Linkage mapping in Haliotis midae using gene-lnked markers

Jansen, Suzaan (2012-03)

Thesis (MSc)--Stellenbosch University, 2012.

Thesis

ENGLISH ABSTRACT: Haliotis midae, or more commonly known as Perlemoen, is an abalone species found along the coast of South Africa. It is the only cultured abalone species in South Africa and has a high demand abroad. Due to its popularity as a seafood delicacy, illegal harvesting has taken its toll on Perlemoen numbers. This increases the need for sustainable farming efforts and efficient implementation of law enforcement practices against poachers. Abalone farms make use of a limited number of broodstock for breeding, so it is necessary to ensure that genetic effects such as inbreeding and bottlenecks do not interfere with the viability of the offspring. Research that focuses on the genetics of Perlemoen will greatly aid the farms to continue sustainable production of this species as well as enhance their breeding efficiency. This study focuses on the construction of a linkage map for H. midae that will allow the future identification of markers associated with genes important to production, such as growth and disease resistance. Identification of these genes will allow breeders to select genetically superior abalone that will be used for breeding programmes in which the phenotype of the offspring will be enhanced. For the construction of a linkage map it is necessary to have enough informative markers for mapping. In this study, gene-linked microsatellite markers were developed by screening a contig assembly of H. midae’s transcriptome. Ninety-eight primer pairs could be developed from the contigs and 60 loci produced amplification products. Twenty-six microsatellites were found to be polymorphic (27% success rate). In addition to these markers, 239 previously developed microsatellites and 48 gene-linked SNPs were used to develop sex-average and sex-specific linkage maps in four full-sib families consisting of approximately 100 offspring each. Of these markers 99 were informative in family DS1 (31% success rate), 81 in family DS2 (26%), 77 in family DS5 (24%) and 71 in family DS6 (23%). These markers were used for linkage analysis (LOD>3). The average number of linkage groups for the sex-average maps ranged from 17-19. The average genome length for these maps ranged from 700cM to 1100cM with an average marker spacing of 8cM. The sex-specific maps’ linkage groups ranged from 13-17 with an average genome length of 600cM to 1500cM. The average marker spacing was approximately 16cM. The integrated map was constructed by merging the sex-average maps. This map contained 25 linkage groups with an average genome length calculation of 1700cM and an average marker spacing of 9.3cM. The linkage maps created in this study are the first to utilize SNPs in H. midae. Further incorporation of SNPs into linkage maps will enhance the density. The maps created in this study are of medium-density (65%) and provide a link to the development of high-density linkage maps to facilitate associations of phenotypic traits to certain markers, to so that QTL mapping can be performed. This information can be used for marker-assisted selection to produce genetically superior abalone.

AFRIKAANSE OPSOMMING: Haliotis midae, of meer algemeen bekend as Perlemoen, is 'n klipkous spesie wat langs die kus van Suid-Afrika voorkom. Dit is die enigste gekweekte klipkous spesie in Suid-Afrika en het 'n hoë aanvraag in die buiteland. As gevolg van sy gewildheid as 'n seekos lekkerny, het onwettige stropery sy tol geneem op Perlemoen getalle. Hierdie verhoog die behoefte vir volhoubare boerdery pogings en doeltreffende implementering van wetstoepassing teen stropers. Perlemoenplase maak gebruik van 'n beperkte aantal broeidiere vir teling, dus is dit nodig om te verseker dat genetiese effekte soos inteling en genetiese bottelnekke nie inmeng met die lewensvatbaarheid van die nageslag nie. Navorsing wat fokus op die genetika van Perlemoen sal grootliks die plase steun om die volhoubare produksie van hierdie spesie voort te sit, sowel as hul teling doeltreffendheid te verbeter. Hierdie studie fokus op die ontwikkeling van 'n genetiese koppelingskaart vir H. midae, wat die toekomstige identifisering van die merkers wat verband hou met die gene wat belangrik is vir die produksie, soos groei en weerstand teen siektes sal verbeter. Identifisering van hierdie gene sal toelaat dat telers genetiese voortreflike Perlemoen kan kies vir teelprogramme waartydens die fenotipe van die nageslag sal verbeter word. Vir die ontwikkeling van 'n genetiese koppelingskaart is dit nodig om genoeg informatiewe merkers vir die kartering te hê. In hierdie studie, is geen-gekoppelde mikrosatelliet-merkers ontwikkel deur ‘contig’ data van H. midae se transkriptoom te ondersoek. Agt en negentig inleier pare kon ontwikkel word uit die ‘contigs’ en 60 loki kon ‘n amplifiseringsproduk lewer. Ses-en-twintig mikrosatelliete was polimorfies (27% suksessyfer). Bykomend tot hierdie ontwikkelde merkers is 239 voorheen ontwikkelde mikrosatelliete en 48 geen-gekoppelde SNPs gebruik om geslagsgemiddelde en geslagspesifieke koppelingskaarte in vier volsib families, wat uit ongeveer 100 nageslag elk bestaan, te ontwikkel. Van hierdie merkers was 99 informatief in familie DS1 (31%), 81 in die familie DS2 (26%), 77 in die familie DS5 (24%) en 71 in die familie DS6 (23%). Hierdie merkers is gebruik vir 'n koppelingsanalise (LOD>3). Die gemiddelde aantal koppelingsgroepe vir die geslagsgemiddelde kaarte het gewissel van 17-19. Die gemiddelde genoom lengte vir hierdie kaarte het gewissel van 700cM tot 1100cM met 'n gemiddelde merker spasiëring van 8cm. Die koppelingsgroepe van die geslagspesifieke kaarte het gewissel van 13-17 met 'n gemiddelde genoom lengte van 600cM tot 1500cM. Die gemiddelde merker spasiëring was ongeveer 16cm. Die geïntegreerde kaart is saamgestel deur die samesmelting van die geslagsgemiddelde kaarte. Die kaart toon 25 koppelingsgroepe met 'n gemiddelde berekende genoom lengte van 1700cM en' n gemiddelde merker spasiëring van 9.3cM. Die genetiese koppelingskaarte wat in hierdie studie ontwikkel is, is die eerste om SNPs te gebruik in H. midae. Verdere insluiting van SNPs in koppelingskaarte sal die digtheid verhoog. Die kaarte wat in hierdie studie ontwikkel is, is van medium digtheid (65%) en bied 'n stap nader aan die ontwikkeling van hoë digtheid koppelingskaarte om fenotipiese eienskappe met sekere merkers te assosieer, vir kwantitatiewe kenmerk lokus kartering. Hierdie inligting kan gebruik word vir merker bemiddelde seleksie om geneties verbeterde Perlemoen te produseer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/20347
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