Development of 30 microsatellite markers for dab (Limanda limanda L.): a key UK marine biomonitoring species

Dab (Limanda limanda) are the principal target fish species in offshore biomonitoring programmes in the UK; however, detailed knowledge of genetic structure and connectivity among sampling locations is unavailable. Here, the isolation and characterization of 30 polymorphic microsatellite loci for dab is described. The number of alleles per locus ranged from 2 to 42, with observed heterozygosities ranging from 0.089 to 1. These loci will enable high resolution of genetic population structure and dynamics of dab around the British Isles.     

Evolutionary mechanisms shaping the genetic population structure of marine fishes; lessons from the European flounder (Platichthys flesus L.)

A number of evolutionary mechanisms have been suggested for generating low but significant genetic structuring among marine fish populations. We used nine microsatellite loci and recently developed methods in landscape genetics and coalescence-based estimation of historical gene flow and effective population sizes to assess temporal and spatial dynamics of the population structure in European flounder (Platichthys flesus L.). We collected 1062 flounders from 13 localities in the northeast Atlantic and Baltic Seas and found temporally stable and highly significant genetic differentiation among samples covering a large part of the species' range (global F(ST) = 0.024, P < 0.0001). In addition to historical processes, a number of contemporary acting evolutionary mechanisms were associated with genetic structuring. Physical forces, such as oceanographic and bathymetric barriers, were most likely related with the extreme isolation of the island population at the Faroe Islands. A sharp genetic break was associated with a change in life history from pelagic to benthic spawners in the Baltic Sea. Partial Mantel tests showed that geographical distance per se was not related with genetic structuring among Atlantic and western Baltic Sea samples. Alternative factors, such as dispersal potential and/or environmental gradients, could be important for generating genetic divergence in this region. The results show that the magnitude and scale of structuring generated by a specific mechanism depend critically on its interplay with other evolutionary mechanisms, highlighting the importance of investigating species with wide geographical and ecological distributions to increase our understanding of evolution in the marine environment.     

Population dynamics of lymphocystis disease in estuarine flounder, Platichthys flesus (L.)

The population dynamics of viral lymphocystis disease in an age-structured population of flounder, Plarichthys flesus (Linnaeus 1758), is studied with a mathematical model. For a single cohort the model illustrates the influence of host density and acquired immunity on length-prevalence profiles. This case corresponds to a host population at demographic equilibrium. When the model is extended to several cohorts, seasonal recruitment of susceptible hosts is shown to drive seasonality in disease prevalence. In both cases, there is good qualitative agreement between model predictions and field data from the Elbe estuary, Germany.     

Isolation and characterization of new microsatellite markers from the Japanese flounder (Paralichthys olivaceus)

The isolation and characterization of eight polymorphic microsatellite loci from a Japanese flounder partial genomic library are reported. The eight markers isolated in this study were highly polymorphic and their positions on the linkage genome map of the Japanese flounder were determined. Therefore, they are useful for ecological studies of wild populations. These markers are more effective than other markers with no information of chromosomal locations.     

Isolation and characterization of microsatellite markers from the hosta species Hosta albomarginata (Liliaceae)

Hostas are very popular ornamental plants in gardens in the United States, Europe, and East Asia. We have developed nine microsatellite loci from an enrichment library of genomic DNA in Hosta albomarginata. We characterized these nine microsatellite loci for 20 individuals from a population of H. albomarginata. The primers developed in this study yielded an average of 12.4 alleles per locus (range five to 20) and an average expected heterozygosity of 0.86 (range 0.65 to 0.95). These markers will be powerful tools for breeding programmes of hosta cultivars, studies of population genetics, and the conservation of wild hosta species.     

Isolation and characterization of eight compound microsatellite markers in a mangrove tree Kandelia candel (L.) Druce

Kandelia candel is an important mangrove tree species of family Rhizophoraceae. Here we isolated eight codominant compound microsatellite simple sequence repeat (SSR) loci from K. candel. Our isolated loci provided compound SSR markers with polymorphism of three to 11 alleles per locus. The expected and observed heterozygosities ranged from 0.230 to 0.887 and from 0.083 to 1.00, respectively. These markers would be the useful tools for analysing questions concerning population genetic structure and mating system of K. candel.     

Development and characterization of eight new microsatellite markers for the haremic sandperch,Parapercis cylindrica (family Pinguipedidae)

Eight di‐, tri‐ and tetranucleotide microsatellite markers were developed for the haremic sandperch Parapercis cylindrica using a linker‐ligated, magnetic bead enrichment protocol. Screening of at least 17 individuals showed these markers to be polymorphic with observed heterozygosity ranging from 0.381 to 1.000 (mean = 0.742) and the numbers of alleles ranging from three to 18. The average polymorphic information content for these eight loci was 0.723. These markers may be used for parentage studies aimed at exploring the complex mating strategies employed by this haremic coral reef fish and will be valuable for population genetic studies.     

Development of 52 new polymorphic microsatellite markers for the olive flounder, Paralichthys olivaceus

We characterized 52 new microsatellite markers isolated from (GT)(n) and (CT)(n) microsatellite-enriched genomic libraries of the olive flounder (Paralichthys olivaceus). All markers were polymorphic, with eight to 30 (mean 15.1) alleles detected in 30 individuals from a single natural population. Observed heterozygosity ranged from 0.20 to 1.0. Segregation analysis within a mapping family revealed non-amplifying null alleles at six loci. These results indicate that these new microsatellite markers will be useful for population genetic, parentage, and genome mapping studies.     

Adaptive differences in gene expression in European flounder (Platichthys flesus)

Population structure was previously believed to be very limited or absent in classical marine fishes, but recently, evidence of weakly differentiated local populations has been accumulating using noncoding microsatellite markers. However, the evolutionary significance of such minute genetic differences remains unknown. Therefore, in order to elucidate the relationship between genetic markers and adaptive divergence among populations of marine fishes, we combined cDNA microarray and microsatellite analysis in European flounders (Platichthys flesus). We demonstrate that despite extremely low levels of neutral genetic divergence, a high number of genes were significantly differentially expressed between North Sea and Baltic Sea flounders maintained in a long-term reciprocal transplantation experiment mimicking natural salinities. Several of the differentially regulated genes could be directly linked to fitness traits. These findings demonstrate that flounders, despite little neutral genetic divergence between populations, are differently adapted to local environmental conditions and imply that adaptation in gene expression could be common in other marine organisms with similar low levels of population subdivision.     

Isolation and characterization of microsatellite markers for Sabatia campestris (Gentianaceae), an Illinois state‐endangered plant

Sabatia campestris is a regionally endangered prairie plant. Eight polymorphic loci have been generated for S. campestris (Gentianaceae) using non‐enriched and enriched libraries. Polymorphism was quantified from an isolated population from central Illinois yielding mean observed and expected heterozygosities of 0.723 and 0.801, respectively. The number of alleles per locus for this population ranged from three to 25 with a median of 15.5. These genetic markers will be used to determine population genetic structure of Illinois populations as well as to estimate fine‐scale gene flow rates.     

Isolation and characterization of polymorphic microsatellite markers from Asian malaria mosquito Anopheles sinensis (Diptera: Culicidae)

Microsatellite-containing region were isolated and characterized in Anopheles sinensis, a primary vector of malaria parasites in Asia. An enrichment protocol yielded 252 microsatellite sequences. We designed primers to amplify 20 unique microsatellites, 14 of which amplify cleanly and were polymorphic. A survey of 24 individuals showed that 12 loci are highly variable with the number of alleles ranging from two to 11, and expected heterozygosity ranging from 0.116 to 0.903. These markers will be useful for population genetic studies and genome mapping in A. sinensis.     

Isolation and characterization of microsatellite loci from Weigela coraeensis (Caprifoliaceae)

Weigela coraeensis var. coraeensis is a deciduous shrub species distributed in Japan on the mainland, Honshu, whereas its variety W. coraeensis var. fragrans is endemic to the Izu Islands located south of Honshu. We isolated eight polymorphic microsatellite loci from the species and characterized these loci for 20 individuals from a population in Honshu. The number of alleles per locus ranged from 7 to 15 and the observed and expected heterozygosities ranged from 0.60 to 0.90 and from 0.65 to 0.90, respectively. These eight polymorphic microsatellites will be useful for examining intraspecific genetic differentiation in W. coraeensis.     

The diet and growth of O-group flounder,Platichthys flesus (L.), in the River Dee,North Wales

Many O-group flounder, Platichthys flesus, live in the lower reaches of rivers in North West Europe; while there is extensive information on the niche that they occupy in estuarine and marine environments, little is known of their life in freshwater. The diet of O-group flounder in the Lower Welsh River Dee was studied by stomach contents analysis of diel and seasonal samples. The dominant prey were chironomid larvae and pupae, tubificid oligochaetes and cyclopoid copepods. There was little quantitative variation in diet temporally, although a weak crepuscular pattern of stomach fullness was shown. There was wide variation in length and weight in all months, but the mean values at age I were 60 mm and 2.9 g respectively.     

Isolation and characterization of microsatellite markers from Robinia pseudoacacia L

Microsatellite markers were isolated from Robinia pseudoacacia L. using an enrichment method. Eleven of the 23 primer pairs designed successfully amplified unambiguous and polymorphic single loci among 39 individual R. pseudoacacia L. from northeastern Japan. The observed and expected heterozygosities of the 11 microsatellite markers ranged from 0.333 to 0.821 and from 0.489 to 0.867, respectively. The polymorphisms observed at the 11 microsatellite loci are useful genetic data for forest ecological studies involving R. pseudoacacia L.     

Kinetic properties of liver pyruvate kinase from the flounder (Platichthys flesus L.)

Pyruvate kinase, purified from flounder liver, in two forms, i.e. PK I and PK II, is characterized by sigmoid kinetics with phosphoenolpyruvate as substrate at pH 6.3, 6.7 and 7.7. K0.5 for PEP increases with increasing pH. PK I and PK II show hyperbolic kinetics with ADP, but are inhibited by ADP concentrations above 1-2 mM. K0.5 for ADP decreases with increasing pH. PK I and PK II differ in their K0.5 values for PEP with a factor of at least 2, showing the highest figures for the latter. K0.5 for ADP is about the same for the two enzyme forms. Other nucleotide diphosphates can replace ADP as the substrate. When the nucleoside diphosphates are arranged in a rank order showing decreasing effectiveness as substrate, different rank orders are obtained for PK I and PK II.     

Isolation and characterization of 11 new microsatellite markers for Macaranga (Euphorbiaceae)

We provide primer sequences for 11 new polymorphic microsatellite markers developed in the tropical ant-plant genus Macaranga (Euphorbiaceae), after enrichment cloning of Macaranga tanarius and Macaranga hypoleuca. Allele numbers per locus ranged from two to 16 among 20 accessions of M. tanarius, and from three to 10 among 22 accessions of M. hypoleuca. Observed and expected heterozygosities ranged from 0.150 to 0.900 and from 0.375 to 0.894 in M. tanarius, and from 0.545 to 1.000 and from 0.434 to 0.870 in M. hypoleuca, respectively. Six of the 11 primer pairs successfully cross-amplified polymorphic polymerase chain reaction products in Macaranga winkleri.     

Isolation and characterization of microsatellite loci in Eurycorymbus cavaleriei,a dioecious endemic tree species in China

Eleven dinucleotide microsatellites were developed and characterized for Eurycorymbus cavaleriei, a dioecious, rare and endemic tree species in China. A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 30 trees from two natural populations. The observed heterozygosity (H_O) ranged from 0.379 to 0.931, with an average of 0.625. These markers provide powerful tools for the ongoing population and conservation genetics studies of E. cavaleriei.     

The effects of handling on flounder (Platichthys flesus L.) and Atlantic salmon (Salmo salar L.)

The effect of 9 min of net confinement on two marine teleost species, the flounder and the Atlantic salmon, was investigated in order to gauge how different species respond to the same stress stimulus. Net confinement in both species induced significant elevations in plasma cortisol, glucose, lactate, osmolality, and monovalent ion levels; the responses to net confinement in salmon were generally of a greater magnitude. In both species, handling induced significant alterations in PFFA levels though there were marked species differences in the qualitative and quantitative aspects of this response. Plasma protein levels were significantly elevated only in the flounders. In general, the two species responded similarly to the net confinement with differences only in the absolute levels and durations of the responses.     

Isolation and characterization of 12 dinucleotide microsatellite loci in the anadromous fish Osmerus eperlanus (L.)

A subtractive hybridization method was used to isolate 12 dinucleotide microsatellite loci for the anadromous European smelt, Osmerus eperlanus (L.). Three to 17 (mean 8.08) alleles per locus were identified in the two populations screened, and observed heterozygosity ranged from 0.067 to 0.933. Loci in both populations showed significant deviations from Hardy-Weinberg expected frequencies. These 12 loci provide a good basis for investigation of O. eperlanus population structure.     

Development and characterization of eight polymorphic microsatellite markers for Daphnia atkinsoni (Crustacea: Ctenodaphnia)

A microsatellite‐enriched genomic library was developed for the water flea Daphnia atkinsoni Baird, 1859, a dominant species of intermittent wetlands in Europe. Eight polymorphic microsatellite markers were successfully optimized. Characterization of 77 individuals from Belgium and Spain showed moderate (in the former) to high (in the latter) levels of polymorphism with two to 11 alleles per locus and an observed heterozygosity ranging from 0 to 0.87. Some of these microsatellite markers were successfully amplified in three other Daphnia species (D. magna n = 4, D. similis n = 6; D. obtusa n = 6).