Thesis (PhD (Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: South African abalone, Haliotis midae, has been the subject of major concern regarding its survival and conservation over the last decade or more. Being the only one of five endemic species with commercial value, there is considerable interest and urgency in genetic management and improvement of this species. Limited genetic information and the increasing conservation concern of this species are considered the key motivations for generating information on the micro- and macro-evolutionary processes of H. midae, the overall objective of this study. This study reported the first microsatellite and Single Nucleotide Polymorphism (SNP) markers developed specifically for Haliotis midae. Both these marker types were applied to elucidate the degree of gene flow in nine natural abalone populations whilst testing for two contrasting hypotheses; panmixia versus restricted gene flow. Data was analysed using a series of methodological approaches ranging from traditional summary statistics to more advanced MCMC based Bayesian clustering methods with and without including spatial information. Using only microsatellite data, the historical demography of the species was also examined in terms of effective population size and population size fluctuations. Finally, the evolutionary positioning and origin of Haliotis midae with regards to other Haliotis species was investigated based on mitochondrial and nuclear sequence data. Both microsatellite and SNP data gave evidence for subtle differentiation between West and East coast populations that correlates with a hydrogeographic barrier in the vicinity of Cape Agulhas. Population substructure was supported by AMOVA, FCA and Bayesian clustering analysis. Clustering utilizing spatial information further indicated clinal variation on both sides of the proposed barrier with a region in the middle coinciding with a secondary contact zone, indicating possible historical isolation during glacial periods. Overall, the similar degree of substructure observed with both microsatellites and SNPs supported the existence of contemporary and/or historical factors with genome-wide effect on gene flow. The population expansion measured with the microsatellites was inconsistent with the known recent decline but taking the species’ life cycle and large effective population size into account, a shrinkage in population size will probably only be apparent in a few generations time. On a macro-evolutionary scale, this study presents the first classification of South African abalone as a monophyletic group within the Haliotidae family. The topology based on the combined mitochondrial and nuclear dataset is highly suggestive of a relatively recent radiation of the SA species from the Indo-Pacific basin. The study concludes by describing the most likely factors that could have affected overall population structure and makes suggestions on how the given genetic information should be incorporated into strategies aimed towards the effective management and conservation of Haliotis midae. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse perlemoen, Haliotis midae, is oor die laaste dekade of meer die onderwerp van groot bekommernis betreffende die spesie se oorlewing en bewaring. Aangesien dit die enigste van vyf endemiese SA spesies is met kommersiёle waarde, is daar besonderse belang en erns in die genetiese beheer en verbetering van die spesie. Beperkte genetiese inligting en ‘n toenemende behoefte om die spesie te bewaar is die hoof motivering agter die generering van informasie rakende mikro- en makro-evolusionêre prosesse in Haliotis midae en is die oorhoofse doel van hierdie studie. Hierdie studie beskryf die eerste mikrosatelliete en enkel basispaar polimorfismes wat ontwikkel is spesifiek vir Haliotis midae. Beide tipe merkers is aangewend om die mate van gene vloei in nege wilde perlemoen populasies te ondersoek terwyl twee hipoteses ondersoek is; panmiksie versus beperkte gene vloei. Data is geanaliseer deur gebruik te maak van ‘n reeks metodieke benaderings wat wissel van tradisionele opsommings statistieke tot meer gevorderde MCMC gebasseerde groeperings metodes met of sonder die gebruik van geografiese data. Mikrosatelliet data is ook aangewend om die historiese demografie van die spesie te bepaal in terme van effektiewe populasie grootte asook veranderinge in populasie groottes. Laastens is die evolusionêre posisionering en oorsprong van Haliotis midae teenoor ander Haliotis spesies ondersoek deur gebruik te maak van mitokondriale en nukleêre DNA volgorde data. Beide mikrosatelliet en enkel basispaar polimorfisme data lewer bewys van ‘n subtiele genetiese verskil tussen wes en ooskus populasies wat verband hou met ‘n hidrografiese skeiding in die omgewing van Kaap Agulhas. Populasie struktuur is ondersteun deur die analise van molekulêre variansie (AMOVA), faktoriale komponente analise asook Bayesiese groeperings analise. Groeperings analise wat geografiese informasie insluit dui klinale genetiese variasie aan beide kante van die skeiding aan met ‘n area in die middel wat ooreenstem met ‘n sekondêre kontak gebied. In totaal, ondersteun die soortgelyke mate van struktuur verkry met beide die mikrosatelliete en enkel basispaar polimorfismes die bestaan van hedendaagse en/of historiese faktore met genoom wye invloed op gene vloei. Die toename in populasie grootte vasgestel deur die mikrosatelliet data stem nie ooreen met die onlangse afname waargeneem in die spesie nie, maar met inagneming van Haliotis midae se lewenssiklus en groot effektiewe populasie grootte, sal die afname in populasie grootte moontlik eers oor ‘n paar generasies na vore kom. Op ‘n makro-evolusionêre skaal lewer hierdie studie die eerste klassifikasie van Suid-Afrikaanse perlemoen as ‘n monofiletiese groep binne die Haliotidae familie. Die topologie gebaseer op ‘n gesamentlike mitkondriale en nukleêre datastel is hoogs aanduidend van ‘n relatiewe onlangse verspreiding van die Suid-Afrikaanse spesies uit die Stille-Indiese Oseaan. Die studie sluit af deur die mees algemene faktore te bespreek wat populasie struktuur kon beïnvloed het en maak voorstelle op watter wyse hierdie genetiese inligting aangewend kan word vir die effekiewe beheer en bewaring van Haliotis midae.

Thesis (PhD (Genetics))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: The South African abalone, Haliotis midae, is a commercially valuable mollusc and is mostly exported to the Far East. Genetics research on H. midae has increased substantially since a genetic improvement programme was introduced in 2006 by collaboration between Stellenbosch University, government and industry partners. The development of molecular markers, QTL-mapping, gene-expression and genome manipulations are the main focuses of the research currently being conducted. The end goal is to create high quality and fast growing animals for the industry. The present study focused on the development of microsatellite markers and the detection of quantitative trait loci (QTL) affecting growth traits (shell length, shell width, wet weight) in this species. A combination of three methods, namely selective genotyping and bulked segregant analysis (pooling analysis), single marker regression and interval mapping were used to identify putative QTL in two full-sib families from two different farmed locations. Additional methods and protocols were developed that can assist the industry in other molecular research aspects. A total of 125 microsatellite loci were characterised. A total of 82 of these loci were isolated using second generation sequencing, a first for any abalone species. A preliminary, low-density framework linkage map was constructed containing 50 loci that mapped to 18 linkage groups. The observed genome length was 148.72cm with coverage of ±47%. QTL analyses revealed two putative QTL for shell width and wet weight, with 17% and 15% variance explained, that mapped on one linkage group in the first family and three putative QTL, for shell length, shell width and wet weight, with 33%, 28.5% and 31.5% variance explained, that mapped on one linkage group in the second family. Additional methods and protocols developed include an automated high-throughput DNA isolation protocol, a real-time PCR assay for H. midae x H. spadicea hybrid verification, a triploid verification microsatellite assay and a pre- and post-PCR multiplex setup and optimisation protocol. Future studies focussing on QTL and marker assisted selection (MAS) should verify the QTL found in this study and also utilise additional family structures and determine QTL-marker phase within the commercial populations. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse perlemoen, Haliotis midae, is ’n kommersieel waardevolle weekdier en word hoofsaaklik na die Verre-Ooste uitgevoer. Genetiese navorsing op H. midae het aansienlik toegeneem sedert ’n genetiese verbeteringsprogram in 2006 deur samewerking tussen die Universiteit van Stellenbosch, die regering en industrievennote ingebring is. Die ontwikkeling van molekulêre merkers, KEL-kartering, geen-uitdrukking en genoom manipulasies is die hooffokusse van die navorsing wat tans uitgevoer word. Die einddoel is om hoë kwaliteit en snelgroeiende diere vir die industrie te skep. Die huidige studie het op die ontwikkeling van mikrosatelliet merkers en die opsporing van groeiverwante (skulplengte, -breedte en nat gewig) kwantitatiewe eienskap lokusse (KEL) in hierdie spesie gefokus. ’n Kombinasie van drie metodes, naamlik selektiewe genotipering en versamelde segregaat analise (samevoegingsanalise), enkel merker regressie en intervalkartering is gebruik om waarskynlike KEL in twee vol-sibbe families van twee verskillende produksiegebiede te identifiseer. Aanvullende metodes en protokolle is ontwikkel wat die industrie in ander molekulêre navorsingsaspekte kan ondersteun. ’n Totaal van 125 mikrosatelliet lokusse is beskryf. ’n Totaal van 82 van hierdie lokusse is deur die gebruik van derde generasie volgordebepaling geïsoleer, ’n eerste vir enige perlemoen spesie. ’n Voorlopige, laedigtheid raamwerkkoppelingskaart is saamgestel met 50 lokusse wat op 18 koppelingsgroepe gekarteer is. Die waarneembare genoomlengte was 148.72cm met ’n dekking van ±47%. KEL-analises het twee waarskynlike KEL vir skulpbreedte en nat gewig blootgelê wat 17% en 15% variasie verduidelik en is op een koppelingsgroep in die eerste familie gekarteer asook drie waarskynlike KEL, vir skulplengte, -breedte en nat gewig wat 33%, 28.5% en 31.5% variasie verduidelik en is op een koppelingsgroep in die tweede familie gekarteer. Aanvullende metodes en protokolle wat ontwikkel is, sluit ’n geoutomatiseerde hoë-deurgang DNS-isolasieprotokol, ’n intydse PKR-proef vir H. midae x H. spadicea hibried verifikasie, ’n triploïed verifikasie mikrosatellietproef en veelsoortige pre- en post-PKR opstelling en optimaliseringsprotokol in. Toekomstige studies wat fokus op KEL en merker ondersteunde seleksie (MOS) behoort die KEL wat in hierdie studie gevind is te verifieer en ook bykomende familie strukture te benut om KEL-merker fases binne die kommersiële populasie te bepaal.

Thesis (MSc)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Haliotis midae, is the only commercially important species of the six abalone species found in South African coastal waters and has become a lucrative commercial commodity. Wild stocks of H. midae are, however, no longer commercially sustainable due to a combination of environmental factors and poaching. The solution to the crisis is artificial production systems in the form of abalone farms. An abalone enhancement programme was initiated in South Africa in 2006, funded by industry and government. This programme focuses on the elucidation of the abalone genome and genetic factors contributing to increased productivity, thereby aiding the commercial production of abalone. The aims of this study, the first of its kind concerning H. midae, were to develop AFLPbased markers (specifically fluorescent AFLP analysis); to monitor the segregation of these markers in a single full-sib family and to use the markers and additional microsatellite markers to generate the first preliminary linkage map for H. midae. Genomic DNA of sufficient quality and purity for fluorescent AFLP analysis was obtained from 3.5-month-old H. midae juveniles. Preliminary linkage maps were constructed using AFLP and microsatellite markers segregating in an F1 family following a pseudo-testcross mapping strategy. Twelve AFLP primer combinations, producing 573 segregating peaks, and 10 microsatellite markers were genotyped in the parents and 108 progeny of the mapping family. Of the 573 segregating AFLP peaks genotyped, 241 segregated in a 1:1 ratio and 332 in a 3:1 ratio. Of these AFLP markers, 90 segregated according to the expected 1:1 Mendelian ratio and 164 segregated according to the expected 3:1 Mendelian ratio at the P = 0.05 level and were used for linkage analysis. Of the 10 microsatellite markers genotyped, nine were informative for linkage mapping analysis. Preliminary male and female genetic linkage maps were developed using markers segregating in the female or male parent. A total of 12 and 10 linkage groups were detected for the female and male maps respectively. The female map covered 1473.5cM and consisted of 56 markers, and the male map covered 738.9cM consisting of 30 markers. Markers with segregation distortion were observed as previously reported in other abalone species and potential homology between one of the linkage groups of the male map and two of the linkage groups of the female map were identified using the 3:1 segregating AFLP markers. In conclusion, the genetic linkage map presented here, despite the fact that it has relatively low genome coverage and low marker density, forms an ideal starting point for more detailed study of the H. midae genome and will provide a scaffold for basic and applied studies in abalone. A high-density linkage map of H. midae should in future be developed with additional co-dominant molecular markers, such as microsatellites, to improve the transferability of the linkage map between different laboratories and among populations. A high-density linkage map will facilitate the mapping of QTL of commercially important traits (i.e. growth) and future MAS breeding programmes. / AFRIKAANSE OPSOMMING: Perlemoenspesie, Haliotis midae, is die enigste spesie van kommersiële belang van die ses wat in die kuswater van Suid-Afrika aangetref word en het ‘n winsgewende handelskommoditeit in Suid-Afrika geword. Die ontginning van natuurlike H. midae populasies is egter, as gevolg van ‘n kombinasie van omgewingsfaktore en stropery nie meer kommersieel volhoubaar nie. Die perlemoenkrisis kan die hoof gebied word deur kunsmatige produksiesisteme op perlemoenplase tot stand te bring. ‘n Perlemoen verbeteringsprogram is in 2006 in Suid-Afrika geïnisieer en word deur die industrie en regering befonds. Die program focus op die ontrafeling van die perlemoen genoom en die genetiese faktore wat bydrae tot verhoogde produksie. Sodanige inligting kan gebruik word om kommersiële perlemoenproduksie te bevorder. Die doel van hierdie studie, die eerste met H. midae, is om AFLP-gebaseerde merkers (spesifiek fluoresserende AFLP analise) te ontwikkel; die segregasie van hierdie merkers te monitor in ‘n enkel volledige verwante familie en die merkers en addisionele mikrosatelliet merkers te gebruik om die eerste voorlopige koppelingskaart vir H. midae te genereer. Genomiese DNS van genoegsame kwaliteit en suiwerheid vir fluoresserende AFLP analise is ge-ekstraeer uit 3.5-maand-oue H. midae individue. Voorlopige koppelingskaart is gekonstrueer deur van segregerende AFLP en mikrosatelliet merkers in ‘n F1 familie gebruik te maak deur ‘n pseudo-kruistoets karteringstrategie te volg. Twaalf AFLP inleier kombinasies, wat 573 segregerende fragmente geproduseer het, en 10 mikrosatelliet merkers is gegenotipeer in die ouers en 108 individue van die nageslag van die karteringsfamilie. Van die 573 segregerende AFLP merkers wat gegenotipeer is, het 241 in ‘n 1:1 verhouding en 332 in ‘n 3:1 verhouding gesegregeer. Van hierdie AFLP merkers, het 90 volgens die verwagte 1:1 Mendeliese verhouding en 164 volgens die 3:1 Mendeliese verhouding by die P = 0.05 gesegregeer vlak en is vir die koppelingsanalise gebruik. Van die 10 mikrosatelliet merkers gegenotipeer, was 9 informatief vir koppeling karteringsanalise. Voorlopige manlike en vroulike genetiese koppelingskaarte is ontwikkel met gebruik te maak van merkers wat in die manlike of vroulike ouer segregeer het. ‘n Totaal van 12 en 10 koppelingsgroepe is onderskeidelik in die vroulike en manlike karate gegenereer. Die vroulike kaart dek 1473.5cM and bestaan uit 56 merkers, terwyl die manlike kaart 738.9cM beslaan het met 30 merkers. Merkers wat segregasie distorsie toon is waargeneem soos voorheen in ander perlemoenspesies gerapporteer. Potensiële ooreenstemming tussen een van die koppelingsgroepe van die manlike kaart en twee van die koppelingsgroepe van die vroulike kaart is aangetoon deur van die 3:1 segregerende AFLP merkers gebruik te maak. Die genetiese koppelingskaarte verskaf wel ‘n relatiewe lae genoomdekking en ‘n lae merkerdigtheid, maar is ‘n ideale vertrekpunt vir meer gedetailleerde studie van die H. midae genoom en dien as ‘n raamwerk vir toekomstige basiese en toegepaste studies in perlemoennavorsing. ‘n Hoëdigtheid koppelingskaart van H. midae moet in die toekoms ontwikkel word met gebruik van bykomstige ko-dominante molekulêre merkers, soos mikrosatelliete. Dit sal die oordraagbaarheid van die koppelingskaart tussen verskillende laboratoria asook tussen populasies verbeter. ‘n Hoëdigtheid koppelingskaart sal die kartering van kwantitatiewe kenmerk loki (KKL) vir kommersieel belangrike kenmerke (onder andere groeikrag) en toekomstige merker bemiddelde seleksie (MBS) teelprogramme moontlik maak.

Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: The five leading abalone producers in South Africa have initiated a genetic enhancement program for Haliotis midae in a collaborative effort to improve economically valuable traits. Several independent objective-specific studies were initiated, including the establishment of a Performance Recording Scheme (PRS), utilised in this study, and necessary to monitor the ongoing performance of individuals as the move from mass-selection to marker assisted selection (MAS) is implemented. The primary objective of this study was parentage assignment of F1 offspring mass-selected for size at approximately one year and allocated to either a “faster” or a “slower” growth group. Nine microsatellite markers were used to genotype juveniles and potential parents, with assignment completed using CERVUS 2.0. Average growth results for Abagold and HIK were comparable for both growth groups. Slight environmental effects, although not statistically significant, were evident as growth advantages for juveniles within the faster growth group at two of the five locations and for juveniles within the slower growth group at one of the five rearing locations. Despite measures to standardise environmental influences, variables are difficult to control within the reality of a production environment; and potential genotype x environment interactions may require further investigation and factoring into future breeding programs. The additional costs associated with MAS often make the technology prohibitive to most aquaculture operations, despite the significant genetic gains to be realised from its implementation. Cost-optimising routine processes such as DNA extractions may be one approach to reduce these additional costs. Chelex®100 appears to be a suitable alternative to the CTAB method – being quick and cost-effective to perform. Applying this method in combination with the high throughput of a robotic platform warrants further evaluation. For the microsatellite development, 50% of positive recombinant clones contained inserts. Sequencing of these clones produced 16% perfect repeats and 47% imperfect repeats for which 52 primer sets were designed and tested. In total, 31 polymorphic microsatellite loci of different motifs and composition were developed. Sixty-one percent of sequenced clones were deemed redundant and pre-screening for both uniqueness and the presence of microsatellites would reduce unnecessary sequencing thus improving the efficiency of the FIASCO method and reducing costs. Nine loci were selected for parentage assignments. Null alleles were present for all the selected markers; however, frequencies were below the critical level of 5%. Parentage yielded 91% and 90% successful assignment for Abagold and HIK respectively; however, observations indicate that a measure of relatedness may exist between breeders. Recommendations with regards to future family breeding include, for both Abagold and HIK, retaining selected breeders based on their respective contributions to the F1 progeny while reassessing the potential of remaining breeding stock under more controlled breeding conditions. No obvious trends were observed for growth with most individuals producing both faster and slower growing offspring. Juveniles will be reassessed at two years to determine whether the size advantage or disadvantages were maintained and to ascertain whether growth advantages/disadvantages may be gender specific. / AFRIKAANSE OPSOMMING: Die vyf mees toonaangewende perlemoen produseerders in Suid Afrika het „n genetiese verbeteringsprogram vir Haliotis midae geinisieer in „n gesamentlike poging om ekonomiese belangrike eienskappe te verbeter. Verskeie onafhanklike fokus-spesifieke studies is geinisieer, insluitend die totstandkoming van „n groeiprestasie aantekenstelsel, soos gebruik in hierdie studie, en wat noodsaaklik is om die aaneenlopende prestasie van individue te moniteer soos daar beweeg word van massa seleksie tot merker bemiddelde seleksie. Die primêre fokus van hierdie studie was die ouerskapsbepaling van F1 nageslag wat massa geselekteer is op ouderdom 1 jaar vir grootte en as of “vinniger” of “stadiger” groeiers geklassifiseer is. Nege mikrosatelliet merkers is gebruik om jong perlemoen individue en moontlike ouers te genotipeer, met die ouerskapstoekenning bereken deur CERVUS 2.0. Groei resultate vir Abagold en HIK was vergelykbaar vir beide groei groepe op drie van die lokaliteite. Geringe omgewingseffekte, alhoewel nie statisties betekenisvol nie, was sigbaar as „n groei voordeel vir jong individue op twee van die vyf lokaliteite. Ongeag maatstawe om omgewingsinvloede te standardiseer, is varieerbares moeilik om te beheer in die produksie omgewing en genotipe x omgewings interaksies mag verdere navorsing vereis en behoort in ag geneem te word in toekomstige telingsprogramme. Die onkoste wat met merker bemiddelde seleksie geassosieer word, maak die tegniek soms onaantreklik vir die meeste akwakultuur operasies; nie teen staande die genetiese voordele wat die gebruik daarvan veroorsaak. Die koste-optimiseering van roetine prosesse, soos byvoorbeeld, DNA ekstraksies, is dalk een aanslag om die addisionele koste te verminder. Chelex®100 blyk „n geskikte alternatief tot die CTAB metode te wees – die tegniek is vinnig en koste-effektief om uit te voer. Die gebruik van hierdie metode in kombinasie met die hoë deurvloei van ‟n robotiese sisteem behoort verder ondersoek te word. Vir die mikrosatelliet ontwikkeling het slegs 50% van die positiewe rekombinante klone invoegings bevat. Nukleotiedvolgorde bepaling van hierdie klone het 16% perfekte herhalings en 47% onderbroke herhalings bevat waaruit 52 inleierstelle ontwikkel en getoets is. In totaal is 31 polimorfiese mikrosatelliet loki van verskillende motiewe en samestelling ontwikkel. Een-en-sestig persent van die volgorde bepaalde klone is oortollig geag en vooraf sifting vir beide uniekheid en die teenwoordigheid van mikrosatelliete sal onnodige volgorde bepaling verhoed, die effektiwiteit van die FIASCO tegniek verhoog sowel as addisionele koste verminder. Nege loki is geselekteer vir ouerskapsbepaling. Nul allele was teenwoordig vir al die geselekteerde merkers, maar die frekwensies was egter laer as die 5% kritieke waarde. Ouerskap is 91% en 90% suksesvol bepaal vir Abagold en HIK onderskeidelik. Waarnemings dui egter daarop dat daar verwantskappe mag wees tussen van die broeidiere. Voorstelle in terme van toekomstige familie teling sluit is, vir beide Abagold en HIK, om geselekteerde broei diere te behou gebaseer op hulle onderskeie bydraes tot die F1 nageslag asook die herevaluaring van die potensiaal van die oorblywende broei diere onder meer beheerde teling toestande. Geen voor-die-handliggende tendense is waargeneem vir groei nie met die meeste individue wat beide vinniger en stadiger groeiende nageslag geproduseer het. Jong individue moet geherevalueer word op tweejarige ouderdom om te bepaal of die groei voordeel of nadele behou is en om te bepaal om groei voordele/nadele geslagspesifiek is.

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 (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The species Haliotis midae is of great commercial value to the South African abalone industry and is mainly exported to Asian markets, specifically China. Up to 50% is sold as canned products with H. midae registering an average canning yield of approximately 35%. The species is presently genetically undomesticated and breeding programmes are being introduced to improve a range of production traits of which growth and yield is of primary importance. The objective of the study was to determine genetic parameters such as heritability, genotypic and phenotypic correlations of yield-related traits to assess the potential genetic improvement through selective breeding. A series of yield-related parameters were identified that is of relevance to the standard abalone canning procedure. Low to moderate heritabilities where recorded for most traits, including pre-shuck/live weight (0.20 ± 0.06), post-shuck weight (0.15 ± 0.05), post-gut weight (0.15 ± 0.05), post-brine weight (0.19 ± 0.06), pre-canning weight (0.19 ± 0.06), post-canning weight (0.21 ± 0.06), shell weight (0.16 ± 0.05), canning yield percentage (0.08 ± 0.03) and shell weight to post-gut weight ratio (SW: PGW) (0.09 ± 0.04). Weight related parameters are phenotypically highly correlated (0.86 ≤ r ≤ 0.99) but show negative correlation with canning yield percentage (-0.38 ≤ r ≤ 0.04). The nett yield of abalone shows a relatively strong positive correlation with the live weight (r = 0.66). Shell length is highly heritable (h2 ≈ 0.48) and show a strong positive correlation with live weight (r = 0.94). Shell weight is also highly correlated with live weight (r = 0.80) and the SW: PGW ratio does not show a significant correlate with live weight (r = 0.03). Weight-related traits show heritability values ranging from 0.15 to 0.20 that could allow a positive genetic response. Shell length (as a linear growth parameter) shows a high heritability (h2 ≈ 0.48) and a strong positive correlation with live weight (r = 0.94) which also makes it suitable for use as a selection criterion in breeding programmes for improved growth rate. Direct selection for canning yield is compromised by the destructive nature of measurement and the low heritability (h2 < 0.10). The negative correlations between yield as a percentage and growth traits (-0.38 ≤ r ≤ 0.04) further complicate its use as a direct breeding objective. Although the canning yield as a percentage shows a decrease with an increase in live weight, the nett canning yield increases (r = 0.66) with the live weight. It is therefore recommended to use shell length as a criterion for selection for increased growth rate and nett yield, thereby optimising profitability. / AFRIKAANSE OPSOMMING: Die spesie Haliotis midae is van groot kommersiёle waarde tot die Suid-Afrikaanse perlemoenindustrie en word meestal uitgevoer na markte in Asiё, spesifiek China. Tot 50% van die perlemoen wat in Suid-Afrika geproduseer en uitgevoer word, word verblik en huidiglik is die verblikkingsopbrengspersentasie ongeveer 35%. Haliotis midae is tans geneties onderontwikkeld en die gebruik van teelprogramme word nou geimplementeer met die doel om 'n verskeidenheid eienskappe te verbeter, waarvan groei en opbrengs van primêre belang is. Die doelwit van die studie was om genetiese parameters soos oorerflikheid en ook die genotipiese en fenotipiese korrelasies van obrengsverwante eienskappe te bepaal om sodoende die potensiёle genetiese verbetering as gevolg van selektiewe teeling te assesseer. 'n Reeks obrengsverwante eienskappe is geidentifiseer wat relevant is binne bestaande en standaard kommersiёle perlemoenverblikkingsprotokolle. Lae tot matige oorerflikheidswaardes is waargeneem en sluit in lewende, of voor-ontskulpingsgewig (0.20 ± 0.06), na-ontskulpingsgewig (0.15 ± 0.05), na-oopvlekkingsgewig (0.15 ± 0.05), na-pekelgewig (0.19 ± 0.06), voor-verblikkingsgewig (0.19 ± 0.06), na-verblikkingsgewig (0.21 ± 0.06), skulpgewig (0.16 ± 0.05), verblikkingsopbrengspersentasie (0.08 ± 0.03) en 'n skulpgewig tot na-oopvlekkingsgewig verhouding (SW: PGW) (0.09 ± 0.04). Gewigsverwante parameters is fenotipies hoogs gekorreleerd met mekaar (0.86 ≤ r ≤ 0.99) maar toon 'n negatiewe korrelasie met die verblikkingsopbrengspersentasie (-0.38 ≤ r ≤ 0.04). Die netto opbrengs van perlemoen dui op 'n relatiewe sterk positiewe korrelasie met lewende gewig (r = 0.66). Skulplengte is hoogs oorerflik (h2 ≈ 0.48) en toon 'n sterk positiewe korrelasie met lewende gewig (r = 0.94). Skulpgewig is ook hoogs gekorreleerd met lewende gewig (r = 0.80) en die SW: PGW verhouding toon geen beduidende korrelasie met lewende gewig nie (r = 0.03). Gewigsverwante eienskappe toon oorerflikheidswaardes wat varieer tussen 0.15 en 0.20 en kan 'n moontlike genetiese respons lewer. Skulplengte (as 'n liniêre groeiparameter) toon 'n hoё oorerflikheid (h2 ≈ 0.48) en 'n sterk positiewe korrelasie met lewende gewig (r = 0.94) wat dit gepas maak vir gebruik as 'n seleksiekriterium in 'n teelprogram met verbeterde groeitempo as doel. Direkte seleksie in terme van verblikkingsopbrengs word ingeboet danksy die destruktiewe natuur van die metingsmetodiek asook 'n lae oorerflikheid (h2 < 0.10). Die negatiewe korrelasies tussen verblikkingsopbrengs (uitgedruk as 'n persentasie) en groeieienskappe (-0.38 ≤ r ≤ 0.04) dien as 'n verdere komplikasie in die gebruik van dié eienskap as direkte teeldoelwit. Alhoewel die verblikkingopbrengs 'n afname toon soos lewende gewig toeneem, is daar steeds 'n positiewe korrelasie tussen die netto verblikkingsopbrengs en die lewende gewig (r = 0.66). Dit word dus aangeraai om skulplengte as seleksiekriterium vir verbeterde groeitempo en netto opbrengs te gebruik om sodoende wins te maksimaliseer.

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 (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The genetic status of H. midae broodstock in the South African aquaculture industry reflects that of random samples originating from undomesticated wild populations. The nature of growth in abalone is very slow, taking between three and four years to reach a marketable size of between 60 to 100 grams. It is therefore of paramount importance to improve this trait in order to ensure global competitiveness and economic viability within the industry. Improving this negative characteristic through conventional selection methods is a long-term venture and alternative means that would yield instantaneous results had to be considered. Crossbreeding was identified as an alternative, short-term strategy to improve growth rate. A crossbreeding experiment was performed between two populations of the abalone, Haliotis midae, from the East (E) and West (W) Coast of South Africa. This was done to investigate the occurrence of heterosis for growth among the crossbred genotypes (East x West, West x East). Fifteen males and females from both the East and the West Coast populations were mated in a complete dialelle crossbreeding experiment to produce four progeny groups (WW, EE, EW and WE). Progeny groups were evaluated for weight (bW) and length gain (bL) over a specific growth period of 9 months. The results provided no evidence of significant differences in weight (P = 0.085) or length gain (P = 0.244) between the four progeny groups, giving no indication of significant heterosis for weight and length gain among the crossbred progenies of these East and West Coast populations. It is recommended that further efforts to obtain improved growth rate in the abalone, Haliotis midae, through crossbreeding only be considered in light of clear evidence of substantial genetic differentiation between such populations. / AFRIKAANSE OPSOMMING: Die huidige status van perlemoen, soos dit voorkom in akwakultuur bedrywighede in Suid Afrika, weerspieël dié van ‘n ewekansige monster vanuit wilde, natuurlike populasies. Perlemoen is inherent ‘n stadig groeiende organisme wat tussen drie en vier jaar neem om tot ‘n bemarkbare grote van 60 tot 100 gram te groei. Dit is dus uiters noodsaaklik om hierdie eienskap te verbeter ten einde die bedryf ekonomies lewensvatbaar en mededingend op wêreld markte te maak. Konvensionele seleksie as ‘n metode om hierdie negatiewe eienskap te verbeter is ‘n langtermyn onderneming wat die identifisering van ‘n korttermyn metode, wat ondmiddellike resultate lewer, noodsaak. Kruisteelt is geïdentifiseer as geskikte korttermyn oplossing aangesien dit onmiddellike resultate lewer. ‘n Kruisteel eksperiment is uitgevoer tussen twee populasies van die perlemoen, Haliotis midae, van die Ooskus (E = East) en die Weskus (W = West) van Suid Afrika. Dit is gedoen om die omvang van heterose vir groeitempo in die gekruisde nageslag (East x West, West x East) te bepaal. Fyftien mannetjies en wyfies van beide die Oos- en Weskus populasies is met mekaar gepaar in ‘n volledige dialleel kruising om vier nageslag groepe (WW, EE, EW en WE) te vorm. Die nageslag is geëvalueer ten opsigte van massa (bW) en lengte (bL) toename oor ‘n spesifieke groei tydperk van 9 maande. Die eksperimentele resultate dui daarop dat die vier nageslag groepe nie betekenisvol van mekaar verskil het ten opsigte van massa (P = 0.085) en lengte (P = 0.244) toename nie en dat daar dus geen aanduiding van heterose vir massa en lengte toename in die nageslag van kruisings tussen die Ooskus en Weskus populasies bestaan nie. Daar word aanbeveel dat kruisteling as ‘n metode van genetiese verbetering van groeitempo in Haliotis midae slegs oorweeg word in die lig van nuwe molekulêre bewyse van genoegsame genetiese differensiasie tussen sulke populasies.