Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Linkage disequilibrium (LD) is defined as the non-random association of alleles at two or more loci within a population. It is sensitive to a variety of locus-specific- and demographic factors, and can thus provide much insight into the micro-evolutionary factors that have shaped species of interest. It can also be exploited to identify the genomic regions determining complex traits of interest, which can then be applied as performance evaluation markers in marker-assisted selection (MAS). The South African abalone, Haliotis midae, supports a rapidly developing aquaculture production industry, in which genetic improvement potential is high. This species also represents an opportunistic model for studying the effects of early domestication in a shellfish species. The aim of this study was therefore to quantify and characterise levels of genome-wide LD within the South African abalone, and to demonstrate its utility within population genetic investigations and the characterisation of complex traits. Estimates of LD between 112 mapped microsatellite markers within wild and cultured H. midae revealed that levels of LD in abalone are high relative to other aquaculture species. This was attributed primarily to small effective population sizes produced by a combination of natural- and anthropogenic factors. The decay of LD with genetic distance was evident in both cultured cohorts, but almost absent in wild cohorts, likely reflecting the differences in size, age and sampling of wild populations relative to cultured. Putative evidence for the effects of recombination, selection, and epistasis were also evident in distinctive locus-specific patterns of LD on some of the linkage groups, many of which could represent the effects of domestication. The effects of selection associated with the domestication event were further investigated using a candidate locus LD mapping approach to determine the proportion of candidate loci under selection associated with artificial selection for faster growth rate in cultured abalone. Two loci (15%) were found to be significantly associated with differences in size of individual animals, both of which could be linked with genes potentially involved in growth and development. These markers could therefore find application in MAS programmes for abalone. Several promising candidates for natural selection were also identified based on similarity with known genes. As the latter represented the majority, natural selection, rather than artificial selection, appears to be predominant during the early stages of domestication in abalone. While some conclusions within the current study were speculative, both the direct and indirect applications of LD were clearly demonstrated. Linkage disequilibrium data can provide a unique perspective on many of the commonly used population genetic estimates, and is therefore of great value in population genetic investigations. Furthermore, these results also highlighted the effectiveness of the candidate locus approach in species with both limited molecular resources and extensive LD. / AFRIKAANSE OPSOMMING: Koppelingsonewewig (KO) word gedefinieer as die nie-lukrake assosiasie van allele by twee of meer lokusse binne 'n populasie. Koppelingsonewewig is sensitief vir 'n verskeidenheid van lokus-spesifieke- en demografiese faktore, en kan dus insiggewend wees m.b.t. mikro-evolusionêre faktore wat spesies van belang beïnvloed het. Dit kan ook benut word om die genoom-gebiede onderligend tot komplekse eienskappe te bespeur; wat dan aangewend kan word vir prestasie-evaluering m.b.v. merkerbemiddelde seleksie (MBS). Die Suid-Afrikaanse perlemoen, Haliotis midae, ondersteun 'n vinnig ontwikkelende akwakultuur produksie bedryf, waarin genetiese verbeteringspotensiaal hoog is. Hierdie spesie verteenwoordig ook 'n opportunistiese model vir die bestudering van die gevolge van vroeë domestiseering in 'n skulpvis spesie. Die doel van hierdie studie was dus om vlakke van genoom-wye KO binne die Suid-Afrikaanse perlemoen te kwantifiseer en te karakteriseer, en om die toepassing hiervan binne populasiegenetiese ondersoeke en die karakterisering van komplekse eienskappe te demonstreer. Ramings van KO tussen 112 gekarteerde mikrosatelliet-merkers binne wilde en gekultiveerde H. midae het aan die lig gebring dat die vlakke van KO in perlemoen hoog was, in vergelyking met ander akwakultuur spesies. Dit word hoofsaaklik toegeskryf aan klein effektiewe populasiegroottes wat deur 'n kombinasie van natuurlike- en antropogeniese faktore teweeg gebring word. Die verval van KO met genetiese afstand was duidelik waarneembaar in gekultiveerde kohorte, maar amper afwesig in die wilde kohorte, waarskynlik a.g.v. verskille in populasiegrootte, ouderdom, en streekproef-neemings metodieke van die verskeie populasies. Vermeende bewyse vir die gevolge van rekombinasie, seleksie en epistase kon ook gesien word a.g.v. lokus-spesifieke patrone van KO op sommige van die koppelingsgroepe, moontlik ‘n gevolg van domestisering. Die gevolge van seleksie wat verband hou met die domestiseringsgebeurtenis is verder ondersoek m.b.v 'n kandidaat-lokus KO karteringsbenadering om die verhouding van kandidaat lokusse wat geassosieer is met kunsmatige seleksie (vir vinniger groeikoers in perlemoen) te bepaal. Twee lokusse (15%) was beduidend geassosieer met verskille in grootte tussen individuele diere. Beide van die lokusse was gekoppel met gene wat potensieel betrokke is by groei en ontwikkeling. Hierdie merkers kan dus moontlik aangewend word in MBS programme vir perlemoen. Verskeie belowende kandidaat lokusse vir natuurlike seleksie is ook geïdentifiseer gebaseer op ooreenkoms met bekende gene. Gegewe dat die laasgenoemde die meerderheid van die merkers verteenwoordig, kan daar afgelei word dat natuurlike seleksie, eerder as kunsmatige seleksie, oorheersend is in die vroeë stadia van domestisering in perlemoen. Terwyl sommige gevolgtrekkings binne die huidige studie spekulatief was, is beide die direkte en indirekte toepassings van KO duidelik gedemonstreer. Koppelingsonewewig-data kan 'n unieke perspektief gee op baie van die algemeen gebruikte populasie genetiese skattings, en is dus van groot waarde in populasie genetiese ondersoeke. Verder demonstreer hierdie resultate ook die doeltreffendheid van die kandidaat lokus benadering in spesies met beide beperkte molekulêre hulpbronne en uitgebreide KO.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Cultured abalone in South Africa is undomesticated. For the local industry to remain competitive on the international markets it is essential to improve production. This study is part of a selective breeding component of a larger genetic programme that aims to enhance productivity of the local industry by genetic improvement of growth rates. Selective breeding programmes are based on genetic variation and correlations. Molecular studies proved genetic differentiation exist between the broodstock- and offspring populations and among the offspring populations used in this study. Five commercial abalone farms from the Walker bay region each entered 3000 randomly selected animals obtained from synchronised mass spawning of conditioned broodstock into a Performance Recording Scheme (PRS). Microsatellite marker analysis proved these broodstock populations to be representative of the wild populations. The five cohorts were assessed over the five locations represented by three replicates per location with 200 randomly assigned animals per replicate. The average growth rate was used as growth performance parameter by measuring shell length and body weight at three month intervals over a period of 24 months. Interaction was observed between cohort and location effects when analysing the full data set. This was unexpected as the cohorts were constructed from parent stick that was randomly sampled from the same geographical area, the larger Walker bay. The factors suspected of causing this observed interaction were considered in a stepwise analysis. Initial and progressive tag loss, differences in initial size of animals entered into the study and on-farm management errors were considered as possible causes of the observed interaction in a stepwise analysis. Statistically significant differences were observed between the five cohorts and between the five locations in terms of length and weight growth rates. Based on these findings it is advised that a central facility is used to effectively compare the growth rates of different cohorts or populations. Any future research in selective breeding to follow this study should involve the integration of molecular techniques and biotechnologies. / AFRIKAANSE OPSOMMING: In Suid-Afrika is gekweekte perlemoen wild. Vir die plaaslike bedryf om op die internasionale markte kompeterend te bly, is dit noodsaaklik om produksie te verbeter. Hierdie studie vorm deel van ’n selektiewe telingskomponent van ‘n groter genetiese program met die doel om die produktiwiteit van die plaaslike bedryf deur die genetiese verbetering van groeitempo’s te verbeter. Selektiewe teelprogramme word gebaseer op genetiese variasie en korrelasies. Molekulêre studies het bewys dat daar genetiese differensiasie bestaan tussen die teel- en nageslagpopulasies en onder die nageslagpopulasies wat in hierdie studie gebruik is. Vyf kommersiële perlemoenplase in die Walkerbaaistreek het elk 3000 ewekansig geselekteerde diere vanaf gesinkroniseerde massa broei van gekondisioneerde teelpopulasies aan ‘n Performance Recording Scheme (PRS) bygedra. Mikrosatelliet merker-analise het bewys dat hierdie teelpopulasies verteenwoordigend is van die wilde populasies. Die vyf kohorte is oor die vyf liggings geassesseer, elkeen waarvan verteenwoordig is deur drie replikate bestaande uit 200 ewekansig toegedeelde diere per replikaat. Die gemiddelde groeitempo is gebruik as die groeiprestasieparameter deur skulplengte en liggaamsgewig elke drie maande oor ‘n tydperk van 24 maande te meet. Daar is interaksie waargeneem tussen kohort- en liggingseffekte toe die volledige datastel geanaliseer is. Hierdie was onverwags, aangesien die kohorte gekonstrueer is uit teelouers waarvan monsters ewekansig vanuit dieselfde geografiese gebied, naamlik die groter Walkerbaai, geneem is. Die faktore wat vermoedelik hierdie waargenome interaksie veroorsaak het, is in ‘n stapsgewyse analise beskou. Aanvanklike en progressiewe merkerverlies, verskille in die aanvanklike grootte van die diere wat in die studie ingesluit is en bestuursfoute op die plaas is as moontlike oorsake van die waargenome interaksie voorgestel. Statisties betekenisvolle verskille is tussen die vyf genotipes en tussen die vyf liggings in terme van lengte en gewigsgroeitempo’s waargeneem. Op grond van hierdie bevindings word daar voorgestel dat ‘n sentrale fasiliteit gebruik word om die groeitempo’s van die verskillende genotipes of populasies doeltreffend te vergelyk. Enige toekomstige navorsing oor selektiewe teelt wat op hierdie studie sou volg, moet die integrasie van molekulêre tegnieke en biotegnologieë behels.

Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Haliotis midae (HM) is one of five indigenous abalone species occurring along the coast of South Africa, and is the only species commercially cultivated in open flow-through systems. Commercial production of HM contributes the most to income generated from aquaculture species in South Africa. Exports in 2011 totalled 1036 ton, which amounts to an income of ZAR 369 million (40 million USD). Commercial production of HM is characterized by a long interval (i.e. up to five years) to harvest. Genetic modification of HM to improve growth rate in HM can potentially decrease the interval from hatch to harvest, which will contribute to optimising the cost-efficient production of HM. However, a major concern in the production of genetically modified (GM) abalone is their escape from production systems, and the potential impact of the GM animals on the natural stocks in the surrounding environment. The implementation of containment methods to prevent the escape from GM abalone from production systems will assist in the management of the potential ecological risks escaped GM abalone may pose. Currently no containment strategies exist for the South African abalone industry, which limits the use of transgenic biotechnologies to optimise the cost-efficient production of HM. The study therefore investigated the potential of biological methods (triploid induction), mechanical methods (polypropylene filter bags and obstructive materials), and chemical methods (sodium hypochlorite and chlorine dioxide) to contain transgenic HM in open flow-through systems. Histological evaluation of gonad tissue samples of 16 triploid male and 15 triploid female HM indicated that triploid induction was unsuccessful in arresting gonad differentiation, development, and function. A combination of immature and mature gametes was observed on both triploid male and female gonads. The fertilizing potential of biopsied sperm obtained from triploid and induced diploid male HM was assessed in fertilization trials using ova obtained from diploid HM. No larvae were observed from the fertilization treatments of triploid male biopsied sperm, however, abnormal larvae (0.01% fertilization) were observed when induced diploid sperm were used to fertilize diploid ova. Polypropylene filter bags (100 μm) were not effective in containing male and female gametes in the broodstock section of the abalone culture system used in this study, and future studies should address specialized methods of containment of HM gametes. Polypropylene filters (100 μm), resulted in effective containment of larvae and settlement larvae, and in this study was not a more effective method of containment than the current on-farm larvae containment protocol. The three obstructive materials were effective in containing HM spat, and did not differ in their ability as containment methods. Sodium hypochlorite and Biox™, chlorine-based chemicals, were effective in containing the five life stages of HM, i.e. sperm, ova, larvae, settlement larvae and spat. The respective life stages were exposed to varying concentrations of the respective chemicals (i.e. 10 ppm, 50 ppm and 100 ppm) over five exposure times (i.e. 4, 6, 8, 10 and 12 minutes). Sodium hypochlorite and Biox™, at the different concentrations and exposure times, resulted in mortalities in all five HM life stages, with higher incidences recorded at the higher concentrations evaluated. Future research on the effect of triploid induction on the endocrine system, and how effective gonad maturation and function is suppressed, needs to be conducted. The aspects of mosaics occurring in induced diploid animals warrant further investigation with a larger sample size to verify the findings of this study. The production of true sterile individuals would improve the results to use of GM animals as a method of biological containment. Incorporation of mechanical containment methods need to be kept in mind when abalone production systems are designed. The use of chemical containment methods warrants additional studies on the practical administration and the economic feasibility of the chemical into the water supply of open flow-through abalone culture systems. / AFRIKAANSE OPSOMMING: Haliotis midae (HM) is een van vyf inheemse perlemoen spesies wat langs die kus van Suid-Afrika voorkom en is die enigste spesie wat kommersieel in oop deurvloeistelsels geproduseer word. Kommersiële produksie van HM dra die meeste tot kommersiële akwakultuur produksie by, met uitvoere van 1036 ton in 2011, wat gelykstaande aan ‘n inkomste van ZAR 369 miljoen (40 miljoen VSA dollar). Kommersiële produksie van HM word gekenmerk deur 'n lang interval (d.i. tot 5 jaar) vanaf uitbroei tot oes. Genetiese modifisering van HM om die groeitempo te verbeter kan potensieel die interval tot slagmassa verkort, wat potensieel tot die optimalisering van die kostedoeltreffende produksie van HM kan bydra. 'n Groot bron van kommer met die produksie van geneties gemodifiseerde (GM) perlemoen is hul ontsnapping uit produksiestelsels en die potensiële impak van die GM diere op die natuurlike perlemoenpopulasies in die omliggende omgewing. Die implementering van beheermetodes om die ontsnapping van GM perlemoen uit produksiestelsels te voorkom sal die bestuur van die potensiële ekologiese risiko's wat GM perlemoen mag inhou, vergemaklik. Tans bestaan daar geen inperking strategieë vir GM diere vir die Suid-Afrikaanse perlemoenbedryf nie, wat die gebruik van transgeniese biotegnologieë vir die kostedoeltreffende produksie van HM beperk. Die studie het dus die potensiaal van biologiese metodes (triploïed induksie), meganiese metodes (polipropileen filters en obstruktiewe materiaal) en chemiese metodes (natriumhipochloriet en Biox™) om as inperk strategieë vir die bestuur van transgeniese HM in oop deurvloei produksiesisteme gebruik te word, ondersoek. Histologiese evaluering van gonadeweefsel monsters van 16 manlike en 15 vroulike triploïed HM het aangedui dat triploïed induksie onsuksesvol was om gonade differensiasie, ontwikkeling, en funksie te onderdruk. 'n Kombinasie van onvolwasse en volwasse gamete is in gonades van beide manlike en vroulike triploïed HM waargeneem. Die bevrugtingsvermoë van sperme wat versamel is met behulp van ʼn biopsie metode van triploïed en geïnduseerde diploïed HM is geëvalueer in bevrugtingsproewe met ova wat van diploïed HM versamel is. Geen larwes is waargeneem met die gebruik van die triploïed sperme nie, maar abnormale larwes (0.01% bevrugting) het wel voorgekom met die evaluering van sperme versamel van geïnduseerde diploïed HM. Die voorkoms van mosaïek diere (d.i. ʼn kombinasie van beide diploïed en triploïed selle in een individu) wat ontstaan a.g.v. onvolledige triploïed induksie noodsaak verdere navorsing met ʼn groter monster diere om die vermoë van triploïed induksie om HM steriel te maak, te verifieer. Polipropileen filters (100 μm) was nie effektief om beide manlike en vroulike gamete wat in die teelafdeling in die perlemoenkultuursisteem in hierdie studie geproduseer is, vas te vang nie. Toekomstige studies moet gespesialiseerde metodes wat insluiting van HM gamete moontlik sal maak, ondersoek. Polipropileenfilters (100 μm) was effektief met die inkamping van larwes en vestigingslarwes, met die filters wat nie meer doeltreffend as die bestaande inkampingsmetodes in die produksiesisteem was nie. Die drie obstruktiewe materiale was doeltreffend ten opsigte van die inkamping van onvolwasse perlemoen individue, met die 3 materiale wat ewe doeltreffend in hulle vermoë was om die onvolwasse HM se ontsnapping uit die sisteem te voorkom. Natriumhipochloriet en Biox™, chloor-gebaseerde chemikalieë, was effektief om die onderskeie 5 lewenstadiums, d.i. HM sperme en ova, larwes, vestigingslarwes en onvolwasse HM se voorkoms tot hulle onderskeie afdelings binne die produksiesisteem te beperk. Die onderskeie lewensstadiums is blootgestel aan verskillende konsentrasies van die onderskeie chemikalieë (d.i. 10 dpm, 50 dpm en 100 dpm) oor vyf tydsintervalle (d.i. 4, 6, 8, 10 en 12 minute). Natriumhipochloriet en Biox™ het ʼn letale effek op al vier lewensstadia gehad, met hoër sterftes wat met hoër konsentrasies aangeteken is. Toekomstige navorsing oor die uitwerking van triploïed induksie op die endokriene stelsel en hoe doeltreffend die rypwording en funksie van die gonades onderdruk word, moet aandag geniet. Die voorkoms van mosaïek diere regverdig ook spesifieke aandag in toekomstige navorsing. Die produksie van ware steriele individue sou die gebruik van GM perlemoen as 'n metode van biologiese inkamping moontlik maak. Inlywing van meganiese inkampingmetodes moet in gedagte gehou word wanneer perlemoen produksiestelsels ontwerp word. Die gebruik van chemiese inkampingmetodes benodig verdere studies om te verseker dat die chemiese middels so bekostigbaar en prakties as moontlik in water van 'n oop deurvloei perlemoen produksiestelsel toegedien kan word.