Identification of growth related quantitative Trait Loci within the abalone using comparative microsatellite bulked segregant analysis

Slabbert, Ruhan (2010-12)

Thesis (PhD (Genetics))--Stellenbosch University, 2010.

Thesis

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.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/5477
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