Kenji Saitoh

Microsatellite-based pedigree tracing in a Japanese flounder Paralichthys olivaceus hatchery strain: implications for hatchery management related to stock enhancement program

Effective population size in captive populations is affected by several factors such as the number of contributing broodstock to the next generation, sex ratio of parents, and variations in family size, which can be accurately evaluated by examination of the pedigree structure in the populations of interest. Here we present an example of microsatellite-based pedigree tracing in a hatchery strain of Japanese flounder Paralichthys olivaceus to be stocked into natural sea areas. We also detail the potential effect of selective operations (size selection) on the pedigree structure. The hatchery strain we screened was founded by using 18 wild captives (6 males and 12 females) through the use of the mesocosm spawning method, and the pedigree of the offspring including 113 individual larvae collected within 24 h after hatching, 216 individuals of 1 month old, and 407 individuals of 4 month old was unambiguously identified. The contribution of candidate broodstock to the next generation was highly skewed as the contribution to almost all of the offspring was monopolized by a single male, and a half of the females did not produce any offspring. The contribution of one family to the released fish selected for larger size (total length) was significantly high, while those of other two families were low (P<0.008).

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.

Mitochondrial gene introgression between spined loaches via hybridogenesis.

Abstract This report deals with an unusual mode of mitochondrial gene introgression between Cobitis hankugensis (C. sinensis) and C. longicorpus which is mediated by a unisexual hybridogenetic system of diploid-triploid C. hankugensis-longicorpus complex. Mitochondrial DNA sequences of 3329-3330bp encompassing from upstream ND6 to 12S rDNA indicated that mitochondrial genomes from the diploid hybrids, triploid hybrids, and their parental species are almost identical. Because triploid hybrids produce haploid ova with C. hankugensis chromosome set, normal diploid C. hankugensis regenerates upon insemination with C. hankugensis sperm. If the hybrid carries C. longicorpus mitochondrial genome, the regenerated C. hankugensis is a nucleo-cytoplasmic hybrid, thus accomplishing the unusual mode of mitochondrial gene introgression.

Identification of Undaria pinnatifida and Undaria undarioides Laminariales, Phaeophyceae using mitochondrial 23S ribosomal DNA sequences

Mitochondrial 23S ribosomal (r) DNA were sequenced from two Undaria species. The 23S rDNA from seven Undaria pinnatifida individuals were 2707 bp in length, whereas the 23S rDNA from four Undaria undarioides individuals were 2708–2709 bp in length. We found 15–20 nucleotide substitutions and 1–2 gaps between U. pinnatifida (the major haplotype) and U. undarioides. Based on the differences in sequences, we carried out PCR/RFLP analyses to distinguish between U. pinnatifida and U. undarioides, which showed different PCR/RFLP patterns upon Hinfl digestion. Sequence differences and PCR/RFLP analyses of mt 23S rDNA are useful for species identification of Undaria species.

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.

Genetic structure in species with shallow evolutionary lineages: a case study of the rare flatfish Verasper variegatus

We examined the genetic population divergence of the spotted halibut Verasper variegatus. A previous report suggested two conservation units for this species along the Japanese Pacific coast. Extending the coverage of the genomes (29 microsatellites and three mitochondrial DNA segments) revealed hitherto-undetected genetic population boundaries. We screened population samples from the major habitats along the Japanese coast and the Yellow Sea coast (East Asian Continent). Significant genetic differentiation was found in every comparison between the habitats. In most cases, the nuclear and mitochondrial population divergences were incongruent, most likely caused by differences between the two genomes in the effects of genetic drift after recent population isolation and bottleneck events. We discuss the ecological and evolutionary mechanisms of the genetic structure as well as the units of conservation. The present study illustrates the merits of wider coverage of genomes in genetic population analysis especially for species with a shallow population history.

Microsatellite markers for a rare species of right-eye flounder Verasper variegatus (Pleuronectiformes, Pleuronectidae)

In the context of stock enhancement for aquatic animals, the risk of negative genetic impacts of releasing hatchery-produced seedlings upon the genetic diversity of indigenous recipient populations becomes a growing concern. This issue is particularly relevant to threatened or rare species such as a member of right-eye flounder, the spotted halibut Verasper variegatus. We present here 29 microsatellite markers for spotted halibut, expecting that these markers could serve as a molecular tool of choice to address a wide range of conservation issues in this species.

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.

Characterization of 12 polymorphic microsatellite DNA loci in the blue shark, Prionace glauca, isolated by next generation sequencing approach

Genetic markers estimating the genetic diversity and population structure of the blue shark (Prionace glauca, Carcharhinidae) have been limited, although this species is one of the most common fishes caught incidentally in the open ocean. A total of twelve novel microsatellite markers in the blue shark were developed using a next generation sequencing approach. All the microsatellite loci isolated were polymorphic with 2–19 alleles, with the observed and expected heterozygosities of 0.25–0.90 and 0.35–0.92, respectively. Cross-species amplification in three other carcharhinid sharks was successful in ten out of twelve loci. The developed microsatellite markers will be useful to analyze the population genetic structure of the carcharhinid sharks including the blue shark.

Tetra-Repeat Microsatellite Markers for the Masu Salmon (Oncorhynchus masou masou) and Its Application in Cross-Subspecies Amplification

We developed tetranucleotide-repeat microsatellite markers for the masu salmon (Oncorhynchus masou) complex. 454 pyrosequencing was used to discover repeat motifs, and seven polymorphic microsatellite-primer sets were identified. The number of alleles detected at each locus ranged from four to 24 and the expected heterozygosity varied from 0.57 to 0.92. Cross-subspecies amplification for O. m. masou, O. m. ishikawae and O. m. subsp. was successful. These microsatellites can be utilized in studies of genetic structure, genetic diversity, and intra- and inter-subspecific hybridization, making a contribution to conservation and management of the Oncorhynchus masou complex.