Mol Biol Rep DOI 10.1007/s11033-014-3847-5 Complete mitogenome of the edible sea urchin Loxechinus albus: genetic structure and comparative genomics within Echinozoa Graciela Cea • Juan Diego Gaita´n-Espitia • Leyla Ca´rdenas Received: 7 May 2014 / Accepted: 25 November 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract The edible Chilean red sea urchin, Loxechinus Hemicentrotus, whereas the other included species of albus, is the only species of its genus and endemic to the Mesocentrotus and Pseudocentrotus. Southeastern Pacific. In this study, we reconstructed the mitochondrial genome of L. albus by combining Sanger Keywords Loxechinus albus Á Mitochondrial genome Á and pyrosequencing technologies. The mtDNA genome Genome architecture Á Echinodermata had a length of 15,737 bp and encoded the same 13 pro- tein-coding genes, 22 transfer RNA genes, and two ribo- somal RNA genes as other animal mtDNAs. The size of Introduction this mitogenome was similar to those of other Echinoder- mata species. Structural comparisons showed a highly Metazoan mitochondrial DNA (mtDNA) is typically a conserved structure, composition, and gene order within circular double-stranded molecule of approximately Echinoidea and Holothuroidea, and nearly identical gene 12–20 kb length that contains 13 protein-coding genes organization to that found in Asteroidea and Crinoidea, (PCGs) involved in oxidative phosphorylation (OXPHOS), with the majority of differences explained by the inversions 22 transfer RNA (tRNA) genes necessary to translate the of some tRNA genes. Phylogenetic reconstruction sup- PCGs, two ribosomal RNA genes (rRNA), and a major AT- ported the monophyly of Echinozoa and recovered the rich non-coding region usually denominated the mito- monophyletic relationship of Holothuroidea and Echinoi- chondrial control region [1, 2]. Although the gene content dea. Within Holothuroidea, Bayesian and maximum like- and organization are notably conserved in most animal lihood analyses recovered a sister-group relationship phyla [3], some differences can be found among taxa; in between Dendrochirotacea and Aspidochirotida. Similarly particular, tRNA genes switch position more frequently within Echinoidea, these analyses revealed that L. albus than do larger protein-coding and rRNA genes [2, 4, 5]. In was closely related to Paracentrotus lividus, both being this context, a wide range of comparative studies have used part of a sister group to Strongylocentrotidae and Echino- mitochondrial genomes (mitogenomes) as phylogenetic metridae. In addition, two major clades were found within markers to resolve deep evolutionary relationships [6]by Strongylocentrotidae. One of these clades comprised all of comparing entire mitogenome sequences [1, 7–10]orby the representative species Strongylocentrotus and analyzing shared genome rearrangements (e.g., inversions and transpositions) at different taxonomic levels [3, 11, 12]. Electronic supplementary material The online version of this Thanks to rapid advances in next generation sequencing article (doi:10.1007/s11033-014-3847-5) contains supplementary material, which is available to authorized users. (NGS) techniques in recent years, the availability and number of entire mitogenome sequences for different tax- G. Cea Á J. D. Gaita´n-Espitia Á L. Ca´rdenas (&) onomic groups have considerably increased [10, 13]. To Facultad de Ciencias, Instituto de Ciencias Ambientales y date, a total of 38 mitogenomes of echinoderms have been Evolutivas, Universidad Austral de Chile, Casilla 567, Valdivia, Chile described and are publicly available in the Organelle e-mail: [email protected] Genome Resources of NCBI (http://www.ncbi.nlm.nih. 123 Mol Biol Rep gov). Of these mtDNA sequences, four represent crinoids, was extracted from gonads of eleven adults using a com- eight ophiuroids, eight asteroids, five holothurians, and 13 mercial RiboPureTMKit (Ambion, Carlsbad, CA, USA). echinoids. Comparative genomics within the phylum RNA quality was analyzed by Agilent 2100 Bioanalyzer Echinodermata have identified a very different gene order RNA assays (Santa Clara, CA, USA) and evaluated by involving at least eight rearrangement events (two inver- calculating the ratio of the 28S and 18S rRNA intensity sions, three transpositions, and three tandem-duplication– peaks. Total RNA was then submitted to Macrogen, Seoul, random-loss events) [12]. This order places crinoids and Korea (http://www.macrogen.com) for cDNA library con- ophiuroids, with the presence of several unassigned struction, library normalization, and cDNA pyrosequencing sequences (UAS) and some pseudogenes, as the most in a 454-GS FLX Titanium System (454 Life Sciences, divergent groups [14]. Additionally, holothurians and Branford, CT, USA). echinoids possess the highest degree of conservation in mtDNA gene order, with few changes in gene structure and Mitogenome assembly and annotation strong similarities in the origin of replication [15]. In particular, the mitogenome architecture of the latter group The raw data was trimmed and assembled using CLC (sea urchins and sand dollars) has been characterized by a Genomics Workbench software (CLC Bio, Aarhus, Den- pattern of organization unique from that of other echino- mark), using the parameters described in [21]. Gene derms [12, 16]. annotation and mapping were performed using Blast2GO Within the class Echinoidea, the edible Chilean red sea software [22] according to the main categories of Gene urchin, Loxechinus albus, is the only species of its genus. Ontology (GO; molecular functions, biological processes This sea urchin is distributed throughout the Chilean coast, and cellular components) [23]. From this database, genes from Arica (18°S) to Tierra del Fuego (55°S) [17]. Loxe- were filtered by homology (Blastx algorithm v2.2.29) to chinus albus is one of the most economically important those of other echinoids (Table 1) using Geneious Pro 5.5.6 species in the littoral benthic systems of the southeastern software [24]. To complete the remaining tRNAs and to Pacific in South America [18]. Harvesting of L. albus verify the intergenic region we design PCR primers for represents the largest extraction volume among world walking amplification (Table S1) using traditional Sanger urchin fisheries [19]. However, overfishing of the red sea sequencing. Similar procedure was used to validate the urchin along Chilean coasts is driving the decline or sequence quality of some genes with ambiguous features depletion of natural populations of this biological resource (e.g. nad4) by using independent primers located in the [20]. Currently, no information exists on the population flanking regions of the target genes (Table S1). genetic background of L. albus or its functional genomics Several PCRs were set up for a total of 10 sea urchins. that could link the metabolic responses of this species (e.g., Each reaction (25 lL total volume) consisted of 2 lLof expression of genes involved in OXPHOS) to the great DNA template, 2.5 lL109 PCR buffer, 0.5 lL BSA, environmental variation experienced along the Chilean 1 lL MgCl2 (2 mM), 1.5 lL of each primer, 0.5 lLof coastal upwelling. In this study, we reconstruct the com- dNTP mix (2.5 mM), 0.6 lL of Taq DNA polymerase plete mitogenome of L. albus by combining Sanger and (Invitrogen, Carlsbad, CA, USA), and 15.4 lL sterile dis- 454 sequencing technologies. Furthermore, we character- tilled water. PCR was performed with the following ized general features such as genome length, nucleotide parameters: an initial cycle of denaturation at 95 °C for biases, and gene structure and compared them with those of 5 min; followed by 30 cycles at 95 °C for 5 min, 50 °C for other echinoderms. Finally, we established the taxonomic 1 min, and 72 °C for 1 min; and terminated by a final relationships of L. albus at the class and family level. This extension at 72 °C for 10 min. PCR products and sequen- work should be useful for studies on evolution and con- ces were developed at Macrogen. servation genetics, as well as for those that integrate functional genomics with the physiology of this species Sequence analysis and genome annotation under environmental variation and climate change scenarios. Protein coding genes (PCGs), rRNA and noncoding sequences were identified by comparing alignments of homologous genes within complete mitogenomes of other Materials and methods echinoids (Table 1) using the BLAST tool implemented in Geneious Pro 5.5.6 [24]. The boundaries of both PCGs and RNA preparation, sequencing, and cDNA library rRNA genes were adjusted manually based on the locations of adjacent genes and the first start and stop codons in Samples of L. albus were collected in Los Molinos frame. All tRNA genes were located and folded into their (39°400S–73°120W), southern Chile. Total L. albus RNA proposed clover-leaf structures to confirm their secondary 123 Mol Biol Rep Table 1 List of the Echinodermata species included in the present study Taxonomic position GenBank accession Genome Length (bp) Class Order Family Species Echinoidea Echinoida Echinidae Loxechinus albus JX888466 15,737 Paracentrotus lividus J04815 15,696 Strongylocentrotidae Strongylocentrotus purpuratus X12631 15,650 Strongylocentrotus fragilis* KC898200 15,748 Strongylocentrotus intermedius* KC898198 15,718 Strongylocentrotus droebachiensis EU054306 15,710 Strongylocentrotus pallidus KC898197 15,552 Mesocentrotus nudus JX263663 15,709 Mesocentrotus franciscanus KJ526170 15,650 Pseudocentrotus depressus* KC898203 15,736 Hemicentrotus pulcherrimus*