Population genetic structure of Semisulcospira gottschei: simultaneous examination of mtDNA and microsatellite markers.

Semisulcospira gottschei is an Asian endemic species inhabiting Korea and China. However, genetic structure analysis of the resource management of this species has not been performed. To investigate the genetic diversity among populations, microsatellites can be used to determine the geographic origins of marine and freshwater species. This study investigated the genetic structures of the Korean and Chinese populations of S. gottschei based on mitochondrial DNA (mtDNA) Cytochrome oxidase subunit I (COI) and polymorphic microsatellite loci developed from Semisulcospira coreana. Analysis of the mtDNA COI sequence revealed 43 haplotypes, which indicated no gene flow between the Korean and Chinese populations. To further elucidate the genetic structures of the Korean and Chinese populations, the population genetics of S. gottschei were analyzed using nine microsatellite markers. The genetic diversity analysis showed an average of 5.25 alleles per locus, with an average allelic richness of 4.02. Excessive homozygosity was found at all loci, which was expected to be due to the presence of null alleles at all loci. Populations of S. gottschei formed two separate clusters according to pairwise FST and AMOVA. Also, the UPGMA tree, PCA, STRUCTURE, and GeneClass indicated separation of the 11 populations into two clusters: Korea and China. These results have potential use in the management, restoration, and distinction of the origin country of populations.

Antiviral effects of extracts from Celosia cristata and Raphanus sativus roots against viral hemorrhagic septicemia virus.

The antiviral activity of an extract mixture from Celosia cristata and Raphanus sativus was tested against viral hemorrhagic septicemia virus (VHSV). Pretreatment of EPC cells with this extract up to 72 h before VHSV infection markedly reduced the virus titer, but it had no effect when added after virus inoculation. In olive flounder that received 5 µg of extract per fish, Mx expression peaked at 48 h after treatment. In contrast, ISG15 and TLR2 expression peaked at 72 h, and that of TLR7 peaked at 48 h, followed by a slight decrease at 72 h, indicating that the antiviral activity was mediated by induction of gene expression involved in the innate immune response.

Cetaceans evolution: insights from the genome sequences of common minke whales.

BACKGROUND: Whales have captivated the human imagination for millennia. These incredible cetaceans are the only mammals that have adapted to life in the open oceans and have been a source of human food, fuel and tools around the globe. The transition from land to water has led to various aquatic specializations related to hairless skin and ability to regulate their body temperature in cold water. RESULTS: We present four common minke whale (Balaenoptera acutorostrata) genomes with depth of ×13 ~ ×17 coverage and perform resequencing technology without a reference sequence. Our results indicated the time to the most recent common ancestors of common minke whales to be about 2.3574 (95% HPD, 1.1521 - 3.9212) million years ago. Further, we found that genes associated with epilation and tooth-development showed signatures of positive selection, supporting the morphological uniqueness of whales. CONCLUSIONS: This whole-genome sequencing offers a chance to better understand the evolutionary journey of one of the largest mammals on earth.

Rapid Origin Determination of the Northern Mauxia Shrimp (Acetes chinensis) Based on Allele Specific Polymerase Chain Reaction of Partial Mitochondrial 16S rRNA Gene.

Acetes chinensis is an economically important shrimp that belongs to the Sergestidae family; following fermentation, A. chinensis' economic value, however, is low in China, and much of the catch in China is exported to Korea at a low price, thus leading to potential false labeling. For this reason, we developed a simple method to identify A. chinensis' origin using allele-specific polymerase chain reaction (PCR). Ten single nucleotide polymorphisms (SNPs) were identified from partial (i.e., 570 bp) DNA sequence analysis of the mitochondrial 16s rRNA gene in 96 Korean and 96 Chinese individual shrimp. Among 10 SNP sites, four sites were observed in populations from both countries, and two sites located in the middle with SNP sites at their 3'-ends were used to design allele-specific primers. Among the eight internal primers, the C220F primer specific to the Chinese A. chinensis population amplified a DNA fragment of 364 bp only from that population. We were able to identify the A. chinensis population origin with 100% accuracy using multiplex PCR performed with two external primers and C220F primers. These results show that the 16S rRNA gene that is generally used for the identification of species can be used for the identification of the origin within species of A. chinensis, which is an important finding for the fair trade of the species between Korea and China.

Population genetic analysis and origin discrimination of snow crab (Chionoecetes opilio) using microsatellite markers.

Major habitats for the snow crab Chionoecetes opilio are mostly found within the northwest Atlantic and North Pacific Oceans. However, the East Sea populations of C. opilio, along with its relative the red snow crab (C. japonicas), are two of the most important commercial crustacean species for fisheries on the east coast of the Korean Peninsula. The East Sea populations of C. opilio are facing declining resources due to overfishing and global climate change. Thus, an analysis of population structure is necessary for future management. Five Korean and one Russian group of C. opilio were analyzed using nine microsatellite markers that were recently developed using next-generation sequencing. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The number of alleles per locus varied from 4 to 18 with a mean of 12, and allelic richness per locus ranged from 4.0 to 17.1 across all populations with a mean of 9.7. The Hardy-Weinberg equilibrium test revealed significant deviation in three out of nine loci in some populations after sequential Bonferroni correction and all of them had higher expected heterozygosity than observed heterozygosity. Null alleles were presumed in four loci, which explained the homozygosity in three loci. The pairwise fixation index (F ST ) values among the five Korean snow crab populations did not differ significantly, but all of the pairwise F ST values between each of the Korean snow crab populations and the Russian snow crab population differed significantly. An UPGMA dendrogram revealed clear separation of the Russian snow crab population from the Korean snow crab populations. Assignment tests based on the allele distribution discriminated between Korean and Russian origins with 93 % accuracy. Therefore, the snow crab populations around the Korean Peninsula need to be managed separately from the populations in Bering Sea in global scale resource management. Also, this information can be used for identification of snow crab origin which is problematic in worldwide crab trade.

Characterizations of Shell and Mantle Edge Pigmentation of a Pacific Oyster, Crassostrea gigas, in Korean Peninsula.

The objectives of this study were to investigate color patterns of shell and mantle edge pigmentation of a Pacific oyster, C. gigas, and to estimate variance components of the two colors. A sample of 240 F0 oysters was collected from six aquaculture farms in Tongyeong, Korea to measure shell color and mantle edge pigmentation. Among the F0s, male and female individuals with black (white) shell and black (white) mantle edge were selected and mated to generate three F1 full-sib black (white) cross families (N = 265). Two and four F2 cross families (N = 286) were also produced from black and white F1 selected individuals, respectively. Variance component estimates due to residuals and families within color were obtained using SAS PROC VARCOMP procedures to estimate heritability of shell and mantle edge pigmentation. In the F0 generation, about 29% (11%) had black (white) color for both shell and mantle edge. However, in the F1 and F2 black (white) cross families, 75% (67%) and 100% (100%) of oysters had black (white) shell colors, and 59% (23%) and 79% (55%) had black (white) mantle edge, respectively. Spearman correlation coefficients between shell and mantle edge color were 0.25, 0.74, and 0.92 in F0, F1, and F2 generations, respectively, indicating that, with generations of selection process, an individual with black (white) shell color is more likely to have black (white) mantle edge pigmentation. This suggests that shell color could be a good indicator trait for mantle edge pigmentation if selection of both the colors is implemented for a couple of generations. Estimates of heritability were 0.41 and 0.77 for shell color and 0.27 and 0.08 for mantle edge pigmentation in the F1 and F2 generations, respectively, indicating that, in general, significant proportions of phenotypic variations for the shell and mantle edge colors are explained by genetic variations between individuals. These results suggest that the two color traits are inheritable and correlated, enabling effective selection on shell and mantle edge color.

Development of Genetic Markers for Triploid Verification of the Pacific Oyster, Crassostrea gigas.

The triploid Pacific oyster, which is produced by mating tetraploid and diploid oysters, is favored by the aquaculture industry because of its better flavor and firmer texture, particularly during the summer. However, tetraploid oyster production is not feasible in all oysters; the development of tetraploid oysters is ongoing in some oyster species. Thus, a method for ploidy verification is necessary for this endeavor, in addition to ploidy verification in aquaculture farms and in the natural environment. In this study, a method for ploidy verification of triploid and diploid oysters was developed using multiplex polymerase chain reaction (PCR) panels containing primers for molecular microsatellite markers. Two microsatellite multiplex PCR panels consisting of three markers each were developed using previously developed microsatellite markers that were optimized for performance. Both panels were able to verify the ploidy levels of 30 triploid oysters with 100% accuracy, illustrating the utility of microsatellite markers as a tool for verifying the ploidy of individual oysters.

Development of microsatellite markers to genetically differentiate populations of Octopus minor from Korea and China.

Of the more than 300 octopus species, Octopus minor is one of the most popular and economically important species in Eastern Asia, including Korea, along with O. vulgaris, O. ocellatus, and O. aegina. We developed 19 microsatellite markers from Octopus minor and eight polymorphic markers were developed to analyze the genetic diversity and relationships among four octopus populations from Korea and three from China. The number of alleles per locus varied from 10 to 49, and allelic richness per locus ranged from 2 to 16.4 across all populations. The average allele number among the populations was 11.1, with a minimum of 8.3 and a maximum of 13.6. The mean allelic richness was 8.7 in all populations. The Hardy-Weinberg equilibrium (HWE) test revealed significant deviation in 19 of the 56 single-locus sites, and null alleles were presumed in five of eight loci. The pairwise F ( ST ) values between populations from Korea and China differed significantly in all pairwise comparisons. The genetic distances between the China and Korea samples ranged from 0.161 to 0.454. The genetic distances among the populations from Korea ranged from 0.033 to 0.090, with an average of 0.062; those among populations from China ranged from 0.191 to 0.316, with an average of 0.254. The populations from Korea and China formed clearly separated into clusters via an unweighted pair group method with arithmetic mean dendrogram. Furthermore, a population from muddy flats on the western coast of the Korean Peninsula and one from a rocky area on Jeju Island formed clearly separated subclusters. An assignment test based on the allele distribution discriminated between the Korean and Chinese origins with 96.9 % accuracy.

Rapid development of microsatellite markers with 454 pyrosequencing in a vulnerable fish, the mottled skate, Raja pulchra.

The mottled skate, Raja pulchra, is an economically valuable fish. However, due to a severe population decline, it is listed as a vulnerable species by the International Union for Conservation of Nature. To analyze its genetic structure and diversity, microsatellite markers were developed using 454 pyrosequencing. A total of 17,033 reads containing dinucleotide microsatellite repeat units (mean, 487 base pairs) were identified from 453,549 reads. Among 32 loci containing more than nine repeat units, 20 primer sets (62%) produced strong PCR products, of which 14 were polymorphic. In an analysis of 60 individuals from two R. pulchra populations, the number of alleles per locus ranged from 1-10, and the mean allelic richness was 4.7. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy-Weinberg equilibrium test showed significant deviation in two of the 28 single-loci after sequential Bonferroni's correction. Using 11 primer sets, cross-species amplification was demonstrated in nine related species from four families within two classes. Among the 11 loci amplified from three other Rajidae family species; three loci were polymorphic. A monomorphic locus was amplified in all three Rajidae family species and the Dasyatidae family. Two Rajidae polymorphic loci amplified monomorphic target DNAs in four species belonging to the Carcharhiniformes class, and another was polymorphic in two Carcharhiniformes species.

Novel microsatellite markers of Meretrix petechialis and cross-species amplification in related Taxa (Bivalvia: Veneroida).

The Asian hard clam, Meretrix petechialis, is an economically important bivalve, but its catch and population sizes are decreasing rapidly, owing to many factors, including large-scale reclamation of its natural habitat on the western coast of the Korean peninsula. Attempts to restore the resources and production of this species require genetic structure and diversity information. In this study, we developed 15 microsatellite markers from a partial genomic library enriched in GT repeats. Nine of these markers were polymorphic, with an average allele number of six, and six were monomorphic in 95 tested individuals. No linkage disequilibrium was found between any pair of loci (p > 0.05), and deviations from the Hardy-Weinberg equilibrium (HWE) test showing excess of heterozygotes was observed in only one of nine loci. In addition, no null alleles or genetic differentiation between two tested populations were detected. A cross-species amplification in 12 species of four families resulted in two M. petechialis-specific loci and three possible universal markers. This information will be useful in the future development of high-quality artificial seedlings and sustainable resource management.

Stepwise Approach for Tracing the Geographical Origins of the Manila Clam Ruditapes philippinarum Using Dual-Element Isotopes and Carbon Isotopes of Fatty Acids.

While there are many studies that have reported methods for tracing the geographical origin of seafoods, most of them have focused on identifying parameters that can be used effectively and not the direct application of these methods. In this study, we attempted to differentiate the geographical origins of the Manila clam R. philippinarum collected from different sites in Korea, the Democratic People's Republic of Korea, and China using a combination of analyses based on dual-element isotopes, fatty acids (FAs), and compound-specific isotopic analysis of FAs. We hypothesized that a stepwise application of new parameters to unclassified samples could achieve this objective by integrating new information while reducing time and labor. The FA profiles and compound-specific carbon isotopic values of FAs were found to enhance the discrimination power of determining the geographic origin up to 100%. Our findings demonstrate the advantageousness of using several parameters simultaneously over the conventional method of employing individual analytical methods when identifying geographic origins of the Manila clam, which could have implications for tracing the origins of different shellfish species or other food products as well.

Classification of Takifugu rubripes, T. chinensis and T. pseudommus by genotyping-by-sequencing.

Takifugu rubripes is more expensive than other species of the genus because of its high protein content and special flavor. However, it is easily confused with imported T. chinensis and T. pseudommus because they have similar morphological characteristics. We identified single nucleotide polymorphism (SNP) markers of T. rubripes by genotyping-by-sequencing (GBS) and evaluated their ability to distinguish among T. rubripes, T. chinensis, and T. pseudommus. In all, 18 polymorphic SNPs were subjected to phylogenetic analyses of the three Takifugu species. Additionally, we subjected a second set of samples to Sanger sequencing to verify that the polymorphic SNPs could be used to evaluate the genetic variation among the three Takifugu species. A phylogenetic tree that included the analyzed sequence of set A, which is referred to as the reference sequence, and a validation sequence of set B with 18 SNPs were produced. Based on this phylogenetic tree and STRUCTURE analyses, T. rubripes, T. chinensis and T. pseudommus have low genetic variation and should be considered the same gene pool. Our findings suggest that further studies are needed to estimate the genetic association of the three Takifugu species.

Cryptic diversity in the inshore hagfish, Eptatretus burgeri (Myxinidae, Pisces) from the northwest Pacific.

The fishery of inshore hagfish (Eptatretus burgeri) is particularly important from the perspective of the eel-skin leather industry in the northwest Pacific. In order to reveal the genetic diversity and population structure of E. burgeri in the northwest Pacific, we analyzed partial nucleotide sequences of three mitochondrial DNA regions (523 bp in COI, 712 bp in ND4 and 617 bp in Cytb) based on specimens collected from six locations in Korea and Japan. The genetic diversities of E. burgeri were higher in Korean locations compared to Japanese ones. AMOVA showed that E. burgeri was completely separated into two groups (group A: southern coast of Korea and western coast of Japan vs. group B: eastern coast of Japan). Furthermore, groups A and B were divided into each two lineages (lineage I: west southern coast of Korea, lineage II: east southern coast of Korea and western coast of Japan, lineage III and IV: eastern coast of Japan). Our molecular results suggest that these two groups and lineages of E. burgeri may be different evolutionary significant unit and management unit, respectively.

Identification of two differentially expressed forms of progranulin mRNA in the teleost fish Paralichthys olivaceus.

Two forms of progranulin mRNA were isolated from kidney and spleen cDNA libraries of the olive flounder, Paralichthys olivaceus. The 3'-untranslated regions (3'-UTRs) of these flounder progranulin (f-pgrn) mRNAs differed in a 20-nucleotide sequence element (5'-AACTGATTACGTTCAACAAC-3') that was present in one mRNA (designated f-pgrn type II) and not in the other (designated f-pgrn type I). Both mRNA sequences contained an open reading frame encoding a 289-amino-acid polypeptide of approximately 33 kDa. Southern blot analysis of the P. olivaceus flounder genome using an f-pgrn cDNA probe and a PCR-based approach identified a single copy of f-pgrn corresponding to the type II mRNA. The expression profiles of the two types of f-pgrn mRNA differed from each other and were tissue- and condition-dependent. The type II mRNA was detected abundantly in studied tissues (gill, kidney, spleen, and intestine) of non-stimulated healthy flounders. The type I mRNA was rarely expressed in any tissues of healthy flounders, but it was continuously increased in the examined tissues of flounders after the intraperitoneal injection of lipopolysaccharide. On the other hand, the expression of type II mRNA was decreased in inverse proportion to the type I mRNA in the LSP-stimulated flounders. These results suggest that type I and type II f-pgrn mRNA are translated into different proteins with different activities in the immune system of flounder.

Development of Species-Specific PCR Primers for the Rapid and Simultaneous Identification of the Six Species of Genus Takifugu.

Pufferfish (Takifugu spp.) are economically important edible marine fish. Mistakes in pufferfish classification can lead to poisoning; therefore, accurate species identification is critical. In this study, we used the mtDNA cytochrome c oxidase subunit I gene (COI) to design specific primers for six Takifugu species among the 21 domestic or imported pufferfish species legally sold for consumption in Korea. We rapidly and simultaneously identified these pufferfish species using a highly efficient, multiplex polymerase chain reaction (PCR) system with the six species-specific primers. The results showed that species-specific multiplex PCR (multiplex species-specific polymerase chain reaction; MSS-PCR) either specifically amplified PCR products of a unique size or failed. MSS-PCR yielded amplification fragment lengths of 897 bp for Takifugu pardalis, 822 bp for T. porphyreus, 667 bp for T. niphobles, 454 bp for T. poecilonotus, 366 bp for T. rubripes, and 230 bp for T. xanthpterus using the species-specific primers and a control primer (ca. 1,200 bp). We visualized the results using agarose gel electrophoresis to obtain accurate contrasts of the six Takifugu species. MSS-PCR analysis is easily performed and provides identification results within 6 h. This technique is a powerful tool for the discrimination of Takifugu species and will help prevent falsified labeling, protect consumer rights, and reduce the risk of pufferfish poisoning..

Quantitative analysis of Alaska pollock in seafood products by droplet digital PCR.

A highly accurate quantitative method, based on the new technique, droplet digital PCR (ddPCR), was applied to determine the content of Alaska pollock (Gadus chalcogrammus) in seafood products. Using this method, we found a linear relationship among raw sample weight, DNA concentration and DNA copy number. We also established a formula to calculate the raw sample weight, based on the number of DNA copies. To confirm the accuracy and applicability of this method, mixed samples of known composition were analyzed. Results from this study indicated that the ddPCR method described is suitable for quantifying Alaska pollock in seafood products and has the potential applied to a variety of tasks in food quality certification.

Molecular cloning and expression analysis of the STAT1 gene from olive flounder, Paralichthys olivaceus.

BACKGROUND: Signal transducer and activator of transcription 1 (STAT1) is a critical component of interferon (IFN)-alpha/beta and IFN-gamma signaling. Although seven isoforms of STAT proteins have been reported from mammals, limited information is available for the STAT genes in fish. We isolated complementary DNA with high similarity to mammalian STAT1 from the olive flounder, Paralichthys olivaceus. RESULTS: A DNA fragment containing the conserved SH2 domain was amplified by RT-PCR using degenerate primers designed based on the highly conserved sequences in the SH2 domains of the zebrafish and mammalian STAT1. The complete cDNA sequence was obtained by 5' and 3' RACE. The flounder STAT1 transcript consisted of 2,909 bp that encoded a polypeptide of 749 amino acids. The overall similarity between flounder STAT1 and other STATs was very high, with the highest amino acid sequence identity to snakehead (89%). Phylogenetic analyses reveal that flounder STAT1 is in the same monophyletic group with snakehead STAT1. Quantitative real time RT-PCR and in situ hybridization revealed that STAT1 was expressed in almost all examined organs and tissues, with high expression in gill, spleen, kidney, and heart. The accumulation of STAT1 mRNA in different developmental stages, as determined by real time RT-PCR, increased with development. CONCLUSION: Recent cloning of various cytokine genes and the STAT1 gene of olive flounder here suggest that fish also use the highly specialized JAK-STAT pathway for cytokine signaling. Identification of other STAT genes will elucidate in detail the signal transduction system in this fish.

Segregation distortion at a microsatellite marker in the olive flounder, Paralichtys olivaceus.

In the course of quantitative trait loci (QTL) analysis of the back cross (BC1) families of olive flounder (Paralichtys olivaceus), we observed significant segregation distortion at a microsatellite marker Poli9-58TUF in two crosses of informative progenies. The family 1 of the random BC1 progenies derived from a cross between a F1 male genotype (A/B) and a F1's female parent genotype (A/C) and the family 2 (A/C x A/C) displayed a strong bias in the locus from the Mendelian inheritance by the elimination of homozygous A/A genotype. The deleterious roles of the AA genotypes are suggested during the metamorphosis and it implies that the parents of these families carried a recessive gene or genes hampering development at an early stage because the offspring of the double heterozygote parents show the reduction in frequency or elimination of one homozygous class, which is an evidence for linkage between the genetic marker and gene(s) with recessive deleterious alleles. This data support a hypothesis that the region contains a recessive lethal gene or genes.

Deciphering the evolutionary signatures of pinnipeds using novel genome sequences: The first genomes of Phoca largha, Callorhinus ursinus, and Eumetopias jubatus.

The pinnipeds, which comprise seals, sea lions, and walruses, are a remarkable group of marine animals with unique adaptations to semi-aquatic life. However, their genomes are poorly characterized. In this study, we sequenced and characterized the genomes of three pinnipeds (Phoca largha, Callorhinus ursinus, and Eumetopias jubatus), focusing on site-wise sequence changes. We detected rapidly evolving genes in pinniped lineages and substitutions unique to pinnipeds associated with amphibious sound perception. Phenotypic convergence-related sequence convergences are not common in marine mammals. For example, FASN, KCNA5, and IL17RA contain substitutions specific to pinnipeds, yet are potential candidates of phenotypic convergence (blubber, response to hypoxia, and immunity to pathogens) in all marine mammals. The outcomes of this study will provide insight into targets for future studies of convergent evolution or gene function.

Complete sequence and polymorphisms of female Ruditapes philippinarum (Mollusca: Bivalvia) mitochondria genome.

Mitogenome of female Ruditapes philippinarum organism was sequenced, and genomic variation and phylogeny were examined in this study. Length of the mitogenome was 22 089 bp showing 94.28% of sequence identity with previously reported sequence. Total 707 single nucleotide polymorphisms, SNPs, were detected and 50 residues were non-synonymous SNPs among the 202 SNPs in protein-coding genes. Deleted genomic fragments with of 265 bp and 322 bp were observed in non-coding regions, ND2 to ND4L and ND4L to tRNA(Ile), respectively. Phylogenic analysis confirmed that used organisms were female R. philippinarum, and the species has closer evolutionary distance with genus Paphia rather than genus Meretrix. Our finding will be help to set an insight for population and evolutionary genomics of Veneroida clams as well as application to marine industry.