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

Low genotypic diversity and long-term ecological decline in a spatially structured seagrass population

In isolated or declining populations, viability may be compromised further by loss of genetic diversity. Therefore, it is important to understand the relationship between long-term ecological trajectories and population genetic structure. However, opportunities to combine these types of data are rare, especially in natural systems. Using an existing panel of 15 microsatellites, we estimated allelic diversity in seagrass, Zostera marina, at five sites around the Isles of Scilly Special Area of Conservation, UK, in 2010 and compared this to 23 years of annual ecological monitoring (1996–2018). We found low diversity and long-term declines in abundance in this relatively pristine but isolated location. Inclusion of the snapshot of genotypic, but less-so genetic, diversity improved prediction of abundance trajectories; however, this was spatial scale-dependent. Selection of the appropriate level of genetic organization and spatial scale for monitoring is, therefore, important to identify drivers of eco-evolutionary dynamics. This has implications for the use of population genetic information in conservation, management, and spatial planning

Coping with Temperature at the Warm Edge – Patterns of Thermal Adaptation in the Microbial Eukaryote Paramecium caudatum

Ectothermic organisms are thought to be severely affected by global warming since their physiological performance is directly dependent on temperature. Latitudinal and temporal variations in mean temperatures force ectotherms to adapt to these complex environmental conditions. Studies investigating current patterns of thermal adaptation among populations of different latitudes allow a prediction of the potential impact of prospective increases in environmental temperatures on their fitness.In this study, temperature reaction norms were ascertained among 18 genetically defined, natural clones of the microbial eukaryote Paramecium caudatum. These different clones have been isolated from 12 freshwater habitats along a latitudinal transect in Europe and from 3 tropical habitats (Indonesia). The sensitivity to increasing temperatures was estimated through the analysis of clone specific thermal tolerances and by relating those to current and predicted temperature data of their natural habitats. All investigated European clones seem to be thermal generalists with a broad thermal tolerance and similar optimum temperatures. The weak or missing co-variation of thermal tolerance with latitude does not imply local adaptation to thermal gradients; it rather suggests adaptive phenotypic plasticity among the whole European subpopulation. The tested Indonesian clones appear to be locally adapted to the less variable, tropical temperature regime and show higher tolerance limits, but lower tolerance breadths.Due to the lack of local temperature adaptation within the European subpopulation, P. caudatum genotypes at the most southern edge of their geographic range seem to suffer from the predicted increase in magnitude and frequency of summer heat waves caused by climate change

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe

Dietary Omega-3 Polyunsaturated Fatty Acids Alter the Fatty Acid Composition of Hepatic and Plasma Bioactive Lipids in C57BL/6 Mice : A Lipidomic Approach

Background Omega (n)-3 polyunsaturated fatty acids (PUFA) are converted to bioactive lipid components that are important mediators in metabolic and physiological pathways; however, which bioactive compounds are metabolically active, and their mechanisms of action are still not clear. We investigated using lipidomic techniques, the effects of diets high in n-3 PUFA on the fatty acid composition of various bioactive lipids in plasma and liver. Methodology and Principal Findings Female C57BL/6 mice were fed semi-purified diets (20% w/w fat) containing varying amounts of n-3 PUFA before mating, during gestation and lactation, and until weaning. Male offspring were continued on their mothers’ diets for 16 weeks. Hepatic and plasma lipids were extracted in the presence of non-naturally occurring internal standards, and tandem electrospray ionization mass spectrometry methods were used to measure the fatty acyl compositions. There was no significant difference in total concentrations of phospholipids in both groups. However, there was a significantly higher concentration of eicosapentaenoic acid containing phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and cholesteryl esters (CE) (p < 0.01) in the high n-3 PUFA group compared to the low n-3 PUFA group in both liver and plasma. Plasma and liver from the high n-3 PUFA group also had a higher concentration of free n-3 PUFA (p < 0.05). There were no significant differences in plasma concentrations of different fatty acyl species of phosphatidylethanolamine, triglycerides, sphingomyelin and ceramides. Conclusions/Significance Our findings reveal for the first time that a diet high in n-3 PUFA caused enrichment of n-3 PUFA in PC, LPC, CE and free fatty acids in the plasma and liver of C57BL/6 mice. PC, LPC, and unesterified free n-3 PUFA are important bioactive lipids, thus altering their fatty acyl composition will have important metabolic and physiological roles

Evidence that oestrogen receptor-alpha plays an important role in the regulation of glucose homeostasis in mice: insulin sensitivity in the liver

Aims/hypothesis: We used oestrogen receptor-alpha (ER alpha) knockout (ERKO) and receptor-beta (ER beta) knockout (BERKO) mice to investigate the mechanism(s) behind the effects of oestrogens on glucose homeostasis. Methods: Endogenous glucose production (EGP) was measured in ERKO mice using a euglycaemic-hyperinsulinaemic clamp. Insulin secretion was determined from isolated islets. In isolated muscles, glucose uptake was assayed by using radiolabelled isotopes. Genome-wide expression profiles were analysed by high-density oligonucleotide microarray assay, and the expression of the genes encoding steroyl-CoA desaturase and the Leptin receptor (Scd1 and Lepr, respectively) was confirmed by RT-PCR. Results: ERKO mice had higher fasting blood glucose, plasma insulin levels and IGT. The plasma leptin level was increased, while the adiponectin concentration was decreased in ERKO mice. Levels of both glucose- and arginine-induced insulin secretion from isolated islets were similar in ERKO and wild-type mice. The euglycaemic-hyperinsulinaemic clamp revealed that suppression of EGP by increased insulin levels was blunted in ERKO mice, which suggests a pronounced hepatic insulin resistance. Microarray analysis revealed that in ERKO mice, the genes involved in hepatic lipid biosynthesis were upregulated, while genes involved in lipid transport were downregulated. Notably, hepatic Lepr expression was decreased in ERKO mice. In vitro studies showed a modest decrease in insulin-mediated glucose uptake in soleus and extensor digitorum longus (EDL) muscles of ERKO mice. BERKO mice demonstrated normal glucose tolerance and insulin release. Conclusions/interpretation: We conclude that oestrogens, acting via ER alpha, regulate glucose homeostasis mainly by modulating hepatic insulin sensitivity, which can be due to the upregulation of lipogenic genes via the suppression of Lepr expression