Transcriptome analysis of the New Zealand sea urchin Kina (Evechinus chloroticus) Gareth B Gillard A thesis submitted for the degree of Master of Science at the University of Otago, Dunedin, New Zealand. February 4th 2014 1 Abstract Sea urchins are studied as model organisms for developmental and systems biology, and the sequencing of one species’ genome revealed many avenues for further studies. Sea urchins’ highly expanded innate immune system is a valuable resource for antimicrobials. Furthermore, the gonads of male and female sea urchins, referred to as the roe, are a highly valued food product internationally, with the largest demand from Asian countries such as Japan. The price of roe is greatly influenced by sensory qualities such as colour, texture and flavour, and research has focused on increasing roe quality to give it higher value. Evechinus chloroticus (Kina) is a sea urchin species that is indigenous to New Zealand. It is the type member of the Evechinus genus based on its morphological characteristics. Previous research has focused on identifying physical factors affecting the commercial roe quality of E. chloroticus, including protein and metabolite analysis, but there is almost no genetic information available for this species. E. chloroticus is the only species in its genus and has yet to be subjected to molecular phylogenetic analysis. This study aimed to increase the amount of genetic data available for the E. chloroticus species through transcriptome assembly and analysis, which would aid current and future research on E. chloroticus. In this study, a de novo transcriptome assembly of Illumina sequencing data was carried out. A total of 123 million 100 base length paired-end reads were generated using RNA-Seq libraries from a range of E. chloroticus tissues from two individuals obtained from Fiordland, New Zealand. The assembly resulted in a set of 75,002 transcripts with an accepted read coverage and length, of which 24,655 transcripts could be functionally annotated using protein similarity. Transcripts were further annotated with Gene Ontology, KEGG Orthology and InterPro terms. The first molecular phylogenetic analysis of E. chloroticus to other species of its family was done using multiple genes. When sequences for the mitochondrial NADH dehydrogenase genes were compared, E. chloroticus remained outside of a family level clade, which indicated E. chloroticus is indeed a genetically distinct genus within its family. A differential gene expression analysis was carried out using the new reference transcriptome data. RNA-Seq libraries were generated from the roe of individual E. chloroticus females that showed a distinct grade of roe colour (light, medium or dark) and the expression of genes between different coloured roe was analysed. The results showed most gene expression 2 in the roe remains consistent regardless of the roe colour, with a small proportion of genes shown to have differential expression. Differentially expressed genes could now be investigated to understand the role of genes whose expression correlates with colour. This study has produced a large set of E. chloroticus transcripts (and predicted proteins) along with functional annotations, vastly increasing the amount of genomic data available for this species. This provides a resource for current and future studies on E. chloroticus, either to increase its commercial value, or its use as a model organism. The phylogenetic results provide a basis for further analysis of relationships between E. chloroticus and its family members, and its evolutionary history. The differential gene expression results provide the first transcriptome analysis of differences in roe colour and the data will be valuable for current and future molecular research on roe colour qualities. 3 Table of contents Abstract .................................................................................. 2 Table of contents .................................................................... 4 List of figures ......................................................................... 7 List of tables ........................................................................... 8 List of Abbreviations .............................................................. 8 Chapter 1 - Introduction ....................................................... 10 1.1 The sea urchin ......................................................................................... 10 1.1.1 The Echinodermata phylum ..................................................................................... 10 1.1.2 The sea urchin as a model organism ........................................................................ 10 1.1.3 Biology of the sea urchin ......................................................................................... 11 1.1.4 Sea urchin roe as a valued food product .................................................................. 13 1.2 Evechinus chloroticus, the New Zealand sea urchin ................................ 14 1.2.1 Ecology of E. chloroticus ........................................................................................ 14 1.2.2 The marketplace and roe quality .............................................................................. 15 1.2.3 Pigmentation in E. chloroticus roe .......................................................................... 16 1.2.4 Phylogeny of E. chloroticus .................................................................................... 18 1.3 Transcriptome analysis study .................................................................. 19 1.3.1 Transcriptome analysis of E. chloroticus ................................................................ 19 1.3.2 RNA-Seq.................................................................................................................. 19 1.3.3 De novo assembly analysis ...................................................................................... 20 1.3.4 Function annotation ................................................................................................. 22 1.3.5 Phylogenetic analysis............................................................................................... 22 1.5.6 Differential expression analysis using RNA-Seq .................................................... 23 1.4 Research goals ........................................................................................ 24 1.5 Aims ....................................................................................................... 24 Chapter 2 - Methods ............................................................. 25 2.1 Transcriptome assembly .......................................................................... 25 2.1.1 Sample collection..................................................................................................... 25 2.1.2 RNA extraction and sequencing .............................................................................. 26 4 2.1.3 Quality analysis ........................................................................................................ 27 2.1.4 De novo assembly..................................................................................................... 29 2.1.5 Post-processing transcripts ....................................................................................... 30 2.1.6 Assembly completeness ........................................................................................... 31 2.2 Functional annotation .............................................................................. 32 2.2.1 Protein similarity search ........................................................................................... 32 2.2.2 Functional term annotations ..................................................................................... 33 2.2.3 Unannotated transcript analysis ............................................................................... 34 2.3 Phylogenetic analysis .............................................................................. 35 2.3.1 Identification of evolutionary conserved genes for E. chloroticus .......................... 35 2.3.2 Sequence alignments ................................................................................................ 36 2.3.3 Phylogenetic trees .................................................................................................... 37 2.4 Gene expression analysis in different coloured roe .................................. 37 2.4.1 Sample selection and RNA sequencing ................................................................... 38 2.4.2 Pooled sample analysis............................................................................................. 38 2.4.3 Quality analysis ........................................................................................................ 39 2.4.4 Read alignment and novel transcript assembly ........................................................ 39 2.4.5 Count data generation............................................................................................... 40 2.4.6 Most abundant transcripts in female roe .................................................................. 41 2.4.7 Differential expression analysis of genes ................................................................. 41 2.4.8 Gene set analysis of functional terms ....................................................................... 42 2.4.9 Cluster analysis of gene expression ........................................................................