Characterization of the mitochondrial genomes of Diuraphis noxia biotypes By Laura de Jager Thesis presented in partial fulfilment of the requirements for the degree Magister Scientiae In the Faculty of Natural and Agricultural Science Department of Genetics University of Stellenbosch Stellenbosch Under the supervision of Prof. AM Botha-Oberholster December 2014 Stellenbosch University http://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. Date: . 25 December 2014. Copyright 2014 Stellenbosch University All rights reserved ii Stellenbosch University http://scholar.sun.ac.za Abstract Diuraphis noxia (Kurdjumov, Hemiptera, Aphididae) commonly known as the Russian wheat aphid (RWA), is a small phloem-feeding pest of wheat (Triticum aestivum L). Virulent D. noxia biotypes that are able to feed on previously resistant wheat cultivars continue to develop and therefor the identification of factors contributing to virulence is vital. Since energy metabolism plays a key role in the survival of organisms, genes and processes involved in the production and regulation of energy may be key contributors to virulence: such as mitochondria and the NAD+/NADH that reflects the health and metabolic activities of a cell. The involvement of carotenoids in the generation of energy through a photosynthesis- like process may be an important factor, as well as its contribution to aphid immunity through mediation of oxidative stress. The complete mitochondrial genome of global Diuraphis noxia populations was characterised using Next Generation sequencing, and was found to be 15 721bp in size and consisting of 38 genes typically found within most insects. Single nucleotide polymorphism (SNP) analyses of the genomes of nine populations revealed 125 SNPs in the protein coding genes with the majority of the SNPs occurring in the ND genes, and the least in the ND4L gene. Low SNP variant frequency was found for the atp6 and atp8 genes, which differed from other reports in the Hemiptera. Variable ND5 expression levels were observed among the biotypes, although no correlation was apparent between ND5 expression and the virulence associated with each biotype. Whereas atp6 transcription was higher in the highly virulent biotype (SAM) under normal and stressful conditions in comparison to the least virulent biotype (SA1). A significantly higher NAD+/NADH ratio was also observed for the SAM biotype under stressful conditions in comparison to the lesser virulent biotypes. UPLC-MS analysis did not reveal any lycopene or β-carotene due to low compound concentrations in the extracted samples but various hydrophobic compounds were present in different concentrations among the biotypes. The carotene desaturase expression profile revealed that SA1 had the lowest relative expression of the gene involved in carotenoid products, while SAM had the highest, under normal and stressful conditions. The results indicate that sequence conservation in mitochondrial genes are associated with key energy processes to maintain a state of homeostasis under variable conditions and that the generation of energy is a contributing factor to the virulence development of D. noxia. The results also show that carotenoids may possibly contribute to fitness of D. noxia through reactive oxygen species scavenging or the production of additional energy, but further investigation is needed for confirmation. iii Stellenbosch University http://scholar.sun.ac.za Uittreksel Diuraphis noxia (Kurdjumov, Hemiptera, Aphididae) algemeen bekend as die Russiese koringluis (RWA), is ‘n klein floëem-voedende pes van koring (Triticum aestivum L). Virulente D. noxia biotipes wat instaat is om op voorheen bestande koring kultivars te voed gaan ontwikkel voortdurend, en daarom is die identifisering van faktore wat kan bydrae tot virulensie so belangrik. Omdat energie-metabolisme ‘n sleutelrol in die oorlewing van organismes speel, kan gene en prosesse wat by die produksie en regulering van energie betrokke is belangrike bydraers tot virulensie lewer: soos onder andere mitokondria en die NAD+/NADH-verhouding wat die gesondheid en metaboliese aktiwiteit van ‘n sel reflekteer. Die betrokkenheid van karotenoïede in die produksie van energie deur 'n fotosintese- verwante proses kan 'n belangrike faktor bydraend tot luis fiksheid wees, asook die bydra daarvan tot plantluis-immuniteit deur bemiddeling van oksidatiewe stres. Die volledige mitochondriale genoom van globale Diuraphis noxia populasies is met behulp van volgende generasie DNA volgordebepaling gekarrakteriseer, en daar is bevind dat dit 15 721 bp in grootte is en uit 38 gene bestaan wat tipies binne insekte voorkom. Enkelnukleotied- polimorfisme (SNP) ontleding van die genome van nege populasies het onthul dat daar 125 SNPs in die proteïen-koderende gene voorkom, met die meerderheid van die SNPs in die ND-gene, en die minste in die ND4L-geen. Lae SNP-frekwensies is gevind vir die atp6- en atp8- gene, wat verskil van verslae oor ander Hemiptera. Veranderlike ND5- uitdrukkingsvlakke onder die biotipes is waargeneem, alhoewel geen korrelasie duidelik was tussen ND5-uitdrukking en die virulensie geassosieer met elke biotipe nie. Die transkripsie van atp6 was hoër in die hoogs virulente biotipe (SAM) onder normale en stresvolle toestande in vergelyking met die minste virulente biotipe (SA1). ‘n Aansienlike hoër NAD+/NADH-verhouding is ook waargeneem vir die SAM-biotipe onder spanningsvolle omstandighede in vergelyking met die minder virulente biotipes. UPLC-MS-analise het geen likopeen of β-karoteen geïdentifiseer nie as gevolg van lae verbinding konsentrasies in die onttrekte monsters, maar verskeie hidrofobiese verbindings was in verskillende konsentrasies tussen die biotipes teenwoordig. Die karoteen desaturase-uitdrukkingsprofiel het aangetoon dat SA1 die laagste relatiewe uitdrukking van gene betrokke by karotenoïed produksie het, terwyl SAM die hoogste relatiewe uitdrukking onder normale en spanningsvolle omstandighede het. Die resultate van die studie dui daarop dat die volgorde bewaring in mitochondriale gene verband hou met die sleutel energie prosesse om 'n toestand van homeostase onder wisselende omstandighede te handhaaf en dat die produksie van energie 'n bydraende faktor tot die ontwikkeling van virulensie in D. noxia is. Die resultate toon ook aan dat karotenoïede moontlik kan bydra tot fiksheid van D. noxia iv Stellenbosch University http://scholar.sun.ac.za deur reaktiewe suurstofspesies te aas of deur die produksie van addisionele energie, maar verdere ondersoeke word benodig ter bevestiging. v Stellenbosch University http://scholar.sun.ac.za Acknowledgements I would like to acknowledge and express my sincere gratitude to the following people and organisation for their support, advice and patience throughout the duration of this study. Professor Anna-Maria Botha- Oberholster whose expertise and vast knowledge made this study a reality. I appreciate the time and effort you put into guiding and helping me with my project, i.e., the writing of this thesis, with lab work, scholarships and the preperation of presentations. I want to especially express my gratitude for the opportunity to have attended an international conference in Morocco, I will forever have fond memories of that experience. Thank you so much. Dr. Marietjie Stander at the Central Analytical Facility for the UPLC-MS analysis of carotenoids in the extracted D. noxia samples. Colleagues in the Cereal Genomic Research Group: Francois Burger, Anandi Bierman, Nadia Fisher, Kelly Breeds, Louis Steyn, Kenneth Mbwanji, Wynand Cloete, Marlon Le Roux and Ilze Visser. Department of Genetics for providing this learning opportunity and infrastructure. The National Research Foundation of South Africa (NRF) for financial assistance during this project. To my parents, Neil and Lauraine de Jager, for their endless love, support and motivation throughout my many years of studying. To my biggest enthusiast and the love of my life, Rickus Visser, for supporting me in achieving my goals. I appreciate your constant love and encouragement the last few years. vi Stellenbosch University http://scholar.sun.ac.za Table of Contents Declaration ii Abstract iii Uittreksel iv Acknowledgements vi Table of Contents vii List of Figures x List of Tables xiii List of Abbreviations xiv Chapter 1 Introduction 1.1 Introduction 2 1.2 Preface 5 1.3 Reference list 6 Chapter 2 Literature Review 2.1 Aphids 8 2.2 Diuraphis noxia (D. noxia) 10 2.2.1 D. noxia –Host 10 2.2.2 D. noxia overview 11 2.2.3 D. noxia – Wheat interaction 12 2.2.4 D. noxia biotypes 14 2.3 Phylogeny 17 2.4 The Mitochondrion 18 2.4.1 Endosymbiont Theory 18 2.4.2 Mitochondrial Structure 19 2.4.3 Mitochondrial Genome Structure 20 2.4.4 Functionality of Mitochondria 22 2.5 Adaptations of aphids 23 2.5.1 Carotenoids 23 2.5.2 Phenotypic polyphenism 26 2.5.3 Wing polyphenism 27 2.5.4 Heat stress 28 2.6 DNA Sequencing Technologies 29 vii Stellenbosch University http://scholar.sun.ac.za 2.7 Reference list 31 Chapter 3 Complete mitochondrial genome of Diuraphis noxia (Hemiptera: Aphididae) from nine populations, SNP variation between populations, and comparison with other Aphididae species 3.1 Introduction 46 3.2 Materials and Methods 48 3.2.1 Aphid sampling and DNA extraction 48 3.2.2 Library construction and sequencing 48 3.2.3 Sequence assembly and annotation 49 3.2.4 Phylogenetic Analyses 51 3.3 Results 52 3.3.1 Phylogenetic analysis of the Hemiptera, Sternorrhyncha 52 3.3.2 Genome structure, organization, and composition 54 3.3.3 Transfer RNA and ribosomal RNA genes 56 3.3.4 Comparisons between D. noxia and its closest relatives 59 S. graminum and A. pisum 3.3.5 Genetic variants amongst D. noxia biotypes 60 3.4 Discussion 61 3.5 Reference list 66 3.6 Appendix A 72 Chapter 4 Association of mitochondrial and carotenoid genes to the energy metabolism of D.