Orientador: Anete Pereira de Souza. / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T05:07:09Z (GMT). No. of bitstreams: 1 Durigan_Mauricio_D.pdf: 7600085 bytes, checksum: 74ae2337a73b6edfb14a403af4ffa590 (MD5) Previous issue date: 2015 / Resumo: Giardia duodenalis é um protozoário flagelado que parasita o homem e diversos animais domésticos e selvagens. Este parasito causa a doença giardiose que é uma das mais prevalentes doenças parasitárias de veiculação hídrica do mundo, responsável por aproximadamente 280 milhões de casos anualmente. Existe uma considerável variabilidade genética em G. duodenalis, de modo que seus isolados foram divididos em oito grupos genéticos (A-H), dois dos quais (A e B) são encontrados tanto em humanos quanto em animais. Os demais grupos (C-H) parasitam outros animais e apresentam maior especificidade a determinados hospedeiros não humanos. A contaminação ambiental por Giardia tem sido amplamente descrita embora esses estudos, em sua maioria, são realizados no nível de identificação de espécie. Há falta de estudos que correlacionam a contaminação ambiental e infecções clínicas na mesma região. O presente trabalho teve como objetivo principal contribuir para o conhecimento da diversidade genética da espécie Giardia duodenalis. Primeiramente, foi realizada a genotipagem multilocos dos principais grupos genéticos de G. duodenalis na região metropolitana de Campinas. Foram encontrados grupos genéticos associados principalmente a infecções humanas bem como isolados com potencial zoonótico em amostras ambientais e obtidas de outros animais. Foi encontrado um alto percentual (25%) de amostras com grupos genéticos mistos e um elevado número de haplótipos distintos, indicando grande diversidade genética do parasito nessa região. Na segunda parte deste trabalho, foi realizado um estudo populacional com amostras clínicas de Giardia provenientes de hospital, creche e centro de controle de zoonoses e amostras ambientais de esgoto hospitalar, efluente de estação de tratamento de esgoto e amostras hídricas de importantes rios e córregos urbanos. As análises populacionais, com exceção das amostras caninas, evidenciaram grande similaridade genética entre essas populações de Giardia. Na terceira parte do presente trabalho, foi realizada uma busca por repetições microssatélites (SSRs) nos genomas publicados de Giardia para desenvolvimento, caracterização e avaliação de polimorfismo de novos marcadores microssatélites. Foram encontrados 506, 438, 402 e 507 microssatélites correspondentes aos genomas AI, AII, B e E, respectivamente. Foram selecionados 80 SSRs específicos aos grupos genéticos A, B e E (40, 20 e 20, respectivamente), além de 36 SSRs compartilhados entre os três genomas. A análise de amplificação confirmou a existência de marcadores específicos aos grupos genéticos A, B e E, além de marcadores compartilhados entre os grupos. A caracterização dos SSRs permitiu a detecção de 12 locos SSRs polimórficos do grupo genético A e sete locos SSRs polimórficos do grupo genético B. Dentre os marcadores compartilhados, o loco GduABE01 apresentou polimorfismo. Os locos polimórficos podem servir para futuros estudos populacionais e os marcadores desenvolvidos podem ser utilizados para identificação dos principais grupos genéticos de G. duodenalis em amostras clínicas e ambientais. Os resultados apresentados contribuem para um melhor entendimento sobre a diversidade genética do parasito bem como sobre a presença de grupos com potencial zoonóticos inter-relacionados em diferentes regiões. Os novos marcadores moleculares disponibilizados podem contribuir para novos estudos populacionais, promovendo melhor discriminação entre os genótipos e possibilitando assim identificar a contaminação e promover o rastreamento da doença / Abstract: Giardia duodenalis is a flagellate protozoan that that parasites humans and several domestic and wild animals. This parasite causes giardiasis, one of the most common waterborne diseases in the world responsible for, approximately 280 million cases per year. There is a great genetic diversity in this species and its isolates have been grouped into eight distinct genetic assemblages (A-H). While groups A and B parasitize different hosts and have zoonotic potential, groups C, D, E, F, G and H usually found in animals and show greater specificity to the parasitized host. Environmental contamination for Giardia has been widely reported however, most of these studies have been performed only at species level. The present study aimed to contribute to the knowledge of the genetic diversity of the species Giardia duodenalis. In the first chapter of this document, multilocus sequence-based genotyping using three gene loci assigned most of the samples as belonging to human genotypes although isolates with zoonotic potential have also been identified in environmental and non-human clinical samples. A high percentage (25%) of mixed assemblages and a high number of different haplotypes were detected, which indicates high genetic diversity of this parasite in this region. In the second chapter, a population genetics study was performed with clinical samples from hospital, day-car center and a center for zoonosis control of the city and environmental samples from hospital sewage, effluent of a wastewater treatment plant and important water samples from rivers and urban streams. With the exception of the canine population, population genetic analysis showed consistent similarity between clinical and environmental populations. In the last chapter, we performed a search for microsatellites (SSRs) in the published genomes of Giardia to develop and characterize the polymorphism of new microsatellite markers. Our group identified 506, 438, 402 and 507 microsatellites of the genomes AI, AII, B and E, respectively. We have selected 80 markers specific to the genetic assemblages A, B and E (40, 20 and 20, respectively) and 36 shared SSRs between the three genomes. Analysis of amplification reactions confirmed the existence of specific loci of each genetic assemblage as well as shared loci among assemblages. Characterization of all loci allowed the detection of 12 polymorphic loci for group A and seven polymorphic loci for group B. Among the shared markers, GduABE01 presented polymorphism. The polymorphic markers can be used in future population genetic studies and the developed markers can contribute to the identification of the main genetic assemblages of G. duodenalis in clinical and environmental samples. The results presented here contribute to a better understanding of the genetic diversity of the parasite as well as the presence of zoonotic potential genotypes, related in different regions. The new molecular markers provided can contribute with population genetic studies in a high level of discrimination that allows identifying the source of contamination and molecular tracking of the disease / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

The aim of this project was to study the population genetics of Echinostoma trivolvis, a parasitic trematode that uses multiple hosts in its lifecycle and has a significant impact on amphibian populations. Microsatellite markers were to be identified and isolated because of their highly variable nature and reported ease of use with PCR. Parasite DNA was extracted from planorbid snails from several locations within California including: Point Reyes National Seashore, Lake Tahoe, and the San Francisco Bay Area. In addition, parasite samples were obtained from Manitoba, Canada. Several microsatellites were identified and 29 PCR primers sets were designed, six of which were capable of amplifying consistently. Sequencing other published molecular markers, COl, NDl, and ITS, unveiled intriguing phylogenetic relationships and potential cryptic species. The echinostome population in central California, as a result of this project, may be much more diverse than has long been reported in the literature.

Advances in aquaculture technologies are being investigated to support the replenishment of local fisheries, develop marine food fish farming opportunities and to increase seafood production globally. In order to promote the expansion and development of aquaculture technologies required to raise new finfish species, a number of key bottlenecks restricting commercial-scale culture need to be addressed, including the ability to control fish reproduction in captivity and to produce high quality seeds. One candidate species for large-scale production, and the focus of this work, is common snook. Prized as a food fish in Mexico, Central and South America and as a popular game fish along the Gulf coast of the United States; common snook are economically important having both a high market value and recreational demand. Despite recent advances in captive spawning, a number of reproductive bottlenecks still need to be addressed such as lack of spontaneous spawning in captivity, poor fertilization rates and inconsistent production of high quality eggs and larvae. Therefore, the overall aim of this thesis was to better understand the reproductive biology of common snook in order to develop protocols to improve the reliability of captive spawning in closed recirculating aquaculture systems and the quality of eggs produced as a basis for commercial scale cultivation. First, this PhD project described oocyte development in common snook and validated a non-invasive method for assessing reproductive condition in wild and captive stocks (Chapter 2). This was done by using a tiered and adaptable staging scheme to compare the wet mount technique with histological preparations of ovarian biopsies. When compared with histology, the wet mount provided an immediate and precise method for determining whether female broodstock were candidates for hormonal induction. In fishery biology, an understanding of fish reproductive success and population reproductive potential is critical for designing and implementing effective fisheries management strategies. The wet mount technique provides a tool for non-lethal, low-cost determination of reproductive status in wild fish stocks. The next research chapter focused on spawning induction of captive snook populations. The first trial compared the effects of slow and regular release GnRHa implants whereas the second trial investigated the effects of GnRHa, alone or in combination with the dopamine antagonist, pimozide (PIM), on milt characteristics and plasma steroid levels in captive male common snook broodstock (Chapter 3). In an effort to better enable reliable control of reproduction under captive conditions, the annual plasma sex steroid profile of captive male and female broodstock maintained under natural photo-thermal conditions was also examined. When possible, milt samples were collected pre and post implantation; sperm density, sperm motility and spermatocrit were documented among individual males. The assigned treatments appeared to have no or little effects on milt production in male broodstock although plasma steroid levels were found to be significantly elevated in individuals treated with GnRHa in combination with the dopamine antagonist, pimozide. At the time this work was performed, no data on spawning dynamics, including individual spawning performance, had been reported for common snook in captivity. Mass spawning tanks are complex systems where fish are left to spawn naturally and fertilized eggs are collected with little or no control over the mating of the animals. Therefore, the third part of this thesis explored the potential of DNA profiling for monitoring mating outcomes in captive broodstock by employing eight microsatellite markers to detect and quantify individual parental contributions for 2,154 larvae obtained from the three broodstock tanks (Chapter 4). The panel of loci was generally robust and allowed unambiguous assignment of 89% of larvae to a single family. Overall, spawn contribution data 1) provided a confirmation of GnRHa treatment efficacy in female snook with a minimum stage of oogenesis (late secondary growth-SGl) required for successful spawning, 2) identified a potential impact of handling on maturation and spawning of captive broodstock and 3) confirmed that, through photothermal conditioning, captive broodstock can spawn over consecutive days and several times per year including outside of their natural spawning season. The exogenous cues that tropical species use to synchronize key life events like reproduction remain largely unstudied, therefore, my PhD project also investigated the influence of tidal cycle on reproductive activity in common snook (Chapter 5). Real-time quantitative RT-PCR assays were developed and validated to measure the temporal expression patterns of gonadotropin genes (fshβ and lhβ) during the reproductive cycle in males and females. These were evaluated in relation to sex steroid production, LH blood plasma levels, gonadal development and tidal cycle. The phylogenetic analysis of the deduced amino acid sequence of common snook for fshβ and lhβ revealed strong identity with other teleosts (75-90%). Additionally, the mRNA profiles of fshβ and lhβ in the pituitary of females displayed a clear pattern of expression concomitant with histological changes in oocyte development. Histological observations of gonads suggested a circa-tidal rhythm of follicular development. The findings, as a whole, provided new information supporting the role of tidal cycle on the entrainment of gametogenesis allowing for a better understanding of the environmental control of reproduction in common snook. Although the primary research emphasis in this PhD was on broodstock spawning and gamete quality, the final chapter focuses on larval ontogeny. The goal of this research was to gain improve understanding of the early life history characteristics of common snook in order to improve larval culture technologies. To do so, a combination of digital photography and histological techniques were used to document the embryonic and early larval development (0 to 14 days post hatch-DPH) of hatchery-reared individuals (Chapter 6). Larvae hatched 15 h after fertilization at 28°C, lacked pigmentation, had a rudimentary digestive tract and undeveloped visual system. Development was rapid and by 3 DPH larvae had almost doubled in length, the yolk sac was nearly exhausted, the mouth was open and eyes were pigmented with a well-structured retinal layer. The alimentary canal was differentiated into three distinct sections including the foregut, midgut and hindgut. Food was observed in the gut (rotifers) and structural epithelium organelles, such as the nucleus, mitochondria, and dark vesicles, were all present in high numbers. The swim bladder was formed and inflated. In summary, understanding early ontogenetic development in common snook can help provide information needed to address key bottlenecks seen in captive cultivation, such as the high incidence of larval mortality observed during the transition from endogenous to exogenous feeding. Overall, this doctoral work 1) validated molecular and endocrine analytical tools for future studies of common snook reproductive physiology, 2) provided a better understanding of both broodfish requirements in tank systems as well as the endocrine control of reproduction and spawning at the level of the brain-pituitary-gonadal axis, 3) increased our knowledge in genetic management of captive broodstock, in terms of parentage assignment and 4) offered new insight into wild population reproductive strategy as well as how reproduction is entrained through environmental cues and the pathways leading to oocyte recruitment and maturation. The new information presented here can be used to conserve wild snook stocks through production of farm raised individuals as a sustainable source of seafood and for fisheries enhancement.

Increasing human pressure combined with sea level rise and increased storminess is threatening coastal ecosystems around the world. Among these ecosystems, saltmarshes are particularly endangered due to their position in temperate areas with low wave action where human density is often high (e.g. estuaries). Around the UK, centuries of land reclamation have led to a substantial decrease of the area of saltmarsh. Over the past decades, restoration schemes have been implemented in numerous coastal locations in an attempt to counteract this loss. Such schemes involve allowing sea water to inundate a previously embanked area and letting the vegetation develop naturally, thereby reverting to saltmarsh through natural colonisation. However, surveys of restored areas that have looked at the recovery of plant species diversity or functional characteristics often show that restored saltmarshes do not reach the state of a natural saltmarsh ecosystem. While there is much data at the species level, recovery of plant intra-specific diversity (genetic diversity) has not been assessed in restored saltmarsh although this component of biodiversity is receiving increasing attention for its effect on ecosystem function. This thesis represents the first attempt to (1) characterize the nation-wide genetic structure of two important north-west European saltmarsh plant species, the common saltmarsh grass (Puccinellia maritima) and the sea arrowgrass (Triglochin maritima) and (2) compare levels of genetic diversity and structure between restored and natural ecosystems. Microsatellite molecular markers were developed for both species. Using innovative methods to analyse the genetic data obtained for these two polyploid species, this thesis highlights that genetic diversity at the national scale is organised regionally for both species, although gene-flow is still restricted between populations within the same region. Gene-flow between populations is determined by different processes depending on the species. While coastal processes mainly influence gene dispersal in P. maritima, overland routes of dispersal are involved for T. maritima. These differences are believed to be due to differences in dispersal ecology between the two species. Although gene-flow exists between distant saltmarshes, the genetic analysis of P. maritima and T. maritima colonists arriving on restored sites highlighted their local origin and reaffirmed that it is preferable to restore saltmarsh where a nearby natural saltmarsh can act as a source of colonists. A multiple paired-site comparison identified similar genetic diversity between restored and natural saltmarshes indicating that restoration of local genetic diversity is rapid for both species. A single site comparison at Skinflats in the Forth estuary compared fine-scale spatial genetic structure between the restored and natural saltmarsh. Interestingly, no structure was detected for T. maritima either in restored or natural saltmarsh. In contrast, a strong genetic structure organised along the elevation gradient was observed in the natural saltmarsh for P. maritima but was absent in the restored saltmarsh. The origin of this structure is not clear but could be due to restricted gene-flow between individuals from different elevations due to strong post-zygotic selection, as suggested in previous work. In any case, this lack of structure in the restored saltmarsh indicates that genetic recovery is incomplete in this respect for P. maritima. This thesis introduces the growing field of restoration genetics to saltmarsh ecology and identifies the principal population genetic trends in two of the species dominating the vegetation of north-west European saltmarshes community. The information given here will be useful for restoration practitioners and provides a strong foundation for future work characterizing the importance of genetic diversity for saltmarsh function.

The three South African crane species, namely, the Wattled Crane (Bugeranus carunculatus), the Blue Crane (Anthropoides paradisea) and the Grey Crowned Crane (Balearica regulorum regulorum) are all threatened. South African legislation protects the cranes, however eggs and/or fledglings are sometimes illegally collected from the wild. These are then sold, often by registered breeders, who falsely claim them as the offspring of their captive breeding pair. DNA fingerprinting is one method to detect this crime. Fifteen RAPD primers were screened for polymorphism in the three species. Seven primers produced polymorphic profiles in the Blue Crane and eight each in the Grey Crowned Crane and Wattled Crane, with an average of 14.57, 12.38 and 5.88 scorable loci per primer, respectively. The Band Sharing Coefficient for unrelated individuals was found to be 0.665, 0.745 and 0.736 for the Blue, Grey Crowned and Wattled Crane respectively. Five microsatellite primers, originally developed for use in Whooping Cranes (Grus american), had previously been shown to be polymorphic in the Wattled Crane. This was also the case in this study with an average of 3.6 alleles per primer. Although all primers cross amplified, only a single primer each showed polymorphism in the Blue Crane (showing 6 alleles) and the Grey Crowned Crane (showing 5 alleles). The RAPDs were found to be irreproducible, show high numbers of novel bands and had parent: offspring BSC values that were not significantly higher than those of unrelated individuals. Statistics showed that, in the Blue Crane, the probability that misassigned parents would be detected was low whilst there was an almost certainty that true parents would be incorrectly excluded. The five microsatellite primers examined gave exclusionary powers of 0.869 and 0.641 where one or two parents were unknown in the Wattled Crane. The exclusionary powers for the Blue Crane and Grey Crowned Crane calculated at only one locus were much lower. It was concluded that RAPDs were totally inappropriate for parentage analyses, however, microsatellites are a suitable technique and recommendations are made that other microsatellites, developed for other species of crane, should be examined for their potential in this respect. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

The blue crane (Grus paradisea) is endemic to southern Africa and has the smallest geographical range of the 15 crane species occurring world-wide. Although this species is still found throughout most of its historic range, it has experienced a significant and rapid decline in numbers over the last 20 years. One factor causing this decline is the illegal removal of chicks from the wild. Permits are required to keep, trade in and breed cranes in captivity. However, birds must be captive bred in order to obtain a permit. Therefore, chicks taken illegally from the wild are fraudulently incorporated into an existing captive population under the pretence that they offspring of a legal captive pair. This study describes the development of a set of microsatellite markers to assist the identification of illegal trade in the blue crane. These markers can ultimately be used to verify the relationship between the offspring and its claimed parents by performing parentage analyses. Forty microsatellite loci were obtained from genomic libraries previously developed in two other crane species and tested for cross-species utility in the blue crane. In addition, 42 loci were developed for this study from a blue crane species-specific genomic microsatellite library, of which 19 were tested for polymorphism in this species. The microsatellite markers characterised here were also tested for their utility in two other crane species: wattled crane (G. carunculatus) and grey-crowned crane (Balearica regulorum). One locus, Gamu007, was found to be sex-linked and therefore excluded from the set of markers. A total of 28 polymorphic loci were tested for the suitability in parentage analysis in the blue crane. Of these, a set of 16 loci were determined to be as suitable for this purpose. These loci were shown to be inherited in a Mendelian fashion in a single blue crane family. In addition, statistical analysis of the loci were identified as exhibiting linkage equilibrium, this was supported by their distant association on a predicted Grus microsatellite map based on the chicken genome. The selected loci were also identified as having a low frequency of null alleles as well as a total first and second parent exclusion power of 0.9999 and 1.0000, respectively. These loci provide a valuable tool for parentage testing in blue crane, and may also be valuable in population genetic studies to assist conservation strategies. In addition, this set may be used to assist legal cases involving the illegal trade in blue cranes upon completion of additional microsatellite marker validation procedures. Twenty-seven loci were polymorphic in the wattled and grey-crowned crane. These could provide a valuable source of micro satellite loci in these species, and could potentially eliminate the need for the development of a species-specific microsatellite library. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

The use of microsatellite markers has matured and become commonplace for plant genome analyses and is now poised for widespread practical application in sugarcane. Sequence Tagged Microsatellite Site (STMS) amplification is the most prevalent microsatellite-based approach and involves the amplification of a microsatellite by designing primers that flank and hence define the microsatellite site, revealing variation in the length of repeat motifs between individuals. Twenty-six microsatellite primer pairs received from the International Sugarcane Microsatellite Consortium (ISMC) were evaluated and the STMS protocol was optimised to ensure robust and reproducible results. The objectives of this study were to use STMS for sugarcane parentage analysis and fingerprinting. Previously, Restriction Fragment Length Polymorphism (RFLP) marker data had suggested that the parentage of a genetic mapping population, sugarcane cross AA40 (N18 x CP57/614), was incorrect. Based on the assertion that the incorrect parentage was as a result of either mislabelling at planting or at seed collection, microsatellite parentage analysis was carried out on eight potential parent pairs (13 cultivars). A total of 75 markers were scored with non-parental bands (12 on average) being observed for all of the potential parent pairs and none could be identified as the true AA40 parents. It has been suggested in other plant species that PCR artefacts could give rise to non-parental bands and to investigate this the marker data of single parent DNA reactions and pooled parent pair DNA reactions or 'synthetic offspring' were compared. The results suggested that either a certain percentage of non-parental bands, perhaps 10% (maximum value observed), should be tolerated in microsatellite parentage analysis or a marker should only be considered to be discriminating for parentage if it is absent in both the parents and the pooled parent pair amplifications. Fingerprinting of 20 cultivars using 14 microsatellite primer pairs was conducted to evaluate the potential of the STMS approach for sugarcane varietal identification. It was found that only two microsatellite primer pairs were required to discriminate between all 20 cultivars with a theoretical number of non-differentiated pairs of cultivars (XK) of only 0.03. This estimator was used to determine the approximate number of microsatellites necessary for large-scale sugarcane fingerprinting. / Thesis (M.Sc.)-University of Natal, Durban, 2002.

[Truncated abstract] In the last two decades, the use of microsatellites has revolutionized the study of ecology and evolution. Microsatellites, or short tandem repeats (STRs), are stretches of DNA repeats, 1 to 5 nucleotides long, where the number of repeats varies between individuals. They are co-dominant, highly variable, neutral markers, and are inherited in a Mendelian fashion. Microsatellite loci were isolated from Dawson’s burrowing bee, Amegilla dawsoni, a large, fast-flying solitary nesting bee endemic to the arid zone of Western Australia. Twelve polymorphic loci were found with an observed number of alleles ranging from two to 24 and observed heterozygosities between 0.17 and 0.85. These loci were used to examine two aspects of this bee’s molecular ecology; its population structure and mating system ... The molecular data were also used to show that the nesting female is the mother of all her offspring and that brood parasitism is unlikely in this species. The data indicate that females make daughters at the beginning of the season followed by large sons in the middle, and then small sons at the end. Females often place one brood cell directly above another. The distribution of sex and morph in these doublets follows a pattern with most containing a female on the bottom and a minor male on the top, followed by almost equal numbers of female on top of female and minor male on top of major male. This pattern is likely favoured by emergence patterns, with males emerging before females and minor males emerging before major males. I suggest that although minor males have low reproductive success, their production may nonetheless be beneficial in that minor males open up emergence tunnels for their larger and reproductively more valuable siblings. In addition, minor males may represent the ‘best of a bad job’ provisioning tactic arising from changes in the costs to nesting females of gathering brood provisions over the course of the flight season. This thesis demonstrates that microsatellites can be used to answer many questions regarding the molecular ecology of a species from the behaviour of the bees on a population scale to the mating behaviour of individual bees and how they allocate resources for the next generation. Many other aspects of the bee’s ecology could also be examined now that suitable molecular markers exist.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The indigenous abalone Haliotis midae is one of the most remarkable and highly exploited species of marine molluscs in South Africa. It is the only species of southern African Haliotidae to be commercially reared and has been successfully cultured for almost two decades. Its short history of domestication along with market demands and the need to develop efficiency in the production process has resulted in an increased interest in the possible genetic improvement of this species. The unhurried growth rate associated with H. midae is a cause of particular concern to the industry, predominantly with regards to profitability and competitiveness in the market place. A modest amount of work has so far been directed at establishing a means of enhancement for selective breeding on the commercial level. Genetics plays a key role in the establishment of successful improvement programmes in various aquaculture species. The aim of this study was to develop species-specific microsatellite markers for the abalone and subsequently perform parentage assignment on farm produced animals entered into a growth performance recording scheme. Animals were obtained from the hatcheries of three commercial abalone farms situated in the Walker Bay region in the Western Cape. Microsatellites were isolated using the enrichment-based FIASCO method, and characterised into perfect, imperfect and compound repeats according to the structural nature of their repetitive units. From the partial gDNA libraries obtained and 365 screened colonies, a total of 54 loci were located. PCR primers were designed for 36 markers and the 15 primer pairs that displayed loci with the highest level of polymorphism were subsequently chosen for fluorescent labelling. The markers were tested on a subset of 32 wild H. midae individuals to determine their usefulness and efficiency in genotyping. Five markers, along with five others that were previously designed, were chosen for assigning parentage to the animals submitted to the performance recording scheme. Three thousand offspring from each of the three participating farms were equally divided and reared at five different locations. From each location 20 fast growing and 20 slow growing juveniles, as well as the broodstocks, were sampled and genotyped using the ten chosen microsatellite loci. Two farms had 60% of offspring unambiguously assigned to a single parental couple. Assignments showed patterns of dominant male and female brooders, but no trend in brooders specifically contributing to fast or slow growing offspring. Parentage assignment for the third farm was, however, unsuccessful due to lack of broodstock data. In future, screening of all available broodstock will ensure acquisition of relevant pedigree information. The results obtained in this study are an initial step in the development of a genetic improvement programme for commercial Haliotis midae. / AFRIKAANSE OPSOMMING: Die inheemse skulpvis Haliotis midae is een van die mees merkwaardige en hoogs oorbenutte mariene slakspesies in Suid-Afrika. Dit is die enigste suidelike Afrika Haliotidae spesie wat kommersieel benut word en dit word al meer as twee dekades suksesvol geteel. Die spesie se kort domestiseringsgeskiedenis, toenemende mark aanvraag en die behoefte om meer effektiewe produksie daar te stel, het gelei tot toenemende belangstelling in die moontlike genetiese verbetering van die spesie. Die stadige groeitempo geassosieer met H. midae is veral ‘n punt van kommer vir die industrie, veral in terme van winsgewendheid en kompetering in die markplek. Minimale werk is sover gedoen in die daarstelling van verbetering deur selektiewe teling op ‘n kommersiële skaal. Genetika speel ’n sleutelrol in die daarstelling van suksesvolle verbeteringsprogramme van verskeie akwakultuur spesies. Die doel van hierdie studie was om spesie-spesifieke mikrosatelliet merkers vir perlemoen te ontwikkel en vervolgens ouerskapsbepaling van kommersiële diere, wat deelneem aan ‘n groeiprestasie aantekenstelsel, uit te voer. Diere is voorsien deur die teelstasies van drie kommersiële perlemoenplase geleë in die Walker Bay omgewing in die Wes-Kaap. Mikrosatelliete is geïsoleer deur die verrykings-gebaseerde FIASCO metode, en gekarakteriseer as perfekte, onderbroke of saamgestelde herhalings gebaseer op die strukturele aard van die herhalings eenhede. Vanaf die gedeeltelik gDNA biblioteke wat bekom is en 365 gesifte kolonies, is ‘n totaal van 54 loki opgespoor. PKR inleiers is ontwerp vir 36 merkers en die 15 inleierpare, wat loki met die hoogste polimorfisme geamplifiseer het, is vervolgens geselekteer vir fluoreserende merking. Die merkers is getoets op ’n kleiner groep van 32 natuurlike H. midae individue om hulle bruikbaarheid en genotiperingseffektiwiteit te bepaal. Vyf merkers is saam met vyf reeds ontwikkelde merkers gekies vir ouerskapsbepaling van die diere in die prestasie aantekenstelsel. Drieduisend nageslag diere vanaf elkeen van die drie deelnemde plase is gelykop verdeel en grootgemaak op die vyf verskillende lokaliteite. ‘n Monster van 20 vinnig groeiende en 20 stadig groeiende jong perlemoen, sowel as broeidiere, is vanaf elke lokaliteit geneem en gegenotipeer deur middel van die 10 geselekteerde mikrosatelliet loki. Sestig persent van twee van die plase se nageslag is onteenseglik toegesê aan ‘n enkele ouerpaar. Ouerskapstoekenning het patrone van dominante vroulike en manlike broeidiere getoon, maar geen tendens in terme van bydrae tot vinnig en stadig groeiende nageslag kon gevind word nie. Ouerskapstoekenning vir die derde plaas was onsuksesvol as gevolg van ’n gebrek aan data vir die broeidiere. In die toekoms sal genotipering van alle beskikbare broeidiere die daarstelling van relevante stamboominligting verseker. Die resultate verkry in hierdie studie verteenwoordig ‘n eerste stap in die ontwikkeling van ’n genetiese verbeteringsprogram vir kommersiële Haliotis midae.

Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Forming part of a conservation programme, this study was concerned with two species of Cichlid fish (Oreochromis mossambicus and O. ni/oticus), which were brought into contact with each other by unnatural ways. They are now hybridizing to some extent and there is also evidence that the foreign O. ni/oticus may out compete the native O. mossambicus. To cast light on what the current distribution is of both these species and the hybrids in Southern Africa, it is important to identify specimens very accurately. In attempting to find genetic markers to distinguish between two species of Cichlids we tested 20 microsatellite dinucleotide (CAn) repeats during a preliminary study and found five of these promising to exhibit little intra-specific genetic diversity but large genetic variation between species. We amplified these five loci in 145 individuals from 10 populations, which included the two species and their hybrids. Exact sizes of the fragments were determined using an automated DNA sequencer. Between the two species, allele sizes were overlapping, but when data were analyzed by statistical models, the differences could be seen for populations, however on individual level there was overlap between the species. The hybrids were found to be intermediate positioned between the two pure species. Our attempt to assign individuals to populations provided doubtful results. Thus, using this set of markers, populations can be ascribed to one of these species, but not individuals by themselves. / AFRIKAANSE OPSOMMING: As deel van 'n natuurbewarings program, word daar in hierdie studie twee spesies van vis ondersoek was in kontak met mekaar gekom het op onnatuurlike wyse. Hierdie twee visspesies vanuit die CICHLIDAEfamilie (Oreochromis mossambicus en 0. ni/oticus) kan hibridiseer wanneer hul saam voorkom, maar dit is ook bekend dat die uitheemse O. ni/oticus die inheemse O. mossambicus kan bedreig in terme van leefruimte, kos en broeispasie. Om die voorkoms van hibriede tussen die twee spesies te ondersoek in Suider Afrika se varswater opvangsgebiede, is dit baie belangrik om individue baie akkuraat te identifiseer. In hierdie poging om genetiese merkers te vind wat die twee spesies van mekaar onderskei, het ons 20 mikrosateliet di-nulkleotied (CAn) herhalende volgordes op verskillende loci ondersoek. Vyf daarvan het belowend voorgekom om as spesie spesifieke merkers te dien. Die fragmente op die vyf loci is ge-amplifiseer in 145 individue vanuit 10 populasies. Presiese groottes van die fragmente is bepaal met behulp van 'n ge-outomatiseerde DNA volgorde bepaler waarna genotiepes vir elke individu toegeken is. Tussen die twee spesies het alleel groottes oorvleuel, maar wanneer data geanaliseer word met behulp van statistiese metodes, was verskille tussen die spesies duidelik op populasie vlak. Die hibriede het intemediêr tussen die twee spesies voorgekom. Dus met behulp van hierdie stel merkers kan onderskei word tussen die twee spesies op populasie vlak, hoewel individue nie op sig self identifiseer kan word nie.