Pheno2Geno - High-throughput generation of genetic markers and maps from molecular phenotypes for crosses between inbred strains

Background

Genetic markers and maps are instrumental in quantitative trait locus (QTL) mapping in segregating populations. The resolution of QTL localization depends on the number of informative recombinations in the population and how well they are tagged by markers. Larger populations and denser marker maps are better for detecting and locating QTLs. Marker maps that are initially too sparse can be saturated or derived de novo from high-throughput omics data, (e.g. gene expression, protein or metabolite abundance). If these molecular phenotypes are affected by genetic variation due to a major QTL they will show a clear multimodal distribution. Using this information, phenotypes can be converted into genetic markers.

Results

The Pheno2Geno tool uses mixture modeling to select phenotypes and transform them into genetic markers suitable for construction and/or saturation of a genetic map. Pheno2Geno excludes candidate genetic markers that show evidence for multiple possibly epistatically interacting QTL and/or interaction with the environment, in order to provide a set of robust markers for follow-up QTL mapping.We demonstrate the use of Pheno2Geno on gene expression data of 370,000 probes in 148 A. thaliana recombinant inbred lines. Pheno2Geno is able to saturate the existing genetic map, decreasing the average distance between markers from 7.1 cM to 0.89 cM, close to the theoretical limit of 0.68 cM (with 148 individuals we expect a recombination every 100/148=0.68 cM); this pinpointed almost all of the informative recombinations in the population.

Conclusion

The Pheno2Geno package makes use of genome-wide molecular profiling and provides a tool for high-throughput de novo map construction and saturation of existing genetic maps. Processing of the showcase dataset takes less than 30 minutes on an average desktop PC. Pheno2Geno improves QTL mapping results at no additional laboratory cost and with minimum computational effort. Its results are formatted for direct use in R/qtl, the leading R package for QTL studies. Pheno2Geno is freely available on CRAN under “GNU GPL v3”. The Pheno2Geno package as well as the tutorial can also be found at: http://pheno2geno.nl.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0475-6) contains supplementary material, which is available to authorized users.     

Uptake and Storage of Choline by Rat Brain: Influence of Dietary Choline Supplementation

In order to elucidate the regulation of the levels of free choline in the brain, we investigated the influence of chronic and acute choline administration on choline levels in blood, CSF, and brain of the rat and on net movements of choline into and out of the brain as calculated from the arteriovenous differences of choline across the brain. Dietary choline supplementation led to an increase in plasma choline levels of 50% and to an increase in the net release of choline from the brain as compared to a matched group of animals which were kept on a standard diet and exhibited identical arterial plasma levels. Moreover, the choline concentration in the CSF and brain tissue was doubled. In the same rats, the injection of 60 mg/kg choline chloride did not lead to an additional increase of the brain choline levels, whereas in control animals choline injection caused a significant increase; however, this increase in no case surpassed the levels caused by chronic choline supplementation. The net uptake of choline after acute choline administration was strongly reduced in the high-choline group (from 418 to 158 nmol/g). Both diet groups metabolized the bulk (greater than 96%) of newly taken up choline rapidly. The results indicate that choline supplementation markedly attenuates the rise of free choline in the brain that is observed after acute choline administration. The rapid metabolic choline clearance was not reduced by dietary choline load. We conclude that the brain is protected from excess choline by rapid metabolism, as well as by adaptive, diet-induced changes of the net uptake and release of choline.     

Stem cells assessed

The increasing momentum of stem cell research continues, with the better characterization of induced pluripotent stem (iPS) cells, the conversion of differentiated cells into different cell types and the use of pluripotent stem cells to generate whole tissues, among other advances. Here, six experts in the field of stem cell research compare different stem cell models and highlight the importance of pursuing complementary experimental approaches for a better understanding of pluripotency and differentiation and an informed approach to medical applications.     

Intraspecific evolution ofMicroseris pygmaea (Asteraceae,Lactuceae) analyzed by cosegregation of phenotypic characters (QTLs) and molecular markers (RAPDs)

The Chilean annual,Microseris pygmaea, has differentiated in distinct coastal and inland series of populations after long-distance dispersal from western North America. Two plants from the most diverse biotypes were crossed, a large F₂ was raised and analysed for segregation of 30 phenotypic characters. Segregation of molecular markers (47 RAPDs, 1 RFLP, 2 isozymes) was determined in a subpopulation of 45 plants which include all extremes for the phenotypic characters. 32 marker/character cosegregations were significant at the 1% level in t-tests between dominant and homozygous recessive marker genotypes. Considering linkage among markers and pleiotropy of certain marker loci, the number of independent quantitative trait loci (QTLs) is reduced to about 18. Interactions among 2 or 3 QTLs affecting one character have been characterized. The phenotypic differentiation ofM. pygmaea during its evolution from a single founder individual begins to be understood at the level of single-gene mutants.     

Technology and data-intensive science in the beginning of the 21st century

This article is a summary of the technology issues and challenges of data-intensive science and cloud computing as discussed in the Data-Intensive Science (DIS) workshop in Seattle, September 19-20, 2010.     

Distinct different contributions of the alternative and classical complement activation pathway for the innate host response during sepsis

Complement activation represents a crucial innate defense mechanism to invading microorganisms, but there is an eminent lack of understanding of the separate contribution of the different complement activation pathways to the host response during sepsis. We therefore investigated different innate host immune responses during cecal ligation and puncture (CLP)-induced sepsis in mice lacking either the alternative (fD(-/-)) or classical (C1q(-/-)) complement activation pathway. Both knockout mice strains showed a significantly reduced survival and increased organ dysfunction when compared with control mice. Surprisingly, fD(-/-) mice demonstrated a compensated bacterial clearance capacity as control mice at 6 h post CLP, whereas C1q(-/-) mice were already overwhelmed by bacterial growth at this time point. Interestingly, at 24 h after CLP, fD(-/-) mice failed to clear bacteria in a way comparable to control mice. However, both knockout mice strains showed compromised C3 cleavage during sepsis. Investigating potential causes for this discrepancy, we were able to demonstrate that despite normal bacterial clearance capacity early during the onset of sepsis, fD(-/-) mice displayed increased inflammatory cytokine generation and neutrophil recruitment into lungs and blood when compared with both control- and C1q(-/-) mice, indicating a potential loss of control over these immune responses. Further in vitro experiments revealed a strongly increased Nf-κB activation capacity in isolated neutrophils from fD(-/-) mice, supporting this hypothesis. Our results provide evidence for the new concept that the alternative complement activation pathway exerts a distinctly different contribution to the innate host response during sepsis when compared with the classical pathway.     

Characterization of an obligate intracellular bacterium in the midgut epithelium of the bulrush bug Chilacis typhae (Heteroptera, Lygaeidae, Artheneinae)

Many members of the suborder Heteroptera have symbiotic bacteria, which are usually found extracellularly in specific sacs or tubular outgrowths of the midgut or intracellularly in mycetomes. In this study, we describe the second molecular characterization of a symbiotic bacterium in a monophagous, seed-sucking stink bug of the family Lygaeidae (sensu stricto). Chilacis typhae possesses at the end of the first section of the midgut a structure which is composed of circularly arranged, strongly enlarged midgut epithelial cells. It is filled with an intracellular endosymbiont. This "mycetocytic belt" might represent an evolutionarily intermediate stage of the usual symbiotic structures found in stink bugs. Phylogenetic analysis based on the 16S rRNA and the groEL genes showed that the bacterium belongs to the Gammaproteobacteria, and it revealed a phylogenetic relationship with a secondary bacterial endosymbiont of Cimex lectularius and free-living plant pathogens such as Pectobacterium and Dickeya. The distribution and ultrastructure of the rod-shaped Chilacis endosymbiont were studied in adults and nymph stages using fluorescence in situ hybridization (FISH) and electron microscopy. The detection of symbionts at the anterior poles of developing eggs indicates that endosymbionts are transmitted vertically. A new genus and species name, "Candidatus Rohrkolberia cinguli," is proposed for this newly characterized clade of symbiotic bacteria.     

Integrating Top-Down and Bottom-Up Sensory Processing by Somato-Dendritic Interactions

The classical view of cortical information processing is that of a bottom-up process in a feedforward hierarchy. However, psychophysical, anatomical, and physiological evidence suggests that top-down effects play a crucial role in the processing of input stimuli. Not much is known about the neural mechanisms underlying these effects. Here we investigate a physiologically inspired model of two reciprocally connected cortical areas. Each area receives bottom-up as well as top-down information. This information is integrated by a mechanism that exploits recent findings on somato-dendritic interactions. (1) This results in a burst signal that is robust in the context of noise in bottom-up signals. (2) Investigating the influence of additional top-down information, priming-like effects on the processing of bottom-up input can be demonstrated. (3) In accordance with recent physiological findings, interareal coupling in low-frequency ranges is characteristically enhanced by top-down mechanisms. The proposed scheme combines a qualitative influence of top-down directed signals on the temporal dynamics of neuronal activity with a limited effect on the mean firing rate of the targeted neurons. As it gives an account of the system properties on the cellular level, it is possible to derive several experimentally testable predictions.