KK2702) and estimated to be three cycles. The optimal number of PCR cycles was determined by a preliminary PCR using KAPA Library Amplification Kit (KAPA, Cat. For DNA purification, we applied 1.8x (after end repair) and 1.0x (after PCR) volumes of Agencourt AMPure XP (Beckman Coulter, Cat. For RNA-seq, using 0.5 μg of each of the extracted total RNAs, strand-specific RNA-seq libraries were prepared with the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, Cat. 5067-1511) were used to measure their RNA integrity number, which yielded the score of 10.0 for all samples ( Fig. For a quality control, the Agilent 2100 Bioanalyzer system and Agilent RNA 6000 Nano Kit (Agilent, Cat. Genomic DNA was removed with gDNA Eliminator columns in this kit. RNAs were extracted with the RNeasy Mini plus kit (QIAGEN, Cat. 1b), and flash-frozen with liquid nitrogen, and stored at −80 ☌. An egg 33 days after deposition was collected, and an about 33 mm-long embryo was dissected into the head, trunk, and tail parts ( Fig. Zebra bullhead shark eggs were incubated at 24.5 ☌, 8.0–8.2 pH in a tank of Ibaraki Prefectural Oarai Aquarium. Of them, about 79,000 protein-coding sequences were predicted from the obtained transcript contigs.Īnimal experiments were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC), RIKEN Kobe Branch. About 900,000 transcripts were assembled from the paired-end libraries of its RNAs produced by Illumina HiSeq. An embryo of the zebra bullhead shark was collected from Ibaraki Prefectural Oarai Aquarium. Thus, the zebra bullhead shark may serve as a reference to characterize the species of this genus in the future. While the zebra bullhead shark is currently classified as Least Concern by the IUCN’s Red List, five out of the nine species are Data Deficient because their biological information is virtually missing 18. The order that this species belongs to is Heterodontiformes, which includes only one living genus with nine species and relatively high ED score 17. The zebra bullhead shark is an elasmobranch species that is common in the Western Pacific ranging from Japan to Australia 16. In this study, we report transcriptome data of the zebra bullhead shark ( Heterodontus zebra Fig. In these respects, molecular information would contribute to making a more effective conservation policy for cartilaginous fishes. In addition, a molecular phylogenetics-based score, “evolutionary distinctness” (ED), which evaluates species uniqueness, is also used for conservation prioritization 14, 15. Recently, transcriptome data is increasingly utilized for population genetics, which can estimate divergence and effective population size of species 12, 13. Therefore, an efficient and precise conservation policy is required for a sustainable interaction between humans and cartilaginous fishes. Owing to the slow growth rate, long generation time, and sparse reproductive cycles, it has been realized that cartilaginous fishes are vulnerable to human impacts 2. In addition, cartilaginous fishes play important roles for marine ecology, bioresources, and aquarium exhibitions 2. Because molecular information of cartilaginous fishes is currently available for a limited number of species, further augmentation of molecular data in this clade would be useful for comparative studies. Therefore, the study of cartilaginous fishes helps us recognize the secondary modifications of model vertebrate species. This comparability is likely attributed to the slower molecular clock of cartilaginous fishes than that of teleosts 1, 10, 11. Indeed, recent studies showed that non-coding sequences are more comparable between the genomes of humans and cartilaginous fishes than between those of humans and zebrafishes 7– 9. However, such convenience-oriented choices of species may lead to accumulation of biased knowledge 4– 6. Instead, animals with a small body and short generation time, such as fruit flies, nematodes, zebrafishes, and mice have been intensely studied as "model organisms", which has accelerated our understandings of biology 3. These factors have distracted researchers from the modern molecular studies of cartilaginous fishes. Long generation cycle, large body size, and slow growth rate are the characteristics of cartilaginous fishes 1, 2, and also the main reasons why they are difficult to keep in laboratories.
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