Yuya Shigenobu

The first symbiont-free genome sequence of marine red alga, Susabi-nori (Pyropia yezoensis).

Nori, a marine red alga, is one of the most profitable mariculture crops in the world. However, the biological properties of this macroalga are poorly understood at the molecular level. In this study, we determined the draft genome sequence of susabi-nori (Pyropia yezoensis) using next-generation sequencing platforms. For sequencing, thalli of P. yezoensis were washed to remove bacteria attached on the cell surface and enzymatically prepared as purified protoplasts. The assembled contig size of the P. yezoensis nuclear genome was approximately 43 megabases (Mb), which is an order of magnitude smaller than the previously estimated genome size. A total of 10,327 gene models were predicted and about 60% of the genes validated lack introns and the other genes have shorter introns compared to large-genome algae, which is consistent with the compact size of the P. yezoensis genome. A sequence homology search showed that 3,611 genes (35%) are functionally unknown and only 2,069 gene groups are in common with those of the unicellular red alga, Cyanidioschyzon merolae. As color trait determinants of red algae, light-harvesting genes involved in the phycobilisome were predicted from the P. yezoensis nuclear genome. In particular, we found a second homolog of phycobilisome-degradation gene, which is usually chloroplast-encoded, possibly providing a novel target for color fading of susabi-nori in aquaculture. These findings shed light on unexplained features of macroalgal genes and genomes, and suggest that the genome of P. yezoensis is a promising model genome of marine red algae.

Evolutionary changes of multiple visual pigment genes in the complete genome of Pacific bluefin tuna.

Tunas are migratory fishes in offshore habitats and top predators with unique features. Despite their ecological importance and high market values, the open-ocean lifestyle of tuna, in which effective sensing systems such as color vision are required for capture of prey, has been poorly understood. To elucidate the genetic and evolutionary basis of optic adaptation of tuna, we determined the genome sequence of the Pacific bluefin tuna (Thunnus orientalis), using next-generation sequencing technology. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified five common fish visual pigment genes: red-sensitive (middle/long-wavelength sensitive; M/LWS), UV-sensitive (short-wavelength sensitive 1; SWS1), blue-sensitive (SWS2), rhodopsin (RH1), and green-sensitive (RH2) opsin genes. Sequence comparison revealed that tunas RH1 gene has an amino acid substitution that causes a short-wave shift in the absorption spectrum (i.e., blue shift). Pacific bluefin tuna has at least five RH2 paralogs, the most among studied fishes; four of the proteins encoded may be tuned to blue light at the amino acid level. Moreover, phylogenetic analysis suggested that gene conversions have occurred in each of the SWS2 and RH2 loci in a short period. Thus, Pacific bluefin tuna has undergone evolutionary changes in three genes (RH1, RH2, and SWS2), which may have contributed to detecting blue-green contrast and measuring the distance to prey in the blue-pelagic ocean. These findings provide basic information on behavioral traits of predatory fish and, thereby, could help to improve the technology to culture such fish in captivity for resource management.

Automated screening and primer design of fish microsatellite DNA loci on pyrosequencing data

In recent years, massive sequencing approaches have allowed us to determine genomic structures of various organisms rapidly, raising novel applicability of the high-throughput sequence data obtained to various fields of biological studies. We present here a pipeline to search for microsatellite DNA and design PCR primers encompassing the microsatellites on genomic sequence data produced by 454 pyrosequencing. We tested this pipeline, called ‘Auto-primer’, on several fish genomic sequences and obtained many and various candidates for microsatellite DNA loci useful for detecting intraspecies genetic variability. This in silico search for microsatellite DNA is superior to conventional cloning methods, since any sequence patterns of repeat unit can be screened.

Effective gene collection from the metatranscriptome of marine microorganisms

BackgroundMetagenomic studies, accelerated by the evolution of sequencing technologies and the rapid development of genomic analysis methods, can reveal genetic diversity and biodiversity in various samples including those of uncultured or unknown species. This approach, however, cannot be used to identify active functional genes under actual environmental conditions. Metatranscriptomics, which is similar in approach to metagenomics except that it utilizes RNA samples, is a powerful tool for the transcriptomic study of environmental samples. Unlike metagenomic studies, metatranscriptomic studies have not been popular to date due to problems with reliability, repeatability, redundancy and cost performance. Here, we propose a normalized metatranscriptomic method that is suitable for the collection of genes from samples as a platform for comparative transcriptomics.ResultsWe constructed two libraries, one non-normalized and the other normalized library, from samples of marine microorganisms taken during daylight hours from Hiroshima bay in Japan. We sequenced 0.6M reads for each sample on a Roche GS FLX, and obtained 0.2M genes after quality control and assembly. A comparison of the two libraries showed that the number of unique genes was larger in the normalized library than in the non-normalized library. Functional analysis of genes revealed that a small number of gene groups, ribosomal RNA genes and chloroplast genes, were dominant in both libraries. Taxonomic distribution analysis of the libraries suggests that Stramenopiles form a major taxon that includes diatoms. The normalization technique thus increases unique genes, functional categories of genes, and taxonomic richness.ConclusionsNormalization of the marine metatranscriptome could be useful in increasing the number of genes collected, and in reducing redundancies among highly expressed genes. Gene collection through the normalization method was effective in providing a foundation for comparative transcriptomic analysis.

Molecular and Morphological Discrimination Between an Invasive Ascidian, Ascidiella aspersa, and Its Congener A. scabra (Urochordata: Ascidiacea)

The solitary ascidian Ascidiella aspersa (Müller, 1776) has sometimes been regarded as conspecific with A. scabra (Müller, 1776), although previous detailed morphological comparisons have indicated that the two are distinguishable by internal structures. Resolution of this taxonomic issue is important because A. aspersa has been known as a notoriously invasive ascidian, doing much damage to aquaculture e.g. in Hokkaido, Japan. We collected many specimens from European waters (including the Swedish coast, near the type localities of these two species) and Hokkaido, Japan (as an alien population) and made molecular phylogenetic analyses using the mitochondrial cytochrome c oxidase subunit I (COI) gene, and found that in terms of COI sequences all the analyzed specimens were clustered into two distinct groups, one of which is morphologically referable to A. aspersa and the other to A. scabra. Thus, these two species should be regarded as distinct from each other.

A new primer for 16S rDNA analysis of microbial communities associated with Porphyra yezoensis

To construct high-quality 16S rDNA clone libraries for microbial communities associated with Porphyra yezoensis and to minimize the detection of rDNA from leafy gametophytes of P. yezoensis, we designed a new 16S rDNA universal primer (75F). Of the clones prepared using 75F, which was designed to distinguish between bacteria and P. yezoensis, 95% were classified into four groups, namely, β-proteobacteria, γ-proteobacteria, Lentisphaerae, and Flavobacteria. PCR-based analysis of the 16S rDNA primer constructed in this study can be used to implement 16S rDNA-based methodologies for the investigation of microbial community composition and diversity related to the Porphyra group.

Complete Genome Sequence Analysis of Two Pseudomonas plecoglossicida Phages, Potential Therapeutic Agents

ABSTRACT Pseudomonas plecoglossicida is a lethal pathogen of ayu (Plecoglossus altivelis) in Japan and is responsible for substantial economic costs to ayu culture. Previously, we demonstrated the efficacy of phage therapy against P. plecoglossicida infection using two lytic phages (PPpW-3 and PPpW-4) (S. C. Park, I. Shimamura, M. Fukunaga, K. Mori, and T. Nakai, Appl Environ Microbiol 66:1416–1422, 2000, http://dx.doi.org/10.1128/AEM.66.4.1416-1422.2000; S. C. Park and T. Nakai, Dis Aquat Org 53:33–39, 2003, http://dx.doi.org/10.3354/dao053033). In the present study, the complete genome sequences of these therapeutic P. plecoglossicida phages were determined and analyzed for deleterious factors as therapeutic agents. The genome of PPpW-3 (myovirus) consisted of 43,564 bp with a GC content of 61.1% and 66 predicted open reading frames (ORFs). Approximately half of the genes were similar to the genes of the Escherichia coli phage vB_EcoM_ECO1230-10 (myovirus). The genome of PPpW-4 (podovirus) consisted of 41,386 bp with a GC content of 56.8% and 50 predicted ORFs. More than 70% of the genes were similar to the genes of Pseudomonas fluorescens phage ϕIBB-PF7A and Pseudomonas putida phage ϕ15 (podoviruses). The whole-genome analysis revealed that no known virulence genes were present in PPpW-3 and PPpW-4. An integrase gene was found in PPpW-3, but other factors used for lysogeny were not confirmed. The PCR detection of phage genes in phage-resistant variants provided no evidence of lysogenic activity in PPpW-3 and PPpW-4. We conclude that these two lytic phages qualify as therapeutic agents.

Stock structure of Japanese flounder inferred from morphological and genetic analyses

Stock structure of Japanese flounder Paralichthys olivaceus has been inferred mainly from either morphological or genetic analyses. However, because the results of both analyses did not always agree with each other, an inclusive conclusion has never been obtained. In this study, the stock structure has been inferred from both morphological and genetic analyses using 722 wild Japanese flounder collected from nine locations along the Japanese coast. The dorsal and anal fin ray counts were larger in the southern than in the northern populations. In total, 1041 bp of mitochondrial NADH dehydrogenase subunit-2 (ND2) and 1830 bp of ND5 sequences were aligned. There are 578 variable sites in the concatenated sequence from the two genes, which defined a total of 490 haplotypes. Both results of morphological and genetic analyses indicated that the western Kyushu group, which included the Nagasaki and Kagoshima populations, was divided from the other seven populations. This is the first report to reveal the heterogeneity of the western Kyushu group based on statistical analysis.

Complete Genome Sequences of Edwardsiella tarda-Lytic Bacteriophages KF-1 and IW-1.

ABSTRACT We report the complete genome sequences of two Edwardsiella tarda-lytic bacteriophages isolated from flounder kidney (KF-1) and seawater (IW-1). These newly sequenced phage genomes provide a novel resource for future studies on phage-host interaction mechanisms and various applications of the phages for control of edwardsiellosis in aquaculture.

Complete Genome Sequence of a Novel Myovirus Which Infects Atypical Strains of Edwardsiella tarda

ABSTRACT We present the genome sequence of a novel Edwardsiella tarda-lytic bacteriophage, MSW-3, which specifically infects atypical E. tarda strains. The morphological and genomic features of MSW-3 suggest that this phage is a new member of the dwarf myoviruses, which have been much less studied than other groups of myoviruses.