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Various genome-scale data have been increasingly published in diverged species, but they can be reused for other purposes by re-analyzing in other ways. As a case study to utilize the published genome data, we developed microsatellite markers from the genome sequence data (assembled contigs and unassembled reads) of the marbled flounder Pseudopleuronectes yokohamae. No microsatellites were identified in the contig sequences, whereas the computer software found 781,773 sequences containing microsatellites with di- to hexa-nucleotide motif in the unassembled reads. For 86,732 unique sequences among them, a total of 331,368 primer pairs were designed. Screening based on PCR amplification, polymorphisms and accurate genotyping resulted in sixteen primer sets, which were later characterized using 45 samples collected in Onagawa Bay, Miyagi, Japan. The presence of null alleles was suggested at four loci in the studied population but no evidence of allelic dropout was found. The observed number of alleles and heterozygosity was 2-20 and 0-0.88889, respectively, indicating polymorphisms and usefulness for population genetic analyses of this species. In addition, a large number of the microsatellite primers developed in this study are potentially applicable also for kinship estimation, individual fingerprint and linkage map construction.

Chlamys farreri , which is naturally distributed along the coasts of East Asia, is a commercially important mollusk in China, and its mariculture has been prevalent since the 1970s with the decrease of natural resources in the 1960s. To conserve its genetic resources and avoid genetic disturbance, the population structure of the natural population in Japan and genetic condition of the stock population in China were investigated using morphological, microsatellite, and mtDNA markers. Analysis of morphological variance in C . farreri revealed no distinct difference between the presumed 2 subspecies ( C . farreri akazara and C . farreri nipponensis ) or between the natural and cultured scallops. Population genetic analysis using 2 DNA markers showed that wild scallops in Japan could be divided into a northern group and a southern group with 2 subgroups. The magnitude of genetic divergence between these groups was below the subspecies level, suggesting that C . farreri comprises 1 species with geographic subpopulations. Analysis of the mtDNA markers indicated the genetic divergence between populations of Japan and China, while that of the microsatellite markers showed that 3 Chinese populations (Qingdao, Weihai, and Yantai) were genetically similar to the northern Japanese group, suggesting the possibility of introduction of foreign genomes from the Tohoku area. On the basis of the present results, the northern and southern groups in Japan and Dalian population in China should ideally be regarded as separate units for management and conserved as the source populations for breeding programs. In addition, the genetic relationship among natural and cultured populations in China and natural populations in Japan should be further investigated for a better genetic management of this species.

Serotonin (5-hydroxytriptamine; 5-HT) is a major neurotransmitter that triggers oocyte maturation and sequential spawning in bivalve mollusks. A proteinous and heat-labile substance that proved to be a novel inhibitor of 5-HT-induced egg release from ovarian tissue was found in the cerebral and pedal ganglia (CPG) of the scallop Patinopecten yessoensis. The same inhibitory activity was also observed in the proteinous fraction from the supernatant of hemolymph. Histological observation demonstrated that the novel inhibitor prevented 5-HT from inducing oocyte maturation in the scallop ovary and that no prostaglandin F2alpha (PGF2alpha) inhibited 5-HT-induced oocyte maturation, although PGF2alpha strongly prohibited 5-HT-induced egg release through the gonoduct from ovarian tissue. The novel inhibitor from the scallop CPG also prohibited 5-HT-induced oocyte maturation of other bivalve species as well as scallops. The novel inhibitor, mediated through a receptor mechanism on oocyte membranes, blocked extracellular Ca2+ uptake into oocytes, which was observed in 5-HT-induced oocyte maturation. It is suggested that the novel inhibitor with a molecular mass of 60 kDa, named oocyte maturation arresting factor, which appears to be a universal substance for bivalve species, may be transported from the CPG to the ovary via hemolymph and may prohibit 5-HT-induced oocyte maturation due to the interference of extracellular Ca2+ influx into oocytes, eventually resulting in the inhibition of spawning. On the other hand, it seems that PGF2alpha inhibits 5-HT-induced transport of mature eggs through the gonoduct.

Five families of gynogenetic diploid Pacific oyster (Crassostrea gigas) were induced by inhibiting the second polar body in meiotic cell division of eggs fertilized with UV-irradiated sperm. Segregation patterns of eight microsatellite loci were investigated in the gynogenetic diploid offspring; the proportion of heterozygous progeny was used to estimate microsatellite–centromere (M–C) distances. Mendelian inheritance was confirmed for the eight loci by examining the genotypic segregation in the control crosses. Three of the eight microsatellite loci showed the existence of null alleles in four control crosses. All gynogenetic offspring only possessed the alleles of the mother, indicating 100% success level for the five families. The M–C recombination frequency estimates ranged from 0.62 to 0.77 (0.72 mean), comparable to those in the oyster based on allozyme markers and suggesting that meiotic gynogenesis does not appear to be a very efficient inbreeding method in the oyster. Recombination frequencies observed were often higher than the theoretical maximum of 0.67, indicating the existence of positive interference after a single chiasma formation in some chromosomes. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution toward assembly of genetic maps in C. gigas.

In order to assess the utility of microsatellite DNA markers for detecting changes of genetic diversity in hatchery strains and for estimating their genetic relationships, we used six microsatellite markers to estimate the level of genetic diversity within three hatchery strains and two wild populations of Pacific abalone, and compared the degree of genetic differentiation between them. High polymorphism at the microsatellite loci was found within both hatchery and wild abalone populations. Compared to wild populations, all the hatchery strains showed less genetic variation as revealed in lower number of alleles and lower expected heterozygosity, indicating that bottleneck effects occurred when each strain was founded. Significant differentiation was found between the hatchery strains, and between the hatchery strains and wild populations (Fst range: 0.059–0.427; Rst range: 0.056–0.351), and no obvious difference was detected between the wild populations (Fst=0.004; Rst=0.007). According to the neighbor-joining tree topology constructed on the basis of genetic distances among individuals, almost all individuals from each hatchery strain were closely clustered, demonstrating the feasibility of microsatellite analysis for discrimination between hatchery strains. The results obtained in this study indicate that it is necessary to genetically characterize the abalone strains that are being released every year in order to monitor the effect on the genetic diversity of wild populations.

Effects of ultraviolet (UV) irradiation on genetic inactivation and morphological structure of sperms were examined in the scallop, Patinopecten yessoensis . Haploid gynogenesis of the scallop was successfully induced by 50–60 s UV irradiation of 720 μW cm −2 s −1 . The fertilization rate apparently decreased with increasing irradiation time, and the development of the eggs fertilized with the genetically inactivated sperms terminated before reaching the D-shaped larvae stage. Scanning electron microscopy (SEM) showed clear destruction of the sperm acrosome and flagellum in the UV-irradiated sperms. As the duration of UV irradiation increased, the acrosome of sperms tended to suffer greater damage, until the sperms eventually lost their flagella. Abnormalities in these structures have appeared to account, at least in part, for the decline of the fertilization rate of eggs inseminated with UV-irradiated sperms.

Ploidy manipulation was used to examine the effects of triploidizatin and gynogenetic diploidization in fancy carp, a race of Cyprinus carpio. Ultraviolet dosages of at least 9000 erg/mm2 were required to inactivates sperm genetically. An interval of 10–12 min before the start of 60-min cold treatments was required to retain the second polar body in fertilized eggs. At optimum conditions, survival rates of gynogenetic diploids were 80% to the eyed embryo stage and 25% to hatching; corresponding values in triploids were 73% and 32%, respectively. The highest success rates in inducing gynogenetic diploids and triploids were 100% and 68.9%, respectively. Triploids with the Japanese strain as the female and European strain as the male parent grew slower than control diploids when they were reared in the same pond for 20 months. However, triploids using the Japanese strain as both parents grew larger in 7 months than control diploids. Triploids at 20 months of age had less developed gonads and more fat around the digestive tract than diploid fish. Male-specific color spots were smaller in triploids than in control diploids. The typical red and white fancy carp colors were found in the gynogenetic diploids in almost the same frequency as in the control diploids. Recombination between gene and centromere was observed at the Gpi-2 locus in gynogenetic diploids.