 & » s a 6 3 TO 3' » H a |>= n ^ ? ° » q TO 3 S. a o O B a S 3 = 3 ° 5. a ^ S I f 5 Si t« O * • = i ° s ^ s 2. 3 IP CHAPTER 3: TAXONOMIC STUDY OF RHODODENDRON SPECIES USING RAPD 3.1 Introduction In addition to D N A fingerprinting, RAPD data have also been analyzed from a taxonomic perspective. The technique has been used to examine and clarify relationships among Lotus species (Campos et al. 1994), poplar species (Castiglione et al. 1993), apple cultivars and wild species (Dunemann et al. 1994) and Allium species(Comincini et al. 1996). Below the species level, RAPDs have been found useful in tea (Wachira et al. 1995), mosquitoes (Ballinger-Crabtree et al. 1992), sorghum (Tao et al. 1993), celery (Yang & Quiros 1993) and many others. Most often, when investigating relationships with RAPDs, analysis is based on band sharing, where two individuals that have a large number of bands in common are assumed to be more closely related than ones that do not. Data matrices are compiled based on similarity, or conversely, dissimilarity coefficients, and this data may be analyzed in a variety of ways. Cluster analysis has been used extensively with R A P D data to generate phenograms illustrating relationships between different taxa. These trees that are produced may be equivalent to the phylogenetic tree of the taxa involved i f certain assumptions are met. These requirements are: first, organisms must be grouped according to genealogical ties; and second, the time of divergence between organisms is estimated ~ assuming constant rates of evolution between lineages (Lewis, 1995). Regardless of whether the tree is phylogenetic or merely a reflection of the similarity present in RAPD banding profiles, it may be useful to taxonomists, in providing additional characters to work with, which reflect 47 similarities in DNA. This in turn may help to determine a better classification for the taxa under study. There is general agreement that classification of the genus Rhododendron will continue to be revised in future years (Luteyn & O'Brien, 1980). The objective of this study was to investigate relationships within the Rhododendron subsection Fortunea using RAPDs. Fortunea was selected for study due to the fact that several species within the subsection are of horticultural importance, and have been used extensively as parents in hybridization. In addition, it has been suggested that some of the species placed in Fortunea may be more closely related to species in other subsections. Accordingly, samples were obtained from species within Fortunea and their proposed relatives in other subsections. Specific relationships that were looked at include: 1. R. hemsleyanum with R. auriculatum (subsection Auriculata) 2. R. orbiculare with R. thomsonii (subsection Thomsonia) R. williamsianum (subsection Williamsiana) 3. R. calophytum with R. rex (subsection Falconera) R. praestans (subsection Grandia) Samples collected from species within subsection Fortunea were subjected to the RAPD protocol with the objective of elucidating relationships within the subsection. One criticism of taxonomic studies using RAPDs has focussed on the assumption that comigrating bands on R A P D gels are homologous (Pillay & Kenny 1995; Thormann et al, 1994). In order to verify that this was indeed the case, and that similarity coefficients were not in fact based on D N A fragments of the same size but unrelated sequence, Southern Blots were performed on a subset of RAPD gels. If comigrating bands are of related sequence, a 48 probe consisting of D N A isolated from that band should hybridize to all bands of the same size. 3.2 Materials & Methods Samples collected for the taxonomic study are listed in Table 8, along with their source and origin. Where possible, samples from 5-7 individuals of each species were obtained. As well, an effort was made to acquire material from wild collected seed rather than garden-bred species. The Rhododendron Species Foundation in Federal Way, Washington proved to be a useful source of plant material, as their mandate is to collect and preserve rhododendron species as they are found in the wild. D N A isolation, quantification, RAPD amplification conditions, and product resolution were as outlined in Chapter 2. The same primers selected for fingerprinting were used for the taxonomic study, with the exception of primer 876, which proved difficult to score. For electrophoresis of RAPD products, the samples were grouped according to species for assays using primers 225, 251, 456, 729, 732, 740, and 792. To facilitate comparison of comigrating bands between different species, a new organization was made, in which at least one of each species was run on one gel and this order was followed for the remaining primers. As well, the original assays utilizing the primers listed above, which had been stored at -20_C were re-electrophoresed using the new order of samples. Additionally, to confirm that certain bands from samples run at opposite sides of the gel were indeed comigrating and not of slightly different sizes, several gels were run in which these samples were run side by side or close together. This procedure served to confirm or refute the identity of several bands, and aided in the scoring of RAPD bands for data analysis. 49 Table 8. Rhododendron species and sources used in the taxonomic study. Species Abbreviation Accession Number Source Rhododendron auriculatum (Subsection Auriculata) A u l Au2 Au3 Au4 rose pink form Au5 Au6 Au7 67698 RSF 76072 RSF 22861-104-82 U B C 82877 V D 19851-55-81 U B C 211-9-75*C ex66 U B C 54-459 PP Rhododendron calophytum var. calophytum (Subsection Fortunea) C l l C12 C13 C14 WJ1523 C15 C16 C17 var. openshawianum Col Co2 77283 82196 64063 78083 82046 77462 13820-104-76 23984-43-83 88013 RSF RSF RSF RSF RSF RSF U B C UBC RSF Rhododendron davidii (Subsection Fortunea) D v l 25296-26-86 U B C Rhododendron decorum (Subsection Fortunea) Rock # (pink) Del (white) Dc2 (pink) Dc3 (pink) Dc4 Rhododendron diaprepes (Subsection Fortunea) Dpi A M Dp2 "gargantua" Dp3 75170 64061 64062 330-26-64 64063 73088 RSF RSF RSF U B C RSF RSF 13597-227-77*Cex75 U B C 50 Table 8. continued Species Abbreviation Accession Source Number Rhododendron fortunei ssp. fortunei (Subsection Fortunea) Ff l sd. China Ff2 FfJ Ff4 "Barto's Favorite" Ff5 Ff7 Ff8 Rhododendron fortunei ssp. discolor Fdl Fd2 Rhododendron fortunei ssp. discolor Houlstonii Group FdHl "random harvest" W. 648a FdFD FdH4 FdH5 FdH6 76340 RSF 82026 RSF 64055 RSF 76147 RSF 69813 RSF 10946-156-74 U B C 76029 RSF 82140 RSF 75064 RSF 75136 RSF 79043 RSF 66562 RSF 80233 RSF 263-51-72 U B C Rhododendron griffithianum (Subsection Fortunea) Gf l Gf2 Gf3 Gf4* Gf5 Gf6 Gf7* Gf8 Gf9 (white) GflO sd. cw Sikkim sd. cw Sikkim sd. cw Sikkim Bowman #2 89031 80154 92053 76033 81115 92054 81113 89032 80099 22983-13-83 RSF RSF RSF RSF RSF RSF RSF RSF RSF U B C Rhododendron hemsleyanum (Subsection Fortunea) Hml Hm2 Hm3 Hm4 Ftm5 73133 RSF 76036 RSF 26282-104-88 U B C unknown U B C 29717-55-91 U B C 51 Table 8. continued Species Abbreviation Accession Source Number Rhododendron orbiculare ssp. orbiculare (Subsection Fortunea) Obi Ob2 Ob3 2 clones collected Ob4 Ob5 Ob6 Ob7 ssp. cardiobasis Ocl Oc2 Oc3 Oc4 66542 RSF 74043 RSF 65350 RSF 67702 RSF 75164 RSF 385-39-64 U B C 340-4-65 U B C 8229 V D 81459 V D 8522 V D 16167-354-77 U B C Rhododendron oreodoxa var. oreodoxa (Subsection Fortunea) Oxl 70395 0x2 80121 0x3 80335 W. 4248 f. haematochelum 0x4 77749 0x5 82152 0x6 65325 Rhododendron oreodoxa var. fargesii Fgl 67686 Fg2 65808 Fg3 65308 Fg4 76160 Fg5 69670 Fg6 75316 Fg7 67681 Fg8 62046 Fg9 11-10-67 RSF RSF V D RSF RSF RSF RSF RSF RSF RSF RSF RSF RSF RSF U B C Rhododendron praestans (Subsection Grandia) Psl Ps2 Ps3 74-094 75117 75074 RSF RSF RSF 52 Table 8. continued Species Abbreviation Accession Source Number Rhododendron praeteritum (Subsection Fortunea) Ptl 75075 RSF Rhododendron praevernum (Subsection Fortunea) Pv l 80147 RSF Pv2 66563 RSF Pv3 82008 RSF Pv4 21521-104-82 U B C "DelJames Select" Pv5 21995-55-82 U B C Rhododendron rex ssp. rex (Subsection Falconera) "Quartz" R.# 18234(03800) R x l 75118 RSF Rx2 81742 V D Rx3 6257-161-73 U B C Rx4 15293-55-77 U B C Rx5 76181 RSF KW#4509 Rx6 62-513 PP ssp. arizelum R z l 120-1-65 U B C Rz2 120-51-72 U B C Rz3 19869-104-81 U B C Rz4 64-16-72 U B C KW#20922 Rz5 24474-497-84 U B C deep pink form Rz6 13818-104-76 U B C ssp. fictolacteum F.#25719 Rf l 79135 RSF rust indumentum Rf2 77417 RSF Rf3 81741 V D Rf4* 13822-104-76 U B C Rf5 15292-55-77 U B C Rock# Rf6 75158 RSF Rf7 77548 RSF Rhododendron sutchuenense (Subsection Fortunea) Sul 77205 RSF Su2 73272 RSF Su3 76330 RSF "Seventh Heaven" W.#1232 Su4 79120 RSF Su5 77224 RSF Su6 77763 RSF Su7 24551-437-85 U B C 53 Table 8. continued Species Abbreviation Accession Source Number R. x geraldii Sgl 77246 RSF Sg2 75192 RSF Sg3 73273 RSF Rhododenron thomsonii ssp. thomsonii (Subsection Thomsonia) L&S#2847 T m l * 313-10-67 U B C clone of Tml Tm2 313-10-75*C ex 67 U B C Tm3 28299-26-89 U B C Tm4 28511-585-90 U B C Tm5 26866-523-88 U B C Tm6 25301-26-86 U B C Tm7 91053 RSF Tm8 67716 RSF Tm9 71617 RSF ssp. lopsangianum T i l 77696 RSF T12 64140 RSF T13 64149 RSF Rhododendron vernicosum (Subsection Fortunea) f. rhantum V n l 75328 RSF Vn2 70353 RSF Vn3 76177 RSF "LochEck" Vn4 75127 RSF R.# 11404 Vn5 82054 RSF H-Sm.#13976 Vn6 79152 RSF R.#l 1328(59599) f. araliaeform Vn7 77760 RSF Vn8 77058 RSF Rhododendron williamsianum (Subsection Williamsiana) Wml 66606 RSF white form Wm2 70332 RSF white form Wm4 25104-227-86 U B C Wm5 74113 RSF WJ1350 Wm6 80055 RSF Wm7 73301 RSF Wm8 77128 RSF 54 Table 8. continued Species Abbreviation Accession Source Number Rhododendron yakushimanum (Subsection Pontica) cv. Greer Angel G A 019905-104-81 U B C cv. Ken Janeck KJa 21992-303-82 U B C cv. Mist Maiden M M 21991-303-82 U B C FCC Form YFa 19907-104-88 U B C Overstreet Form Y O 24646-303-85 U B C cv. Yakity Yak Y Y 21576-303-82 U B C Yk#19 21506-104-82 UBC Yk#20 21537-104-82 UBC natural collection Yka 66-143 PP natural collection Ykb 66-143 PP natural collection Ykc 62-68 PP Ykd 62-68 PP FCC form Yke 64-203 PP FCC form Y k f 203 RSF VD3 82492 V D Exbury Form VD9 87138 V D Notes on Table 8: Collector's numbers are listed to the left of abbreviations. Collector's names are abbreviated as follows: F. = George Forrest H-Sm. = ? K W . = Frank Kingdon-Ward L & S = Frank Ludlow & George Sherriff R. = Joseph Rock W. = Ernest Henry Wilson Source Abbreviations: PP = U B C Physical Plant Nursery RSF = Rhododendron Species Foundation Botanical Garden U B C = U B C Botanical Gardens V D = Van Dusen Gardens 55 3.2.1 Southern Blotting In sections dealing with Southern blots, "hybridization" refers to complementary binding of the probe to D N A fixed to the blot, and not the offspring of a cross between two rhododendron species, as in Chapter 2. In order to verify the homology of comigrating RAPD bands, Southern hybridization was performed on a subset of fragments. Seven RAPD gels, on which at least one of each species was run, were selected for analysis after ethidium bromide staining and visualization under U V light. These are listed in Table 9, and are henceforth referred to using the primer that was used to generate the RAPD patterns as reference - eg. Blot 740 was derived from the gel in which R A P D samples generated from primer 740 were run. D N A was transferred to a Hybond-N+ nylon membrane by the capillary transfer method under alkali conditions (Sambrook et al., 1989). The gel was first cut to minimize the blotting area, incubated in .25M HC1 for 20 minutes, and rinsed in distilled water for a further 20 minutes. The blotting apparatus was assembled as follows: approximately 500 mis of 0.4M NaOH was poured into a tray, and a plexiglass support was laid across the top. A wick made of three pieces of 3 M M Whatman paper cut so as to drape the support with ends sitting in the solution was wetted with NaOH and air bubbles were removed with a pipette. The gel was placed on the wick, saran wrap was placed over the whole, and a hole was cut in the saran with a razor blade to allow the gel to be exposed. The membrane was cut to the same size as the gel, soaked in NaOH and placed on top of the gel, followed by four layers of 3 M M Whatman paper cut to gel dimensions as well. A stack of paper towels topped by a weight was placed on the whole, and transfer was allowed to proceed over night. Blots were then labelled in pencil, and the 56 Table 9. Southern Blots: Samples and Probes. Primer Samples1 Probe Size (bp) Probe Source 232 Au4-Ykc 1100 Oc l 416 Au3-Yk#20 790 Rx2 729 Au4-Ykc 1150 0x6 792 Au l -KJa 800 Rf l 456 Au7-YFa 930 Su7 225 Au7-YFa 520 Su7 740 Aul -KJa 1075 Ob6 57 D N A was fixed in a vacuum oven for two hours at 80°C. After wrapping with Saran Wrap, blots were stored at 4°C or room temperature. 3.2.2 Probe Isolation and Labelling Probes were isolated directly from the RAPD gel. The band of interest was cut out of the ethidium bromide stained gel with a razor blade, and the D N A was extracted using the QIAEX II kit from QIAGEN. In a preliminary experiment using RAPDs generated from primer 232, the probe was labelled with 3 2PdCTP. In this case, hybridization of the probe to the blot was so strong that non-radioactive methods of labelling were explored as a safer alternative. Consequently, the DIG High Prime Kit (Boeringher Mannheim) was used for subsequent labelling and hybridizations. The D N A to be labelled was denatured in a Perkin Elmer 480 thermal cycler for 10 minutes at 100°C, and then chilled in an ice/ethanol bath. Four ml of DIG-High Prime (containing random primers, dNTPs, Klenow, and digoxigenin (DIG) labelled dUTP) were added, and each tube was mixed and centrifuged briefly, then incubated for 15 hours at 37°C. To stop the random-primed labelling reaction, 2 ml of 0.2M EDTA was added to each tube. Although quantification of labelling efficiency was unsuccessful using the test strips provided with the kit, subsequent hybridization and detection proved that the probes were sufficiently labelled. 3.2.3 Hybridization Membranes were prehybridized at least ninety minutes at 68 °C with shaking in hybridization buffer [5X SSC 1 , 0.1% (w/v) sarcosyl, 0.02% (w/v) SDS, 1% blocking reagent (provided with kit)]. Probes were denatured for 5 minutes at 100°C in a Perkin Elmer 480 thermal cycler 'All SSC solutions were diluted from a stock solution of 20X SSC (3M NaCl, 300mM sodium citrate, pH 7.0) 58 thermal cycler, cooled on ice, then added to 7 ml of hybridization buffer, preheated to 68 °C. The membranes were placed in hybridization bags, sealed on three sides, and the hybridization solution was added. Air bubbles were removed, and the bags were sealed completely. These were then incubated overnight at 68°C with agitation. Following hybridization, the membranes were placed in a container and washed to remove any unbound or poorly bound probe. A l l membranes were subjected to two 5 minute washes in 2X SSC, 0.1% SDS at room temperature. Blot 416 was then washed twice in 0.5X SSC and 0.1% SDS at 68°C, which is a medium stringency wash. Since the signal detected from this blot was very strong, the remaining blots were washed at high stringency in 0.1X SSC, 0.1% SDS at 68°C. 3.2.4 Antibody Binding and Detection After washing, the membranes were rinsed 5 minutes in wash buffer (lOOmM maleic acid, 150 mM NaCl, 0.3% (v/v) Tween 20; pH 7.5), then incubated for a minimum of one hour in 2% blocking solution [2% blocking reagent (10X solution supplied with kit), lOOmM maleic acid, 150mM NaCl; pH 7.5]. For antibody binding, the anti-DIG-alkaline phosphatase (AP) conjugate was centrifuged for 30 seconds, then diluted 1/10 000 in blocking solution. Membranes were again placed in a hybridization bag and 20 ml of the antibody solution was added. The bags were placed on a rocker and incubated for 30 minutes. Following antibody binding, the membranes were washed three times for 10 minutes in 100 ml wash buffer and then equilibrated for three minutes in detection buffer (0.1 M Tris base, 0.1 M NaCl, 50mM MgCl 2 ; pH 9.5). The above procedures were all performed at room temperature with agitation. For detection, the membranes were placed D N A side up on a development folder and about 20 drops of ready-to-use CSPD® were applied to the membrane surface. The membrane was covered with the second sheet of the folder, air bubbles were removed, and the folder was allowed to sit at room temperature for 5 minutes. The excess liquid was then squeezed out, and the edges of the folder were sealed with tape. Incubation then followed at 37°C for 15 59 minutes to allow the light emission to reach a steady state. The folders were then placed in a film cassette and exposed to X-ray film for 15 minutes at room temperature. 3.2.5. Data Analysis RAPD data were considered as discrete variables, and accordingly a binary data matrix was created, with the presence of a band being scored as 1, and absence of a band 0. Seventy-five bands are included in the data matrix, and the approximate sizes of these are listed in table 4. In general, only reproducible, discrete, strong bands were scored. The Numerical Taxonomy System for personal computer (NTSYS-PC) version 1.80 (Rohlf, 1993) was used for statistical analysis. A similarity matrix based on Jaccard's coefficient (Sneath & Sokal, 1973) was generated using the similarity for qualitative data (SIMQUAL) program. Cluster analysis was performed employing the sequential, agglomerative, hierarchical, nested (SAHN) program, and several methods of clustering were examined. These included the unweighted pair group arithmetic mean method, single linkage, and complete linkage. The dendrogram with the best fit to the similarity matrix was identified using the cophenetic value (COPH) and matrix comparison (MXCOMP) programs in NTSYS-PC to calculate the cophenetic correlation. 3.3 Results 3.3.1 Southern Blots The samples, primers and probes involved in each blot are summarized in Table 9. The blot derived from the gel generated from primer 232 and samples Au4 - Ykc was the first to be completed. The probe used was approximately 1100 bp in length, and was isolated from the RAPD reaction using D N A from Rx2 as the template. This was the only probe to be labelled with 3 2P-dCTP. As expected, the probe hybridized to all comigrating bands in the photograph. Somewhat surprisingly, however, the probe also hybridized to a second band of -1375 bp in one sample — Tm4 (Figure 8). 60 Results from blot 416 were puzzling (Figure 8.1). The probe was observed to bind to two bands instead of one in all samples where hybridization was expected. As well, no hybridization was observed in one sample, Tm3, which possessed a band that comigrated with the probe. Reasons for this aberrant result are discussed below. The respective probes for blots 729, 792, 225 (Figure 8) and 740 all hybridized to a single band on each blot, these bands comigrating with the band from which the probe was derived. The probe for blot 456 hybridized to an extra band of approximately 1130 bp in the lane in which C16 was run. 3.3.2 Cluster Analysis Several methods of sequential, agglomerative, hierarchical, nested (SAHN) clustering techniques were explored using the similarity matrix based on Jaccard's coefficient. Three phenograms were generated initially, using the single linkage, complete linkage, and unweighted pair-group method, arithmetic mean. Results of the goodness of fit test are shown in Table 10. Based on the matrix comparison - goodness of fit tests, the tree with the highest cophehetic correlation coefficient was selected for further examination, as this tree gives the best representation of the similarity matrix. The correlation coefficient for the U P G M A method of 0.81 indicates a good fit (Rohlf, 1993). As the S A H N program in NTSYS indicated that several ties were detected during the clustering process with U P G M A , the FIND option was employed to identify other possible trees. Over 25 alternative trees were found, but upon closer examination most of the ties were due to the high degree of similarity found within the R. yakushimanum samples. When these were eliminated, only four alternative trees were detected. The differences in these trees all involved clustering within species and did not involve between species clustering. 61 C I) I) 4 4 1 Blot 232 P K K 4 4 V V W n n m 4 8 4 Y V k k a b • • I t 1500 bp -600 bp -B. F A C C d F <; O u I I H f f b 7 6 7 6 7 6 6 Blot 225 <> ( ) F F !| x S R 7 6 6 7 R S R R R x z f f u 6 6 6 7 6 S T T V V u m in n n 7 6 7 6 7 v V W W V ^ A m m A 9 6 7 # 1500 bp -600 bp -Figure 8. Southern blots and the RAPD gels they were generated from. A. Blot 232; B. Blot 225. Arrows indicate the size of band from which the probe was isolated. 62 B l o t 4 1 6 M D D d F G H O O O F P P c p H f f m b c x g s v x z f 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Y Y R R R S S T T V W V V k k g m 1 n m D D 1 2 3 3 3 3 3 3 9 9 0 M I 1500bp 600bp Figure 8.1. Southern Blot 416 and the R A P D gel it was generated from. Arrow indicates the size of band from which the probe was isolated. M -100 base pair DNA ladder 63 Table 10. Results of Goodness of Fit Tests comparing Dendrograms to the Similarity Matrix Clustering Method Matrix Correlation Coefficient Single Linkage (SLINK) 0.61 (very poor fit)1 Complete Linkage (CLINK) 0.71 (poor fit) Unweighted Pair Group Method with 0.81 (good fit) Arithmetic Average (UPGMA) 'interpretations of degree of fit taken from Rohlf, 1993. 64 Ambiguities were detected in the cluster encompassing Ff2 - Ff8 and these involved the placement of Ff4. Two possibilities were presented; one in which Ff4 grouped with Ff3, and one in which it grouped with Ff8. Other than this small exception, the topology of the tree was not affected by ties. Figure 9 illustrates the phenogram generated by U P G M A on the similarity matrix created from the RAPD data. Two major clusters are formed at a level of 34% similarity. R. yakushimanum compose one cluster, while the remainder of species make up the other, larger group. This large cluster is divided into several smaller clusters which all separate from the main cluster at a similarity of 42-53%. These groups include R. calophytum, R. sutchuenense, and R. praevernum making up one cluster, which separates at a similarity of 42%. R. rex and R. praestans form a cluster that divides from the main cluster at a similarity of 44%, while R. orbiculare separates from the main cluster at a similarity of 46%. R. oreodoxa, R. thomsonii and R. williamsianum make up a cluster that is separated from the main group at a similarity coefficient 48%. A group encompassing R. griffithianum and R. vernicosum separates from the main cluster at a level of 50% similarity, and the R. hemsleyanum cluster divides at 52% similarity. What remains is a heterogeneous group including R. auriculatum, R. fortunei, R. decorum, R. diaprepes, and R. vernicosum. Within this group, however, R. auriculatum is well separated from the rest, with all individuals of this species grouping at a minimum of 79% similarity, and separating from R. fortunei ssp. discolor at a level of 65% similarity. Although most individuals of the same species clustered together on the final dendrogram, some intermingling of species occurred. Most notably, R. decorum, R. vernicosum and the R. fortunei subspecies were not separated into well defined groups. As well, three individuals of R. vernicosum grouped with the R. decorum/R. fortunei ssp. discolor cluster, while the remaining individuals of this species clustered with R. griffithianum. 65 0.2 0.4 0.6 0.8 H ' 1 1 - c I — - — I 0 1 A u l Au3 Au6 Au2 Au7 Au4 Au5 F d ! F d 2 O c l F l l D p i Dp2 Op3 FH1 FH3 FI2 FI3 I FI5 FI7 FI4 F(8 Dc2 Un3 Dc3 D e l FFW FH5 FH6 Un5 Hml Hm3 Hm-4 Hm2 Hm5 G i l GI8 GI9 GI6 Gf5 GI2 GI3 GIO U n l Un2 Un4 Un8 Un6 O v l O x l 0 x 3 0 x 5 0x6 F q ! F g 2 F q 3 F q l Fq5 Fq7 0x4 . P i ! 0 x 2 Fq8 Fq9 Fq6 Tm2 Tm3 Tml Tm8 Tm6 Tm5 Tm7 Tm9 T l ! TI2 T13 Urn I Um4 Um2 Pm7 Um5 Um6 Um3 Um8 Obi 0b2 • 0b3 O O Ob1! 0b6 0 b 5 • 0b7 • O c l • Oc3 • Oc2 • P s l • P s 3 • P s 2 • Q z l • Rz2 • Rz3 • Rz5 • Rz4 • Rz6 • R x l • Rx6 • Rx4 • RI3 • Rx2 • Rx3 • Rx5 • R(6 • R17 - R l ! • RI2 - RI5 • C l l • C12 • C o l • C13 • C l b - C17 • C M • Ci 5 • Co2 • Pvl • Pv2 - P v l - P v 3 - Su2 - Su3 - Su5 - S q l • P v 5 - Sq2 • Sq3 • S u l - S u l . - Su6 • Su7 i GA 1 Y k d • Ykb tin U03 Yke Y U Yk 2 Ykc Y k a UD1 UD9. YY YO Sk K J a YA YFa Figure 9. Dendrogram generated by the UPGMA clustering method on the similarity matrix created using Jaccard's coefficient. 66 3.4 Discussion 3.4.1 Southern Blots In general, the Southern blots verified that comigrating RAPD bands were indeed homologous. Of the seven blots completed, unexpected results were observed in three. There may be several explanations for this. Firstly, hybridization of the probe to an additional band on blot 416 could be due to the fact that a high stringency wash was not performed. This was the first probe to be labelled with digoxigenin and detected with chemiluminescence, and as no labelling was observed using the test strips provided with the kit, the blot was subjected to a low stringency wash only. Therefore, the probe could have bound to bands in the blot that had only slight homology. This double band seems to be an artifact of the hybridization process, as it is seen in several instances where there is no accompanying band on the ethidium bromide stained gel (Figure 8.1, lanes 3, 8). In addition, it only appears where hybridization of the 790 bp probe is expected despite the fact that a band of approximately the same size (850bp) as the "contaminating" band is present in several lanes which do not have the 790 bp band (Figure 8.1, Lanes 12, 13, 16). The probe has also hybridized to several larger bands on the blot, but these do not seem to correspond to presence or absence of bands in the gel photo, again, this may be attributed to non-specific binding of probe to blot due to the absence of a high stringency wash. It is possible that the hybridization of the probe to two bands in blot 416 indicates that some contamination of the probe occurred. Supporting this hypothesis is the fact that the two bands in question are fairly close to each other on the gel. However, this alone cannot explain the results because the larger band never appears alone. The fact that Tm3, whose RAPD pattern did contain a band that comigrated with the one from which the probe was isolated did not hybridize the 67 probe seems to indicate that this band is not homologous with the others. Because this could not be verified in the time available, this band was not included in the cluster analysis. Hybridization of probe 232 to two bands in sample Tm4 and of probe 456 to two bands in sample C16 may indicate several things. First of all, there is a chance that the probe was contaminated. Since the probe was obtained by cutting the band out of the agarose gel and the D N A isolated from the gel slice was used directly as a probe, there is a very real possibility that a few fragments were left behind as the sample was electrophoresed, or that some of the larger fragments were sheared and migrated farther than the rest. Some of these fragments would then have been cut out with the band of interest, labelled and hybridized, resulting in an additional band being detected on the film. While this may explain the hybridization of the probe to two bands in several samples, it is less easy to explain with blot 232 because the only hybridization to that particular band is observed in the lane in which sample Tm4 was run; where in fact a band of equal size is present in virtually every other lane. If the aberrant band is due to probe contamination, one would expect that the probe would also at least hybridize to the same sized band in some of the R. rex species since the probe was isolated from a R. rex ssp. rex individual. The fact that there is no hybridization in any other lane suggests a difference in D N A present in the lane containing Tm4. A more reasonable explanation than probe contamination is that the locus in question, which gives rise to an ~1100 bp band in all the individuals looked at, is codominant and in fact the individual Tm4 is heterozygous at that locus, while all other species looked at are homozygous. The second allele at the locus happens to be the same size as another locus amplified by the R A P D primer. Thus, the ethidium bromide stained band at -1375 bp in sample Tm4 would be composed of at least two different types of D N A fragment. This explanation seems to be supported by the fact that the intensity of the 1375 base pair band in 68 the lane in which Tm4 was run is more intense than the other samples (Figure 8). A similar explanation may be given for the extra band in blot 456, only in this case probe contamination is a more viable explanation as there is a band present in the lane C16 on the agarose gel that does not appear to be present in the others, and the contaminating band is not particularly bright. It should also be noted that R. thomsonii individuals were involved in both cases in which hybridization of the probe did not confirm homology of comigrating bands. Other than R. yakushimanum, this species is classified by Cullen (1982) as furthest from the subsection Fortunea of the species looked at in this study; a fact which may be reflected here. Thus, out of seven bands under scrutiny, one out of the seven for R. thomsonii was not homologous (approximately 14%). If this fraction is representative of all bands sampled, this could have a substantial impact on similarity coefficients and subsequently clustering. That is, up to 14% of bands which are scored as the same between R. thomsonii individuals and those of other species may not be homologous. This fact should be taken into account when scrutinizing the clustering. 3.4.2 Cluster Analysis of Samples of Rhododendron Species As stated in the introduction, the relationships of subsection Fortunea were examined in this study. It has been hypothesized that some species within Fortunea are more closely related to species in other subsections. In particular, the relationships to be looked at were: 1. R. hemsleyanum and R. auriculatum 2. R. orbiculare and R. thomsonii, R. williamsianum 3. R. calophytum and R. rex, R. praestans 69 As seen in the dendrogram presented in Figure 9, there is no evidence as to support the hypothesis of any of these relationships. Data from Southern blots seem to corroborate this observation. Whereas homology of certain shared bands were verified between species, those species which did share bands were not necessarily all of the same subsection, nor did all species within one subsection share the band. While R. hemsleyanum does cluster with the group containing R. auriculatum at a level of 53% similarity, R. auriculatum is more closely grouped with R. decorum, R. fortunei, and R. diaprepes. According to the D N A data, it may be more feasible to place auriculatum within the Fortunea subsection. While R. thomsonii and R. williamsianum, the putative relatives of R. orbiculare, did cluster together, the nearest group of species to this cluster is R. oreodoxa. R. orbiculare is no more closely related to R. thomsonii or R. williamsianum than many of the other species studied, including R. auriculatum or R. rex. Finally, R. calophytum is observed to group with R. sutchuenense and R. praevernum in a cluster which is quite discrete from the rest of the species. The RAPD data suggests these species should be removed from subsection Fortunea. While the hypothesized relatives of R. calophytum, namely R. rex and R. praestans cluster together, this cluster is actually more similar to the other species in Fortunea than the R. calophytum/sutchuenense group. The similarity of R. rex and R. praestans suggests that the "large-leaved species" subsections Grandia and Falconera should be merged. R. yakushimanum, which was selected as a sort of outgroup by virtue of being quite distinct morphologically from any of the other species under study is separated from the rest of the species quite distinctly in the cluster analysis. While all individuals within this species cluster at a minimum similarity of 68%, separation from the rest of the species is at a similarity of 34%. 70 It is interesting to note that species from other subsections, ie. R. auriculatum, R. thomsonii, R. williamsianum, R. rex, and R. praestans are not distinctly separated from the species within Fortunea, but are intermingled within the Fortunea species. Indeed, as stated above, some of these species show more phenetic similarity to Fortunea species than species actually placed within the subsection, such as R. praevernum and R. sutchuenense. Chamberlain (1982) states that within subsection Fortunea there is a central group of species around R. fortunei which have 6-7 lobed corollas and glandular styles in common, and have affinities with subsection Auriculata. This group includes R. fortunei, R. vernicosum, R. decorum and R. diaprepes. The phenogram produced in this study supports this hypothesis, as the aforementioned four species form a cluster with similarity of about 65%, and with R. auriculatum being the closest group, joining the cluster at a similarity of 59%. While Chamberlain's proposal that R. calophytum, with its 5-7 lobed, mortar shaped corolla, glabrous style, and large capitate stigma may be more closely allied to subsections Grandia or Falconera was not supported by the RAPD data, the idea that the campanulate to open campanulate corollas of R. orbiculare, R. oreodoxa, and R. griffithianum suggest an affinity with subsections Campylocarpa and Williamsiana was at least partially substantiated. It may be seen in Figure 9 that R. oreodoxa is the closest member of Fortunea to the R. thomsonii/R. williamsianum cluster. R. orbiculare has been described as an aberrant member of the subsection by several authors (Cox, 1979; Tagg, 1930), and indeed, it does form a distinct cluster, dividing from the main group of species at a similarity of 46% and before R. thomsonii and R. williamsianum. The placement of the R. sutchuenense/calophytum/praevernum cluster is somewhat surprising. Cox (1979) has suggested that R. calophytum does show some similarity to R. 71 sutchuenense, but he and most authors also agree that R. calophytum shows definite affinities to the Grandia or Falconera subsections. The RAPD data suggest, however, that R. calophytum is more closely allied to R. sutchuenense and R. praevernum than to either R. praestans, which is of the Grandia subsection, or R. rex, which belongs to subsection Falconera. Cox (1979) has also observed that some plants in cultivation appear to lie between the two species R. calophytum and R. sutchuenense, however, this may be due to hybridization of the two in cultivation. The sources of R. calophytum used in this study are given in Table 8. Only one specimen, C15, was grown from wild collected seed. The fact that this individual clusters with the others of its kind and not with the R. praestans/rex group suggests that the clustering shown is valid, but it may be possible that hybridization and backcrossing in cultivation has given rise to this similarity between the species in question here. Further work using wild collected specimens is required to clarify the relationships. The dendrogram also highlights some relationships that were not the initial focus of the study. Firstly, R. rex ssp. arizelum shows more similarity to R. praestans than to R. rex ssp. rex and R. rex ssp. fictolacteum, grouping with praestans at a level of 64% similarity, as compared to 53% similarity with the other R. rex subspecies. Cox (1979) suggests that R. rex ssp. arizelum is of hybrid ancestry, possibly with R. praestans, and this seems to be supported by the RAPD data. However, it has been reported that arizelum merges with R. rex ssp. fictolacteum in the wild, but this may be due to local hybridization (Chamberlain, 1982) rather than representing a geographical cline within R. rex. The data here suggest that R. rex ssp. arizelum should be classified with R. praestans, and this evidence may generate more study on this group. The placement of R. vernicosum on the phenogram is something of an enigma. It is not surprising that some individuals are quite similar to R. decorum and R. fortunei, however, the 72 majority of individuals of this species cluster most closely to R. griffithianum. R.. griffithianum is considered a distinct species without close allies by Chamberlain (1982) and Cox (1979) by virtue of its very large, well developed calyx. As previously expressed, several clustering methods were examined. Rohlf (1993) states that clusters that are common in phenograms generated from different clustering methods are the most distinct. Although the U P G M A technique gave the best goodness-of-fit to the similarity matrix, the dendrogram generated from the complete-linkage approach (Figure 10), which had a cophenetic correlation of 0.71 was compared to the U P G M A phenogram to determine the most well defined clusters. Typically, clusters which grouped individuals of the same species together were well defined. That is, individuals of one species consistently clustered together; although in some cases the internal grouping within these clusters may have been slightly different. The only exception to this rule was R. vernicosum. Whereas four individuals of this species, Vn 2,4,6, and 8 clustered together consistently in both phenograms, the placement of this cluster differed between the two. In the U P G M A tree, this cluster is grouped with R. griffithianum, while in the CLINK tree it is joined with R. williamsianum. This indicates that the clustering is not well defined. Therefore, no conclusions may be drawn regarding the relationships of R. vernicosum.. The large cluster that includes R. auriculatum, R. fortunei, R. decorum and R. diaprepes and a few individuals of R. vernicosum appears to be extremely well defined, as these species are grouped together consistently using the two clustering methods. Also well defined is the addition of R. hemsleyanum as the next closest group to this large cluster. Other well defined clusters include, R. thomsonii with R. williamsianum, R. calophytum with R. sutchuenense and R. praevernum, and R. praestans and R. arizelum with R. rex. As the level of similarity 73 0.25 0.50 0.75 LOO A u l j Au3 1 Au6 Au2 Au7 Au4 Au5 F d l F d 2 FH1 FH3 D e l F l l D p i Dp2 Dp3 F I 2 F I 3 ; | F I 5 1 FI7 F M F I 8 Dc2 Un3 Dc3 Dc4 FH4 FH5 FH6 Un5 Un7 Hml Hm3 Hr»2 Hut Hm5 D v l - 0x1 0 x 3 0x4 - 0 x 5 - 0 x 6 - P t l - F g l - T q 2 - F g 3 - Fgl - F g 5 - F g 7 - Fg6 - 0 x 2 - F g 8 — . F g 9 - P s l - P s 3 - P s 2 - R z l - fiz2 - R z 3 - R z 5 - Rzl - R z 6 - R x l - Rx6 - Rx4 - R x 2 - Rx3 - RI3 - Rx5 - R f l - RI2 - RI5 - RI6 —-RI7 - C l i - C12 - C o l - C16 - C17 - C14 - C15 - Co2 - C13 - P v l - P v 2 - Pv4 - P v 3 - P v 5 - S q 2 - Sq3 - S u l - Su4 - Su2 • Su3 • Su5 ' Su6 • S g l • Su7 • G i l • GI8 • G O GI5 GI2 GI3 GIO GI6 U n l Tm2 T « 3 Tm4 Tm8 Tm6 Tm5 U7 Tm9 T12 T13 Un2 Un4 Un8 Un6 Urn I Um2 Um7 - Um4 - Um3 - Um8 - Um5 - Um6 - O b i - 0 b 2 - 0 b 3 - 0 c 4 - O c l - 0 c 3 - 0 c 2 - O b i - 0b6 - 0b7 - 0 b 5 i GA 1 Y k d • U09 • Ykb • Y k c • K J a • YA YFa nn UD3 Yke Y k l Yk2 YY Y k a YO Sk DDI Dendrogram generated by the complete linkage clustering method on the similarity matrix created using Jaccard's coefficient. 7 4 decreases, so too does the distinctiveness of the clustering. It may be observed that the levels of similarity at which several of the species specific clusters diverge from the central, large cluster are very similar in the U P G M A phenogram. Thus, it is not completely surprising that the order of this separation is somewhat different using the complete-linkage method of clustering. Instead of clustering closely with the R. hemsleyanum/ fortunei central cluster as in the U P G M A tree, the griffithianum group is placed more closely to R. thomsonii in the CLINK tree. Indeed, it appears that R. oreodoxa, which was grouped with the R. thomsonii/williamsianum cluster under U P G M A has switched places with R. griffithianum. As the placements of these clusters are somewhat ambiguous and dependent on the method of clustering, no conclusions may be drawn with respect to their relationships. It is interesting to note that in the CLINK phenogram, R. orbiculare clusters more closely with R. williamsianum and R. thomsonii than under UPGMA. While it seems clear that species such as R. calophytum, R. arizelum and R. auriculatum are placed in distinctive clusters, the remaining groupings do not give a clear picture of relationships. Where some dispute arises, the U P G M A phenogram should be given more weight due to its higher cophenetic correlation value. It should also be noted here that there were not distinct differences between subsection Fortunea and any of the other subsections under study, with the exception of subsection Pontica. 75 C H A P T E R 4: G E N E R A L CONCLUSIONS The objective of this thesis was to apply the RAPD technology to rhododendrons. In terms of methodology, the technique was found to be reproducible when reaction conditions were optimized and held constant. That is, the concentrations of M g + 2 , Taq D N A polymerase and D N A template that yield the best results should be determined prior to beginning any study in a new organism, as well as the PCR cycling temperatures and times. The parameters used in this study may be regarded as a starting point from which to begin. With respect to D N A fingerprinting, the technique was quite successful, as 105 cultivars were distinguished using information from only five primers. Limitations do exist, however, when trying to identify unique fingerprinting patterns for sibling cultivars, as seen with individuals belonging to the Loderi grex used in this study. In all probability, the RAPD technique should be able to distinguish such cultivars - provided that the proper primers are identified. Further investigation is required to determine exactly which primers to use. While RAPDs do provide a quick and simple method of cultivar identification, the technique can not be recommended for general use in identifying parents of a cultivar from a large number of possible parents due to the relatively high frequency of nonparental bands seen in cultivars of known parentage. RAPDs may be of use in determining parentage when one parent is known and there are a small number of possibilities for the second parent. With respect to taxonomy, cluster analysis based on the data set generated by scoring RAPD bands tended to group individuals of the same species together, but did not support the current classification of subsection Fortunea, nor did it support the specific hypotheses 76 about relationships that were tested. However, some interesting relationships were highlighted between species not originally under this sort of scrutiny. Rhododendron auriculatum was grouped closely with R. fortunei, diaprepes and decorum, while R. calophytum, R. sutchuenense and R. praevernum were clustered in a distinct group. In addition, grouping between the R. rex subspecies and R. praestans raises some question as to the classification of R. rex ssp. arizelum and subsections Grandia and Falconera. Comparison of the U P G M A clustering method with the complete linkage method allowed identification of the most well defined clusters, which were listed above. Any other groupings require further study before clear conclusions may be made. Also of note is the fact that while several of the species selected for the study belonged to different subsections, such as Thomsonia, Grandia, Auriculata, etc., no distinction was seen on the dendrogram with respect to these. Indeed, species of all different subsections were clustered together, some appearing more closely related to species within Fortunea than many of the other Fortunea species themselves. The exception to this was R. yakushimanum, of subsection Pontica, all individuals of which were grouped into a very distinct cluster. The fact that R. yakushimanum, which was used as a sort of standard to indicate a species which is acknowledged not to be closely related to subsection Fortunea, was clustered distinctly away from the other species indicates that there is some validity to this method of analysis. It appears that subsection Fortunea as currently defined is very heterogeneous. It may be that the current classification of Rhododendron needs to be revised to reflect the high degree of similarity seen in the species of different subsections studied here. Certainly, further studies on these taxa are warranted. Other methods such as D N A sequencing or PCR-RFLPs 77 may provide further information as to the relationships between these species and clarify some of the indications of relationship that were found in this study. Recommendations 1. The RAPD technique has the potential to be an extremely useful tool in cultivar identification within nurseries, provided that the reaction parameters are standardized. 2. RAPDs will be of limited use in parentage analysis, due to the high frequency of nonparental bands. 3. In this investigation, RAPDs did show some potential for use in classification and study of relationships between rhododendron species. However, the large number of bands often generated by the technique makes scoring of bands difficult. It is recommended that primers be selected with care. 4. Other molecular methods should be utilized to confirm some of the relationships uncovered here, and also to extend the information available on relationships within subsection Fortunea. 78 B I B L I O G R A P H Y Arnold, M.L. , C M . Buckner & J.J. Robinson. 1991. Pollen mediated introgression and hybrid speciation in Louisiana irises. Proc. Natl. Acad. Sci. USA 88:1398-1402. Aruna, M . , P. Ozias-Akins & M.E. Austin. 1993. 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A revision of Rhododendron II. subgenus Hymenanthes. Notes, Royal Botanical Garden, Edinburgh 39(2):209-486. Comincini, S., M . Sironi, C. Bandi, C. Giunta, M . Rubini & F. Fontana. 1996. R A P D analysis of systematic relationships among the Cervidae. Heredity 76:215-221. Connolly, A .G. , I.D. Godwin, M . Cooper & I.H. DeLacy. 1994. Interpretation of randomly amplified polymorphic D N A marker data for fingerprinting of sweet potato (Ipomoea batatas L.) genotypes. Theoretical and Applied Genetics 88:332-336. Cox, P.A. 1979. The Larger Species of Rhododendron B.T. Basford Ltd., London. Cullen, J. 1980. A revision of Rhododendron I. subgenus Rhododendron sections Rhododendron and Pogonanthum. Notes, Royal Botanical Garden, Edinburgh 39(1): 1-207. Davidian, H.H. 1982. The Rhododendron Species. Volume I: Lepidotes. Timber Press, Portland, Oregon. Davidian, H.H. 1988. The Rhododendron Species. Volume II: Elepidotes. Timber Press, Portland, Oregon. 79 Demeke, T., R.P. Adams & R. Chibbar. 1992. Potential taxonomic use of random amplified polymorphic DNA (RAPD): a case study in Brassica. Theoretical & Applied Genetics 84:990-994. Doyle, J.J., & Doyle, J.L. 1990. Isolation of D N A from small amounts of plant tissue. BRL Focus 12:13-15. Dunemann, F., R. Kahnau, & H. Schmidt. 1994. Genetic relationships in Malus evaluated by RAPD "fingerprinting" of cultivars and wild species. Plant Breeding 113:150-159. Hu, J., & C.F. Quiros. 1991. Identification of broccoli and cauliflower cultivars with R A P D markers. Plant Cell Reports 10:505-511. Iqbal, M.J. , L .E. Gray, D.W. Paden & A . L . Rayburn. 1994. Research note: Feasibility of rhododendron DNA profiling by R A P D analysis. Plant Varieties and Seeds 7:59-63. Iqbal, M.J., D.W. Paden, & A.L . Rayburn. 1995a. Clonal stability of R A P D markers in three Rhododendron species. Journal of Environmental Horticulture 13(l):43-46. Iqbal, M.J. , D.W. Paden, & A . L . Rayburn. 1995b. Assessment of genetic relationships among rhododendron species, varieties and hybrids by R A P D analysis. Scientia Horticulturae. 63:215-223. Joshi, C P . , & H.T. Nguyen. 1993. RAPD (random amplified polymorphic DNA) analysis based intervarietal genetic relationships among hexaploid wheats. Plant Science 93:95-103. Knuttel, A.J . 1990. Tissue culture troubles. Journal of the American Rhododendron Society Summer, 1990. Lewis, K .G. 1995. Genetic variation among populations of Pissodes strobi (white pine weevil) reared from Picea and Pinus hosts as inferred from RAPD markers. Master's Thesis. University of British Columbia. Luteyn, J.L., & M.E. O'Brien, eds. 1980. Contributions towards a classification of Rhododendron The New York Botanical Garden New York, New York. Mailer, R.J., R. Scarth, & B. Fristensky. 1994. Discrimination among cultivars of rapeseed (Brassica napus L.) using D N A polymorphisms amplified from arbitrary primers. Theoretical and Applied Genetics 87:697-704. 80 Marsolais, J.V., J.S. Pringle & B.N. White. 1993. Assessment of random amplified polymorphic D N A (RAPD) as genetic markers for determining the origin of interspecific lilac hybrids. Taxon 42:531-537. Orozco-Castillo, C , K.J. Chalmers, R. Waugh & W. Powell. 1994. Detection of genetic diversity and selective gene introgression in coffee using RAPD markers. Theoretical and Applied Genetics 87:943-940. Philipson, W.R. & M . N . Rhilipson. 1986. A revision of Rhododendron III. Subgenera Azaleastrum, Mumeazalea, Candidastrum, and Therorhodion. Notes Royal Botanical Garden, Edinburgh 44:1-23. Pillay, M . , & S.T. Kenny. 1995. Anomalies in direct pairwise comparisons of RAPD fragments for genetic analysis. BioTechniques 19:694-698. Prince, J.P., V . L . Lackney, C. Angeles, J.R. Blauth, & M . M . Kyle. 1995. A survey of D N A polymorphism within the genus Capsicum and the fingerprinting of pepper cultivars. Genome 38:224-231. 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D N A polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18(22):6531. Wolff, K. & J. Peters-Van Rijn. 1993. Rapid detection of genetic variability in chrysanthemum (Dendranthema grandiflordTzwe\ev) using random primers. Heredity 71:335-341. 82 Yang. X . & C. Quiros. 1993. Identification and classification of celery cultivars with R A P D markers. Theoretical and Applied Genetics 86:205-212. Yu. L . X . & H.T. Nguyen. 1994. Genetic variation detected with RAPD markers among upland and lowland rice cultivars (Oryza sativa L.). Theoretical and Applied Genetics 87:668-672. 83 APPENDIX I DNA Isolation Protocol for Small Amounts of Tissue 1. Preheat extraction buffer (3% C T A B , 20mM EDTA, 1.4M NaCl, lOOmM Tris-HCl, pH 8.0, 1% polyvinylpyrollidone and 0.5% P-mercaptoethanol, added just prior to use) per sample at 65 °C. 2. Measure approximately lOOmg finely ground leaf tissue into a 1.5 ml microcentrifuge tube. 3. Add 450pJ of hot extraction buffer, vortex, and incubate at 65°C for -30 minutes. 4. Add 800(xl of chloroform/isoamyl alcohol (24:1); vortex and invert to mix. 5. Spin tubes 10 minutes in a microcentrifuge and remove the aqueous phase to a new tube. 6. Repeat the chloroform/isoamyl alcohol extraction (may be omitted). 7. Add -335pi isopropanol to each tube, invert several times and let sit at room temperature for 30 minutes to several hours. 8. Spin 15-30 minutes in a refrigerated microcentrifuge and remove supernatant. 9. Wash the pellet with 70% ethanol. If necessary, spin another 30 minutes to stick the pellet to the tube. 10. Remove ethanol with aspirator and invert to dry. 11. Resuspend the pellet in ~150[xl of TE buffer. 84 e ii o U X) C a X d, X o o o -a a a o CN ON > X! 4> oo (D X PH PHX> - ° o o -st o o^ 2=3 ° d m © S: W-) - G «5 O eo c o T3 C a X I •St m oo Z H Z a: OH « X O 5 * Zta £ z •< O O H > m o 00 •St" in 00 co" 00 DH o" CL oo 00 o ci, ^-T O P L * " v-i 1 pa ON" H -Q < c_ O , ^ 60 13 41 U X H in o 00 • S t m 00 co" oo OH o" r~-CL oo" oo" o PH in m PQ C O o •4—> O OB C N > O in ^r t • ' ^ H H PQ < 8>o c 4> ii C L (A Tvt o •4—* o 60 CN > S H O « e Vi ii C L « B "S 41 C Vi ii 00 o 60 X E-i co" oo OH o" NO OH 0 oo -fcJ ^ H 60 „ 1 0 0 00 m PQ m o oo pq —TON PH < v (3 a <« U HO CL « o O 60 - S105 Fl, S54 -Tt • • present absent -present absent 06 OH OH H X P J z o ON O 60 X H o" r-0H oo" o o <N-i oo oo" pq o" PQ ON < ii S V) ii ii VI u Xi CL A O 60 2 o x > i n H pq ON"0" < pq 41 Vi U HO CL A ii G VJ ii ii Vi § 2 So. xw P J z o H o o ft. z o H O 0 1 oo «5 CQ 41 S 4) Vi U X> CL CQ <N CN L. T3 C c3 X ) PH X O m co T3 C X PH X o m O CN T3 C 03 X PH X o O co -a c a x PH X o •St T3 B X PH X o » n mi T 3 C C3 X PH X o •St NO 13 -a C -a c a3 c 03 X r3 X PH X PH X PH X in X o r-- o o o -st Tt CN f-l> od ON T3 C X PH X O m NO -a e a X PH X o O 00 85 T 3 i n G 5 <D £-2 § c/3 o3 2 <u C/3 Ctf C/3 U C O S 03 O > S3 4= NO co t/T Q OH ^ m CN 5 o o CO C O U co m - CM 00 CN Z o -4—) n ° ro O O CO co" 2 e 3^ o 0 OJO 1 2 to . U " b «3rj - O H „0H d vo" MH VO SO ^ co i—l ffl „ „co U U O H £ m PQ J fN 0 g> i 1 ^ O H > CO „ CO --co NO co <2 ^ CO <N < J OH Q > n o NO - o Q « i—> o £ • r>) O ^ CN ^> CN o fN N O O O C O co o o co co o H CN Q £ CN fl - w fl * H S e <u fl vi cu vi cu V « cu vi b X) b X ! Q> A DH CQ v fl V! CU CU V3 b ja OH CQ V fl VI CU CU VI b Xi e CU VI CU b a e CU V) CU VI CU bl a fl cU VI A 03 cu B vi cu cu vi b A a. « cu C V! CU CU V> b A Cu C3 fl *i CU fl VI cu CU VI b A a a e CU VI CU cu cs Ml in cs C N b cU bi OH T3 C as X > PH X ) IT) PH o m o CN PH X ) o o CO co PH X ) o CO i n I T ) C N f N b cU b OH PH X ) o I D r-i o co PH PH X I PH PH PH X ) o X ) X X ) O I T ) o o O o o o i n m ON T _ 1 NO i n NO CN CO i n NO 86 m c o O c o 00 O N O -4—> O O O CO 0 bO 1 ^ O C O C, —r o s • CN O b O "3-co C N # " N O N O o " oo" l O C N h-) HJ r-»" r-»" C N C O J o ^ oo" N O C N ( j ^H £ o ^ - > o rC o co" o M ooco oo U . < . 5 a c/J V cu CA b & cu CQ o oo i n o C O ~ „ o CN bO CL, , N O N O c o O oo" W o c o C L & oo" CN r ceo U co cu S w cu cu U X ! a s3 ro N O o b O oo" CN o " r o C L s H-CU S C/3 CU CU C/3 o b O O co O H oo" CN cu S C/3 CU CU V3 H - ° OH CQ © 0 0 f N O N J cu S c/> cu CU Cfl a CQ O N O b O m c o CN C L NO" N O co O U cu C C/3 CU CU Ul L. X ! OH CQ CU X ) O o O C N A OH X ) o i n o 00 PH X ) o i n r--O N PH X ) o PH PH PH PH PH co X ) X ) X ) PH X ) PH X ) PH o o o X ) o X ) O X ) 1 o o o o o o m m o o m T j - C O i n •«* r - - o C O , — 1 i n ^ r - o o d CN c o i n N O l > 00 87 o H C> O o O CN ° « ft, • r tj NO O S CN O O • s t H a s o ^ oo"£:~ "I C N i-J Q ^ " - N O co CN O NO co 0 0 0 0 " U OH NO CO C0 o4 OH H X w z 0 H O 0 1 - J 1 NO CN O O • s t >n Q CN W o o" 6 0 in ^ C N 06 o i OH H X w z 0 H O O 1 - J o " >n - J NT § a . -1 0 0 -VO NO" ^ OH H H e i c N pq 1 0 0 " o 0 0 NO O N C N ^ OH O N . 2 2 OH O - M O N s i n o " m O O N 3 o " •St p q u u o 0 0 C Q CN CN P Q CN ^ Oo" >H co" Z S o C U ° o O N ^ OH >>. „t> P Q ^ E CN pq ^ H f^ . C N OH C O P Q O - f—1 - C N ' " J 0 0 " . 0 0 ' - ' „>—> pq CN S 0 0 O i n T-H co 'pq>< Wit—1 1 -ON co" o 0 0 N O " _-oo" CN # P< «t - p q OH O •St C N O i P Q o CN C N ^ I D & P S -0H e"^ W i n ^ -ON „ 0 0 0 0 -NO r* C N r -OH U v C! cn <u a CQ A « a G vi a ii Vi U £t a CQ A "B ii ii Vi fa * a, CQ G a Vi A -a ii G Vi a ii Vi U £ Q , CS ii G Vi ii ii Vi a cs fl "S <u G Vi a a vi G ^ a, CQ s • H G a> e a G a (A H Vi ii Vi ii Vi a Vi ii U £ LH a, CQ a CQ a e CQ PH HO o i n ON O m o CN PH o ON NO I o 0 0 NO CN PH PH PH PH X X X X PH O O O O X O O m O m • S t m 0 rt ^ r -CN co • s t i n NO CN CN CN CN CN I T ) C N C N u ii PH X o m o PH X o O ON CN 88 ~0 X> NO o bi) o w CN o CO 00 bJO 2 • s -.« r~ 0. i-i „ - 0 0 NO in o bJO OH O H O © -NO OH ^ 0-0-oo 2 P 8)2 1 o O bO CN , £ 2 t> in U S o r) c/3 rj NO ON CO oo" 01 CN _ 0< CN ON" W CN O OH ~ „ O T}- bO OH ^ .ON" -co ' - i NO ;> W |-| t> o i d g o ffi bO in co > O H g ^ c m _-co „ 2 - C N :* o CN W co pq U o bO O 00 m CN" CN CQ 8 ^ 00 f N c CO o" CO NO" ON CO oo" 0. ON" 2 CN O o •J 2 ° £ ^PO ON" CO »-» >H 2 „>: boW CN °i ^ r-OiQco ^d H - f^N CN m " C- o o w *•* I O vo • >" ON .. >H CO vo" -ON 2 co H oo" C CN/CO ON CN - 1—5 m _ ffi o^ 2 d sa CA CU CU CA fc- Xi OH CO CU fl CA CU cu cn U Xi OH C3 A "S fl *2 CU fl cu fl CA CU CA CU cu CA o in «- Xi U X> OH S3 OH CS CU fl CA cu cu w t. Xi OH 03 C -cu fl CA CU CU CO >- X) a 03 CU C/3 cu I i a fl CU c/> Xi 03 cu fl co cu CU C/3 2 Xi a 03 cu C c/> CU CU CA t-H Xi a 03 fl a cu C CA CU CU CA a -° a 03 fl a CU fl CA CU CU CA a -° a 03 fl a CU & CA CU CU CA t- Xi a c3 OH OH OH X) . X) OH OH X) OH OH O o x> X> O X) X) o O o in in o O m in r- in m oo ON in m CO in NO 00 . ON OH OH OH X) X) X> m O o CN m o CO oo ^ d ,_; CN 1-H r—< OH X O o CO OH X O o 89 o bO i ON >* to (19; NO" ON 00 o 00 - go to (16 P29*, R8, go to (18) , S96, Y9 -• OL, T37 G49, H5, O H • • • H5, R8 -G49, P29 : oo m present • absent -present absent present • absent -present absent o CN O OO ON NO" ON 00 ON „ CN ON H 13 tu S V) 41 CU VI H ^ ON 03 OH H X w z o H O O NO . ON ON S3 - M cu B i / l 4 ) 4 ) V) U £1 DM CQ CN CN O co o w OO o • NO o r—i 60 >H i cn" NO CN" CN co" co" H H o" o" 00 00 •sf 1-H t-H OH OH PQ PQ7 i—> 1—> NO" NO" 1—1 I—I B O ro • ->• © > rt • • B B <-4> B v B VI 4> Vi 4> 4 ) VI 4> V) t- £ a CQ a CQ OH W o i ^ ^ O H C i C i X o o « ~ ~ Z O O A 00 bO^ -1 1 i H NO CO O CN r-1 [o P OH E-H <*> - .'<t • O O r t PQ > PH 4 ) fi V S3 V) V VI 4 1 4 ) v i 4 ) v i OH CQ OH CQ 0< W 2 OH H X w z o H O 0 1 S 00 B -4> B V) 4> 4 ) VI a CQ OH O O PH X O o O CN A NO PH PH PH PH PH PH PH X PH X X PH X X X X o X o o • X o o o o o m o o m o o CN lO CO CO NO ir> 00 •St 00 00 ON d CN co CN CN CN CN CN CN 90 Wl H X w z o H O o T3 c cn O N O 0< s cn o o CN cn O oo as o bo oo W cn W oo r--cn U CN" m CM r-- 3 Q w o n 05 O u NO ,<3 0H §R3 00 CM cn oo W < Oi < g e CU cn cu t -QH c cu co X) 93 m o o 60 r--cn 05 0< cn" o Z ^ . c n - o * bO O S CN CQ < cu M V) cu CU VI S Xi o •4—* O 60 r--go to (4) 03, R31, V - go to (6) - go to (8) W7 - go to • go to (10) - R6 A7, M7 -• D51, Nl, B21, R4 Tt fN • i • D51, 03 Nl, R31, m 1 • • Nl, W7 -- R31 • • present absent -present absent present • absent -present absent present • absent -present absent present • absent present absent CN" s NO* NO OH ON CN -- H v ' t—I 2 00 as 1 oo" oo Q CN M m - O ^ * «-• PH cn 60' < < . 1 • • H-> cu O Vi cu cu v i LH A a CQ cn o o -4—> o o bO w ,—c 00 CM cn -cu H vi cu CU V! £g X O i CQ m CM fN i -CU OH PH x> o CM ON PH PH PH PH PH X) PH X) PH X PH ' X) X) o X) o X) o X) o O o O O CN o m i n "3- o i n m cn i n r -m ON CM cn i n NO 00 ON PH X ) O m CN PH X) O m m PH X ) O CN 91 e a CA CU CU </> CM a O b O r -m E-i H * " m o C o sO o S So ^"co CM „ CN , -H CQpq cu C CA CU CU CA i n o Co" o w b O O ' cu B CA CU CU CA a x a co oi w S s CL, H X w o H 0 a 1 •< s - a cu e CA CU CU CA a - ° OH X> O i n OH OH OH C O X) - O XI o o O O o o o o o C O '— 1 C O i n o o u cu 2-OH X) o o o OH X) O O so CN OH OH ' OH X> X) X) m O O t-- i n i n as CO i n 92 % X o e T3 XI §3 0 X O N 8 rt O cn © s s • J ? OH =3 .9 X a & X 5 in c3 Q x ON "O O T37* -T16, *" in 0 # " NO /-> CSS M66, go to H16, LW -B22*, H19, 3a • • present absent • present -absent -present absent O 60 VO o m C - © o 0 0 0 NO" 60 Q> 1 NO" CN NO CN cu H cn cu cu cn s -° OH 03 H S cu C «) cu cu cn a -° OH 03 fl "B cu s cn cu cu cn a OH 03 © C N 1-H C N 5 5 -B cu B cn cu cu cn U £t OH 03 inf - 1 ~>n ON c/3 O NO C O 0 0 co -W C O -00 C O Q . 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H 0 0 1 — 1 CLH £ . o cn ""5 OH cn H m 5 a CU CA CU U i OH J OH Q CQ OH OH 0 0 - H NO OH CN OH ooo o C N 0 0 ^ „ C N W —1 o oo *-• „ o cn oo o o NO C § ^ OH X ) o o NO HJ 0 0 oo"cn" NO h J o o ON" OH O v o S O H CN „ Q ^ „ 0 0 OH U O H ^ a-ON" U JN .OH o CQO o — o ^ i N O " ^ CQ d . C N CQ J oo"3 cn -< 3 r-"cn" O > ^ o c i m r j o o cn CN o> - 1 m oo CN oq" pq" oo" cn" OH < H J H * H c * H 0 cu 0 cu CA cu CA cu CA Xi U i X CQ a CQ OH X ) o i n 0 0 CN o CN cn o o o CN 1 3 U oo"2 r-"0n -U N 0 " Z do< /• oo . N ; CO 0 0 (—1 * " 0 H ^ CQ^CQ OS . < OH < a CU CA c u u OH C CU CA X CQ OH X ) O O cn 1 o 532 S"5" A B c u O CA CU CU CA U X! a CQ OH x> o o 0 0 OH W oi 0H H X W £ 9 2 O o i ba * i S oo U l Z l 1 I ^ U • i * H 0 B B B c u C cu 0 IA CU CA a > CU CA CU CA U Xi U X OH CQ a CQ OH X OH o X o 1—1 o i n NO 94 oi w oi pq oi CM H X pq Z oo H o O 0 0 1 • * c< •st -PQ °o o oo fn oo -st-PQ PH O b O 0 0 vo" Oi -m oo 5 ? 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Thesis/Dissertation
eng
Genetics
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Graduate
DNA fingerprinting in Rhododendrons using random amplified polymorphic DNA
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