Transcriptome sequencing in an ecologically important tree species: assembly, annotation, and marker discovery

<p>Abstract</p> <p>Background</p> <p>Massively parallel sequencing of cDNA is now an efficient route for generating enormous sequence collections that represent expressed genes. This approach provides a valuable starting point for characterizing functional genetic variation in non-model organisms, especially where whole genome sequencing efforts are currently cost and time prohibitive. The large and complex genomes of pines (<it>Pinus </it>spp.) have hindered the development of genomic resources, despite the ecological and economical importance of the group. While most genomic studies have focused on a single species (<it>P. taeda</it>), genomic level resources for other pines are insufficiently developed to facilitate ecological genomic research. Lodgepole pine (<it>P. contorta</it>) is an ecologically important foundation species of montane forest ecosystems and exhibits substantial adaptive variation across its range in western North America. Here we describe a sequencing study of expressed genes from <it>P. contorta</it>, including their assembly and annotation, and their potential for molecular marker development to support population and association genetic studies.</p> <p>Results</p> <p>We obtained 586,732 sequencing reads from a 454 GS XLR70 Titanium pyrosequencer (mean length: 306 base pairs). A combination of reference-based and <it>de novo </it>assemblies yielded 63,657 contigs, with 239,793 reads remaining as singletons. Based on sequence similarity with known proteins, these sequences represent approximately 17,000 unique genes, many of which are well covered by contig sequences. This sequence collection also included a surprisingly large number of retrotransposon sequences, suggesting that they are highly transcriptionally active in the tissues we sampled. We located and characterized thousands of simple sequence repeats and single nucleotide polymorphisms as potential molecular markers in our assembled and annotated sequences. High quality PCR primers were designed for a substantial number of the SSR loci, and a large number of these were amplified successfully in initial screening.</p> <p>Conclusions</p> <p>This sequence collection represents a major genomic resource for <it>P. contorta</it>, and the large number of genetic markers characterized should contribute to future research in this and other pines. Our results illustrate the utility of next generation sequencing as a basis for marker development and population genomics in non-model species.</p

Carbon Monoxide Abrogates Ischemic Insult to Neuronal Cells via the Soluble Guanylate Cyclase-cGMP Pathway

Purpose Carbon monoxide (CO) is an accepted cytoprotective molecule. The extent and mechanisms of protection in neuronal systems have not been well studied. We hypothesized that delivery of CO via a novel releasing molecule (CORM) would impart neuroprotection in vivo against ischemia-reperfusion injury (IRI)-induced apoptosis of retinal ganglion cells (RGC) and in vitro of neuronal SH-SY5Y-cells via activation of soluble guanylate-cyclase (sGC). Methods: To mimic ischemic respiratory arrest, SH-SY5Y-cells were incubated with rotenone (100 nmol/L, 4 h) ± CORM ALF186 (10–100 µmol/L) or inactivated ALF186 lacking the potential of releasing CO. Apoptosis and reactive oxygen species (ROS) production were analyzed using flow-cytometry (Annexin V, mitochondrial membrane potential, CM-H2DCFDA) and Western blot (Caspase-3). The impact of ALF186± respiratory arrest on cell signaling was assessed by measuring expression of nitric oxide synthase (NOS) and soluble guanylate-cyclase (sGC) and by analyzing cellular cGMP levels. The effect of ALF186 (10 mg/kg iv) on retinal IRI in Sprague-Dawley rats was assessed by measuring densities of fluorogold-labeled RGC after IRI and by analysis of apoptosis-related genes in retinal tissue. Results: ALF186 but not inactivated ALF186 inhibited rotenone-induced apoptosis (Annexin V positive cells: 25±2% rotenone vs. 14±1% ALF186+rotenone, p<0.001; relative mitochondrial membrane potential: 17±4% rotenone vs. 55±3% ALF186+rotenone, p<0.05). ALF186 increased cellular cGMP levels (33±5 nmol/L vs. 23±3 nmol/L; p<0.05) and sGC expression. sGC-inhibition attenuated ALF186-mediated protection (relative mitochondrial membrane potential: 55±3% ALF186+rotenone vs. 20±1% ODQ+ALF186+rotenone, p<0.05). ALF186 protected RGC in vivo (IRI 1255±327 RGC/mm2 vs. ALF186+IRI 2036±83; p<0.05) while sGC inhibition abolished the protective effects of ALF186 (ALF186+IRI 2036±83 RGC/mm2 vs. NS-2028+ALF186+IRI 1263±170, p<0.05). Conclusions: The CORM ALF186 inhibits IRI-induced neuronal cell death via activation of sGC and may be a useful treatment option for acute ischemic insults to the retina and the brain

Recent Hybrids Recapitulate Ancient Hybrid Outcomes

Genomic outcomes of hybridization depend on selection and recombination in hybrids. Whether these processes have similar effects on hybrid genome composition in contemporary hybrid zones versus ancient hybrid lineages is unknown. Here we show that patterns of introgression in a contemporary hybrid zone in Lycaeides butterflies predict patterns of ancestry in geographically adjacent, older hybrid populations. We find a particularly striking lack of ancestry from one of the hybridizing taxa, Lycaeides melissa, on the Z chromosome in both the old and contemporary hybrids. The same pattern of reduced L. melissa ancestry on the Z chromosome is seen in two other ancient hybrid lineages. More generally, we find that patterns of ancestry in old or ancient hybrids are remarkably predictable from contemporary hybrids, which suggests selection and recombination affect hybrid genomes in a similar way across disparate time scales and during distinct stages of speciation and species breakdown

The genetic architecture of divergence between threespine stickleback species.

The genetic and molecular basis of morphological evolution is poorly understood, particularly in vertebrates. Genetic studies of the differences between naturally occurring vertebrate species have been limited by the expense and difficulty of raising large numbers of animals and the absence of molecular linkage maps for all but a handful of laboratory and domesticated animals. We have developed a genome-wide linkage map for the three-spined stickleback (Gasterosteus aculeatus), an extensively studied teleost fish that has undergone rapid divergence and speciation since the melting of glaciers 15,000 years ago. Here we use this map to analyse the genetic basis of recently evolved changes in skeletal armour and feeding morphologies seen in the benthic and limnetic stickleback species from Priest Lake, British Columbia. Substantial alterations in spine length, armour plate number, and gill raker number are controlled by genetic factors that map to independent chromosome regions. Further study of these regions will help to define the number and type of genetic changes that underlie morphological diversification during vertebrate evolution

Caterpillars on a Phytochemical Landscape: The Case of Alfalfa and the Melissa Blue Butterfly

Modern metabolomic approaches that generate more comprehensive phytochemical profiles than were previously available are providing new opportunities for understanding plant‐animal interactions. Specifically, we can characterize the phytochemical landscape by asking how a larger number of individual compounds affect herbivores and how compounds covary among plants. Here we use the recent colonization of alfalfa (Medicago sativa) by the Melissa blue butterfly (Lycaeides melissa) to investigate the effects of indivdiual compounds and suites of covarying phytochemicals on caterpillar performance. We find that survival, development time, and adult weight are all associated with variation in nutrition and toxicity, including biomolecules associated with plant cell function as well as putative anti‐herbivore action. The plant‐insect interface is complex, with clusters of covarying compounds in many cases encompassing divergent effects on different aspects of caterpillar performance. Individual compounds with the strongest associations are largely specialized metabolites, including alkaloids, phenolic glycosides, and saponins. The saponins are represented in our data by more than 25 individual compounds with beneficial and detrimental effects on L. melissa caterpillars, which highlights the value of metabolomic data as opposed to approaches that rely on total concentrations within broad defensive classes

The Impact of Response to MTX on the Illness and Treatment Beliefs of Parents of Children with JIA

Background: Research on adult patients with chronic illness has shown that their illness beliefs are associated with patient reported outcomes. There is limited research focusing on the illness and treatment beliefs of parents of ill children. The aim of this study was to discern if treatment success, as measured by the ACR Pediatric (Ped) criteria has an impact upon parents’ beliefs about their child’s illness and treatment. Methods: The data of 157 mothers and 75 fathers was used in this cross-sectional study. Parents were asked to complete a series of questionnaires including the Illness Perception Questionnaire Revised (IPQ-R) and the Treatment Representation Inventory (TRI). Parents of children, who reached the ACR Ped 70 level of improvement in arthritis were compared to parents of non-responders using Chi-Square Tests and ANOVAs. Results: A number of significant differences in parents’ illness perceptions were found. Mothers of non-responders had a stronger belief in the unpredictable nature of the arthritis (p = 0.01) and mothers of ACR Ped 70 responders showed a stronger belief in their ability to control the arthritis (p = 0.03). Fathers of ACR Pediatric 70 responders had a stronger belief in their child’s understanding of the arthritis (p = 0.01) and in the arthritis being an acute condition (p = 0.03). Only one significant difference was found in parents’ treatment representations. Mothers of ACR Ped 70 responders had a stronger belief in the ability of the treatment to cure the arthritis. The means of the scale assessing the value of treatment were high for all mothers and fathers. Other treatment and illness beliefs were in the mid-range reflecting uncertain beliefs. Conclusions: With the exception of mothers’ beliefs in the ability of the treatment to cure the arthritis, no differences were found in the treatment beliefs between parents of ACR Ped 70 responders and parents of non-responders. This suggests that with exception of beliefs in cure, the treatment success is not generally reflected in parents’ treatment beliefs. However, treatment success influenced a number of parents’ beliefs about their child’s illness. These differences were all in the expected direction. Notably, on a number of the scales mothers and fathers consistently showed a degree of uncertainty in their beliefs about their child’s illness and treatment, regardless of treatment success. This uncertainty may reflect the fact that currently there is no cure for JIA. Illness uncertainty has been found to be positively associated with negative psychological outcomes in patients with chronic illness. The findings in this study suggest that parents of children with JIA may require additional support to deal with the uncertainty in relation to their child’s illness and treatment

Geographically multifarious phenotypic divergence during speciation

Abstract Speciation is an important evolutionary process that occurs when barriers to gene flow evolve between previously panmictic populations. Although individual barriers to gene flow have been studied extensively, we know relatively little regarding the number of barriers that isolate species or whether these barriers are polymorphic within species. Herein, we use a series of field and lab experiments to quantify phenotypic divergence and identify possible barriers to gene flow between the butterfly species Lycaeides idas and Lycaeides melissa. We found evidence that L. idas and L. melissa have diverged along multiple phenotypic axes. Specifically, we identified major phenotypic differences in female oviposition preference and diapause initiation, and more moderate divergence in mate preference. Multiple phenotypic differences might operate as barriers to gene flow, as shown by correlations between genetic distance and phenotypic divergence and patterns of phenotypic variation in admixed Lycaeides populations. Although some of these traits differed primarily between species (e.g., diapause initiation), several traits also varied among conspecific populations (e.g., male mate preference and oviposition preference)

Genome-Wide Association Mapping of Phenotypic Traits Subject to a Range of Intensities of Natural Selection in Timema cristinae*

abstract: The genetic architecture of adaptive traits can reflect the evolutionary history of populations and also shape divergence among populations. Despite this central role in evolution, relatively little is known regarding the genetic architecture of adaptive traits in nature, particularly for traits subject to known selection intensities. Here we quantitatively describe the genetic architecture of traits that are subject to known intensities of differential selection between host plant species in Timema cristinae stick insects. Specifically, we used phenotypic measurements of 10 traits and 211,004 single-nucleotide polymorphisms (SNPs) to conduct multilocus genome-wide association mapping. We identified a modest number of SNPs that were associated with traits and sometimes explained a large proportion of trait variation. These SNPs varied in their strength of association with traits, and both major and minor effect loci were discovered. However, we found no relationship between variation in levels of divergence among traits in nature and variation in parameters describing the genetic architecture of those same traits. Our results provide a first step toward identifying loci underlying adaptation in T. cristinae. Future studies will examine the genomic location, population differentiation, and response to selection of the trait-associated SNPs described here