Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: The slow growth rate of Haliotis midae impedes the optimal commercial production of this most profitable South African aquaculture species. To date, no comprehensive effort has been made to identify genes associated with growth variation in farmed H. midae. The aim of this study was therefore to investigate growth variation in H. midae and to identify and quantify the expression of selected growth-related genes. Towards this aim, molecular methodologies and cell cultures were combined as a time-efficient and economical way of studying abalone transcriptomics and cell biology. Modern Illumina sequencing-by-synthesis technology and subsequent sequence annotation were used to elucidate differential gene expression between two sibling groups of abalone demonstrating significant growth variation. Following transcriptome sequencing, genes involved in growth and metabolism, previously unknown in H. midae, were identified. The expression of selected target genes involved in growth was subsequently analyzed by quantitative real-time PCR (qPCR). The feasibility of primary cell cultures for H. midae was furthermore investigated by targeting embryo, larval and haemolymph tissues for the initiation of primary cell culture. Larval cells and haemocytes could be successfully maintained in vitro for limited periods. Primary haemocyte cultures demonstrated to be a suitable in vitro system for studying gene expression and were subsequently used for RNA extraction and qPCR, to evaluate differential growth induced by bovine insulin and epidermal growth factor (EGF). Gene expression was thus quantified in fast and slow growing abalone and in in vitro primary haemocyte cultures treated with different growth stimulating factors. The results obtained from transcriptome analysis and qPCR revealed significant differences in gene expression between large and small abalone, and between treated and untreated haemocyte cell cultures. Throughout in vivo and in vitro qPCR experiments, the up-regulation of genes involved in the insulin signaling pathway provides evidence for the involvement of insulin in enhanced growth rate for various H. midae tissues. Besides the regulation of target genes, valuable knowledge was also gained in terms of reference genes, during qPCR experimentation. By quantifying the stable expression of two genes (8629, ribosomal protein S9 and 12621, ornithine decarboxylase) in various tissues and under various conditions, suitable reference genes, that can also be used in future H. midae qPCR studies, were identified. By providing evidence at the transcriptional level for the involvement of insulin, insulin-like growth factors (IGFs) and insulin-like growth factor binding proteins (IGFBPs) in improved growth rate of H. midae, the relevance of investigating ways to stimulate insulin/IGF release in aquaculture species was again emphasized. As nutritional administration remains the most probable route of introducing agents that can stimulate the release of insulin-related peptides, continuous endeavours to stimulate abalone growth through a nutritional approach is encouraged. This is the first time next generation sequencing is used towards the large scale transcriptome sequencing of any haliotid species and also the first time a comprehensive investigation is launched towards the establishment of primary cell cultures for H. midae. A considerable amount of sequence data was furthermore annotated for the first time in H. midae. The results obtained here provide a foundation for future genetic studies exploring ways to optimise the commercial production of H. midae. / AFRIKAANSE OPSOMMING: Die stadige groeitempo van Haliotis midae belemmer die optimale kommersiele produksie van hierdie mees winsgewende Suid-Afrikaanse akwakultuur spesie. Tot op hede is geen omvattende poging aangewend om gene verwant aan groeivariasie in H. midae te identifiseer nie. Die doel van hierdie studie was dus om groeivariasie in H. midae te ondersoek en om spesifieke groei-gekoppelde gene te identifiseer en hul uitdrukking te kwantifiseer. Ter bereiking van hierdie doel is molekulêre metodes en selkulture gekombineer as 'n en tydsbesparende en ekonomiese manier om perlemoen transkriptomika en selbiologie te bestudeer. Moderne Illumina volgordebepaling-deur-sintese tegnologie en daaropvolgende annotasie is gebruik om verskille in geenuitdrukking tussen naby-verwante groepe perlemoen, wat noemenswaardige groeivariasie vertoon, toe te lig. Na afloop van die transkriptoom volgordebepaling is gene betrokke by groei en metabolisme, vantevore onbekend in H. midae, geïdentifiseer. Die uitdrukking van uitgesoekte teikengene betrokke by groei is vervolgens ge-analiseer deur kwantitatiewe "real-time PCR" (qPCR). die lewensvatbaarheid van 'n primêre selkulture vir H. midae is ook ondersoek deur embrio, larwe en hemolimf weefsels te teiken vir die daarstelling van primêre selkulture. Larweselle en hemosiete kon in vitro suksesvol onderhou word vir beperkte periodes. Primêre hemosietkulture het geblyk 'n gepaste in vitro sisteem te wees om geenuitdrukking te bestudeer en dit is vervolgens gebruik vir RNS ekstraksie en qPCR, om differensiële groei, geïnduseer deur insulien en epidermale groeifaktor (EGF), te evalueer. Geenuitdrukking is dus gekwantifiseer in vinnig- en stadiggroeiende perlemoen en in in vitro primêre hemosiet selkulture wat behandel is met verskillende groei stimulante. Die resultate wat verkry is van transkriptoomanalise en qPCR het noemenswaardige verskille in geenuitdrukking tussen groot en klein perlemoen, en tussen behandelde en onbehandelde hemosiet selkulture uitgelig. Die op-regulering van gene betrokke by die insulien sein-padweg, tydens in vivo en in vitro qPCR eksperimente, bied getuienis vir die betrokkenheid van insulien in die verhoogde groeitempo van verskeie H. midae weefsels. Benewens die regulering van teikengene is waardevolle kennis ook ingewin in terme van verwysingsgene tydens qPCR eksperimentering. Deur die stabiele uitdrukking van twee gene (8629, ribosomale proteien S9 en 12621, ornitien dekarboksilase) te kwantifiseer in verskeie weefsels en onder verskeie kondisies is gepaste verwysingsgene, wat ook in toekomstige H. midae qPCR eksperimente aangewend kan word, geïdentifiseer. Deur getuienis vir die betrokkenheid van insulien, insuliensoortige groeifaktor en insuliensoortige groeifaktor-bindingsproteïene by verbeterde groei van H. midae op transkripsievlak te bied, is die toepaslikheid van bestudering van maniere om insulienvrystelling in akwakultuurspesies te stimuleer, beklemtoon. Aangesien voeding die mees waarskynlike roete is om middele wat insuliensoortige peptiedvrystelling stimuleer daar te stel, word vogehoue pogings om perlemoengroei deur die regte voeding te stimuleer, aangemoedig. Hierdie is die eerste studie wat volgende generasie volgordebepaling (“next generation sequencing”) gebruik vir die grootskaalse transkriptoom volgordebepaling van enige haliotied spesie. Dit is ook die eerste keer dat ‘n omvattende ondersoek geloods word na die daarstelling van primêre selkulture vir H.midae. ‘n Aansienlike hoeveelheid volgorde data is ook vir die eerste keer geannoteer in H. midae. Die resultate wat hier verkry is bied ‘n basis vir toekomstige genetiese studies wat maniere ondersoek om die kommersiële produksie van perlemoen te optimiseer.

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The abalone Haliotis midae is the most important aquaculture species in South Africa. The industry is dependent on export to Far Eastern markets in a variety of forms, including live, frozen, canned or dried. The species is considered undomesticated in the sense that the current commercial broodstock has been obtained from natural populations through a process of random collection. Global competition has necessitated the South African industry to introduce a genetic improvement program to increase biological productivity and financial profitability. The objective of this study was to assess the genetic variation and to estimate key parameters in terms of growth and yield related traits, between family groups that form part of the breeding program. The study reports on heritability estimates of growth rate (0.14 ± 0.05), canning yield (0.08 ± 0.03), and drip loss during live export (0.03 ± 0.02). The high genetic correlation (0.94 ± 0.34) between shell length and live weight enables industry to utilise either weight or shell length as a criteria during operational practices such as sorting, grading and harvesting. The correlation of 0.85 ± 0.01 between live weight and canning loss indicates that animals that weigh more have a lower dressing percentage. Based on these low heritability values obtained for yield related traits it is recommended not to include these traits in the selection program at this stage. The findings of the study were however, compromised by the availability of a limited number of family groups, the age differences between families and the effect of different locations on the variance in phenotypes. Further investigation is needed to confirm the credibility of the results. / AFRIKAANSE OPSOMMING: Die perlemoen Haliotis midae is die belangrikste akwakultuurspesie in Suid Afrika. Die industrie is afhanklik van uitvoere na markte in die Verre Ooste. ‘n Verskeidenheid van produkte word uitgevoer, insluitend lewendige, gevriesde, gedroogte en verblikte perlemoen. Die spesie word as ongedomestikeerd beskou aangesien die huidige teeldiere op ‘n lukrake wyse uit natuurlike populasies versamel is. Globale kompetisie het die Suid Afrikaanse industrie genoodsaak om ‘n genetiese verbeteringsprogram in werking te stel om sodoende die biologiese produktiwiteit en finansiële winsgewendheid te verbeter. Die studie poog om genetiese variasie in groei en opbrengsverwante kenmerke tussen familiegroepe wat deel uitmaak van die teelprogram te ondersoek. Oorerflikheid van groeitempo (0.14 ± 0.05), opbrengs na verblikking (0.08 ± 0.03), en vogverlies na lewendige uitvoer (0.03 ± 0.02) is beraam. Die hoë korrelasie (0.94 ± 0.34) tussen gewig en skulplengte stel die industrie in staat om beide massa en skulplengte as kriteria te geruik tydens operasionele praktyke van sortering, groepering en oes. Die korrelasie van 0.85 ± 0.01 tussen gewig en verlies na verblikking dui aan dat swaarder diere ‘n laer uitslagpersentasie het. As gevolg van die lae oorerflikheidswaardes vir opbrengsverwante kenmerke word daar aanbeveel dat hierdie kenmerke nie op hierdie stadium ingesluit word in die seleksieprogram nie. Resultate is egter beïnvloed deur ‘n beperkte aantal familie groepe, ouderdomsverskille tussen families en die effek van verskillende lokaliteite op die variansie in die fenotipes. Verdere ondersoeke is nodig om die geloofwaardigheid van die bevindinge te bevestig.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science. / ENGLISH ABSTRACT: Haliotis midae, or Perlemoen, is the only cultured species of abalone in South Africa and is under great international demand. This species is considered endangered, making sustainable farming practises and law enforcement against poaching essential for maintaining wild stocks. A limited amount of broodstock animals are provided to each farm from which thousands of offspring are grown and exported. The prevention of inbreeding and preservation of genetic diversity within farmed stocks is necessary for future sustainable farming and production of genetically stable offspring. Further research into the genetic dynamics of Perlemoen will provide the knowledge for advanced management programs for optimal farming practises and essentially sustainable production. This study focuses on genetic linkage map development with the intention of future identification of markers associated with genes of economic importance, such as growth rate. Identification of markers linked to genes responsible for such phenotypic traits will ultimately allow farming practises to select naturally genetically superior animals for breeding, thereby enhancing production. For the construction of a genetic linkage map of H. midae, microsatellite markers were developed using two strategies: FIASCO and screening of next generation sequence-bysynthesis contig data. The FIASCO-derived markers were characterised by genotype screening in 32 individuals from a full-sib family and analysed using Mendelian segregation expectations. The Illumina-derived markers were characterised by genotype screening in 32 individuals from wild populations and analysed against Hardy-Weinberg expectations. Forty four microsatellite-family combinations were obtained from FIASCO of which 28 provided informative genotype results (32% success). Twenty two markers were developed from sequence-by-synthesis screening. Fourteen provided reliable genotypes (37%) and six conformed to Hardy-Weinberg expectations. These markers were used, in addition to 156 previously developed markers, to develop sex-specific and sex-average linkage maps in two full-sib families consisting of approximately 100 offspring each. One hundred and six polymorphic loci were used for linkage analysis (LOD>3) in both families. The number of linkage groups obtained from sex-specific maps ranged from 13-16. The average genome length ranged from 500 cM to 800 cM with an average marker spacing of 10 cM. The sex-average linkage map provided 18 linkage groups with an average genome length calculation of 1800 cM and average marker spacing of approximately 13 cM. The linkage maps created in this study are preliminary but provide a stepping stone towards a high density map incorporating high throughput markers. This also provides a base for QTL mapping studies, in which phenotypic traits of interest can be identified and associated to specific locations in the H. midae genome for marker-assisted selection. / AFRIKAANSE OPSOMMING: Haliotis midae, ook bekend as Perlemoen, is in groot internasionale aanvraag en is ook die enigste klipkous spesie waarmee in Suid Afrika geboer word. Hierdie spesie word as bedreig beskou en daarom is volhoubare boerdery bedrywe en wetstoepassing teen stroping noodsaaklik om wilde populasies te beskerm. Elke perlemoenplaas word met ‘n beperkte aantal broeidiere verskaf, waarvan die nageslag dan gekweek en uitgevoer word. Voorkoming van inteling en handhawing van genetiese diversiteit binne gekweekte populasies is noodsaakllik vir toekomstige volhoubare kweking en produksie van ń geneties stabiele nageslag. Verdere ondersoeke na die genetiese dinamika van Perlemoen sal die nodige kennis verskaf om sodoende gevorderde bestuursprogramme te ontwikkel, wat tot optimale kweek praktyke en effektiewe volhoubare produksie sal lei. Hierdie studie fokus op die ontwikkeling van ‘n genetiese koppelingskaart met die voorneme om toekomstige merkers te identifiseer wat met gene van ekonomiese belang, soos byvoorbeeld groei tempo geassosieerd is. Identifisering van merkers wat vir sulke fenotipiese eienskappe verantwoordelik is sal sodoende toelaat dat boerdery praktyke kan selekteer vir diere vir verbeterde teling en produksie. Mikrosatelliet merkers is ontwikkel om die genetiese koppelingskaart saam te stel. Die volgende twee strategieë is benut: FIASCO en sifting van volgende generasie volgordebepaling-deur-sintese “contig” data. Die FIASCO-afgeleide merkers is gekarakteriseer deur genotipiese sifting in 32 individue van ‘n volsib familie en is deur Mendeliese segregasie verwagtinge ge-analiseer. Die Illumina-afgeleide merkers is gekarakteriseer deur genotipiese sifting in 32 individue van wilde populasies en is met Hardy-Weinberg ewewig ge-analiseer. Vier en veertig mikrosatelliet-familie kombinasies is deur FIASCO verky, waarvan 28 informatiewe genotipiese resultate gelewer het (32% sukses). Twee en twintig merkers is vanaf volgordebepaling-deur-sintese sifting ontwikkel. Veertien van hierdie merkers het betroubare genotipes (37%) verskaf en ses het aan Hardy-Weinberg verwagtinge voldoen. Hierbenewens is 156 voorheen ontwikkelde merkers gebruik om geslagspesifieke en geslagsgemiddelde koppelingskaarte in twee volsib families saam te stel. Hierdie volsib families het uit ń naslag van 100 elk bestaan. Een honderd en ses polimorfiese lokusse is vir koppelingsanalise gebruik, waar ‘n LOD waarde groter as drie statisties betekenisvol geag was. Die aantal koppelingsgroepe verkry van geslagspesifieke kaarte het tussen 13 en 16 gewissel. Die gemiddelde genoom lengte het van 500 cM tot 800 cM met ‘n gemiddelde merker spasiëring van 10 cM. Die geslagsgemiddelde koppelingskaart het 18 koppelingsgroepe gehad met ‘n gemiddelde genoom lengte berekening van 1800 cM en ‘n gemiddelde merker spasiëring van ongeveer 13 cM. Die koppelingskaarte wat in hierdie studie geskep is, is voorlopig en verskaf ‘n grondslag vir die ontwikkeling van ‘n hoër digtheidskaart, wat hoë deurset merkers inkorporeer. Dit verskaf ook ‘n basis vir kwantitatiewe kenmerk lokus karteringstudies. Hierdie karteringstudies kan fenotipiese eienskappe van belang identifiseer en assosieer met spesifieke posisies binne die H. midae genoom vir merker bemiddelde seleksie.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Haliotis midae (commonly referred to as perlemoen) is the only one of five endemic species in South Africa that is commercially valued both locally and internationally. Unfortunately, natural perlemoen populations have become a dwindling resource due to commercial exploitation, poaching and the influx of natural threats, such as the West Coast rock lobster, Jasus lalandii. To preserve the natural diversity and sustainability of natural populations as well as commercial stocks, genetic management and improvement of perlemoen is critical. Genetic management requires the utilisation of molecular markers, which aid in the construction of linkage maps and the identification of quantitative trait loci (QTL) associated with economically significant traits. This will allow improvement of commercial stock management in terms of broodstock selection as well as provide valuable insight into natural population dynamics. Single Nucleotide Polymorphisms (SNPs) were selected as the marker of choice due to their successful employment as molecular markers and their wide distribution and abundance within the genomes of various marine species. This study focuses on the characterisation of novel SNPs from transcript sequences generated by Next Generation Sequencing technology. Approximately 40% of the transcripts facilitated the isolation of 105 putative markers, indicating a SNP frequency of ~1% within the H. midae genome. A subset of 24 markers, in addition to 24 previously developed markers, was characterised using the Illumina GoldenGate genotyping assay with the VeraCode technology, a medium to high-throughput genotyping technology. This is the first reported medium- to highthroughput characterisation of SNPs in H. midae. The selected markers were used to determine the efficiency and overall success rate of the GoldenGate platform. Marker characterisation was completed in both natural and commercial populations to determine the utility of these markers for genetic diversity and population structure inference. An 85% genotyping success rate was achieved with the platform. Statistical analysis indicated that the markers developed in this study are suitable for applications including population genetic structure inference, genetic diversity estimation and possibly other downstream applications such as linkage mapping. These markers are considered to be invaluable for future work regarding the genetic management and conservation of H. midae. / AFRIKAANSE OPSOMMING: Haliotis midae (ook bekend as perlemoen) is die enigste van vyf inheemse spesies in Suid-Afrika wat noemenswaardige kommersiële waarde toon plaaslik sowel as internasionaal. Ongelukkig het kommersiële uitbuiting, wildstropery en natuurlike bedreiging (bv. die Weskus kreef Jasus lalandii), wilde perlemoen populasies noemenswaardig verminder. Dus, om natuurlike diversiteit en die voortbestaan van beide wilde en kommersiële populasies te beskerm, is genetiese bestuur en verbetering absoluut noodsaaklik. Genetiese bestuur vereis die gebruik van molekulêre merkers as ’n hulpmiddel in die opstellingvan koppelingskaarte, en die identifisering van die relevante kwantitatiewe eienskap loki (QTL) tipies geassosieer met ekonomies belangrike eienskappe. Die laasgenoemde beoog om kommersiële voorraad bestuur te verbeter, kragtens deur broeidier seleksie sowel as om insig te verskaf m.b.t. wilde bevolking dinamika. Enkel Nukleotied Polimorfismes (SNPs) is gekies as die toepaslike merker vanweë die omvattende toepaslikheid van hierdie merkers binne die genome van verskeie mariene spesies. Hierdie studie fokus op die karakterisering van nuwe SNPs vanuit transkript volgordes ontwikkel deur middel van Volgende Generasie Volgordebepaling (“Next Generation Sequencing”). ’n Beraamde 40% van transkripte het gelei tot die ontwikkeling van 105 potensiëlemerkers, aanduidend van ’n SNP frekwensie van ~1% binne die H. midae genoom. ’n Sub-versameling van 24 merkers, tesame met 24 bestaande merkers, is gekarakteriseer deur die Illumina GoldenGate genotiperings toets met die VeraCode tegnologie, ’n medium tot hoë deurvloei genotiperingstegnologie. Hierdie is die eerste berig van medium tot hoë deurvloei karakterisering van SNPs in H. midae. Die geselekteerde merkers is gebruik om die doeltreffendheid van die GoldenGate platform te bepaal. Merker karakterisering is uitgevoer in beide wilde en kommersiële bevolkings om die effektiewe bruikbaarheid van hierdie merkers m.b.t. genetiese diversiteit, en bevolking struktuur bepaling, te ondersoek. Die platform het ’n 85% genotiperingsukses syfer getoon. Statistiese analise dui daarop dat merkers ontwikkel tydens hierdie studie toepaslik is vir bevolking genetiese struktuur bepaling, genetiese diversiteitberaming en moontlik ook genetiese koppelingskartering. Hierdie merkers word bestempel as onmisbaar vir toekomstige navorsing in genetiese bestuur en bewaring van H. midae.