Isolation and characterization of microsatellite markers for blue marlin, Makaira nigricans

In the Unites States, blue marlin, Makaira nigricans, is managed as separate Atlantic and Indo-Pacific stocks. To facilitate the discrimination of the two stocks for enforcement purposes, we developed 10 new microsatellite markers and screened collections of 20 individuals each from Ghana and Hawai’i. Observed heterozygosities (H_O) ranged from 0.700 to 1.000 among loci at the two collections. The number of alleles per locus ranged from 14 to 24, there were no significant deviations from the expectations of Hardy–Weinberg equilibrium, and there was no evidence of linkage disequilibrium among the loci. These new markers will provide more power to assign marketed blue marlin products to ocean origin.     

Photoreceptor layer of salmonid fishes: transformation and loss of single cones in juvenile fish

The retinas of many vertebrates have cone photoreceptors that express multiple visual pigments. In many of these animals, including humans, the original cones to appear in the retina (which express UV or blue opsin) may change opsin types, giving rise to new spectral phenotypes. Here we used microspectrophotometry and in situ hybridization with cDNA probes to study the distribution of UV and blue cones in the retinas of four species of Pacific salmon (coho, chum, chinook, and pink salmon), in the Atlantic salmon, and in the rainbow/steelhead trout. In Pacific salmon and in the trout, all single cones express a UV opsin at hatching (lambda(max) of the visual pigment approximately 365 nm), and these cones later transform into blue cones by opsin changeover (lambda(max) of the blue visual pigment approximately 434 nm). Cones undergoing UV opsin downregulation exhibit either of two spectral absorbance profiles. The first is characterized by UV and blue absorbance peaks, with blue absorbance dominating the base of the outer segment. The second shows UV absorbance diminishing from the outer segment tip to the base, with no sign of blue absorbance. The first absorbance profile indicates a transformation from UV to blue phenotype by opsin changeover, while the second type suggests that the cone is undergoing apoptosis. These two events (transformation and loss of corner cones) are closely associated in time and progress from ventral to dorsal retina. Each double cone member contains green (lambda(max) approximately 510 nm) or red (lambda(max) approximately 565 nm) visual pigment (double cones are green/red pairs), and, like the rods (lambda(max) approximately 508 nm), do not exhibit opsin changeover. Unlike Pacific salmonids, the Atlantic salmon shows a mixture of UV and blue cones and a partial loss of corner cones at hatching. This study establishes the UV-to-blue cone transformation as a general feature of retinal growth in Pacific salmonids (genus Oncorhynchus).     

Isolation and characterization of 29 polymorphic microsatellite markers for the endangered Atlantic goliath grouper (Epinephelus itajara), and the Pacific goliath grouper (E. quinquefasciatus)

We isolated 40 microsatellite markers for the endangered Atlantic, and Pacific goliath grouper, Epinephelus itajara and E. quinquefasciatus. Loci were characterized using samples of E. itajara (n = 52) and E. quinquefasciatus (n = 10). Twenty-nine loci were polymorphic. For E. itajara and E. quinquefasciatus, respectively, mean numbers of alleles per locus were 5.2 and 4.3 and mean observed heterozygosities were 0.46 and 0.63. Three loci departed from Hardy–Weinberg equilibrium expectations in the E. itajara sample. Allele frequencies differed significantly between the two taxa for each polymorphic locus.     

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.     

Twenty-two novel microsatellite loci for lumpfish (Cyclopterus lumpus)

Lumpfish (Cyclopterus lumpus L. 1758) are widely distributed on both sides of the North Atlantic. They are a commercially important species, but stock size estimates have declined since the mid-1980s in Canada, Norway and Iceland. Little is known about the biology of this species, in particular the breeding migrations and population structure which are fundamental for effective management. This paper describes the development and characterization of twenty-two polymorphic microsatellite loci using next generation sequencing. The number of alleles per locus ranged from 3 to 27 in two geographically distant North Atlantic populations, with observed and expected heterozygosities ranging between 0.0625–0.979 and 0.0618–0.946, respectively. These loci are an important resource that will allow assessment of the population genetic structure of this species, and contribute to its appropriate management.     

Development and characterization of ten polymorphic microsatellite loci in the yellowtail flounder (Limanda ferruginea)

We isolated and characterized a total of 10 microsatellite loci from the Yellowtail flounder, Limanda ferruginea. Loci were screened in 24 individuals, 8 each, from the Georges Bank, Southern New England/Mid Atlantic, and Cape Cod/Gulf of Maine, management areas. The number of alleles per locus ranged from 5 to 23, observed heterozygosity ranged from 0.375 to 0.875, and the probability of identity values ranged from 0.102 to 0.505. These new loci will provide tools for examining existing management stock designations and for determining levels of gene flow between these stocks.     

Development of EST-SSR PCR primers within three North Pacific rockfishes of the genus Sebastes

Severe declines in abundance of Pacific Ocean Perch (Sebastes alutus) and other rockfishes have led to catch restrictions along the US west coast. The stock structure of many important commercial members of Sebastes spp remains a major source of uncertainty in stock assessment models. Here we report development of PCR primer pairs from 13 novel EST-SSR loci obtained from public Sebastes EST databases, and redesigned primer pairs from six published EST-SSR loci. Amplification and diversity data from these and 27 published EST-SSR loci are reported for S. polyspinis, S. alutus, and two cryptic sub-types of S. aleutianus. Loci were moderately polymorphic, with heterozygosities averaging 0.44, and allele number averaging 5.2 across all loci and species. Divergence between the two S. aleutianus sub-types was high (mean F _ST = 0.33) allowing for unambiguous identification. The loci will be informative for resolving stock structure issues and possible local adaptation along the species’ ranges.     

Isolation and characterization of 12 microsatellites for the commercially important sablefish, Anoplopoma fimbria

Sablefish, Anoplopoma fimbria, are long-lived, highly migratory, bathydemersal, commercially important fishes that inhabit continental slope waters of the North Pacific Ocean. Here we describe 12 microsatellite loci developed for sablefish, and cross-species amplification in skilfish, Erilepis zonifer. Microsatellites were developed from one sablefish and characterized using 55 juveniles collected in the eastern Gulf of Alaska. The number of alleles ranged from 3 to 27 per locus, and observed heterozygosity ranged from 0.074 to 0.964. There was no significant evidence for linkage disequilibrium or departure from Hardy?CWeinberg Equilibrium. Ten of the 12 microsatellite loci were successfully amplified in skilfish. These new microsatellites were developed for use in sablefish fishery management and conservation applications including selecting broodstock for aquaculture operations and defining population boundaries for stock assessments.     

Isolation and characterization of thirty microsatellite loci for Atlantic mackerel (Scomber scombrus L.)

Atlantic mackerel (Scomber scombrus L.) is a widely distributed commercially important pelagic species. Little is known about the stock structure of this species, but it is thought to be undergoing a range extension due to environmental changes. Knowledge of the stock structure under these changing conditions is fundamental for effective management. In this paper, 30 highly polymorphic microsatellite loci developed with next generation sequencing are described. The number of alleles per locus ranged from 4 to 39 in two geographically distant populations, observed and expected heterozygosities ranged between 0.370–0.978 and 0.426–0.962, respectively. These loci are an important resource that will allow assessment of the current population genetic structure of this species, and enable monitoring of climate related changes in the species range and distribution.     

Characterization of 18 polymorphic microsatellite loci from Bathymodiolus manusensis (Bivalvia, Mytilidae) from deep-sea hydrothermal vents

Species in the genus Bathymodiolus are mytilid mussels found at deep-sea hydrothermal vents and cold seeps. Next-generation sequencing techniques were employed to identify eighteen unlinked polymorphic microsatellite loci for Bathymodiolus manusensis from Manus Basin in the western Pacific. Allele frequencies for eight loci conform to Hardy–Weinberg expectations and observed heterozygosities ranged from 0.04 to 0.90 (mean H _O = 0.45, SD = 0.25). Ten of eighteen loci cross-amplified in Bathymodiolus heckerae from Atlantic seeps. Microsatellites developed for Bathymodiolus manusensis are being deployed to study connectivity among populations of this species colonizing geographically discrete back-arc basin vent systems.     

Isolation and characterization of microsatellite loci in <Emphasis Type="Italic">Rhinogobio ventralis</Emphasis>

Over-exploitation of traditional coastal stocks and a rising demand for seafood have resulted in the shift of commercial fishing towards less-known, deep-sea species in many parts of the world. Yet, the lack of knowledge of the biology, ecology and life-history of these species represents a serious impediment for establishing sound stock management plans. With the aim of providing tools that will allow assessment of the population genetic structure of Macrourus berglax, we have isolated and characterised a suite of novel microsatellite loci for this deep sea grenadier. Eight of these markers showed between 4 and 11 alleles per locus in two distant North Atlantic populations, with observed and expected heterozygosities between 0.17–0.83 and 0.35–0.87, respectively. Importantly, eight of these loci also cross-amplify in other Macrourid species.     

Development of SNP and microsatellite markers for goldsinny wrasse (<Emphasis Type="Italic">Ctenolabrus rupestris</Emphasis>) from ddRAD sequencing data

Wrasse (Labridae) species have been used as parasite cleaners in Atlantic salmon farming since the 1980s. However, their use has recently escalated, with millions now being introduced into salmon cages each year. Most fish are of wild origin, their exploitation potentially impacting native populations. Genetic information is urgently required to inform management decisions. We identified 174 microsatellite and 149 SNP markers from ddRAD sequence data. From these, 17 and 48 microsatellite and SNP markers, respectively, were validated by genotyping 150 goldsinny wrasse collected from five locations along the Norwegian and Swedish coasts. Two to 30 alleles were identified at the microsatellite loci, while gene diversity (H _e ) ranged 0.101–0.907. All SNP loci were biallelic, with averaged H _e per locus ranging between 0.063 and 0.495.     

Complete mitochondrial genome of the Pacific white-sided dolphin <Emphasis Type="Italic">Lagenorhynchus obliquidens</Emphasis> (Cetacea: Delphinidae)

The Pacific white-sided dolphin (Lagenorhynchus obliquidens Gill, 1865) is one of the most abundant, widely distributed delphinids in the North Pacific Ocean, whose existence is threatened by fisheries and environmental contamination. Herein, we report the first complete mitochondrial genome of L. obliquidens. The 16,392-bp sequenced genome exhibited typical cetacean mitochondrial gene arrangement, consisted of the typical set of 37 genes, one replication origin, and a D-loop. As expected, the genome displayed the highest similarity with that of Cephalorhynchus heavisidii and was distinct from that of L. albirostris. Multigene phylogeny also revealed that L. obliquidens was closely related to C. heavisidii, thus suggesting that the genus Lagenorhynchus is polyphyletic, in accordance with the results of recent molecular phylogenetic studies. The results provide information fundamental for genetic and conservation studies for L. obliquidens.     

Establishing a genetic system to distinguish between domestic Mallards,Pacific Black Ducks and their hybrids

Dabbling ducks are subject to many threatening processes. Hybridisation with introduced Mallards (Anas platyrhynchos) is a threat that is commonly overlooked. Mallards, both domestic and wild, have been introduced in Australia and New Zealand. While hybridisation with Mallards caused significant decline in the New Zealand populations of Pacific Black (Grey) Ducks (Anas superciliosa), the degree of hybridisation between Mallard and the Pacific Black Duck in Australia is currently unknown, largely because hybrid backcrosses are difficult to visually identify. We screened 27 cross-amplifying waterfowl microsatellite markers and developed a set of 9 markers that can be used for genotyping and assignment tests to identify cryptic hybrids. Assignment tests, performed with the program Structure 2.3.4, had a 99 % likelihood that specimens of a known species (Mallard or Pacific Black Duck) were assigned to the correct group. The system was applied used to confirm the hybrid status of two putative hybrids identified phenotypically. The successful application of this system demonstrates its potential use in determining the rate of hybridisation between introduced Mallards and Pacific Black Ducks throughout Australia.     

Development of microsatellite markers for blue duiker (Cephalophus monticola) using next-generation sequencing and cross-amplification in other duikers

We developed nine microsatellite markers for duikers by next-generation sequencing using a muscle tissue sample of blue duiker (Cephalophus monticola). For blue duikers, the number of alleles ranged from 4 to 14 (mean 9.4), and the observed and expected heterozygosities were 0.333–0.833 (mean 0.716) and 0.625–0.878 (mean 0.817), respectively. Cumulative probability of identity was low (1.5 × 10^?12), indicating that these markers are enough for individual discrimination. All nine markers were successfully amplified in three other duikers (C. dorsalis, C. ogilbyi and C. sylvicultor), and almost all loci were polymorphic. These markers will be useful for clarifying genetic structure of duikers.     

Comparison of radio-iodinated serum albumin and blue dextran as indicators to measure rate of formation of cerebrospinal fluid.

The characteristics of two nondiffusible indicators, ¹²⁵I-labeled albumin (RISA-125) and blue dextran, were compared by using them simultaneously to measure rate of formation of cerebrospinal fluid (CSF) in in vitro experiments and in a series of ventriculocisternal perfusion experiments in rhesus monkeys. Mean CSF formation calculated from the dilution of RISA-125 was 37.6 μl/min (±0.74 sem); the CSF formation calculated from the corresponding dilution of blue dextran was 37.1 μl/min (±0.70 sem). The difference is not statistically significant. As measured by counts per minute, the concentration of RISA-125 in aliquots of a dilute RISA-125 solution varied considerably. In the in vitro experiments, simulated CSF formation calculated from these counts varied around the mean by more than ± 10% at the 95% confidence limits. Counting variability is probably related to the low energy levels of the ¹²⁵I isotope. Cerebrospinal fluid formation calculated from the dilution of blue dextran had one-tenth the coefficient of variation of that calculated from the dilution of RISA-125. We have concluded that blue dextran is preferable to RISA-125 to measure the rate of formation of cerebrospinal fluid in the experimental setting because it is easier to detect accurately small changes of CSF formation with blue dextran.     

Thirty-seven additional microsatellite loci in the Pacific lion-paw scallop (Nodipecten subnodosus) and cross-amplification in other pectinids

We characterized 37 new microsatellite markers in the Pacific lion-paw scallop (Nodipecten subnodosus) and tested for cross-amplification in four other species. Genetic diversity was estimated using 24 individuals from the Lagoon Ojo de Liebre, B.C.S., Mexico. Allelic richness varied from 5 to 27 alleles per locus and the average expected heterozygosity was 0.76. Ten loci exhibited significant departure from Hardy–Weinberg equilibrium likely due to the presence of null alleles. Sixteen of these markers cross-amplified in closely related N. nodosus, while little or no amplification was observed in three Argopecten species.     

Isolation and characterization of microsatellite loci in a mangrove species, Rhizophora stylosa (Rhizophoraceae)

Ten microsatellite markers were isolated from a mangrove species, Rhizophora stylosa (Rhizophoraceae), a widely distributed mangrove plant in the Indo-West Pacific. We tested the variability of the markers on 15–16 individuals of R. stylosa from each of four populations (totaling 63 individuals) and found one to six alleles per locus and population. The range of expected heterozygosity of these markers was 0.000–0.754 in these populations. Cross-species amplification was examined for four congeneric species, R. mucronata, R. apiculata, R. mangle and R. racemosa. Of the ten markers, nine could be amplified in R. mucronata and R. apiculata, and seven in R. mangle and R. racemosa. Seven of them could be used for both Indo-West Pacific species and Atlantic-East Pacific species. Amplification by polymerase chain reaction was optimized under the same parameters across loci, thereby facilitating multiplexing and rapid multi-locus genotyping.     

Isolation and characterization of eleven novel polymorphic microsatellite loci in the spiny softshell turtle (Apalone spinifera)

We isolate and characterize 11 microsatellite loci for the spiny softshell turtle (Apalone spinifera) from a partial genomic library obtained using 454 sequencing technology. Genotypes of 15 individuals from southern Ontario and 30 individuals of unknown origin contain 6?C20 alleles per locus and the level of heterozygosity ranges from 0.229 to 0.800. These loci will be useful for population genetics studies and enforcement activities such as assignment of illegally traded individuals to their population of origin.     

Characterization of blue tit Cyanistes caeruleus microsatellite loci, and assessment of sequence homology among species to predict cross-species utility and chromosome locations

We isolated 54 unique loci from a microsatellite-enriched blue tit (Cyanistes caeruleus) genomic library. Thirty-eight loci were tested for polymorphism in four unrelated blue tits from a population in Chaddesley Woods National Nature Reserve, Worcestershire, UK. This identified 23 scorable polymorphic loci that were then characterized in 29 unrelated individuals from the same population. We assigned the location of the orthologue of each blue tit sequence in the assembled zebra finch (Taeniopygia guttata) and chicken (Gallus gallus) genomes. The polymorphic loci were all predicted to be autosomal and this was confirmed by the presence of heterozygotes in a proportion of both the male and female individuals. The sequences of five loci (Pca14, Pca23, Pca27, Pca30 and Pca32) displayed high sequence homology to the genetically distant chicken and are therefore expected to amplify in the widest range of species, including those of conservation concern. Design of primer sequences which are a consensus between species would enhance cross-species utility further.