Molecular aspects of exploited sharks in South Africa : multiple paternity and identification of novel molecular markers

Rossouw, Charne (2015-12)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: Sharks have existed for the past 400 million years and play an important role in the oceanic ecosystem as they occupy the upper categories of the food web. Since the 1920s they have been fished on a commercial scale as the demand for shark related products increased. This placed considerable pressure on shark populations, resulting in a global decline of many populations and an increased risk of population extinction. As the genetic diversity of a population determines it’s resilience to changing environmental factors, including such information has become paramount for short and long-term management and conservation of individual species. This study therefore aimed to add to the growing body of biological and genetic data by investigating mating strategy through assessing the presence of multiple paternity (MP) in three commercially important shark species: the common smoothhound Mustelus mustelus, dusky shark Carcharhinus obscurus and the scalloped hammerhead shark Sphyrna lewini; and by identifying potential microsatellite and single nucleotide polymorphism (SNP) markers in two of the species, M. mustelus and C. obscurus, through the use of Next-Generation Sequencing (NGS) platforms. The first aim of this study was achieved by cross-amplifying microsatellite markers developed in closely related species to the study species. A total of 22 microsatellite markers were initially tested on four litters of M. mustelus and C. obscurus in order to determine the most informative markers for parentage analysis. Reduced marker panels of five to six microsatellites were selected and parentage analysis in GERUD and COLONY revealed the presence of MP in all three species. Mustelus mustelus had the highest frequency of MP (67%), followed by S. lewini (46%) and C. obscurus (35%). The second aim of this study entailed reduced genome sequencing of one M. mustelus and one C. obscurus individual using the HiSeq Illumina and Ion Proton platforms, respectively. For M. mustelus, 51,5 million reads with an average read length of 250bp were obtained, whereas C. obscurus yielded 27,6 million reads with an average read length of 213bp. Contigs were constructed for both species in order to search for perfect repeat motifs. In total, 2 700 and 1 255 microsatellite-containing regions were identified for M. mustelus and C. obscurus respectively. In order to search for SNP-containing regions, both sample species were aligned to previously assembled scaffolds of the ghost shark Callorhinchus milii, which served as a reference genome. After quality filtering, only 767 SNP-containing regions were identified for M. mustelus, whereas the identification of potential SNPs for C. obscurus was not successful. The insights gained into the mating strategies of M. mustelus, C. obscurus and S. lewini as well as the identification of potential species-specific molecular markers add to the growing body of information and genetic resources available for exploited species. In future, this information could be used for further molecular assessment of shark populations and a more intergrated approach to conservation and management of these already vulnerable sharks.

AFRIKAANSE OPSOMMING: Haaie bestaan reeds vir die afgelope 400 miljoen jaar en speel 'n belangrike rol in die oseane se ekosisteme omdat hulle die boonste kategorie van die voedselweb in beset neem. Sedert die 1920’s word haaie op 'n kommersiele vlak gevis soos die aanvraag vir haai verwante produkte toeneem. Dit het aansienlike druk op haai populasies geplaas wat ‘n globale afname van verskeie populasies se getalle tot gevolg gehad het en hul kans op uitwissing vergroot het. Omdat die genetiese diversiteit van 'n populasie die weerstandigheid van 'n populasie teen veranderinge in die omgewing bepaal, het dit toenemend belangrik geword om sogenaamde inligting in te sluit vir kort en langtermyn bestuur en bewaring van die spesie. Hierdie studie het daarom gepoog om tot die groeiende liggaam van biologiese sowel as genetiese data by te dra deur die voortplantingsstrategieë te ondersoek in terme van die teenwoordigheid van veelvoudige vaderskap (VV) in drie kommersiele belangrike haai spesies: die algemene hondhaai Mustelus mustelus, donkerhaai Carcharhinus obscurus en die skulprand-hammerkophaai Sphyrna lewini te asseseer; en deur moontlike mikrosatelliet en enkel-nukleotied-polimorfiese merkers te identifiseer in M. mustelus en C. obscurus deur die gebruik van volgende-generasie volgorde bepalings platforms (NGS). Die eertse doelwit van die studie is bereik deur die kruisamplifisering van mikrosatelliet merkers wat ontwikkel is in nabyverwante spesies tot die studiespesies. 'n Totaal van 22 mikrosatelliet merkers is op vier werpsels M. mustelus en C. obscurus getoets om die mees informatiewe mikrosatelliete vas te stel vir ouerskaptoetse. Verkleinde merkerpanele van vyf tot ses mikrosatelliete is geselekteer en ouerskap analises in GERUD en COLONY het die teenwoordigheid van VV in al drie spesies vasgestel. Mustelus mustelus het die hoogste frekwensie van VV gehad (67%), gevolg deur S. lewini (46%) en C. obscurus (35%). Die tweede doelwit van die studie het die verkleinde genoom volgorde bepaling van een M. mustelus en een C. obscurus individu behels deur gebruik te maak van beide 'n HiSeq Illumina en Ion Proton platvorm. Vir M. mustelus is 51,5 miljoen lesings met 'n gemiddelde lengte van 250bp en 27,6 miljoen lesings met 'n gemiddelde lengte van 213bp vir C. obscurus verkry. Contigs is vir beide spesies gebou om vir perfekte mikrosatellietstreke te soek. In totaal is 2 700 en 1 255 mikrosatellietstreke is vir M. mustelus en C. obscurus geidentifiseer. Beide spesies is tot die voorafontwikkelde Callorhinchus milii stelasies belyn (wat as 'n verwysingsvolgorde gedien het) om vir SNP-streke te soek. Na kwaliteitsfiltering is slegs 767 SNP-streke vir M. muselus geidentifiseer terwyl SNP-streek identifikasie vir C. obscurus nie suksesvol was. Die insig wat oor die voortplantingsstrategieë vir M. mustelus, C. obscurus en S. lewini verkry is asook die identifisering van moontlike spesie-spesifieke molekulêre merkers sal bydra tot die groeiende liggaam van data en genetiese bronne wat beskikbaar is vir hierdie uitgebuite haai spesies. In die toekoms kan hierdie inligting vir die verdere molekulêre assessering van haai populasies gebruik word om sodoende ‘n meer geïtegreerde benadering tot die bewaring en bestuur van hierdie reeds kwesbare haaie te weeg te bring.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/97758
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