The transcriptome of the NZ endemic sea urchin Kina (Evechinus chloroticus)
© Gillard et al.; licensee BioMed Central Ltd. 2014
Received: 29 October 2013
Accepted: 30 December 2013
Published: 20 January 2014
Sea urchins are studied as model organisms for developmental and systems biology and also produce highly valued food products. 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 commercial roe quality of E. chloroticus, but there is almost no genetic information available for E. chloroticus. E. chloroticus is the only species in its genus and has yet to be subject to molecular phylogenetic analysis.
In this study we performed a de novo transcriptome assembly of Illumina sequencing data. 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 could be further annotated with Gene Ontology, KEGG Orthology and InterPro terms. With this sequence data we could perform the first phylogenetic analysis of E. chloroticus to other species of its family using multiple genes. When sequences for the mitochondrial nitrogen 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.
This study has produced a large set of E. chloroticus transcripts/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, its family members, and its evolutionary history.
New Zealand coastal zones contain an abundant population of the native sea urchin species Evechinus chloroticus, locally known by the Maori name, Kina. It is the type member of its genus, but there has been little molecular analysis performed on this species to date. Sea urchin roe (gonads of male and female sea urchins) is a highly valued food product internationally, with the largest demand coming from Japan where the roe, locally known as “uni”, is used for sushi. The demand for sea urchin roe has grown as Japanese food increases in popularity in the North American food industry . Sea urchin roe is considered a high quality food product with the price greatly influenced by factors such as appearance, colour, texture, and flavour . E. chloroticus is fished off the coasts of New Zealand both commercially and recreationally for its roe. The reported commercial catch of E. chloroticus for 2013 was 875,031 kilograms, mainly obtained off the South Coast . The majority of E. chloroticus roe is sold in New Zealand as a local delicacy, with some exports sent to Australia .
There has been research interest in enhancing the quality and yield of E. chloroticus roe to increase the return value for the domestic market. This would also potentially create opportunities for exportation to overseas markets that demand specific qualities in roe . Although most assessments of sea urchin roe quality have previously been on physical differences, recent studies sought to identify protein and metabolite differences contributing to variation in quality for E. chloroticus roe, specifically to variations in colour . It was hypothesised from the results that binding proteins targeting carotenoid molecules, the major source of pigmentation in roe, might affect colour. Efforts have thus been made to identify carotenoid binding proteins in the roe . Despite the research interest in E. chloroticus, very little genetic information was available for this species in public databases. There was an opportunity to develop genetic data for E. chloroticus, which would facilitate a genomic based analysis. For this purpose, we conducted a whole transcriptome sequencing project aiming to characterise transcripts from various tissues. This would provide a source of genetic information to aid current and future research involving E. chloroticus.
As well as a valued food product, sea urchins have long been used as a model organism in areas such as developmental and systems biology. Their importance as a research model system for modern molecular, evolutionary, and cell biology led to the genome sequencing project for the sea urchin species Strongylocentrotus purpuratus. The genome was estimated to encode around 23,300 genes, and was shown to share many pathways with humans including orthologs to human disease genes. Also discovered was the lack of an adaptive immune system, and instead the possession of a large innate immune system that contained a diverse range of pathogen-binding motifs . The many innate immune proteins encoded in the sea urchin genome are considered a valuable resource for antimicrobial applications and for furthering our understanding of the human innate immune system . Gene structure in the S. purpuratus genome has subsequently been further defined by transcriptome analysis , but aside from S. purpuratus there is little genomic data available for any other sea urchin species from public databases. Genomic data produced for the E. chloroticus species would be a novel resource in addition to the S. purpuratus data for any research involving the sea urchin as a model system.
E. chloroticus is currently placed as the single species of its genus Evechinus under the Echinometridae family of sea urchins, which belong to the marine phylum Echinodermata. The Echinometridae family includes species that are geographically close to E. chloroticus such as Heliocidaris located off the south coast of Australia and Echinometra located in the Indo-West Pacific and Pacific to Atlantic oceans. The Echinometridae family had recently been placed in a superfamily called Odontophora with the Strongylocentrotidae and Toxopneustidae families based phylogenetic and morphological data . The placement of E. chloroticus with the Echinometridae species had been based on morphological evidence and was described as morphologically close to species from the Heliocidaris genus, specifically H. tuberculata. The genus Evechinus, the following history of which was described by McRae (1958) , was first placed in the Echinidae family by H. L. Clark (1912) who later (1925) shifted Evechinus and Heliocidaris to the Strongylocentrodidae family based on the polyporus ambulacral plates and circular ambitus of Heliocidaris, and the larval specialisation and the pedicellariae of Evechinus. Their placement was later contended by Mortensen (1943) who moved the genera to their current family Echinometridae based on the strongly developed single lateral tooth of the gemmiform pedicellariae, the paired nature of the poison glands, and the structure of the larval forms . The established relationships of E. chloroticus to other sea urchin species has been based solely on morphological evidence, and has yet to be analysed at a genomic level. Obtaining transcriptome data would provide an opportunity to compare the sequence similarity of E. chloroticus genes to other sea urchins species and infer new information about its phylogenetic relationships.
Here we describe the extraction of RNA from multiple tissue types of E. chloroticus followed by their sequencing using Next Generation Sequence (NGS) technology. Transcripts were reconstructed by de novo assembly and annotated by sequence similarity to public protein databases to provide a set of transcripts along with functional annotations. Lastly, we describe the relationship of E. chloroticus to other sea urchin species based on the sequence similarity of selected genes.
Results and discussion
Sequencing and quality control
Libraries (cDNA) were constructed using RNA extracted from selected tissue samples from a male and female E. chloroticus. These animals were taken from Doubtful Sound, Fiordland, New Zealand, and housed feeding on kelp at the New Zealand Marine Studies Centre, Portobello, Dunedin, for 3 years. Tissues used for sequencing included the roe, muscle, gut tissue, water vascular system and also a sample of the coelomic fluid. Samples were harvested, snap frozen and total RNA extracted using a RNAeasy kit with a Qiagen® shredder. Equal amounts of total RNA from each tissue sample were combined to give the mixed RNA sample. RNA from an individual male and female, consisting of a mixed total tissue sample, a roe tissue sample and a coelomic fluid sample, were used to generate six libraries.
Sequencing was carried out on an Illumina HiSeq-2000 machine, which generated 123 million pairs of 100 base length paired-end reads (24.8 Gb). The raw sequence data in FASTQ format was submitted to the National Centre for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database and accessible through the BioProject [Accession: PRJNA190637]. Quality control was carried out on the raw reads prior to assembly. This involved the removal of adapter sequences, trimming of low quality bases (Q < 20) from both ends of reads and discarding reads less than 25 bases in length. Reads from potential contaminating species were also removed. Quality control resulted in a total of 118,016,465 high quality reads (95.3% of total raw reads) with 94.5% remaining paired-end. These reads were then used for transcript assembly.
De novo assembly
Number of transcripts
Total transcripts length (bp)
Mean transcript length (bp)
Median transcript length (bp)
Minimum transcript length (bp)
Maximum transcript length (bp)
Total read alignment %
The distribution of E-values for the top hits showed that 72% of transcripts had a strong match to a database protein (E < 10-50) (Figure 3B). The species distribution for the top hits showed 85% of top hits to proteins from S. purpuratus (Figure 3C). Previous research on S. purpuratus, including the genome sequencing project, has provided a set of annotated proteins for this sea urchin species. This resource provided the majority of best annotations for the E. chloroticus transcripts. The next species most represented in the top hits at 2% was Saccoglossus kowalevskii. S. kowalevskii is a species of acorn worm, which are closely related to members of the Echinodermata phylum . Out of the 458 transcripts with protein top hits to S. kowalevskii, most appeared to have less significant matches to S. purpuratus. The relatively large proportion of S. kowalevskii top hits could be due to longer, more complete protein sequence available for S. kowalevskii over S. purpuratus for those transcripts. The proportion of others (9%) represented all other specific species with top hits to less than 1% of the transcripts and of these 24 transcripts had ‘unknown’ species as their top hit.
Summary of repeating elements
Reduced set (Unannotated set)
Number of transcripts
75, 002 (50,347)
Total sequence length (MB)
Number of elements
Percentage of total sequence (%)
ncRNA fragments (mainly rRNA)
Total interspersed repeats
The redundancy of unannotated transcripts was assessed by clustering transcripts based on 90% or more sequence similarity. Out of 50,347 transcripts, this resulted in 44,687 clusters of non-redundant transcripts. The proportion of nucleotide sequence matches to the unannotated transcripts was assessed by using BLASTN to search against the NCBI non-redundant nucleotide database with a cut-off E-value of 10-6. Only 3,254 (6.5%) transcripts had a match to a database nucleotide sequence. These results for the unannotated transcripts showed that a large number of transcripts in the reduced set contained novel sequence without a protein or nucleotide match to the NCBI non-redundant databases. These transcripts are not due to overly redundant sequences or an increased proportion of repeated sequence.
There are challenges to providing a complete annotation of transcripts from a de novo assembly. High-throughput annotation approaches such as multiple BLAST searches provide a practical means to giving automated annotations to transcriptome datasets, but the broad nature of this search is limited by the detail and sensitivity of the annotation. From the proportion of unannotated E. chloroticus transcripts there will be biologically important transcripts that could not be annotated by this approach.
As an example we looked to identify a non-coding transcript for telomerase RNA. Telomerase RNA is a non-coding RNA that together with the telomerase reverse transcriptase protein forms a ribonucleoprotein enzyme that is essential for synthesising the telomeric DNA repeats at the ends of chromosomes . The first invertebrate telomerase RNA sequence was recently identified using a targeted strategy in S. purpuratus, which gave an opportunity to attempt to identify this transcript in E. chloroticus. A BLASTN search using the 535 bp telomerase RNA sequence from S. purpuratus identified an unannotated 520 bp transcript from E. chloroticus with an E-value of less than 10-114. The E. chloroticus and S. purpuratus sequences were aligned and conserved domains identified (Additional file 2). The E. chloroticus sequence showed close similarity to the S. purpuratus sequence with 76% identity. The universal template-pseudoknot domain and the vertebrate specific H/ACA domain could be identified. However, while the Box H and CAB box could be identified, the terminal Box ACA motif was not covered, suggesting that the end of the E. chloroticus assembly was truncated. While a number of transcripts could not be annotated with multiple BLASTX/BLASTN searches, this unannotated set still contains biologically important transcripts, such as the telomerase RNA, that can be discovered through such a detailed search.
Gene ontology (GO) annotation
EC and KEGG Orthology annotations
The resulting trees were very consistent with those from Kinjo et al., (2008). The ND1-ND2 trees (Figure 9A-C) supported the monophyly of each genus and the relationships between genera reported in Kinjo et al., (2008) were represented in these trees. The grouping of all Echinometridae species into a single clade from the two outgroup species was not supported for the ML tree, but was achieved with low support for the NJ tree and higher support for the Bayes tree. Analysing the placement of E. chloroticus in the ND1-ND2 trees, E. chloroticus did not form any clades with any of the other Echinometridae species. The ML tree placed E. chloroticus in the same position as the outgroup species S. purpuratus, separate from the Echinometridae clades formed. The NJ tree placed E. chloroticus outside of the Echinometridae family clade and in a clade with S. purpuratus. The Bayes tree, which had the highest supporting values for each Echinometridae clade, placed E. chloroticus outside of a highly supported family level clade for Echinometridae. These results showed E. chloroticus is phylogenetically distinct from the other species of its family.
Kinjo et al. (2008) described ND2 as having higher rates of mutation compared to other mitochondrial genes and therefore useful for determining close relationships between species. The ND1 had a slower rate of mutation and was described as being useful for determining distant relationships. In the separate ND2 trees (Figure 9G-I) E. chloroticus was placed in the same positions as in the ND1-ND2 trees. The ND2 sequence has therefore been significant in constructing the ND1-ND2 trees and the faster mutating gene could not determine any relationships between E. chloroticus and the other Echinometridae. In the ND1 trees (Figure 9D-F) E. chloroticus was placed in a clade with the two genera clades for Heliocidaris and Echinostrephus with low support in the ML tree and higher support in the Bayes tree. The Bayes tree though unexpectedly placed S. purpuratus as a sister to one of the Echinometra clades. This result may have been the ability of the ND1 gene to detect distant relationships as these species belonged to the same superfamily. The results from the ND1 ML and Bayes trees suggested a distant relationship between E. chloroticus and the Heliocidaris and Echinostrephus genera.
The results of the phylogenetic comparison of these two mitochondrial genes showed E. chloroticus as a distinct species that had significant genetic difference to other species of its family. This greater difference in genetic sequence in E. chloroticus compared with that of other Echinometridae species may be attributed to the fact that E. chloroticus had lived in isolation, solely around the coasts of New Zealand, for millions of years since its speciation . The geographical isolation of E. chloroticus would have prevented genetic exchange through fertilisation with other Echinometridae. Mutations over time would cause genetic differences to build up within the E. chloroticus species, and due to its isolation they would remain unique, unable to be shared with other Echinometridae. This could have led to the early speciation of E. chloroticus. The other species of Echinometridae remained in contact longer and could have taken longer to speciate and become genetically different from each other through unique mutations. The earlier speciation of E. chloroticus would explain the greater number of genetic differences in its gene sequences compared to other Echinometridae species, which led to its distinct place in the phylogenetic trees. Although separated, E. chloroticus remained morphologically similar to other Echinometridae species. This could be due to the similar environments the species share, specifically with the Heliocidaris species off the southern coasts of Australia, and there had not been pressures for greater morphological difference. E. chloroticus therefore had remained morphologically similar to species within its family, but had become significantly different phylogenetically due to its early separation and long period of isolation around New Zealand. A comprehensive phylogenetic analysis of the Echinometridae family is limited by the range of species for which genomic data is available. Such a study would be valuable in uncovering the distant relationships between species within this family and to predict the time of the geographical isolation of E. chloroticus.
We report the first genome wide dataset for the New Zealand sea urchin species E. chloroticus. High-throughput sequencing of RNA extracted from multiple tissue samples followed by de novo transcriptome assembly has produced a dataset of 75,002 transcripts for E. chloroticus. A total of 24,655 transcripts had a significant protein match to the NCBI non-redundant database. Transcripts were annotated with GO terms, InterPro domains and KO terms to provide additional functional information. This new data allowed the first phylogenetic analysis of E. chloroticus to other species of its family Echinometridae. Comparison of ND1 and ND2 sequence revealed E. chloroticus as being genetically distinct from its family, as it did not form any strong clades with other family members and remained outside of family level clades. There is an opportunity to further analyse the phylogenetic relationship of E. chloroticus to other sea urchin species and uncover its evolutionary history. This transcriptome data will provide a valuable resource of genomic information for this unique sea urchin species for studies either looking to improve the commercialisation of E. chloroticus or using the sea urchin as a model system. Additional datafiles including transcript datasets and functional annotation data are available from the project site: http://mRNA.otago.ac.nz/Kina/.
Sample collection and RNA extraction
E. chloroticus specimens were collected from Doubtful Sound, Fiordland, New Zealand. The animals were housed at the New Zealand Marine Studies Centre, Portobello, Dunedin, for 3 years and feed a natural diet of kelp (Macrocystis pyrifera). Tissues were harvested from a male and female individual and snap frozen using liquid nitrogen. Using an RNAeasy kit with a Qiagen® shredder RNA was extracted from tissues which included the roe, muscle, gut and the water vascular system and also from a sample of the coelomic fluid (containing total coelomocyte cell components). Equal concentrations of RNA were combined from all five sample types to produce a mixed RNA sample. Six RNA samples of equal concentrations consisted of a male mix, roe and coelomic fluid sample, along with a female mix, roe and coelomic fluid sample. Samples with high RNA quality (an RNA Integrity Number (RIN) of over 6) were processed for library construction. E. chloroticus is an endemic species and is of particular interest to southern Maori. Consultation with Maori included the University of Otago Ngai Tahu Consultation Committee.
Sequencing and quality control
Libraries (cDNA) were constructed for the six samples using the Illumina TruSeq RNA protocol. Sequencing was done on an Illumina HiSeq-2000 sequencer, generating 100 base paired-end reads. Reads saved in FASTQ format were quality assessed using FASTQC v0.10.1 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) for base quality and adapter sequence. Adapter sequences detected were trimmed from read ends using FastqMcf from ea-utils v1.1.2 (https://code.google.com/p/ea-utils). Bases with low quality phred scores were trimmed from either ends of reads using DynamicTrim with a phred score cut-off of 20. Length filtering of reads was performed using LengthSort with a minimal length of 25 bases. Both DynamicTrim and LengthSort are part of the SolexaQA package v2.2 (http://solexaqa.sourceforge.net) . Bowtie2 v2.1.0 (http://bowtie-bio.sourceforge.net/bowtie2)  was used to align reads to genomic sequences of possible contaminating species (e.g. Homo sapiens). Potential contaminating reads were then aligned back to sea urchin genomic sequence, retaining any likely E. chloroticus reads.
De novo assembly
De novo assembly of the processed reads into transcripts was carried out using the Trinity assembly program release 2013-02-25 (http://trinityrnaseq.sourceforge.net)  using default parameters. Minimum length for reported transcripts was 200 bases. To assess transcript abundance paired reads were aligned to the transcripts through a Trinity script, which used Bowtie v0.12.9 (http://bowtie-bio.sourceforge.net)  to align reads. RSEM v1.2.3 (http://deweylab.biostat.wisc.edu/rsem) was then used to generate isoform percentages and FPKM values. Transcripts of the same trinity component could be treated as different sequence isoforms. Transcripts that had less than 1% of the total reads for their component were considered unsupported isoforms and removed from the set. Transcripts were then filtered based on a minimal FPKM value of 0.5 which corresponded to 41 reads per kilobase to give a reduced set of transcripts. This was to remove transcripts from the assembly that had relatively low read support and were therefore less likely to be complete. The majority of transcripts removed were short, being less than 500 bases in length. The reduced set was then selected for functional annotation. Transcript statistics were computed using in-house scripts. Bowtie2 v2.1.0 was used to align all reads used in the assembly to the total and reduced transcript sets for read representation statistics. Trinity scripts were used to generate the protein coverage statistics by extracting predicted E. chloroticus proteins from transcript ORFs, followed by BLASTP  searches to S. purpuratus proteins obtained from the NCBI database. The CEGMA v2.4 program (http://korflab.ucdavis.edu/datasets/cegma)  was used to provide KOG annotations of transcripts and reports for completeness of Core Eukaryotic Genes.
The Blast2GO v2.6.6 program (http://www.blast2go.com)  was used extensively for the functional annotation of transcripts. BLASTX  searches were used within Blast2GO to the NCBI non-redundant protein database to identify similar proteins using an E-value cut-off of 10-3 and the top 20 hits for each transcript were recorded. The top hit for each transcript was selected for E-value and species distributions. Transcript abundance was calculated by aligning reads from each of the tissue samples to the transcripts using Bowtie2 v2.1.0 followed by RSEM for FPKM values. Using the Trinity transcript component level for gene distinction, the top 20 genes from each tissue sample with the highest FPKM values were selected to show expression levels of the most abundant transcripts. The pheatmap package v0.7.4  within R v3.0.1  was used to create a heatmap using the log2 of FPKM values of each top gene across each tissue. Rows were clustered by Euclidean distance. The RepeatMasker v2.2.27+ (http://www.repeatmasker.org) program was used along with the latest (22-04-2013) Repbase database (http://www.girinst.org/repbase)  to identify repeating elements. Default settings were used and the query species was set as Echinoidea. RepeatMasker used a Smith-Waterman-Gotoh type algorithm to search for matching sequences to known interspersed repeats (retroelements, DNA transposons), simple repeats (microsatellites), and fragments of ncRNAs, that were represented in the Repbase database. Low complexity sequences (polypurine, polypyrimidine, and regions of extremely high AT (>87%) or GC (>89%) content) were also identified with RepeatMasker. Clustering of transcripts was performed using CD-HIT-EST (http://weizhong-lab.ucsd.edu/cd-hit)  at 90% similarity to assess transcript redundancy. For nucleotide sequence matches, BLASTN  searches were used to the NCBI non-redundant nucleotide database using an E-value cut-off of 10-6. An increased E-value cut-off was used to provide more unique and meaningful nucleotide matches from the BLASTN search. S. purpuratus nucleotide sequence [Accession: JQ684708] was used in a local BLASTN  search to identify an E. chloroticus telomerase RNA transcript and the sequences were aligned in Geneious v6.1.4 program (http://www.geneious.com) using the Geneious aligner. GO annotations were assigned to transcripts by Blast2GO using the BLASTX results. InterProScan  searches were used within Blast2GO to InterPro databases  to provide InterPro annotations of conserved protein domains and functional sites. Blast2GO then used InterPro results to add additional GO annotations to transcripts based on associated terms. Enzyme codes were assigned to transcripts by Blast2GO based on associated GO terms providing results for EC numbers and their KEGG pathways. The KEGG Automatic Annotation Server (KAAS) (http://www.genome.jp/tools/kaas)  was used to annotate transcripts with KO codes.
For the phylogenetic analysis of E. chloroticus and the Echinometridae species, the ND1 and ND2 sequence data used in Kinjo et al., (2008)  was downloaded from the NCBI database [Accession: AB178488 - AB178518]. A total of 14 species from the Echinometridae family were used, which included the six Echinometra, E. sp. A, E. sp. C, E. lucunter, E. mathaei, E. oblonga, E. vanbrunti; the two Colobocentrotus, C. atratus, C. mertensii; the two Heterocentrotus, H. mammillatus, H. trigonarius; the two Echinostrephus, E. aciculatus, E. molaris; and the two Heliocidaris, H. crassispina, H. tuberculata. The two outgroup species used were Tripneustes gratilla from the sister family Toxopneustidae and S. purpuratus from the sister family Strongylocentrotidae. Sequence data for S. purpuratus was obtained from NCBI [Accession: NC_001453]. The E. chloroticus ND1 and ND2 sequence was obtained by a BLASTN search with the S. purpuratus genes against the assembled transcripts. The E. chloroticus mitochondrial genome was almost fully reconstructed across several transcripts. The transcript identified containing the ND1 and ND2 genes was aligned with all the previous sequences and 819 matching bases for ND1 and 1059 matching bases for ND2 were extracted for the following analysis. The phylogenetic trees were constructed within the Geneious v6.1.4 program. ND1, ND2, as well as combined ND1-ND2 sequences from each species were aligned to each other. All alignments were carried out using the Geneious Aligner within Geneious, set at 70% identity. The NJ trees were constructed with the Geneious Tree Builder using the HKY85 model  with 1000 bootstrap repeats. The ML trees were constructed with the PhyML  Geneious module using the HKY85 model with 1000 bootstrap repeats and estimated gamma distribution and invariable site proportion. The Bayes trees were constructed with the MrBayes  Geneious module using the HKY85 model with gamma distribution and invariable site estimations, gamma categories of 4, chain length of 50,000, heated chains at 4, subsampling frequency of 10, and a burn-in length of 1000. All trees were set at a bootstrap threshold of 50% and rooted using T. gratilla as the outgroup.
We gratefully thank Campbell McManaway of Cando Fishing Ltd for providing their time and services to acquire E. chloroticus specimens. Jodi Pilbrow and Alan Carne for helpful discussions on Kina. NZ government subsidised sequencing services were provided by New Zealand Genomics Limited (http://www.nzgenomics.co.nz). Funding for this study was provided by the University of Otago.
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