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Mosquito pathogenic strains of Bacillus sphaericus are indistinguishable from nonpathogenic strains based on simple phenotypic tests. DNA-DNA hybridizations performed in 1980 placed the 7 pathogens included in that study in a distinct homology group separate from 5 groups of nonpathogens. The overall homology of the pathogenic strains to the species type strain was only 19% indicating that these pathogens should be a separate species. Since the DNA homology study was published in 1980, many more pathogenic strains have been isolated worldwide. Pathogenic strains have been differentiated from other strains of B. sphaericus by rRNA sequencing, fatty acid analysis, and isozyme analysis. The pathogens have been further classified by type of toxin produced, serotyping, and phage typing. I have used random amplified polymorphic DNA (RAPD) fingerprinting to determine the phenetic relationships among 31 pathogenic and 14 nonpathogenic strains of B. sphaericus. DNA Bands in agarose gel migrating the same distance were verified as being homologous using PCR-generated probes made from the RAPD bands. Band patterns resulting from 8 10-mer primers were examined by three coefficients, Jaccard, Dice, and simple matching. Each coefficient was able to distinguish DNA homology groups, although the relative similarity values differed. In agreement with DNA homology studies, pathogenic strains showed less than 10% similarity to nonpathogens using Jaccard and Dice coefficients. This value was 68% based on the simple matching coefficient. Individual serotypes were clearly indicated among the pathogenic strains by each coefficient. This suggests an overall genetic homogeneity among strains within serotypes. It also parallels the uniform toxicity pattern found within each serotype (unlike the toxin diversity found within B. thuringiensis serotypes). These results together with DNA homology data support the establishment of a new species for the pathogenic strains. / Master of Science

No

Horses have been domesticated for thousands of years. Through selection practices horses have been separated into groups that pass on desired traits. We have viewed what the breeders have done at the genotypic level to accomplish their breeds. By using a relatively new technique, DNA fingerprinting, Thoroughbred inbreeding and eight other breeds (Standardbreds, Quarter horses, American Saddlebreds, National Show Horses, Arabians, Morgans, Mustangs and Belgians) have been studied. The probes CAC5 and YNZ 132 gave the best probability (ranging from 1.9x10⁻¹⁰ to 4.8x10⁻¹⁵) that two unrelated individuals would not have the same DNA fingerprint out of the probes screened. The level of inbreeding in Thoroughbreds has been estimated by comparing the number of bands shared among these animals to a quasi-natural population (Mustang) and a theoretically known genetic relationship (a sire and his offspring). Using the probes CAC5 and YNZ 132 Thoroughbreds share 20% more bands than the Mustang and 30-50% less than the sire and offspring. To compare the nine breeds, blood from ten horses from nine different breeds was mixed and DNA fingerprinted. Each lane on the autoradiograph therefore represents one breed. The two probes produced data with a rank correlation of .75 (Kendall's tau) (Ostle,B., 1963). Selection practices have been divided into, narrow selection regimes (where one or two traits have been selected for) and broad selection regimes (where numerous traits have been selected for). The amount of bands shared between the breeds was calculated and applied to a computer program named Gendiv (Gentzbittel and Nicolas, 1989,1991). Three consensus trees were derived showing that the narrow selection regime breeds, Thoroughbreds and Standardbreds, were the most genetically distanced from broad selection regime breeds, Mustangs and Morgans. / Master of Science

In this study, I used restriction fragment length polymorphism (RFLP) analysis (DNA fingerprinting) to study the genetic population structure of wood frogs, Rana sylvatica, collected as egg masses from vernal pools within the Massachusetts Audubon Society Lincoln Woods Wildlife Sanctuary in Leominster, MA. The average genetic relatedness of sibling individuals, non-sibling individuals from within the same pool, and individuals from pools of close (5 m), far (200 m) and distant (40 km) spatial separations was calculated. The goal was to use genetic relatedness to estimate the breeding patterns of R. sylvatica and use that information to make general management recommendations that could be applied to other vernal pools breeders. I detected relative differences in genetic similarity between individuals from pools only 5 meters apart, however over a larger distance of 200 meters no significant genetic differences were present. This suggests that although wood frogs are known to be highly philopatric, they may use information other than simply proximity to their natal pool when choosing breeding sites. Factors such as species composition, water chemistry and heterogeneity of the landscape between pools may influence breeding site choice. Also, contrary to the findings of recent studies, the distance between vernal pools may not be the best indicator of the genetic similarity of the individuals they host. Pools in close proximity to one another may host genetically distinct populations, and therefore management decisions should be made on a pool-by-pool basis. Consequently, when managing populations of R. sylvatica, and possibly other vernal pool breeders, taking into account parameters other than simply the spatial separation of pools within an array may avoid decisions that could result in the loss of genetic diversity.

Cranes are large elegant birds that occur on all continents of the world except for South America and Antarctica. Of the fifteen species of crane worldwide, three predominantly occur in southern Africa; the Wattled crane (Bugeranus carunculatus), the Blue crane (Anthropoides paradisea) and the Crowned crane (Balearica regulorum). Crane numbers throughout the world are diminishing, mostly because of the destruction of their habitat and illegal bird trading. Efforts are underway to prevent species extinction, legally and through the compilation of a studbook that contains descriptions of physical attributes, ownership, location and possible kinships of birds in captivity . This investigation, first of its kind, WdS undertaken to assess whether twelve published and unpublished microsatellite primers developed for the related Whooping crane and Red-Crowned crane could be used to fingerprint the southern African crane species using cost effective polyacrylamide gel electrophoresis. The results obtained were then used to determine the extent of genetic variation within species and distance between species. All primer sets amplified heterologous microsatellite loci in the three crane species, however, the unpublished primers produced poorly defined fingerprints even after extensive optimization. Of the twelve microsatellite loci investigated, the Blue crane and the Wattled crane revealed a high level of polymorphism. The Blue crane displayed 76% polymorphism and the Wattled crane 92%. In contrast, for the Crowned crane, that belongs to a different subfamily, Balearicinae, only 50% of the loci were polymorphic. The alleles displayed sizes similar to that of the species for which the primers were developed. Little variation in size, less than 10 bp, was noted for the different alleles of the polymorphic loci. The number of alleles, on the other hand, at each of the polymorphic loci was found to be low. The frequency of the most prevalent allele at most of the loci was generally reasonably high. These results therefore suggest that these primer sets are not suitable for individual identification and differentiation using polyacrylamide gel electrophoresis. Xll The observed heterozygosity of the three crane species was low; 12% in Blue crane; 7% in Crowned crane; and 13% in Wattled crane. Nei's identity further confirmed the high similarity between individuals; 66-100% for Blue crane; 55-100% for Crowned crane and 41-95% for Wattled crane. This low genetic variation is attributed to possible relatedness between birds supplied by aviculturists whom have a limited number of birds in captivity. A Hardy-Weinberg test for equilibrium revealed that most of the microsatellite loci displayed a deficiency of heterozygotes, while a few loci displayed an excess of heterozygotes. In general, the Hardy Weinberg test of equilibrium supported the notion that the individuals within each of the species might have been related. Differentiation between the three crane species ranged from 3-5%, with Blue and Wattled crane displaying a higher degree of genetic similarity when compared to the Crowned crane, known to be the oldest extant crane species. The limited allelic variation within the microsatellite loci tested, as well as the extensive genetic similarity between individuals suggests that a wide-ranging search for additional microsatellite loci that are more polymorphic and contain a larger number of alleles should be undertaken for the southern African crane species. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

M.Sc. (Botany) / Global efforts to protect the world’s forests from unsustainable and inequitable exploitation have been undermined in recent years by rampant illegal logging in many timber-producing countries. A prerequisite for efficient control and seizure of illegally harvested forest product is a rapid, accurate and tamper proof method of species identification. DNA barcoding is one such a tool, relatively simple to apply. It is acknowledged to bring about accuracy and efficiency in species identification. In this study a DNA barcode reference library for traded and protected tree species of southern Africa was developed comprising of 81 species and 48 genera. Four primary analyses were conducted to assess the suitability of the core barcodes as a species identification tool using the R package Spider 1.2-0. Lastly, to evaluate this identification tool, query specimens independently sampled at a Mozambican logging concession were identified using DNA barcoding techniques. The nearest neighbour (k-NN) and best close match (BCM) distance based parameter yielded 90% and 85% identification success rate using the core plant barcodes respectively. DNA barcoding identification of query specimens maintained a constant 83% accuracy over the single marker dataset and the combined dataset. This database can serve as a backbone to a control mechanism based on DNA techniques for species identification and also advance the ability of relevant authorities to rapidly identify species of timber at entry and exit points between countries with simple, fast, and accurate DNA techniques.

Ha Wai-Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 166-180). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Abstract --- p.iii / Abbreviations --- p.vi / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- "Hstory, cultivation and trade" --- p.2 / Chapter 1.2 --- Botany --- p.4 / Chapter 1.3 --- Chemical Constituents and Pharmacological effects --- p.8 / Chapter 1.4 --- Authentication of Chinese herbal materials --- p.13 / Chapter 1.4.1 --- Morphological marker --- p.15 / Chapter 1.4.2 --- Histological marker --- p.18 / Chapter 1.4.3 --- Chemical marker --- p.20 / Chapter 1.4.4 --- Molecular markers --- p.24 / Chapter 1.4.4.1 --- Protein marker --- p.24 / Chapter 1.4.4.2 --- DNA-based markers --- p.26 / Chapter 1.4.4.2.1 --- PCR-based markers --- p.27 / Chapter 1.4.4.2.1.1 --- Random-primed PCR --- p.28 / Chapter 1.4.4.2.1.2 --- Simple Sequence Repeats (SSR) --- p.30 / Chapter 1.4.4.2.1.3 --- Polymerase Chain Reaction Fragment Length Polymorphism (PCR-RFLP) --- p.31 / Chapter 1.4.4.2.2 --- Hybridization-based markers --- p.33 / Chapter 1.4.4.2.3 --- Sequencing-based markers --- p.35 / Chapter 1.5 --- Objectives and Strategies of the studies --- p.39 / Chapter Chapter 2 --- General Materials and Methods --- p.40 / Chapter 2.1 --- Reagents and Buffers --- p.41 / Chapter 2.1.1 --- Media for bacterial culture --- p.41 / Chapter 2.1.2 --- Reagents for preparation of competent cells --- p.42 / Chapter 2.1.3 --- Reagents for plasmid DNA preparation --- p.42 / Chapter 2.1.4 --- Reagents for agarose gel electrophoresis --- p.43 / Chapter 2.1.5 --- Reagents for polyacrylamide gel electrophoresis --- p.43 / Chapter 2.1.6 --- Reagents for Southern hybridization --- p.44 / Chapter 2.2 --- Agarose Gel electrophoresis of DNA --- p.46 / Chapter 2.3 --- Purification of PCR products --- p.46 / Chapter 2.3.1 --- From agarose gel using Geneclean® II kit --- p.46 / Chapter 2.3.2 --- Using Microspin´ёØ Column --- p.47 / Chapter 2.4 --- End modification of PCR amplified DNA --- p.47 / Chapter 2.5 --- Preparation of Escherichia coli Competent Cells --- p.48 / Chapter 2.6 --- "Ligation and Transformation of E, coli" --- p.49 / Chapter 2.7 --- Plasmid Preparation --- p.50 / Chapter 2.7.1 --- Minipreparation of plasmid DNA --- p.50 / Chapter 2.7.2 --- Preparation of plasmid DNA using Wizard® Plus SV Minipreps DNA Purification Kit (Promega) --- p.50 / Chapter 2.8 --- Screening for the Presence of insert in plasmid --- p.51 / Chapter 2.8.1 --- Rapid alkaline lysis --- p.51 / Chapter 2.8.2 --- PCR screening --- p.52 / Chapter 2.8.3 --- Restriction digestion of plasmid DNA --- p.53 / Chapter 2.9 --- DNA sequencing --- p.53 / Chapter 2.9.1 --- Plasmid sequencing using T7 Sequencing Kit --- p.53 / Chapter 2.9.2 --- Cycle Sequencing from PCR products or plasmid --- p.54 / Chapter 2.10 --- DNA Sequencing electrophoresis --- p.55 / Chapter 2.10.1 --- Preparation of 6 % polyacrylamide gel solution --- p.55 / Chapter 2.10.2 --- Gel casting --- p.55 / Chapter 2.10.3 --- Electrophoresis of Sequencing Gel --- p.56 / Chapter 2.10.4 --- Autoradiography --- p.57 / Chapter 2.11 --- DNA elution from dried sequencing gel --- p.57 / Chapter 2.12 --- Southern blot analysis --- p.58 / Chapter 2.12.1 --- Restriction digestion of genomic DNA --- p.58 / Chapter 2.12.2 --- Purification of digested DNA and agarose gel electrophoresis --- p.58 / Chapter 2.12.3 --- Capillary transfer of DNA to a Hybond´ёØ N+ nylon membrane --- p.59 / Chapter 2.12.4 --- DNA radiolabeling by nick translation --- p.60 / Chapter 2.12.5 --- Purificaiton of radiolabeled probe by NICK® Spin Column --- p.60 / Chapter 2.12.6 --- Hybridization of DNA --- p.61 / Chapter Chapter 3 --- Plant DNA extraction --- p.62 / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Reagents and buffer for total DNA extraction --- p.66 / Chapter 3.3 --- Extraction methods --- p.70 / Chapter 3.3.1 --- Sample preparation --- p.70 / Chapter 3.3.2 --- CTAB extraction method --- p.70 / Chapter 3.3.3 --- Potassium acetate/ SDS extraction method --- p.71 / Chapter 3.3.4 --- GIBRO Plant DNAzol® reagent for genomic DNA isolation --- p.72 / Chapter 3.4 --- Qualitative and quantitative analysis of DNA --- p.74 / Chapter 3.5 --- Results --- p.75 / Chapter 3.6 --- Discussion --- p.78 / Chapter Chapter 4 --- Amplified Fragment Length Polymorphism (AFLP) analysis of P. ginseng and P. quinquefolius --- p.81 / Chapter 4.1 --- Introduction --- p.82 / Chapter 4.2 --- Materials and methods --- p.88 / Chapter 4.2.1 --- Plant materials --- p.88 / Chapter 4.2.2 --- Choice of Primers and radiolabeling --- p.89 / Chapter 4.2.3 --- AFLP assay --- p.90 / Chapter 4.2.4 --- Electrophoresis of AFLP fingerprint --- p.91 / Chapter 4.2.5 --- Similarity Index (S.I.) analysis of AFLP profile --- p.91 / Chapter 4.2.6 --- Re-amplification of polymorphic DNA fragments isolated from dried sequencing gel --- p.92 / Chapter 4.2.7 --- Cloning and Sequencing of the AFLP fragments --- p.93 / Chapter 4.2.8 --- Conversion of AFLP marker into Directed Amplification of Minisatellite-region DNA polymorphism (DAMD) marker --- p.93 / Chapter 4.3 --- Results --- p.95 / Chapter 4.4 --- Discussion --- p.102 / Chapter Chapter 5 --- Direct Amplification of Length Polymorphisms (DALP) analysis of P. ginseng and P. quinquefolius --- p.107 / Chapter 5.1 --- Introduction --- p.108 / Chapter 5.2 --- Materials and methods --- p.112 / Chapter 5.2.1 --- Plant materials --- p.112 / Chapter 5.2.2 --- Choice of Primers --- p.113 / Chapter 5.2.3 --- Alternative labelled Amplification reaction --- p.114 / Chapter 5.2.4 --- Electrophoresis of the multi-locus amplification products --- p.114 / Chapter 5.2.5 --- Isolation and Re-amplification of polymorphic DALP fragments from dried sequencing gel --- p.115 / Chapter 5.2.6 --- Cloning and Sequencing --- p.115 / Chapter 5.2.7 --- Conversion of DALP marker to Sequence Tagged Site (STS) marker --- p.116 / Chapter 5.3 --- Results --- p.117 / Chapter 5.4 --- Discussion --- p.135 / Chapter Chapter 6 --- Sequence-characterized amplified region (SCAR): the sequel of random amplified polymorphic DNA (RAPD) --- p.137 / Chapter 6.1 --- Introduction --- p.138 / Chapter 6.2 --- Materials and methods --- p.140 / Chapter 6.2.1 --- Plant materials --- p.140 / Chapter 6.2.2 --- PCR reaction --- p.141 / Chapter 6.2.3 --- Cloning and sequencing --- p.143 / Chapter 6.3 --- Results --- p.144 / Chapter 6.4 --- Discussion --- p.157 / Chapter Chapter 7 --- Outlook --- p.159 / Chapter 7.1 --- Molecular authentication of Chinese medicinal materials --- p.160 / Chapter 7.2 --- Development of molecular markers for Ginseng --- p.161 / Appendix I --- p.164 / Appendix II --- p.165 / References --- p.166

Poon Wing-sem. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 163-171). / Abstracts in English and Chinese. / ABSTRACT I-IV --- p.I-IV / ACKNOWLEDGMENTS V --- p.V / TABLE OF CONTENT --- p.VI-VIII / LIST OF FIGURES AND TABLES --- p.IX-XI / LIST OF ABBREVIATIONS --- p.XII / Chapter CHAPTER ONE --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Rutaceae --- p.1 / Chapter 1.1.1 --- Introduction --- p.1 / Chapter 1.1.2 --- taxonomy of Rutaceae --- p.2 / Chapter 1.1.3 --- Controversial taxonomic issues --- p.4 / Chapter 1.1.3.1 --- Subfamilies Rutoideae and Toddalioideae --- p.4 / Chapter 1.1.3.2 --- "Euodia, Melicope and Tetradium" --- p.7 / Chapter 1.1.3.2.1 --- History --- p.7 / Chapter 1.1.3.2.2 --- Arguments based on morphology --- p.10 / Chapter 1.2 --- Molecular Approach --- p.12 / Chapter 1.2.1 --- Introduction to molecular systematics --- p.12 / Chapter 1.2.2 --- DNA sequence markers --- p.14 / Chapter 1.2.3 --- Applications --- p.18 / Chapter 1.3 --- Traditional Chinese Medicine (TCM) --- p.21 / Chapter 1.3.1 --- Introduction --- p.21 / Chapter 1.3.2 --- Fructus Evodiae --- p.22 / Chapter 1.3.3 --- Functional chemicals and pharmacological effects of Fructus Evodiae --- p.23 / Chapter 1.3.4 --- Problem in authentication --- p.25 / Chapter 1.4 --- Objectives --- p.27 / Chapter CHAPTER TWO --- METHODOLOGY AND MATERIALS --- p.29 / Chapter 2.1 --- Plant and Herb Materials --- p.29 / Chapter 2.2 --- DNA extraction --- p.44 / Chapter 2.2.1 --- Modified cetyltriethylammonium bromide (CTAB) extraction --- p.44 / Chapter 2.2.2 --- Kit extraction --- p.45 / Chapter 2.2.2.1 --- DNeasy® Plant MiniKit of Qiagen --- p.45 / Chapter 2.2.2.2 --- GenElute´ёØ Plant Genomic DNA Miniprep Kit of Sigma® --- p.46 / Chapter 2.3 --- Polymerase chain reaction (PCR) reaction --- p.47 / Chapter 2.4 --- DNA gel electrophoresis --- p.49 / Chapter 2.5 --- PCR product purification --- p.49 / Chapter 2.5.1 --- Rapid Gel Extraction System of Marligen Biosciences INC --- p.50 / Chapter 2.5.2 --- Gel-M´ёØ Gel Extraction System --- p.50 / Chapter 2.6 --- Ligation and transformation --- p.51 / Chapter 2.6.1 --- Ligation and transformation --- p.51 / Chapter 2.6.2 --- Cell culture --- p.52 / Chapter 2.6.3 --- Plasmid extraction --- p.52 / Chapter 2.7 --- Determination of DNA concentration --- p.54 / Chapter 2.8 --- Cycle Sequencing --- p.54 / Chapter 2.9 --- Sequence Analysis --- p.55 / Chapter 2.10 --- Materials --- p.56 / Chapter CHAPTER THREE --- MOLECULAR AUTHENTICATION OF FRUCTUSEVODIAE --- p.60 / Chapter 3.1 --- Results and data analysis --- p.60 / Chapter 3.1.1 --- Authentication based on ITS-1 region --- p.60 / Chapter 3.1.1.1 --- Phylogram study --- p.60 / Chapter 3.1.1.2 --- Sequence alignment --- p.65 / Chapter 3.1.1.3 --- ITS-1 region nucleotide differences significant in authentication of Fructus Evodiae --- p.71 / Chapter 3.1.1.4 --- Comparison of sequences --- p.74 / Chapter 3.1.2 --- Authentication based on ITS-2 region --- p.78 / Chapter 3.1.2.1 --- Phylogram study --- p.78 / Chapter 3.1.2.2 --- Sequence alignment --- p.82 / Chapter 3.1.2.3 --- ITS-2 region nucleotide differences significant inauthentication of Fructus Evodiae --- p.86 / Chapter 3.1.2.4 --- Comparison of sequences --- p.89 / Chapter 3.2 --- Discussion --- p.93 / Chapter 3.2.1 --- Molecular markers --- p.93 / Chapter CHAPTER FOUR --- PHYLOGENETIC STUDIES ON RUTACEAE --- p.96 / Chapter 4.1 --- Results and data analysis --- p.96 / Chapter 4.1.1 --- Chloroplast trnL intron region --- p.96 / Chapter 4.1.1.1 --- Sequence alignment --- p.96 / Chapter 4.1.1.2 --- Phylogenetic analysis --- p.107 / Chapter 4.1.2 --- Chloroplast trnL-F intergenic spacer region --- p.116 / Chapter 4.1.2.1 --- Sequence alignment --- p.116 / Chapter 4.1.2.2 --- Phylogenetic analysis --- p.126 / Chapter 4.1.3 --- Nuclear ITS-1 region --- p.132 / Chapter 4.1.3.1 --- Sequence alignment --- p.132 / Chapter 4.1.3.2 --- Phylogenetic analysis --- p.143 / Chapter 4.2 --- Discussion --- p.152 / Chapter 4.2.1 --- "Euodia, Melicope and Tetradium" --- p.152 / Chapter 4.2.2 --- Tetradium --- p.153 / Chapter 4.2.3 --- Tetradium and Phellodendron --- p.155 / Chapter 4.2.4 --- Zanthoxylum and Toddalia --- p.156 / Chapter 4.2.5 --- Rutoideae and Toddalioideae --- p.156 / Chapter 4.2.6 --- Tree constructing methods --- p.158 / Chapter CHAPTER FIVE --- CONCLUSION --- p.161 / REFERENCES --- p.163