A Mutant Brassica napus (Canola) Population for the Identification of New Genetic Diversity via TILLING and Next Generation Sequencing

We have generated a Brassica napus (canola) population of 3,158 EMS-mutagenised lines and used TILLING to demonstrate that the population has a high enough mutation density that it will be useful for identification of mutations in genes of interest in this important crop species. TILLING is a reverse genetics technique that has been successfully used in many plant and animal species. Classical TILLING involves the generation of a mutagenised population, followed by screening of DNA samples using a mismatch-specific endonuclease that cleaves only those PCR products that carry a mutation. Polyacrylamide gel detection is then used to visualise the mutations in any gene of interest. We have used this TILLING technique to identify 432 unique mutations in 26 different genes in B. napus (canola cv. DH12075). This reflects a mutation density ranging from 1/56 kb to 1/308 kb (depending on the locus) with an average of 1/109 kb. We have also successfully verified the utility of next generation sequencing technology as a powerful approach for the identification of rare mutations in a population of plants, even in polyploid species such as B. napus. Most of the mutants we have identified are publically available.     

Predicting expected progeny difference for marbling score in Angus cattle using artificial neural networks and Bayesian regression models

Background

Artificial neural networks (ANN) mimic the function of the human brain and are capable of performing massively parallel computations for data processing and knowledge representation. ANN can capture nonlinear relationships between predictors and responses and can adaptively learn complex functional forms, in particular, for situations where conventional regression models are ineffective. In a previous study, ANN with Bayesian regularization outperformed a benchmark linear model when predicting milk yield in dairy cattle or grain yield of wheat. Although breeding values rely on the assumption of additive inheritance, the predictive capabilities of ANN are of interest from the perspective of their potential to increase the accuracy of prediction of molecular breeding values used for genomic selection. This motivated the present study, in which the aim was to investigate the accuracy of ANN when predicting the expected progeny difference (EPD) of marbling score in Angus cattle. Various ANN architectures were explored, which involved two training algorithms, two types of activation functions, and from 1 to 4 neurons in hidden layers. For comparison, BayesCπ models were used to select a subset of optimal markers (referred to as feature selection), under the assumption of additive inheritance, and then the marker effects were estimated using BayesCπ with π set equal to zero. This procedure is referred to as BayesCpC and was implemented on a high-throughput computing cluster.

Results

The ANN with Bayesian regularization method performed equally well for prediction of EPD as BayesCpC, based on prediction accuracy and sum of squared errors. With the 3K-SNP panel, for example, prediction accuracy was 0.776 using BayesCpC, and ranged from 0.776 to 0.807 using BRANN. With the selected 700-SNP panel, prediction accuracy was 0.863 for BayesCpC and ranged from 0.842 to 0.858 for BRANN. However, prediction accuracy for the ANN with scaled conjugate gradient back-propagation was lower, ranging from 0.653 to 0.689 with the 3K-SNP panel, and from 0.743 to 0.793 with the selected 700-SNP panel.

Conclusions

ANN with Bayesian regularization performed as well as linear Bayesian regression models in predicting additive genetic values, supporting the idea that ANN are useful as universal approximators of functions of interest in breeding contexts.     

Antagonist minigenes identify genes regulated by parathyroid hormone through G protein-selective and G protein co-regulated mechanisms in osteoblastic cells

Parathyroid hormone (PTH) is the major hormone regulating bone remodeling. Binding of PTH to the PTH1 receptor (PTH1R), a heterotrimeric G protein coupled receptor (GPCR), can potentially trigger multiple signal transduction pathways mediated through several different G proteins. In this study, we employed G protein antagonist minigenes inhibiting Gα_s, Gα_q or Gα₁₂ to selectively dissect out which of these G proteins were responsible for effects of PTH(1-34) in targeted signaling and osteogenesis arrays consisting of 159 genes. Among the 32 genes significantly regulated by 24 h PTH treatment in UMR-106 osteoblastic cells, 9 genes were exclusively regulated through G_s, 6 genes were solely mediated through G_q, and 3 genes were only controlled through G₁₂. Such findings support the concept that there is some absolute specificity in downstream responses initiated at the G protein level following binding of PTH to the PTH1R. On the other hand, 6 PTH-regulated genes were regulated by both G_s and G_q, 3 genes were regulated by both G_s and G₁₂, and 3 genes were controlled by G_s, G_q and G₁₂. These findings indicate potential overlapping or sequential interactions among different G protein-mediated pathways. In addition, two PTH-regulated genes were not regulated through any of the G proteins examined, suggesting that additional signaling mechanisms may be involved. Selectivity was largely maintained over a 2-48-hour time period. The minigene effects were mimicked by downstream inhibitors. The dissection of the differential effects of multiple G protein pathways on gene regulation provides a more complete understanding of PTH signaling in osteoblastic cells.     

Do purely capital layers exist among flying birds? Evidence of exogenous contribution to arctic-nesting common eider eggs

The strategy of relying extensively on stored resources for reproduction has been termed capital breeding and is in contrast to income breeding, where needs of reproduction are satisfied by exogenous (dietary) resources. Most species likely fall somewhere between these two extremes, and the position of an organism along this gradient can influence several key life-history traits. Common eiders (Somateria mollissima) are the only flying birds that are still typically considered pure capital breeders, suggesting that they depend exclusively on endogenous reserves to form their eggs and incubate. We investigated the annual and seasonal variation in contributions of endogenous and exogenous resources to egg formation in eiders breeding at the East Bay colony in the Canadian Arctic. We collected prey items along with females and their eggs during various stages of breeding and used two complementary analytical approaches: body reserve dynamics and stable isotope [δ(13)C, δ(15)N] mixing models. Indices of protein reserves remained stable from pre-laying to post-laying stages, while lipid reserves declined significantly during laying. Similarly, stable isotope analyses indicated that (1) exogenous nutrients derived from marine invertebrates strongly contributed to the formation of lipid-free egg constituents, and (2) yolk lipids were constituted mostly from endogenous lipids. We also found evidence of seasonal variation in the use of body reserves, with early breeders using proportionally more exogenous proteins to form each egg than late breeders. Based on these results, we reject the hypothesis that eiders are pure capital layers. In these flying birds, the fitness costs of a strict capital breeding strategy, such as temporary loss of flight capability and limitation of clutch and egg size, may outweigh benefits such as a reduction in egg predation rate.     

Allometry, bilateral asymmetry and sexual differences in the vocal tract of common eiders Somateria mollissima and king eiders S. spectabilis

Intraspecific sexual differences, high variation, and positive allometry of sexually-selected external display structures are common. Many sexually-selected anatomical specializations occur in the avian vocal tract but intraspecific variation and allometry have been investigated little. The tracheal bulla bulla syringealis occurs in males of most duck species. We quantified variation and size-scaling of the bulla, plus sexual differences in size of trachea, bronchi, and vocal muscles, for 62 common eiders Somateria mollissima and 51 king eiders S. spectabilis. Trends were similar in both species. Bullar ossification and definitive size occurred early in life: bullar size did not differ between first-year and older males. Bullar size did not vary more than size of other body parts (CVs of 3.4–7.0% for bullar length and breadth). Bullar size scaled to body size with negative allometry or isometry. Vocal muscles were 10–50% thicker in males than females, a much greater sexual difference than in body size (CVs of 3–6% on linear body-size variables). Vocal muscles were larger on the left side in both sexes and bilateral asymmetry was slightly more pronounced in males. Low variation and a trend towards negative allometry suggest that bullar size is under stabilizing selection; if bullar size affects vocal attributes of voice, then the latter cannot be condition-dependent. We recommend comparative research on vocal communication, vocal individuality and vocal-tract anatomy and function in eiders and other ducks.     

Modeling within-host HIV-1 dynamics and the evolution of drug resistance: trade-offs between viral enzyme function and drug susceptibility

There are many biological steps between viral infection of CD4(+) T cells and the production of HIV-1 virions. Here we incorporate an eclipse phase, representing the stage in which infected T cells have not started to produce new virus, into a simple HIV-1 model. Model calculations suggest that the quicker infected T cells progress from the eclipse stage to the productively infected stage, the more likely that a viral strain will persist. Long-term treatment effectiveness of antiretroviral drugs is often hindered by the frequent emergence of drug resistant virus during therapy. We link drug resistance to both the rate of progression of the eclipse phase and the rate of viral production of the resistant strain, and explore how the resistant strain could evolve to maximize its within-host viral fitness. We obtained the optimal progression rate and the optimal viral production rate, which maximize the fitness of a drug resistant strain in the presence of drugs. We show that the window of opportunity for invasion of drug resistant strains is widened for a higher level of drug efficacy provided that the treatment is not potent enough to eradicate both the sensitive and resistant virus.     

Measurement of intracellular strain on deformable substrates with texture correlation

Mechanical stimuli are important factors that regulate cell proliferation, survival, metabolism and motility in a variety of cell types. The relationship between mechanical deformation of the extracellular matrix and intracellular deformation of cellular sub-regions and organelles has not been fully elucidated, but may provide new insight into the mechanisms involved in transducing mechanical stimuli to biological responses. In this study, a novel fluorescence microscopy and image analysis method was applied to examine the hypothesis that mechanical strains are fully transferred from a planar, deformable substrate to cytoplasmic and intranuclear regions within attached cells. Intracellular strains were measured in cells derived from the anulus fibrosus of the intervertebral disc when attached to an elastic silicone membrane that was subjected to tensile stretch. Measurements indicated cytoplasmic strains were similar to those of the underlying substrate, with a strain transfer ratio (STR) of 0.79. In contrast, nuclear strains were much smaller than those of the substrate, with an STR of 0.17. These findings are consistent with previous studies indicating nuclear stiffness is significantly greater than cytoplasmic stiffness, as measured using other methods. This study provides a novel method for the study of cellular mechanics, including a new technique for measuring intranuclear deformations, with evidence of differential magnitudes and patterns of strain transferred from the substrate to cell cytoplasm and nucleus.     

Oocyte maturation: emerging concepts and technologies to improve developmental potential in vitro

Oocyte in vitro maturation (IVM) is an important reproductive technology that generates mature oocytes that are capable of supporting preimplantation embryo development and full development to term. There is great clinical and commercial incentive to improve the efficiency of the technology, however, progress has been slow over the past decade. A critical challenge is to understand what constitutes oocyte developmental competence and the mechanisms governing it. We have taken the approach of studying in detail oocyte-somatic cell interactions; including, oocyte-cumulus cell (CC) gap-junctional communication, and bidirectional paracrine signalling between the two cell types. It is becoming clear that, compared to oocytes matured in vivo, IVM oocytes undergo maturation prematurely as they are still in the process of acquiring developmental competence in vivo, and the molecular cascade reinitiating meiosis differs entirely to that in vivo. Attempts to enhance oocyte developmental competence by attenuating the spontaneous meiotic resumption of oocytes in vitro have been met with mixed success. Kinase inhibitors that prevent maturation-promoting factor activity have, in general, been ineffectual on promoting oocyte developmental potential post-IVM. In contrast, agents that modulate oocyte cAMP during IVM show greater potential, possibly as these compounds extend oocyte-CC gap-junctional communication. An important concept that is now emerging is that the oocyte secretes potent growth factors that regulate fundamental aspects of CC function and thereby determine the distinctive phenotype of the cumulus-oocyte complex. The capacity of an oocyte to regulate its own microenvironment by oocyte-secreted factors (OSFs) may constitute an important component of oocyte developmental competence. In support of this notion, we have recently demonstrated that supplementing IVM media with exogenous OSFs improves oocyte developmental potential, as evidenced by enhanced pre- and post-implantation embryo development.     

Modeling Within-Host Evolution of HIV: Mutation,Competition and Strain Replacement

Virus evolution during infection of a single individual is a well-known feature of disease progression in chronic viral diseases. However, the simplest models of virus competition for host resources show the existence of a single dominant strain that grows most rapidly during the initial period of infection and competitively excludes all other virus strains. Here, we examine the dynamics of strain replacement in a simple model that includes a convex trade-off between rapid virus reproduction and long-term host cell survival. Strains are structured according to their within-cell replication rate. Over the course of infection, we find a progression in the dominant strain from fast- to moderately-replicating virus strains featuring distinct jumps in the replication rate of the dominant strain over time. We completely analyze the model and provide estimates for the replication rate of the initial dominant strain and its successors. Our model lays the groundwork for more detailed models of HIV selection and mutation. We outline future directions and application of related models to other biological situations.