Comprehensive Primer Design for Analysis of Population Genetics in Non-Sequenced Organisms

Nuclear sequence markers are useful tool for the study of the history of populations and adaptation. However, it is not easy to obtain multiple nuclear primers for organisms with poor or no genomic sequence information. Here we used the genomes of organisms that have been fully sequenced to design comprehensive sets of primers to amplify polymorphic genomic fragments of multiple nuclear genes in non-sequenced organisms. First, we identified a large number of candidate polymorphic regions that were flanked on each side by conserved regions in the reference genomes. We then designed primers based on these conserved sequences and examined whether the primers could be used to amplify sequences in target species, montane brown frog (Rana ornativentris), anole lizard (Anolis sagrei), guppy (Poecilia reticulata), and fruit fly (Drosophila melanogaster), for population genetic analysis. We successfully obtained polymorphic markers for all target species studied. In addition, we found that sequence identities of the regions between the primer sites in the reference genomes affected the experimental success of DNA amplification and identification of polymorphic loci in the target genomes, and that exonic primers had a higher success rate than intronic primers in amplifying readable sequences. We conclude that this comparative genomic approach is a time- and cost-effective way to obtain polymorphic markers for non-sequenced organisms, and that it will contribute to the further development of evolutionary ecology and population genetics for non-sequenced organisms, aiding in the understanding of the genetic basis of adaptation

The long-term consequences of hybridization between the two Daphnia species, D. galeata and D. dentifera, in mature habitats

<p>Abstract</p> <p>Background</p> <p>Ecological specializations such as antipredator defense can reinforce morphological and distributional divergence within hybridizing species. Two hybridizing species of <it>Daphnia </it>(<it>D. galeata </it>and <it>D. dentifera</it>) are distributed in both Japan and North America; however, these populations have a longer history in Japan than in North America due to the differing impact of the last glaciation on these two regions. We tested the hypothesis that this longer coexistence in Japan would lead to extensive genetic admixture in nuclear and mitochondrial DNA whilst the distinct morphological traits and distributional patterns would be maintained.</p> <p>Results</p> <p>The high level of correspondence among morphological traits, distribution, and mitochondrial and nuclear DNA types for the specimens with <it>D. dentifera </it>mtDNA indicated that the species distinction has been maintained. However, a discordance between mtDNA and nuclear ITS-1 types was observed for most specimens that had <it>D. galeata </it>mtDNA, consistent with the pattern seen between the two species in North America. This observation suggests nuclear introgression from <it>D. dentifera </it>into <it>D. galeata </it>without mitochondrial introgression.</p> <p>Conclusions</p> <p>The separation of morphological traits and distribution ranges of the two hybridizing species in Japan, as well as in North America, has been maintained, despite large differences in climatic and geographical histories of these two regions. Variations in environmental factors, such as predation pressure, might affect maintenance of the distribution, although the further studies are needed to confirm this.</p

Classification of Inhibitors of Hepatic Organic Anion Transporting Polypeptides (OATPs): Influence of Protein Expression on Drug–Drug Interactions

ABSTRACT: The hepatic organic anion transporting poly-peptides (OATPs) influence the pharmacokinetics of several drug classes and are involved in many clinical drug−drug interactions. Predicting potential interactions with OATPs is, therefore, of value. Here, we developed in vitro and in silico models for identification and prediction of specific and general inhibitors of OATP1B1, OATP1B3, and OATP2B1. The maximal transport activity (MTA) of each OATP in human liver was predicted from transport kinetics and protein quantification. We then used MTA to predict the effects of a subset of inhibitors on atorvastatin uptake in vivo. Using a data set of 225 drug-like compounds, 91 OATP inhibitors were identified. In silico models indicated that lipophilicity and polar surface area are key molecular features of OATP inhibition. MTA predictions identified OATP1B1 and OATP1B3 as major determinants of atorvastatin uptake in vivo. The relative contributions to overall hepatic uptake varied with isoform specificities of the inhibitors

Functional classes of genes with primers.

a<p>The estimated <i>P</i> values were adjusted by Bonferroni correction. Only statistically significant functional classes are shown.</p

Association between presence of polymorphisms in PCR products and identities between reference genomic regions.

<p>The x-axis indicates the proportion of mono- and polymorphic markers amplified by using the primer pairs designed for <i>Rana ornativentris</i>. Only readable sequences were included in the analysis. The y-axis indicates the identity between reference genomic regions predicted to be amplified by the primers. The number of primer pairs is shown at right of each bar.</p

Association between the success-rate of PCR amplification and sequence identities between corresponding reference genomic regions.

<p>The x-axis indicates the success rates of PCR amplifications of <i>Rana ornativentris</i> sequences using the primer pairs designed from the reference genomes. The y-axis indicates the identity between orthologous reference DNA regions predicted to be amplified by using the primers. “Single band” indicates a successful PCR with a single band amplified; “multiple bands” indicates an unsuccessful PCR with more than two bands or smeary band; and “no band” indicates amplification failure. The number of primer pairs is shown at right of each bar.</p

Distribution of sequence identities between orthologous reference genomic regions predicted to be amplified by the primer pairs.

<p>The x-axis indicates the identity between orthologous DNA regions predicted to be amplified by primer pairs in the following reference genomes: (A) <i>Oryzias latipes</i> and <i>Gasterosteus aculeatus</i>; (B) <i>O. latipes</i> and <i>Tetraodon nigroviridis</i>; (C) <i>O. latipes</i> and <i>Takifugu rubripes</i>; (D) <i>O. latipes</i> and <i>Danio rerio</i>; (E) <i>Drosophila melanogaster</i> and <i>Drosophila ananassae</i>; (F) <i>Anolis carolinensis</i> and <i>Gallus gallus</i>; and (G) <i>Xenopus tropicalis</i> and <i>G. gallus</i>.</p

A novel canine model of immune thrombocytopenia: has immune thrombocytopenia (ITP) gone to the dogs?

Canine immune thrombocytopenia (ITP) is analogous to human ITP, with similar platelet counts and heterogeneity in bleeding phenotype among affected individuals. With a goal of ultimately investigating this bleeding heterogeneity, a canine model of antibody-mediated ITP was developed. Infusion of healthy dogs with 2F9, a murine IgG2a monoclonal antibody to the canine platelet glycoprotein GPIIb (a common target of autoantibodies in ITP) resulted in profound, dose-dependent thrombocytopenia. Model dogs developed variable bleeding phenotypes, e.g. petechiae and haematuria, despite similar degrees of thrombocytopenia. 2F9 infusion was not associated with systemic inflammation, consumptive coagulopathy, or impairment of platelet function. Unexpectedly however, evaluation of cytokine profiles led to the identification of platelets as a potential source of serum interleukin-8 (IL8) in dogs. This finding was confirmed in humans with ITP, suggesting that platelet IL8 may be a previously unrecognized modulator of platelet-neutrophil crosstalk. The utility of this model will allow future study of bleeding phenotypic heterogeneity including the role of neutrophils and endothelial cells in ITP