Medium-throughput SNP genotyping and linkage mapping in Haliotis midae

Du Plessis, Jana (2012-12)

Thesis (MSc)--Stellenbosch University, 2012.

Thesis

ENGLISH ABSTRACT: Haliotis midae (locally also known as perlemoen) is the largest of five endemic species found along the coast of South Africa. It is the only species with commercial value contributing to the exploitation of these animals. Due to declines of natural stocks, farming practices were established during the early 1990s in order to supply the international demand. To facilitate efficient breeding methods and ensure the sustainability of these commercial populations, genetic management, which can be accomplished with the use of molecular markers such as single nucleotide polymorphisms (SNPs), is necessary. Single nucleotide polymorphisms have become the markers of choice in various applications in aquaculture genetics due to their abundance in genomes, reduction in developmental costs and increased throughput of genotyping assays. Identification of SNPs in non-model species such as H. midae can be achieved by in silico approaches. In silico methods are suitable for de novo SNP identification and are both cost- and time-efficient. It is based on the analysis of multiple alignments where mismatches may be reported as candidate SNPs. Various medium-throughput genotyping methods are available to confirm putative SNPs, but the ideal method depends on factors such as cost, accuracy and multiplexing capacity. Although SNP markers can have various applications within the aquaculture environment the focus for this current study was saturating the linkage map of H. midae with additional markers. This would assist in the identification of quantitative trait loci associated with economically important traits, which in turn could ultimately be employed for marker-assisted selection and improved molecular breeding programs. In order to identify in silico SNPs, sequenced transcriptome data from a previous study was used and subjected to a series of criteria: minor allele frequency 10%, minimum coverage 80, 60 bp flanking regions. Selected loci were genotyped using a 192-plex assay with the Illumina GoldenGate genotyping assay with the VeraCode technology on the BeadXpress platform, in individuals from six mapping families. A conversion rate of 69.35% and global success rate of 76.34% was achieved. Polymorphic loci were subjected to linkage analysis using JoinMap® v.4.1 to create sex-average and sex-specific maps and to saturate the current linkage map for H. midae. Along with previously developed markers, 54% of the newly developed SNPs could be successfully incorporated into the linkage map of H. midae. A total of 18 linkage groups were observed with an average marker spacing of 6.9 cM and genome coverage of 79.1%. Bioinformatic analyses and setting stringent criteria to identify SNPs from sequenced transcriptomic data proved to be an efficient way for SNP discovery in the current study. Genotyping of the identified loci with the GoldenGate genotyping assay demonstrated a high success rate; providing a genotyping assay adequate for species with little genomic information. The linkage map created in this study illustrated the utility of SNP markers in conjunction with microsatellite markers for linkage map construction and the adequate marker spacing obtained provides a step closer to quantitative trait loci mapping in this species.

AFRIKAANSE OPSOMMING: Haliotis midae (plaaslik ook bekend as perlemoen) is die grootste van vyf inheemse spesies wat langs die kus van Suid-Afrika aangetref word. Dit is die enigste spesie van kommersiële waarde wat bydraend is tot die uitbuiting van hierdie diere. As gevolg van die afname in hierdie natuurlike hulpbron het boerdery praktyke gedurende die vroeë 1990's ontstaan om in die internasionale aanvraag te voorsien. Ten einde doeltreffende teelmetodes te beoefen en die volhoubaarheid van hierdie kommersiële populasies te verseker is genetiese bestuur, wat bewerkstellig kan word deur die gebruik van molekulêre merkers soos enkel nukleotied polimorfismes (ENPs), baie belangrik. Enkel nukleotied polimorfismes is gewilde merkers in verskeie toepassings in akwakultuur genetika as gevolg van hul oorvloed in genome, verlaagde ontwikkelingskoste en verhoogde deurset van ENP-genotiperingstoetse. Identifisering van ENPs in nie-model spesies soos H. midae kan uitgevoer word deur in siliko benaderings te gebruik wat geskik is vir de novo ENP identifisering en ook tyd- en koste-effektief is. Dit word gebaseer op die analise van veelvuldige inlynstellings waar nukleotiedes wat nie ooreenstem nie as kandidaat ENPs gerapporteer kan word. Om kandidaat ENPs te bevestig, kan verskeie medium-deurset genotiperingsmetodes uitgevoer word, maar die ideale metode word bepaal deur faktore soos koste, akkuraatheid en multipleks kapasiteit. Alhoewel ENP merkers in verskeie toepassing binne die akwakultuur omgewing gebruik kan word was die fokus van die huidige studie om die koppelingskaart van H. midae te versadig. Dit sal bydrae tot die identifisering van kwantitatiewe eienskap lokusse wat gekoppel kan word aan ekonomies belangrike eienskappe wat dan op die beurt weer vir merkerbemiddelde seleksie gebruik kan word en uiteindelik ten opsigte van die verbetering van molekulêre teelprogramme aangewend kan word. Ten einde in siliko ENPs te identifiseer is transkriptoomdata van 'n vorige studie gebruik en onderwerp aan 'n reeks kriteria: geringste alleelfrekwensie 10%, minimum dekking 80, 60 bp gebiede weerskante van polimorfisme. Geïdentifiseerde lokus-genotipering is met behulp van 'n 192-pleks toets uitgevoer met die Illumina GoldenGate genotiperingstoets met die VeraCode tegnologie op die BeadXpress-platform, in individue afkomsitg vanaf ses karteringsfamilies. 'n Omskakelingskoers van 69.35% en 'n algehele sukseskoers van 76.34% is bereik. Polimorfiese lokusse is onderwerp aan koppelings-analise met behulp van JoinMap® v.4.1 om geslags-gemiddelde en geslags-spesifieke kaarte te skep asook om die kaart wat beskikbaar is vir H. midae te versadig. Saam met voorheen ontwikkelde merkers is 54% van die nuut ontwikkelde ENPs suksesvol opgeneem in die kaart van H. midae. 'n Totaal van 18 koppelingsgroepe is verkry met 'n gemiddelde merker-spasiëring van 6.9 cM en 'n genoomdekking van 79.1%. Die gebruik van bioinformatiese analises en streng kriteria om ENPs vanaf transkriptoomdata te identifiseer blyk doeltreffend te wees in hierdie studie. Genotipering van die geïdentifiseerde lokusse met die GoldenGate genotiperingstoets dui op 'n hoë suksessyfer en verskaf 'n voldoende genotiperingstoets aan spesies met min genomiese inligting. Die koppelingskaart in hierdie studie het geïllustreer dat die ENP merkers suksesvol saam met mikrosatelliet merkers gebruik kan word vir koppelingskaart konstruksie en dat die voldoende merker-spasiëring verkry 'n stap nader aan kwantitatiewe eienskap lokus kartering in hierdie spesie bied.

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