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Characterisation of Snps Associated with Growth Rate in Dusky Kob (Argyrosomus Japonicus), Using Exome Sequencing

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Characterisation of Snps Associated with Growth Rate in Dusky Kob (Argyrosomus Japonicus), Using Exome Sequencing Characterisation of SNPs associated with growth rate in dusky kob (Argyrosomus japonicus), using exome sequencing by Tassin Jackson Thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Stellenbosch University Supervisor: Clint Rhode, Ph.D., Pr.Sci.Nat. Department of Genetics March 2020 Stellenbosch University https://scholar.sun.ac.za Declaration By submitting this thesis electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), that reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. March 2020 Copyright © 2020 Stellenbosch University All rights reserved I Stellenbosch University https://scholar.sun.ac.za Abstract Marine living-resources such as dusky kob, (Argyrosomus japonicus) are particularly vulnerable to overfishing as this species has been targeted for decades by commercial, recreational and subsistence fisheries, which has led to the steady decline in the natural populations. A shift towards aquaculture as a sustainable alternative supply to the market has been initiated, with considerable efforts being made to understand the fundamental role that genes play in the biological processes influencing complex traits such as growth rate. Although a few studies have been conducted on the species, they have been hindered by the limited number of genomic resources, which is an issue that affects many non-model species. Therefore, this study aimed to investigate the transferability of a model organism’s exon capture kit in a non-model species for the development of SNP markers associated with growth. By using 16 dusky kob individuals for exome sequencing this study was able to capture 6,623 of the 346,263 exons found within the model organisms, zebrafish, as well as a large number of exons that could potentially be species-specific. Overall, the exome data proved to be a valuable resource for the identification of variants, with variant detection identifying 4.5 million potential molecular makers with a total of 2.8 million putative SNPs and 3,276 tandem repeats. These variants were spread across the exome regions with a SNP occurring approximately every 1000 nt. Using the candidate gene approach and a selection of 15 gene regions, 263 putative SNPs were identified, of which 38 SNPs in nine genes were confirmed using Sanger sequencing and identified as having a potential association to the trait of interest. Association of these markers was analysed by performing both case-control and quantitative analyses using 80 individuals (classified as large and small) of dusky kob. These analyses were able to identify eight SNPs in three key genes. This study demonstrated the ability of exon capture to be customised for cross-species capture to assist in molecular marker discovery for non-model organisms with limited or no genomic resources. Resources which could be used for the development of markers which could assist in the implementation of marker assisted selection (MAS), which will aid in the development and effective utilisation of the species. II Stellenbosch University https://scholar.sun.ac.za Opsomming Mariene lewende hulpbronne soos die boerkabeljou (Argyrosomus japonicus) is veral kwesbaar vir oorbevissing, aangesien hierdie spesie al dekades lank deur kommersiële, ontspannings- en bestaansvisserye geteiken word, wat gelei het tot die bestendige afname van die natuurlike populasie. Akwakultuur bied 'n volhoubare alternatiewe oplossing aan die mark, en toenemende pogings word aangewend om die fundamentele rol van gene in biologiese prosesse van komplekse eienskappe, soos groeitempo, te verstaan. Ongelukkig word studies in hierdie spesie, net soos in ander nie-modelspesies, belemmer deur die beskikbaarheid van ‘n beperkte aantal genomiese hulpbronne. Daarom het hierdie studie ten doel gehad om die oordraagbaarheid van die eksonvangsstel (“exon capture kit”) van 'n modelorganisme in 'n nie-modelspesie te ondersoek, met die oog op die ontwikkeling van ENP-merkers wat met groeitempo geassosieer word. Hierdie studie het deur middel van eksoomvolgordebepaling op 16 boerkabeljou individue daarin geslaag om 6,623 uit 346,263 eksone van die model organisme, zebravis, sowel as ʼn groot aantal moontlike spesie- spesfieke eksone vas te vang. Die ontdekking van 4.5 miljoen potensiele molekulere merkers, waarvan 2.8 miljoen moontlike ENP merkers en 3,726 tandem herhalings, dui daarop aan dat die eksoomdata ʼn waardevolle hulpbron vir die identifisering van genetiese variasie is. Hierdie variante was verspreid oor die eksoomareas, met ʼn ENP wat ongeveer elke 1000 nt voorkom. Met behulp van die kandidaatgeenbenadering en ʼn seleksie wat 15 geenstreke behels, is 263 veronderstelde ENPs geidentifiseer, waarvan 38 ENPs in nege gene van sanger-volgorde bevestig was, en getoon het om moontlike assosiasie met die eienskap van belangstelling, groei, te toon. Bimodale gevallestudie en kwantitatiewe analises is uitgevoer deur gebruik te maak van 80 boerkabeljou individue (wat geklassifiseer is as klein en groot) om die assosiasie tussen merkers en groei te ondersoek. Hierdie analises het gelei tot die identifisering van ag ENPs in drie sleutelgene. Hierdie studie het getoon dat dit moontlik is om ʼn eksonvangsstel aan te pas vir gebruik in ander spesies om te help met die ontdekking van molekulere merkers in nie-model organismes met beperkte of geen genomiese hulpbronne. Daarmee help hierdie studie om genomiese hulpbronne op te bou, wat kan lei tot die ontwikkeling van molekulere merkers wat gebruik kan word om merker bemiddelde seleksie (MBS) toe te pas, om sodoende die optimale benutting van hierdie spesie te bereik. III Stellenbosch University https://scholar.sun.ac.za Acknowledgements I would like to extend my gratitude to the Department of Science and Technology, the National Research Foundation of South Africa, and Stellenbosch University for financial support. My gratitude also goes out to the members of the Molecular Breeding and Biodiversity research group for all their help and support. To my supervisor Dr Clint Rhode who always pushed me to be the best that I can be. Thank you for all the knowledge and inspiration over the last few years. Finally, I would like to thank my family, partner and friends for their support, particularly during the final stages of thesis writing, I could not have done it without each and every one of you. IV Stellenbosch University https://scholar.sun.ac.za Table of Contents CHAPTER 1 Introduction: Literature Review, Aims and Objectives ..................................... 1 1.1) Species biology: An introduction to dusky kob (Argyrosomus japonicus) .................... 1 1.1.1) Classification and Evolution of Dusky Kob ...................................................... 1 1.1.2) Ecology, Distribution and Life-History in South Africa ..................................... 2 1.2) Aquaculture of the Finfish, Dusky Kob ......................................................................... 5 1.2.1) Classification and Evolution of Dusky Kob ........................................................... 5 1.2.2) Ecology, Distribution and Life-History in South Africa .......................................... 7 1.3) Molecular Markers ..................................................................................................... 10 1.4) SNP development strategies and genotypic technologies ......................................... 13 1.5) Application of SNPs in aquaculture............................................................................ 17 1.5.1) Individual identification, Pedigree inference and Population Assessments ....... 17 1.5.2) Loci Associated with Complex Traits in Aquaculture and Marker-Assisted Selection ...................................................................................................................... 19 1.6) Study rationale, aims and objective ........................................................................... 21 1.6.1) Problem Statement ............................................................................................ 21 1.6.2) Aims and Objectives .......................................................................................... 22 References ........................................................................................................................ 23 CHAPTER 2 Transferability of a model organisms’ solution-based exon-capture kit, in the non-model organism, dusky kob ........................................................................................ 45 Abstract .............................................................................................................................. 45 2.1) Introduction ................................................................................................................ 46 2.2) Methods and Materials .............................................................................................. 47 2.2.1) Study populations and DNA extraction .............................................................. 47 2.2.2) Library construction and sequencing ................................................................. 48 V Stellenbosch University https://scholar.sun.ac.za 2.2.3) Assembly and analysis pipeline ........................................................................
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