[Truncated abstract] Traditional medicines are used by millions of people throughout the world as their primary source of medical care. A range of materials are in used traditional medicines including plant and animal parts. Even though the traditional medicine trade is estimated to be worth sixty billion dollars annually the trade remains largely unregulated. Unscrupulous practices by vendors to increase their profit margins such as substituting and adulterating expensive material with cheaper varieties go unchecked. This can be dangerous to consumers because some substitutions involve poisonous material. Also, animal parts from endangered species can find their way into traditional medicines, therefore there needs to be a way to identify them in traditional medicines to prosecute poachers. The traditional techniques used for the identification of material used in Traditional Chinese Medicine (TCM) include, morphological, histological, chemical and immunological analysis. However, these techniques have their limitations. This makes applying multiple techniques essential to provide thorough authentication of the material. DNA profiling provides a technique well suited to analysing material used in TCM. DNA profiling is advantageous over other techniques used to authenticate material used in TCM because it requires only a small sample amount, can determine the cultivator, be used on all forms of TCM and potentially distinguish the components of mixtures. ... Therefore, profiles of different species/individual are different and species? can be distinguished. Commercially sold traditional medicines are processed which is likely to degrade the DNA of the sample making extraction and amplification difficult. Here an organic Phenol:Chloroform extraction technique extracted DNA from commercial dried root samples. The extracted DNA was amplifiable using RAPD primers. The RAPD primers used here produced enough polymorphic bands to distinguish different plant species. They were used to distinguish commercial samples that were sold as three different species within the Panax genus, Panax ginseng, Panax quinquefolium and Panax notoginseng and genetically unrelated plant material; Potato and Eleutherococcus senticosus. Liquid samples and mixtures were also profiled with the RAPD primers to determine whether the RAPD primers provide enough distinguishing ability to analyse these forms of TCM. DNA was extracted from the liquid samples, one a ginseng drink and the other an ginseng extractum. However, there was no reliability in the production of PCR products. The analysis of the mixture samples found that not enough polymorphic bands were produced by the RAPD primers used here to identify Panax species within mixtures of two Panax species. While when P. ginseng was mixed with a genetically unrelated sample there was enough polymorphism to differentiate the two samples in the mixture. The results of this research show that RAPD analysis provides a simple and inexpensive technique to begin analysis of materials used in TCM. Using RAPD analysis it is possible to distinguish Panax plant species from each other. However, the RAPD primers used here did not provide enough reproducibility or polymorphism to analyse liquid and mixtures of Panax species plants.

The work reported here was done at the Wheeler Orchid Collection and Species Bank and the Department of Biology at Ball State University. We have developed a research teaching program with two applied research goals: genetically transforming and DNA fingerprinting orchid tissue. As part of their molecular biology education, students have investigated the genetic transformation of orchids for mitigating viral symptoms and the identification of unknown orchids by DNA fingerprinting. In a second application of the technology, DNA fingerprinting has been used to determine evolutionary relationships and to quantify genetic diversity among orchids.This dissertation details the background and need for this project and the research that was done to start it. As the early work has, developed and students have added their contributions, the data have developed into two papers formatted for submission to scientific journals. They are included as results.The first is a project designed to insert exognenous DNA into orchid tissue. The soil microbe Agrobacterium tumefaciens causes crown-gall tumors to develop in its plant hosts by inserting DNA into their cells which then controls the biosynthesis of development-controlling hormones. A. tumefaciens which has been disarmed has been routinely used to bioengineer dicotyledonous plants but its use has been rare on monocotyledons. In this paper, we report that A. tumefaciens transformed embryonic orchid tissue and caused alteration in its normal developmental course.The second paper details the DNA fingerprinting of tissue from Aplectrum hymale, a terrestrial orchid native to this climate. Three populations of A. hymale have been sampled and DNA extracted from the tissue samples. RAPD primers were used to prime PCR amplifications of random sequences of the DNA and the amplified DNA was visualized by gel electrophoresis. Loci of the resulting bands were treated as potentially multiallelic gene loci and heterozygosity between and within subpopulations was calculated. We report that the three populations could be partially differentiated by this procedure and that the two populations located nearest to each other yielded the least between -ubpopulation heterozygosity. We report very high levels of genetic diversity between individuals within small subpopulations in spite of the fact that these subpopulations are considered to be primarily clonal in reproductive nature. / Department of Biology

Thesis (MSc (Genetics))--University of Stellenbosch, 2007. / The aim of this study was to develop a DNA-based diagnostic system that can be used to distinguish between genotypes in the wheat breeding program at the University of Stellenbosch. Known marker systems were investigated and the chosen marker system would then be implemented to determine its utility in the breeding program. Three marker systems were considered, i.e. microsatellites, Amplified Fragment Length Polymorphisms (AFLPs) and various retrotransposon-based markers. Each system is based on polymerase chain reaction (PCR) amplification from specific primer pairs. The multitude of primer options was narrowed down during a review of published literature regarding wheat molecular markers. Thirty nine microsatellite primer pairs and nine AFLP primer combinations were chosen for the initial genotype evaluation. Four different retrotransposonbased techniques were investigated; namely Inter-Retrotransposon Amplified Polymorphism (IRAP), REtrotransposon-Microsatellite Amplified Polymorphism (REMAP), Sequence- Specific Amplified Polymorphism (SSAP) and, a derivative of these developed in this study, Wis-2 Retrotransposon Amplification. The study started with twenty genotypes which included varieties/breeding lines from five breeding programmes. The genotypes were chosen as representative of the respective breeding populations and were used in the initial testing of the marker systems. Eighteen microsatellites were evaluated using the panel of twenty genotypes. From this, six primer pairs (Xgwm190, Xgwm437, Xgwm539, Xwmc11, Xwmc59 and Xwmc177) were chosen to test the semi-automated DNA sequencer detection system. A single band/peak in each microsatellite profile was used for genotyping. Four of the primer pairs were labelled with different fluorochromes which enabled them to be multiplexed. The differences in amplification products of the six microsatellites meant that all six could be detected in one electrophoresis run. The banding pattern produced by microsatellite Xwmc177 was complex and highly polymorphic and was therefore also analysed in the same way as the AFLP patterns. When analyzed in this manner it proved to be more informative than the combination of six microsatellites (with a single prominent band scored in each). Three AFLP primer combinations could also be multiplexed and visualised together. The three EcoRI selective primers were labelled with different dyes and used with one MseI selective primer. The SSAP system also used fluorescently labelled primers and proved to be the most useful of the retrotransposon-based methods. However, this system produced such a large amount of data that it made analysis too time consuming. Therefore the six microsatellites and three AFLP primer combinations (MseI-CTC and EcoRI-ACA, -AAC, - AGG) were selected for routine genotyping. Due to the numerous highly polymorphic bands produced by the SSAP system it could be very useful to differentiate very closely related genotypes that cannot be distinguished with the markers proposed for routine use. A panel of 119 breeding lines were then used to implement the two chosen marker systems. The results obtained for these markers were used to produce a dendrogram of the lines using the SAS cluster analysis function. The clusters showed that most of the lines could be distinguished from each other. The MseI-CTC and EcoRI-AGG primer combination was the most informative. It produced the largest number of clusters (53) and could therefore discriminate between more of the lines than any other method. The dendrograms and clusters allowed sixteen of the breeding lines to be selected to test the optimal number of seeds to represent an entire population (variety/breeding line) as one seed was not sufficient. It was decided that eight seeds could provide a good representation of the intra-line variability.

M.Sc. / Invasive species are a considerable threat to ecosystems globally, especially on islands where species diversity can be relatively low. Understanding the drivers of invasion is the first step towards an adequate management plan. Although Darwin’s naturalisation hypothesis has fuelled our understanding in this regard, several studies provided mixed results, suggesting that invasion success might be context-dependent. The main objectives of this study are two-fold: (1) testing Darwin hypothesis on Robben Island, and (2) investigating the relative role of invasive alien plants on phylogenetic diversity (PD) loss in native community. I sampled extensively the flora of the island, and using a Bayesian analysis, I reconstructed its phylogeny based on two plastid DNA loci, rbcLa and matK. I also surveyed a total of 127 plots of 50 x 50 m (i.e. local communities) where species presence/absence was recorded. Analysing phylogenetic patterns of the native and invasive floras at both regional (phylogeny level) and smaller scales (plots level), I found that invasive species are, on average, more distantly related to the native communities, giving strong support to the hypothesis tested. Furthermore I found that native communities have accumulated lower PD than alien communities; and that local communities are more overdispersed than expected. These findings suggest that competitive interactions might be the major ecological forces shaping plant communities, with the possibility of alien being higher competitors than native, and therefore decreasing native plant diversity. The implications of these findings for the recovery of native plants are also discussed. Key words: Invasion biology - Darwin’s naturalisation hypothesis - Phylogenetic diversity - Community structure - Conservation - Robben Island, South Africa.

M.Sc. (Botany) / The market and public demand for medicinal plants over the past few decades has increased dramatically with more than 1 000 plant species actively traded for medicinal purposes throughout South Africa. Intensive harvesting of wild material is now acknowledged as a serious threat to biodiversity in this country. Also the substitution of a valuable commodity (medicinal plant) by a cheaper alternative (other plant species), either inadvertently due to misidentification, or deliberately to cheat consumers, raises some serious concerns as these adulterants may not be as effective or may even be toxic and cause harm to consumers. To add to the problem many species are either traded as dried leaf, root, bark products, or extracts and their identification becomes problematic. Therefore, DNA barcoding can help to provide a rapid and accurate identification tool for medicinal plants. In the current study I targeted the most commonly used medicinal plants in South Africa and produced a set of barcodes for fast and easy DNA-based species identification (rbcLa & matK). I tested the efficiency of core barcodes in the identification of medicinal plants using four main analyses, in the R package Spider 1.1-1. Here the extent of specific genetic divergence, DNA barcoding gap, BLAST test, and the ability to discriminate between species were assessed. Overall, the matK region was found to be a more useful tool for the species identification of medicinal plants in South Africa.

Please read the abstract in the section 00front of this document / Dissertation (MSc (Botany))--University of Pretoria, 2005. / Plant Science / unrestricted

Characterization studies on the genetic diversity among cultivated cowpea (Vigna unguiculata (L.) varieties are valuable tools to optimize the use of available genetic resources by farmers, local communities, researchers and breeders. Eight cowpea (Vigna unguiculata (L.) genotypes ( Vegetable cowpea, Ivory grey, Okhalweni, Fahari, Fahari dark, 97K-1069-8, IT93K-73h, and 129-3) were subjected to molecular, morphological and agronomical characterization. DNA amplification fingerprinting markers were used to evaluate the genetic diversity among the eight genotypes. Nine random arbitrary primers were used to screen the eight genotypes to assess their ability to reveal polymorphisms in cowpea, and seven of them were selected for use in characterizing the total sample. A total of 43 bands were generated which are all polymorphic. On the average, the primers generated a total of 6.1 polymorphic bands. The resulting data-matrix included 43 analysed bands with a total of 344 characters. Neighbour joining analysis was used to generate the dendrogram, clustering the genotypes into two groups at an agglomerate coefficient of 0.30 irrespective of their geographical origins. The results also showed the presence of significant differences in morphological and quality traits among the genotypes. Fahari yielded the highest concentration of crude protein (46.51 mg/mg dry leaf) while Vegetable cowpea yielded the lowest (24.41 mg/mg dry leaf). The influence of manure was also found to be effective by increasing the crude protein content of the genotypes as shown by Fahari dark with an average of 53.53 mg/mg dry leaf as opposed to 39.85 mg/mg dry leaf without manure application. Although some small clusters grouped accessions of the same growth habits, a general lack of agreement between clustering and morphological features was observed. It can therefore be concluded that the significant differences between the molecular genetic analysis using DAF-PCR markers, morphologic characters and yield traits can be important tools to identify and discriminates the different cowpea genotypes.

Maize (Zea mays L.) is an important agronomic crop with the maize industry forming an important component of the South African economy. Considerable effort has been directed towards the genetic improvement of maize through both conventional breeding and biotechnology. Genotype identification by DNA fingerprinting is becoming an important activity in plant breeding. A widely used molecular based and relatively inexpensive method for DNA fingerprinting is the randomly amplified polymorphic DNA (RAPD) technique. The RAPD technique was tested in this study for its potential use in maize breeding programmes. Initial results using the technique showed a low degree of reproducibility, therefore both the DNA isolation and RAPD protocols were extensively optimised. DNA quality and quantity, and choice of Taq polymerase buffer were three of the variables found to be influential in ensuring reproducibility. The ability of the RAPD technique to characterise seven maize genotypes was evaluated. Sixty random oligonucleotide primers were screened. Forty two primers scored a total of 233 fragments (an average of 5.5 per primer), but not all primers gave reproducible profiles. Eighteen primers scored a total of 110 loci for the presence (1) and absence (0) of DNA fragments. RAPD markers were able to distinguish between all seven genotypes with five primers producing specific fragments for four genotypes. Genetic similarity matrices were calculated using two software programmes i.e. Genstat 5™ release 4.1 (1993) and PAUP (Phylogenetic Analysis Using Parsimony) 4.0 beta version (Swafford, 1998). Cluster analysis was used to generate dendrograms to visualise the genetic relationships of the seven maize genotypes (only minor differences were observed between the Genstat or PAUP method of analysis). Genetic diversity ranged from 0.62 to 0.96. The estimation of genetic relationship was in accordance with the presumed pedigree of the genotypes showing that the RAPD technique demonstrates potential for genome analysis of maize. The applicability of the technique for marker assisted selection was also evaluated. Near-isogenic lines (NILs) for leaf blight (Helminthosporium spp.) were screened for polymorphisms using a total of 120 primers. Ten primers identified polymorphisms between the NILs. Four primers produced five polymorphic fragments present in the resistant inbred K0315Y and absent in the susceptible inbred D0940Y. A small F2 population of 14 individuals was produced by selfing the F1 of a cross between K0315Y and D0940Y. To speed up the generation time, the F1 and F2 plants were cultured by embryo rescue from 18d old harvested seed. One fragment of 627 base pairs produced by primer OPB-01 (5' GTTTCGCTCC 3') showed a 3: 1 segregation in the small F2 population and was considered putatively linked to the HtN gene for leaf blight resistance. This study shows that the RAPD technique does have application in maize breeding programmes. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Pigeonpea (Cajanus cajan (L.) Millsp.) is a drought tolerant legume of the Fabaceae family in the order Fabales and the only cultivated species in the genus Cajanus. It is mainly cultivated in the semi-arid tropics of Asia and Oceania, Africa and America. In Malawi, one of the top producers of pigeonpea in Africa, it is grown by small scale farmers as a source of food and income and for soil improvement in intercropping systems. However, varietal contamination due to natural outcrossing causes significant yield losses for farmers. In this study, 48 polymorphic SSR markers were used to assess diversity in all pigeonpea varieties cultivated in Malawi with the aim of developing a genetic fingerprint to distinguish the released varieties. SSR alleles were separated by capillary electrophoresis on an ABI 3700 automated sequencer and allele sizes determined using GeneMapper 4.0 software. Allelic data was analysed with PowerMarker. A total of 212 alleles were revealed averaging 5.58 alleles per marker with a maximum number of 14 alleles produced by CCttc019 (Marker 40). Polymorphic information content (PIC) ranged from 0.03 to 0.89 with an average of 0.30. DARwin software was used to generate a neighbour-joining tree that displayed three major clusters with two sub clusters in Cluster I. The released varieties were scattered across all the clusters observed, indicating that they generally represent the genetic diversity available in Malawi, although it was observed that there is substantial variation that can still be exploited through further breeding. Screening of the allelic data associated with five popular pigeonpea varieties for which a DNA fingerprint was to be developed, revealed 6 markers – CCB1 (Marker 1), CCB7 (Marker 2), Ccac035 (Marker 7), CCttc003 (Marker 15), Ccac026 (Marker 37) and CCttc019 (Marker 40)– which gave unique allelic profiles for each of the five varieties. With further tests needed for its robustness, this genetic fingerprint can be used for seed certification to ensure only genetically pure seeds are delivered to Malawi farmers. / Agriculture and  Animal Health / M. Sc. (Agriculture)

Pigeonpea (Cajanus cajan (L.) Millsp.) is a drought tolerant legume of the Fabaceae family in the order Fabales and the only cultivated species in the genus Cajanus. It is mainly cultivated in the semi-arid tropics of Asia and Oceania, Africa and America. In Malawi, one of the top producers of pigeonpea in Africa, it is grown by small scale farmers as a source of food and income and for soil improvement in intercropping systems. However, varietal contamination due to natural outcrossing causes significant yield losses for farmers. In this study, 48 polymorphic SSR markers were used to assess diversity in all pigeonpea varieties cultivated in Malawi with the aim of developing a genetic fingerprint to distinguish the released varieties. SSR alleles were separated by capillary electrophoresis on an ABI 3700 automated sequencer and allele sizes determined using GeneMapper 4.0 software. Allelic data was analysed with PowerMarker. A total of 212 alleles were revealed averaging 5.58 alleles per marker with a maximum number of 14 alleles produced by CCttc019 (Marker 40). Polymorphic information content (PIC) ranged from 0.03 to 0.89 with an average of 0.30. DARwin software was used to generate a neighbour-joining tree that displayed three major clusters with two sub clusters in Cluster I. The released varieties were scattered across all the clusters observed, indicating that they generally represent the genetic diversity available in Malawi, although it was observed that there is substantial variation that can still be exploited through further breeding. Screening of the allelic data associated with five popular pigeonpea varieties for which a DNA fingerprint was to be developed, revealed 6 markers – CCB1 (Marker 1), CCB7 (Marker 2), Ccac035 (Marker 7), CCttc003 (Marker 15), Ccac026 (Marker 37) and CCttc019 (Marker 40)– which gave unique allelic profiles for each of the five varieties. With further tests needed for its robustness, this genetic fingerprint can be used for seed certification to ensure only genetically pure seeds are delivered to Malawi farmers. / Agriculture and  Animal Health / M. Sc. (Agriculture)