Program

The ray-finned fishes (Actinopterygians; hereafter referred to as ‘fishes’) are the most diverse and successful group of vertebrates. With approximately 30,000 species, fishes constitute half of all extant vertebrates. They exhibit a wide variation in their genome size, morphology, adaptation, behavior and breeding habits. Fishes are also an important source of protein-diet and contribute substantially to the economy of many countries through capture fisheries, aquaculture, recreational fisheries, and ornamental fish industries. Several fish species are under threat of extinction due to overexploitation and destruction of habitat. To better understand the genetic basis of phenotypic diversity and adaptations of fishes and to generate genomic resources that can help in devising efficient strategies for management of fish stocks, genomic sequences of many fish species are being sequenced. One of the concerted efforts to sequence fish genomes was initiated by the Genome 10K project which aims to catalogue genome sequences of 10,000 vertebrates across mammals, birds, non-avian reptiles, amphibians and fishes (https://genome10k.soe.ucsc.edu/). In line with the abundance and diversity of fishes, Genome 10K has targeted to catalogue the genomes of 3,000 fishes. Priority is given to representatives of different taxonomic groups, families with most diverse species, threatened species, economically important species, potential genetic models and phenotypically highly derived species. At present more than 60 fish genomes have been sequenced and published by Genome 10K members and other groups. Comparative analyses of fish genomes have provided novel insights into the diversity of fish genomes as well as into the genetic bases of phenotypic diversity of fishes.

[Objective] Genetic linkage maps facilitate genome assembly and are essential for genetic linkage studies. Anchoring and ordering contigs/scaffolds to chromosomes according to linkage maps are highly effective process for genome assembly. Thus, the present study is aiming to develop a method to construct ultra high resolution linkage map effectively based on gynogenetic fish and whole-genome sequencing.
[Methods] Double haploid (DH) embryos were generated from artificial gynogenetic process of a wild female torafugu. Each gynogenesis embryo was subject to sequencing by Illumina Sequencing Technology. SNPs calling was processed by software BWA and Samtools. Subsequently, the SNPs data matrix was further processed to complement missing data. Finally, linkage map was constructed by software MSTmap and ASmap.
[Results] We obtained about 74 Gb of genomic sequence of 192 individuals, then the sequence of each read was mapped on torafugu reference genome. About 1 million of SNPs sites were found and, and typing data at the SNP sites were obtained. A high-resolution linkage map was constructed with identification of more than 4,023 crossover breakpoints and total linkage distance of 2073 cM. Based on this linkage map, 90 Mb (23% of the whole genome) of unplaced scaffolds were mapped to the chromosomes (10% were located and oriented; 13% were located but not-oriented). Consequently, The total length of draft genome based on located and oriented sequence was improved from 72% to 82% of the whole genome. Because millions of SNPs are utilized, the limit of resolution of linkage map depends not on number of polymorphic makers but on number of total crossover sites of the examined individuals. These results showed the usefulness of our strategy.

Immunoglobulin novel antigen receptor (IgNAR) is a unique antibody found only in cartilaginous fish. It is a heavy chain homodimer with smallest variable region (around 12 kDa) identified to date. Affinity maturation of IgNAR was tested in few shark species but the results were not always promising. Therefore current study aimed to determine IgNAR in vivo affinity maturation against specific antigen in brownbanded bamboo shark (Chiloscyllium punctatum). Mature bamboo sharks were immunized with hen egg lysozyme antigen for four times at monthly intervals. Collection of around 10 ml of blood from the caudal vein was performed under general anaesthesia prior to each immunization. Whole blood was centrifuged and plasma samples were separated for the analysis of IgNAR antibody using enzyme linked immunosorbant assay (ELISA). SDS-page and western blotting analysis was also performed to determine the plasma protein content. Anti-horn shark IgNAR (rabbit polyclonal) and anti-rabbit IgG antibodies were used as primary and secondary antibodies respectively. According to western blot analysis presence of IgNAR antibody in both unimmunized and immunized shark plasma was identified. ELISA results indicated an increase of antigen specific IgNAR titer upon antigen exposure by fourth immunization. Therefore ELISA revealed affinity matured IgNAR from brownbanded bamboo shark. In conclusion, C. punctatum develops antigen specific IgNAR upon immunization, which can be used for future immunotherapeutics.

【Objective】 Cetaceans have lost their hind limbs during the transition from terrestrial to aquatic environments. However, the molecular mechanism of hind limbs reduction during their evolution remains unclear. In 2006, one common bottlenose dolphin Tursiops truncatus which has a pair of hind limbs was found and kept alive. It was expected to elucidate molecular machanism of loss/regeneration of hind limbs of cetacean through the research of the four-finned dolphin. Nevertheless, this dolphin died without generating offspring. Here, we focused our studies on establishments of (1) cell lines derived from the four-finned dolphin to conserve its genome resources, and (2) induced pluripotent stem (iPS) cells to utilize them for varous studies.
【Methods】 (1) Four-finned dolphin fibroblasts were cultured on 6 well dish and infected with recombinant lentivirus encoding immortalizing factor SV40T or hTERT. These lentivirus also have a blasticidin S-resistance gene. In infection experiments, six conditions were attempted, either individually or mixed virus solutions of 0.1mL or 1mL per well. Two days after transfection, blasticidin S was added to select infected cells. (2) Four-finned cells were co-transfected by electroporation with human derived reprogramming factors Oct3/4, Sox2, L-Myc, Klf4, Lin28. A week after electroporation, the cells were re-seeded on feeder cells and cultured for 3 weeks.
【Results】 (1) Blasticidin S-resistance cells were obtained under conditions with SV40T alone, or mixture of SV40T and hTERT. These cells have been passaged more than 50 times. Then, we collected single cell clones and made growth curves. All transfected clones showed remarkable increases in growth rate compared to the non-transfected cells. Also, we detected SV40T expression from these clones by Western blotting. (2) A month after electroporation, iPS cell-like colonies appeared. In addition, these colonies showed positive reactions by ALP staining suggesting the colonies may be in a de-differentiated state.

RNA-Seq, like other state-of-the-art techniques, produces new questions while overcoming some problems, on the basic and applied research of fishery and marine biotechnology. The bias of analyzing RNA-Seq data caused by dynamics of RNA is one of such issues in the transcriptome investigations of fishery species, such as shellfish, shrimps, and fish. The present study reviews the effects of physiologically producing and degrading RNA on the analysis of RNA-Seq data. Equations representing RNA abundance are derived from synthesis and degradation of RNA. Accumulation of RNA influences the quantification of RNA and causes overestimation of transcription levels, whereas RNA decay causes underestimation of them. RNA degradation, moreover, affects proper identification of RNA sequences, which depends on the research target. How to deal with RNA abundance is discussed, linking with the general viewpoint that transcription levels are affected by reaction of organisms to environment changes.