Traditional Genetic Models Research Articles

DMKPs provide a generalizable strategy for studying genes required for reproduction or viability in nontraditional model organisms.

The advent of CRISPR/Cas9-mediated genome editing has expanded the range of animals amenable to targeted genetic analysis. This has accelerated research in animals not traditionally studied using molecular genetics. However, studying genes essential for reproduction or survival in such animals remains challenging, as they lack the tools that aid genetic analysis in traditional genetic model organisms. We recently introduced the use of distinguishably marked knock-in pairs (DMKPs) as a strategy for rapid and reliable genotyping in such species. Here we show that DMKPs also facilitate the maintenance and study of mutations that cannot be maintained in a homozygous state, a group which includes recessive lethal and sterile mutations. Using DMKPs, we disrupt the zero population growth locus in Drosophila melanogaster and in the dengue vector mosquito Aedes aegypti. In both species, DMKPs enable the maintenance of zero population growth mutant strains and the reliable recovery of zero population growth mutant animals. Male and female gonad development is disrupted in fly and mosquito zero population growth mutants, rendering both sexes sterile. In Ae. aegypti, zero population growth mutant males remain capable of inducing a mating refractory period in wild-type females and of competing with wild-type males for mates, properties compatible with zero population growth serving as a target in mosquito population suppression strategies. DMKP is readily generalizable to other species amenable to CRISPR/Cas9-mediated gene targeting, and should facilitate the study of sterile and lethal mutations in multiple organisms not traditionally studied using molecular genetics.

Identifying genetic variants underlying medication-induced osteonecrosis of the jaw in cancer and osteoporosis: a case control study

BackgroundBisphosphonate-induced osteonecrosis of the jaw (BRONJ) presents with a typical pattern of jaw necrosis in patients who have been prescribed bisphosphonates (BPs) and other antiangiogenetic drugs to treat osteoporosis or bone-related complications of cancer.MethodsThis study divided 38 patients with BRONJ into two groups according to the prescribing causes: cancer (n = 13) and osteoporosis (n = 25), and underwent whole exome sequencing and compared them with normal controls (n = 90). To identify candidate genes and variants, we conducted three analyses: a traditional genetic model, gene-wise variant score burden, and rare-variant analysis methods.ResultsThe stop-gain mutation (rs117889746) of the PZP gene in the BRONJ cancer group was significantly identified in the additive trend model analysis. In the cancer group, ARIDS, HEBP1, LTBP1, and PLVAP were identified as candidate genes. In the osteoporosis group, VEGFA, DFFA, and FAM193A genes showed a significant association. No significant genes were identified in the rare-variant analysis pipeline. Biologically accountable functions related to BRONJ occurrence-angiogenesis-related signaling (VEGFA and PLVAP genes), TGF-β signaling (LTBP1 and PZP genes), heme toxicity (HEBP1) and osteoblast maturation (ARIDS)-were shown in candidate genes.ConclusionThis study showed that the candidate causative genes contributing to the development of BRONJ differ according to the BP dose and background disease.

A genetic model based on evapoconcentration for sediment-hosted exotic-Cu mineralization in arid environments: the case of the El Tesoro Central copper deposit, Atacama Desert, Chile

Although the formation of exotic-Cu deposits is controlled by multiple factors, the role of the sedimentary environment has not been well defined. We present a case study of the El Tesoro Central exotic-Cu deposit located in the Atacama Desert of northern Chile. This deposit consists of two mineralized bodies hosted within Late Cenozoic gravels deposited in an arid continental environment dominated by alluvial fans with sub-surficial ponded water bodies formed at the foot of these fans or within the interfan areas. Both exotic-Cu orebodies mostly consist of chrysocolla, copper wad, atacamite, paratacamite, quartz, opal, and calcite. The most commonly observed paragenesis comprises chrysocolla, silica minerals, and calcite and records a progressive increase in pH, which is notably influenced by evaporation. The results of stable isotope analyses (δ13C and δ18O) and hydrogeochemical simulations confirm that evapoconcentration is the main controlling factor in the exotic-Cu mineralization at El Tesoro Central. This conclusion complements the traditional genetic model based on the gradual neutralization of highly oversaturated Cu-bearing solutions that progressively cement the gravels and underlying bedrock regardless of the depositional environment. This study concludes that in exotic-Cu deposits formed relatively far from the source, a favorable sedimentary environment and particular hydrologic and climatic conditions are essential to trap, accumulate, evapoconcentrate, neutralize and saturate Cu-bearing solutions to trigger mineralization. Thus, detailed sedimentological studies should be incorporated when devising exploration strategies in order to discover new exotic-Cu resources, particularly if they are expected to have formed relatively far from the metal sources.

Positional RNA-Seq identifies candidate genes for phenotypic engineering of sexual traits.

IntroductionRNA interference (RNAi) of trait-specific genes permits the manipulation of specific phenotypic traits (“phenotypic engineering”) and thus represents a powerful tool to test trait function in evolutionary studies. The identification of suitable candidate genes, however, often relies on existing functional gene annotation, which is usually limited in emerging model organisms, especially when they are only distantly related to traditional genetic model organisms. A case in point is the free-living flatworm Macrostomum lignano (Lophotrochozoa: Platyhelminthes: Rhabditophora), an increasingly powerful model organism for evolutionary studies of sex in simultaneous hermaphrodites. To overcome the limitation of sparse functional annotation, we have performed a positional RNA-Seq analysis on different body fragments in order to identify organ-specific candidate transcripts. We then performed gene expression (in situ hybridization) and gene function (RNAi) analyses on 23 candidate transcripts, both to evaluate the predictive potential of this approach and to obtain preliminary functional characterizations of these candidate genes.ResultsWe identified over 4000 transcripts that could be expected to show specific expression in different reproductive organs (including testis, ovary and the male and female genital systems). The predictive potential of the method could then be verified by confirming organ-specific expression for several candidate transcripts, some of which yielded interesting trait-specific knock-down phenotypes that can now be followed up in future phenotypic engineering studies.ConclusionsOur positional RNA-Seq analysis represents a highly useful resource for the identification of candidate transcripts for functional and phenotypic engineering studies in M. lignano, and it has already been used successfully in several studies. Moreover, this approach can overcome some inherent limitations of homology-based candidate selection and thus should be applicable to a broad range of emerging model organisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12983-015-0106-0) contains supplementary material, which is available to authorized users.

Development of a markerless gene replacement system in Corynebacterium glutamicum using upp as a counter-selection marker.

Corynebacterium glutamicum is well-established for industrial and biotechnological applications. However, its genetic manipulation has generally lagged behind traditional genetic models. In this study, a counter-selectable marker gene upp was firstly confirmed to be more efficient than traditional sacB. Furthermore, a markerless gene replacement system was developed by combining upp with double-strand break repair caused by the exogenous endonuclease I-SceI. Finally, genetic modification using a dsDNA PCR fragment was carried out with the expression of recombinase/exonuclease RecE/RecT. Our results show that the genetic modification system allows precise and markerless gene replacement without altering the chromosome, with a simplified screening procedure to generate its modification.

Establishment of a Markerless Mutation Delivery System in Bacillus subtilis Stimulated by a Double-Strand Break in the Chromosome

Bacillus subtilis has been a model for gram-positive bacteria and it has long been exploited for industrial and biotechnological applications. However, the availability of facile genetic tools for physiological analysis has generally lagged substantially behind traditional genetic models such as Escherichia coli and Saccharomyces cerevisiae. In this work, we have developed an efficient, precise and scarless method for rapid multiple genetic modifications without altering the chromosome of B. subtilis. This method employs upp gene as a counter-selectable marker, double-strand break (DSB) repair caused by exogenous endonuclease I-SceI and comK overexpression for fast preparation of competent cell. Foreign dsDNA can be simply and efficiently integrated into the chromosome by double-crossover homologous recombination. The DSB repair is a potent inducement for stimulating the second intramolecular homologous recombination, which not only enhances the frequency of resolution by one to two orders of magnitude, but also selects for the resolved product. This method has been successfully and reiteratively used in B. subtilis to deliver point mutations, to generate in-frame deletions, and to construct large-scale deletions. Experimental results proved that it allowed repeated use of the selectable marker gene for multiple modifications and could be a useful technique for B. subtilis.

The evolutionary potential of paramutation: A population-epigenetic model

Paramutation involves an interaction between homologous alleles resulting in a heritable change in gene expression without altering the DNA sequence. Initially believed to be restricted to plants, paramutation has recently been observed in animal models, and a paramutation-like event has been noted in humans. Despite the accumulating evidence suggesting that trans-acting epigenetic effects can be inherited transgenerationally and therefore generate non-genomic phenotypic variation, these effects have been largely ignored in the context of evolutionary theory. The model presented here incorporates paramutation into the standard model of viability selection at one locus and demonstrates that paramutation can create long-term biological diversity in the absence of genetic change, and even in the absence of the original paramutagenic allele. Therefore, if paramutation is present, attributing evolution to only a traditional genetic model may fail to encompass the broad scope of phenotypic differences observed in nature. Moreover, we show also that an unusual mathematical behaviour, analogous to “Ewens’ gap” of the two-locus two-allele symmetric-selection model, occurs: when the rate of one parameter–for example, the rate of paramutation–is increased, a pair of equilibria may disappear only to reappear as this parameter increases further. In summary, by incorporating even the simplest epigenetic parameters into the standard population-genetic model of selection, we show how this type of inheritance system can profoundly alter the course of evolution.

Sequencing and characterization of the guppy (Poecilia reticulata) transcriptome

BackgroundNext-generation sequencing is providing researchers with a relatively fast and affordable option for developing genomic resources for organisms that are not among the traditional genetic models. Here we present a de novo assembly of the guppy (Poecilia reticulata) transcriptome using 454 sequence reads, and we evaluate potential uses of this transcriptome, including detection of sex-specific transcripts and deployment as a reference for gene expression analysis in guppies and a related species. Guppies have been model organisms in ecology, evolutionary biology, and animal behaviour for over 100 years. An annotated transcriptome and other genomic tools will facilitate understanding the genetic and molecular bases of adaptation and variation in a vertebrate species with a uniquely well known natural history.ResultsWe generated approximately 336 Mbp of mRNA sequence data from male brain, male body, female brain, and female body. The resulting 1,162,670 reads assembled into 54,921 contigs, creating a reference transcriptome for the guppy with an average read depth of 28×. We annotated nearly 40% of this reference transcriptome by searching protein and gene ontology databases. Using this annotated transcriptome database, we identified candidate genes of interest to the guppy research community, putative single nucleotide polymorphisms (SNPs), and male-specific expressed genes. We also showed that our reference transcriptome can be used for RNA-sequencing-based analysis of differential gene expression. We identified transcripts that, in juveniles, are regulated differently in the presence and absence of an important predator, Rivulus hartii, including two genes implicated in stress response. For each sample in the RNA-seq study, >50% of high-quality reads mapped to unique sequences in the reference database with high confidence. In addition, we evaluated the use of the guppy reference transcriptome for gene expression analyses in a congeneric species, the sailfin molly (Poecilia latipinna). Over 40% of reads from the sailfin molly sample aligned to the guppy transcriptome.ConclusionsWe show that next-generation sequencing provided a reliable and broad reference transcriptome. This resource allowed us to identify candidate gene variants, SNPs in coding regions, and sex-specific gene expression, and permitted quantitative analysis of differential gene expression.

From Gene Expression to Phenotype in Insects: Non-microarray Approaches for Transcriptome Analysis

Transcripts and their expression levels link an organism's genotype and phenotype, so understanding this relationship can aid our understanding of phenotypic evolution at the gene-expression level. The emerging field of functional genomics is concerned primarily with understanding how allelic and gene-expression variation is linked to observable, biologically relevant phenotypes. Insects are particularly well studied in this area because they are good laboratory systems and have incredible biodiversity and agricultural and public-health importance. Technology developed over the last decade or so permits gene expression studies in any insect system, thus advancing the field of functional genomics beyond traditional genetic model systems such as Drosophila. In this article we provide an overview of commonly used non-microarray gene-expression techniques in insect systems and review several empirical studies that use each technique. We also discuss RNA interference as a means to test the link between gene ex...

EVALUATING SIGNATURES OF GLACIAL REFUGIA FOR NORTH ATLANTIC BENTHIC MARINE TAXA

A goal of phylogeography is to relate patterns of genetic differentiation to potential historical geographic isolating events. Quaternary glaciations, particularly the one culminating in the Last Glacial Maximum approximately 21 ka (thousands of years ago), greatly affected the distributions and population sizes of temperate marine species as their ranges retreated southward to escape ice sheets. Traditional genetic models of glacial refugia and routes of recolonization include these predictions: low genetic diversity in formerly glaciated areas, with a small number of alleles/haplotypes dominating disproportionately large areas, and high diversity including "private" alleles in glacial refugia. In the Northern Hemisphere, low diversity in the north and high diversity in the south are expected. This simple model does not account for the possibility of populations surviving in relatively small northern periglacial refugia. If these periglacial populations experienced extreme bottlenecks, they could have the low genetic diversity expected in recolonized areas with no refugia, but should have more endemic diversity (private alleles) than recently recolonized areas. This review examines evidence of putative glacial refugia for eight benthic marine taxa in the temperate North Atlantic. All data sets were reanalyzed to allow direct comparisons between geographic patterns of genetic diversity and distribution of particular clades and haplotypes including private alleles. We contend that for marine organisms the genetic signatures of northern periglacial and southern refugia can be distinguished from one another. There is evidence for several periglacial refugia in northern latitudes, giving credence to recent climatic reconstructions with less extensive glaciation.

Maintenance of clinal variation for shell colour phenotype in the flat periwinkle Littorina obtusata

Clines can signal spatially varying selection and therefore have long been used to investigate the role of environmental heterogeneity in maintaining genetic variation. However, clinal patterns alone are not sufficient to reject neutrality or to establish the mechanism of selection. Indirect, inferential methods can be used to address neutrality and mechanism, but fully understanding the adaptive significance of clinal variation ultimately requires a direct approach. Ecological model systems such as the rocky intertidal provide a useful context for direct experimentation and can serve as a complement to studies in more traditional genetic model systems. In this study, we use indirect and direct approaches to investigate the role of environmental heterogeneity in the maintenance of shell colour polymorphism in the flat periwinkle snail, Littorina obtusata. We document replicated clines in shell colour morph frequencies over thermal gradients at two spatial scales, contrasting with patterns at previously reported microsatellite loci. In addition, experimental results demonstrate that that shell colour has predictable effects on shell temperature and that these differences in temperature, in turn, coincide with patterns of survivorship under episodic thermal stress. Direct manipulation of shell colour revealed that shell colour, and not a correlated character, was the target of selection. Our study provides evidence that spatially varying selection via thermal regime contributes to the maintenance of shell colour phenotype variation in L. obtusata in the sampled areas of the Gulf of Maine.

SASQuant: A SAS Software Program to Estimate Genetic Effects and Heritabilities of Quantitative Traits in Populations Consisting of 6 Related Generations

Plant breeders are interested in the analysis of phenotypic data to measure genetic effects and heritability of quantitative traits and predict gain from selection. Measurement of phenotypic values of 6 related generations (parents, F(1), F(2), and backcrosses) allows for the simultaneous analysis of both Mendelian and quantitative traits. In 1997, Liu et al. released a SAS software based program (SASGENE) for the analysis of inheritance and linkage of qualitative traits. We have developed a new program (SASQuant) that estimates gene effects (Hayman's model), genetic variances, heritability, predicted gain from selection (Wright's and Warner's models), and number of effective factors (Wright's, Mather's, and Lande's models). SASQuant makes use of traditional genetic models and allows for their easy application to complex data sets. SASQuant is freely available and is intended for scientists studying quantitative traits in plant populations.

Combined Effects of Thrombosis Pathway Gene Variants Predict Cardiovascular Events

The genetic background of complex diseases is proposed to consist of several low-penetrance risk loci. Addressing this complexity likely requires both large sample size and simultaneous analysis of different predisposing variants. We investigated the role of four thrombosis genes: coagulation factor V (F5), intercellular adhesion molecule 1 (ICAM1), protein C (PROC), and thrombomodulin (THBD) in cardiovascular diseases. Single allelic gene variants and their pair-wise combinations were analyzed in two independently sampled population cohorts from Finland. From among 14,140 FINRISK participants (FINRISK-92, n = 5,999 and FINRISK-97, n = 8,141), we selected for genotyping a sample of 2,222, including 528 incident cardiovascular disease (CVD) cases and random subcohorts totaling 786. To cover all known common haplotypes (>10%), 54 single nucleotide polymorphisms (SNPs) were genotyped. Classification-tree analysis identified 11 SNPs that were further analyzed in Cox's proportional hazard model as single variants and pair-wise combinations. Multiple testing was controlled by use of two independent cohorts and with false-discovery rate. Several CVD risk variants were identified: In women, the combination of F5 rs7542281 × THBD rs1042580, together with three single F5 SNPs, was associated with CVD events. Among men, PROC rs1041296, when combined with either ICAM1 rs5030341 or F5 rs2269648, was associated with total mortality. As a single variant, PROC rs1401296, together with the F5 Leiden mutation, was associated with ischemic stroke events. Our strategy to combine the classification-tree analysis with more traditional genetic models was successful in identifying SNPs—acting either in combination or as single variants—predisposing to CVD, and produced consistent results in two independent cohorts. These results suggest that variants in these four thrombosis genes contribute to arterial cardiovascular events at population level.

Sexual Selection and the Sex Ratio: An ESS Analysis

Should attractive fathers have more sons? According to widespread verbal arguments, the answer is in the affirmative, but formal models are lacking. We investigate the question by means of an ESS analysis of sexual selection and sex ratio selection. The purpose of this paper is twofold: firstly, we show how an ESS approach can be used to model the coevolution of female mate choice and male secondary sexual traits. In the ESS approach, the genetic covariances of traditional genetic models of sexual selection are replaced by covariances between strategies. In comparison with population genetic and quantitative genetic models of sexual selection, the ESS approach is simpler and yields more insight in the underlying selective forces. Secondly, we show that whether attractive fathers should have more sons depends on the mechanism of sexual selection: sexual selection driven by the Fisherian runaway process alone, without costs of choosiness, does not select for sex ratio specialization according to paternal attractiveness, but the good-genes process causes selection to favor such specialization.

Self-Ordering and Complexity in Epizonal Mineral Deposits

Epizonal base and precious metal deposits makeup a range of familiar deposit styles including porphyry copper-gold, epithermal veins and stockworks, carbonate-replacement deposits, and polymetallic volcanic rock-hosted (VHMS) deposits. They occur along convergent plate margins and are invariably associated directly with active faults and volcanism. They are complex in form, variable in their characteristics at all scales, and highly localized in the earth’s crust.More than a century of detailed research has provided an extensive base of observational data characterizing these deposits, from their regional setting to the fluid and isotope chemistry of mineral deposition. This has led to a broad understanding of the large-scale hydrothermal systems within which they form. Low salinity vapor, released by magma crystallization and dispersed into vigorously convecting groundwater systems, is recognized as a principal source of metals and the gases that control redox conditions within systems. The temperature and pressure of the ambient fluid anywhere within these systems is close to its vapor-liquid phase boundary, and mineral deposition is a consequence of short timescale perturbations generated by localized release of crustal stress.However, a review of occurrence data raises questions about ore formation that are not addressed by traditional genetic models. For example, what are the origins of banding in epithermal veins, and what controls the frequency of oscillatory lamination? What controls where the phenomenon of mineralization occurs, and why are some porphyry deposits, for example, so much larger than others?The distinctive, self-organized characteristics of epizonal deposits are shown to be the result of repetitive coupling of fracture dilation consequent on brittle failure, phase separation (“boiling”), and heat transfer between fluid and host rock. Process coupling substantially increases solute concentrations and triggers fast, far-from-equilibrium depositional processes. Since these coupled processes lead to localized transient changes in fluid characteristics, paragenetic, isotope, and fluid inclusion data relate to conditions at the site of deposition and only indirectly to the characteristics of the larger-scale hydrothermal system and its longer-term behavior. The metal concentrations (i.e. grade) of deposits and their internal variation is directly related to the geometry of the fracture array at the deposit scale, whereas finer-scale oscillatory fabrics in ores may be a result of molecular scale processes.Giant deposits are relatively rare and develop where efficient metal deposition is spatially focused by repetitive brittle failure in active fault arrays. Some brief case histories are provided for epithermal, replacement, and porphyry mineralization. These highlight how rock competency contrasts and feedback between processes, rather than any single component of a hydrothermal system, govern the size of individual deposits. In turn, the recognition of the probabilistic nature of mineralization provides a firmer foundation through which exploration investment and risk management decisions can be made.