Detecting major genetic loci controlling phenotypic variability in experimental crosses

Traditional methods for detecting genes that affect complex diseases in humans or animal models, milk production in livestock, or other traits of interest, have asked whether variation in genotype produces a change in that trait’s average value. But focusing on differences in the mean ignores differences in variability about that mean. The robustness, or uniformity, of an individual’s character is not only of great practical importance in medical genetics and food production but is also of scientific and evolutionary interest (e.g., blood pressure in animal models of heart disease, litter size in pigs, flowering time in plants). We describe a method for detecting major genes controlling the phenotypic variance, referring to these as vQTL. Our method uses a double generalized linear model with linear predictors based on probabilities of line origin. We evaluate our method on simulated F₂ and collaborative cross data, and on a real F₂ intercross, demonstrating its accuracy and robustness to the presence of ordinary mean-controlling QTL. We also illustrate the connection between vQTL and QTL involved in epistasis, explaining how these concepts overlap. Our method can be applied to a wide range of commonly used experimental crosses and may be extended to genetic association more generally.QUANTITATIVE trait locus (QTL) analysis has traditionally focused on detection of major genes controlling the expected mean of a phenotype. But there is substantial evidence that not only the mean but also the variance, that is, the stochastic variability of the phenotype about its average value, may itself be under genetic control. The identification of such variance-controlling loci, which we call vQTL, can be helpful in a variety of contexts, including selection of livestock for uniformity, evaluating predictability of response to medical treatment, identification of key biomolecular stabilizers, and assessment of population resilience in ecology and evolution.One way of interpreting an increase in variability is as a decrease in stability. Waddington (1942) described the concept of canalization, whereby natural selection favors the relative constancy of some attributes, for example, well-formed organs and limbs, and thereby leads to the evolution of heritable architectures that buffer the impact of environmental or background genetic variation that would otherwise cause development to go astray. These architectures create virtual “canals” down which developmental programs flow. For a canalized phenotype, which modern usage expands to include nondevelopmental traits, the “zone of canalization” is the range of underlying liability over which potentially disruptive variation may be absorbed without serious consequence to the expressed trait value (Lynch and Walsh 1998). A well-studied example of a stabilizing architecture is that provided by heat-shock protein 90 (Hsp90), which buffers genetic and stochastic variation in the development of plants and flies (Rutherford and Lindquist 1998; Queitsch et al. 2002; Sangster et al. 2008).But in absorbing variation, such stabilizing architectures also hide it from view, and a sensitizing change in the stabilizer that shifts liability outside the zone of canalization can have a dramatic effect on the phenotype. Such shifts release the combined effects of previously “cryptic” genetic variation: now decanalized, the phenotype is more sensitive to internal (including genetic) and external environment, and as a result varies more greatly between individuals (Dworkin 2005; Hornstein and Shomron 2006). In this vein, decanalization has been proposed to explain why the genetic architectures of some diseases in human populations seem more amenable than others to genetic dissection through genome-wide association (Gibson and Goldstein 2007). Specifically, whereas some disease phenotypes in homogeneous populations may be heavily canalized and thereby harder to dissect, others may have been decanalized by modern living conditions (e.g., inflammatory diseases) or modern admixture, while yet others are simply too recent in evolutionary history for buffering networks to have evolved (e.g., response to HIV).Increased variability can also be adaptive. In natural populations disruptive selection favors diversity, with increased “capacitance” (Rice 2008) or “bet-hedging” (Beaumont et al. 2009) spreading risk over a variable fitness landscape. Feinberg and Irizarry (2010) recently proposed a heritable and selectable mechanism for this based on stochastic epigenetic variation. In controlled populations, variability can be increased through directional selection. For example, in a Drosophila selection experiment Clayton and Robertson (1957) reported increased bristle number variance, which is consistent with the idea that genotypes associated with higher environmental variance have a greater chance of being selected under directional selection (Hill and Zhang 2004). Moreover, genetic differences have been observed for phenotypic variability in body weight for chickens (Rowe et al. 2006) and snails (Ros et al. 2004) and litter size in rabbits (Ibanez-Escriche et al. 2008), sheep (Sancristobal-Gaudy et al. 1998), and pigs (Sorensen and Waagepetersen 2003).In natural populations with stabilizing selection we should expect to find alleles minimizing variance for fitness traits (Lande 1980; Houle 1992), whereas directional selection during domestication will favor alleles that increase variance. One may therefore expect to find vQTL in experimental crosses between wild and domestic animals (see Andersson 2001). Nonetheless, genetic buffering that leads to phenotypic robustness need not require an evolutionary explanation to be observed, nor to be useful in medicine and agriculture. Plainly, detecting vQTL and inferring how they arose are separate questions; here we concentrate on the first.

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

High-resolution genetic mapping using the Mouse Diversity outbred population

The JAX Diversity Outbred population is a new mouse resource derived from partially inbred Collaborative Cross strains and maintained by randomized outcrossing. As such, it segregates the same allelic variants as the Collaborative Cross but embeds these in a distinct population architecture in which each animal has a high degree of heterozygosity and carries a unique combination of alleles. Phenotypic diversity is striking and often divergent from phenotypes seen in the founder strains of the Collaborative Cross. Allele frequencies and recombination density in early generations of Diversity Outbred mice are consistent with expectations based on simulations of the mating design. We describe analytical methods for genetic mapping using this resource and demonstrate the power and high mapping resolution achieved with this population by mapping a serum cholesterol trait to a 2-Mb region on chromosome 3 containing only 11 genes. Analysis of the estimated allele effects in conjunction with complete genome sequence data of the founder strains reduced the pool of candidate polymorphisms to seven SNPs, five of which are located in an intergenic region upstream of the Foxo1 gene.     

Salt-avoidance tropism in Arabidopsis thaliana

The orientation of plant root growth is modulated by developmental as well as environmental cues. Among the environmental factors, gravity has been extensively studied because of its overpowering effects in modulating root growth direction. However, our knowledge of the effects of other abiotic signals that influence root growth direction is largely unknown. Recently, we have shown that high salinity can modify root growth direction by inducing rapid amyloplast degradation in root columella cells of Arabidopsis thaliana. By exploiting salt overly sensitive (sos) mutants and PIN2 expression analyses, we have shown that the altered root growth direction in response to salt is mediated by ion disequilibrium and is correlated with PIN2 mRNA abundance and expression and localization of the protein. Our study demonstrates that the SOS pathway may mediate this process. Here we discuss our data from broader perspectives. We propose that salt-induced modification of root growth direction is a salt-avoidance behavior, which is an active adaptive mechanism for plants grown under saline conditions. Furthermore, high salinity also stimulates alteration of gravitropic growth of shoots. These findings illustrate that plants have a fine and sophisticated sensory and communication system that enable plants to dynamically and efficiently cope with rapidly changing environment.Key words: abidopsis, adaptation, avoidance, root, salt stress, tropic growthOwing to their sessile nature, plant roots are constantly bombarded with various environmental stimuli from the soil, such as gravity, physical obstacles and imbalanced distribution of water and/or nutrients and high salinity. Where to grow is an important developmental decision in the life cycle of a plant that is crucial for its adaptation and the subsequent reproductive success. The proper orientation of root growth is shaped by both the developmental inputs and external signals.^1,2 The overwhelming environmental factor that modulates root growth direction is gravity, and plant primary roots grow downward toward the gravity vector. This directed growth of root in response to gravity is named as tropic growth to gravity or gravitropism. Studies of gravity perception and signaling pathway of the root cap at the primary root of Arabidopsis strongly support the starch statolith hypothesis.³ In this hypothesis, the columella cells in the root cap, which contain sedimentable amyloplasts, are the gravity-perceptive site in roots. The inner columella cells of the second tier have been proposed as making the greatest contribution to root gravitropism.⁴ Upon gravity stimulation, cytosolic ions such as Ca²⁺ and rapid cytoplasmic alkalization may be involved in gravity signal transduction.^5–7 Asymmetric distribution of auxin in roots caused by basipetal transport mainly through the auxin efflux carrier PIN-FORMED2 (PIN2), which is distributed asymmetrically within the cells, results in gravitropic root response of the root elongation zone.^8,9In contrast to our understanding of gravitropism of root, our knowledge of tropistic responses of root to other major environmental stimuli, such as water availability, imbalanced nutrient distribution and high salinity, and the interplay between these stimuli in determining the directional growth of root remains enigmatic. Recent studies have confirmed the existence of hydrotropism and the molecular genetic basis of the tropistic growth of root to water in determining the final direction of root growth starts to be deciphered.^10–12 Hydrotropic growth of roots is an important trait for plants to actively find water and to optimize their fitness under drought condition. Salinity is another major constraint to root system development, and limits the productivity of agricultural crops and the distribution of plant species.^13–15 It is known that salt stress-induced disturbed balance of ions is the primary cause for inhibition of plant growth and subsequent yield reduction. How does root minimize entrance of harmful ions and subsequently avoid salt injury? Does plant have capacity to sense salt signal, and prevent potentially harmful ions reaching root and shoot?Previous studies have shown that plant use different strategies to avoid salt injury at various levels. After Na⁺ enters the root cells, the Casparian strip can restrict the movements of the harmful ion into the xylem.¹⁶ Root cells also avoid salt injury by extruding Na⁺ actively back to the outside solution. This energy-dependent ion efflux from cytosol across the plasma membrane is mediated by SOS1 gene, a Na⁺-H⁺ antiporter, which is regulated by at least other genes, SOS3 (calcium binding protein) and SOS2 (serine/threonine kinase). This is the well characterized SOS (Salt Overly Sensitive) signaling pathway.^17,18 Another way for plant root cells to avoid ion injury is to accumulate Na⁺ into vacuole. Vacuolar compartmentation of Na⁺ is also in part regulated by Na⁺-H⁺ antiporters, such as AtNHX1.¹⁹ These findings reveal mechanisms of how plants avoid Na⁺ injury after passive entrance of sodium ions into root cells. We questioned whether a plant is capable of actively preventing the harmful ions from reaching root cells or escape from high salinity in the environment, and how plant roots respond to changing salt conditions, because salt distribution is unbalanced under natural saline conditions, especially after rain and irrigation. With a new assay that allows us to specifically address how plant roots respond to changing salt levels, we discovered an alternative adaptive mechanism for plant root to avoid salt injury.²⁰We set up a two-layer medium assay in which a sodium ion gradient would be generated. A normal nutrient agar medium is at the top of the growth bottle and an agar with salt-stressed medium is in the bottom of the bottle. This simple assay allows us to monitor root growth and orientation. The roots of the wild type seedlings penetrated the interface of the layers and grew straight downwards exhibiting gravitropism, when both layers were MS media. In contrast, when the bottom medium contained NaCl, roots of seedlings grew downward first, and then curved and grew upward toward the lower levels of salt. Roots started to bend upward at an early stage even before contacting high-salt medium (250 mM NaCl) on the bottom. The results indicate that roots can sense ion gradients in the growing environment and transduce the signal, combine with internal signals to make decisions that enable roots to stay away from high salt.^21,22 Here, we would like to propose this salt-induced tropic growth as a salt-avoidance tropism, which is an important adaptive behavior for plant roots to avoid salt injury and direct them toward their goal of optimal fitness.²³ Because salt stress inhibits root elongation, we tested impact of salt-induced negative gravitropism on the root growth. The results showed that inhibitory effect of salt on root growth was largely alleviated with this tropic curve (Fig. 1), further verifying our hypothesis that the salt-induced developmental plasticity is a salt-avoidance behavior (Fig. 2).Open in a separate windowFigure 1Effects of salt on root elongation of Arabidopsis thaliana seedlings from different salt treatments. The inhibitory effect of salt stress on root growth was greatly alleviated in the wild type (Col-0) when root growth of the seedlings was analyzed using a two-layer medium assay (black bars). The MS nutrient medium is on the top, and NaCl concentrations in the media on the bottom are 0, 150 and 250 mM. More severe inhibition of root growth of the seedlings by various levels of NaCl in a root bending assay (white bars) was observed. Data represents means of measurements from >40 individuals from three independent experiments. Bars represent standard error.Open in a separate windowFigure 2An illustrative model of the sensing and response by the plant root when grown under different saline conditions. This model proposes two major mechanisms of salt responses by plants, where salt tolerance is the ability to function while stressed; Salt avoidance is the capacity to stay away from salt stress when growing in changing saline conditions.Another important point that we would like to bring out based on our observation in this work is that salinity also stimulated shoot positive gravitropism or negative phototropism. The observation implicates long-distance communication from root to shoot during plant salt response in the stressed plants. The exact biological function of shoot tropic growth, the signals in this long-distance communication, and underlying molecular mechanism still remains unknown.In conclusion, our study has revealed a novel complex adaptive mechanism that provides plants a capacity for avoiding injury from salt. The hypothesis we have proposed here should provide novel insights into plant stress avoidance. Further analysis using a combinatorial approach, mutant analysis and genomics, is required to decipher the molecular network underlying this salt-avoidance behavior.     

Transgenic nonhuman primates for neurodegenerative diseases

Animal models that represent human diseases constitute an important tool in understanding the pathogenesis of the diseases, and in developing effective therapies. Neurodegenerative diseases are complex disorders involving neuropathologic and psychiatric alterations. Although transgenic and knock-in mouse models of Alzheimer's disease, (AD), Parkinson's disease (PD) and Huntington's disease (HD) have been created, limited representation in clinical aspects has been recognized and the rodent models lack true neurodegeneration. Chemical induction of HD and PD in nonhuman primates (NHP) has been reported, however, the role of intrinsic genetic factors in the development of the diseases is indeterminable. Nonhuman primates closely parallel humans with regard to genetic, neuroanatomic, and cognitive/behavioral characteristics. Accordingly, the development of NHP models for neurodegenerative diseases holds greater promise for success in the discovery of diagnoses, treatments, and cures than approaches using other animal species. Therefore, a transgenic NHP carrying a mutant gene similar to that of patients will help to clarify our understanding of disease onset and progression. Additionally, monitoring disease onset and development in the transgenic NHP by high resolution brain imaging technology such as MRI, and behavioral and cognitive testing can all be carried out simultaneously in the NHP but not in other animal models. Moreover, because of the similarity in motor repertoire between NHPs and humans, it will also be possible to compare the neurologic syndrome observed in the NHP model to that in patients. Understanding the correlation between genetic defects and physiologic changes (e.g. oxidative damage) will lead to a better understanding of disease progression and the development of patient treatments, medications and preventive approaches for high risk individuals. The impact of the transgenic NHP model in understanding the role which genetic disorders play in the development of efficacious interventions and medications is foreseeable.     

Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity

The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.     

Recent developments in statistical methods for detecting genetic loci affecting phenotypic variability

ABSTRACT: A number of recent works have introduced statistical methods for detecting genetic loci that affect phenotypic variability, which we refer to as variability-controlling quantitative trait loci (vQTL). These are genetic variants whose allelic state predicts how much phenotype values will vary about their expected means. Such loci are of great potential interest in both human and non-human genetic studies, one reason being that a detected vQTL could represent a previously undetected interaction with other genes or environmental factors. The simultaneous publication of these new methods in different journals has in many cases precluded opportunity for comparison. We survey some of these methods, the respective trade-offs they imply, and the connections between them. The methods fall into three main groups: classical non-parametric, fully parametric, and semi-parametric two-stage approximations. Choosing between alternatives involves balancing the need for robustness, flexibility, and speed. For each method, we identify important assumptions and limitations, including those of practical importance, such as their scope for including covariates and random effects. We show in simulations that both parametric methods and their semi-parametric approximations can give elevated false positive rates when they ignore mean-variance relationships intrinsic to the data generation process. We conclude that choice of method depends on the trait distribution, the need to include non-genetic covariates, and the population size and structure, coupled with a critical evaluation of how these fit with the assumptions of the statistical model.     

Simulation of stent deployment in a realistic human coronary artery

Background  

The process of restenosis after a stenting procedure is related to local biomechanical environment. Arterial wall stresses caused by the interaction of the stent with the vascular wall and possibly stress induced stent strut fracture are two important parameters. The knowledge of these parameters after stent deployment in a patient derived 3D reconstruction of a diseased coronary artery might give insights in the understanding of the process of restenosis.