Natural Variation for Lifespan and Stress Response in the Nematode Caenorhabditis remanei

Genetic approaches (e.g. mutation, RNA interference) in model organisms, particularly the nematode Caenorhabditis elegans, have yielded a wealth of information on cellular processes that can influence lifespan. Although longevity mutants discovered in the lab are instructive of cellular physiology, lab studies might miss important genes that influence health and longevity in the wild. C. elegans has relatively low natural genetic variation and high levels of linkage disequilibrium, and thus is not optimal for studying natural variation in longevity. In contrast, its close relative C. remanei possesses very high levels of molecular genetic variation and low levels of linkage disequilibrium. To determine whether C. remanei may be a good model system for the study of natural genetic variation in aging, we evaluated levels of quantitative genetic variation for longevity and resistance to oxidative, heat and UV stress. Heritability (and the coefficient of additive genetic variation) was high for oxidative and heat stress resistance, low (but significant) for longevity, and essentially zero for UV stress response. Our results suggest that C. remanei may be a powerful system for studying natural genetic variation for longevity and oxidative and heat stress response, as well as an informative model for the study of functional relationships between longevity and stress response.     

Sporulation Genes Associated with Sporulation Efficiency in Natural Isolates of Yeast

Yeast sporulation efficiency is a quantitative trait and is known to vary among experimental populations and natural isolates. Some studies have uncovered the genetic basis of this variation and have identified the role of sporulation genes (IME1, RME1) and sporulation-associated genes (FKH2, PMS1, RAS2, RSF1, SWS2), as well as non-sporulation pathway genes (MKT1, TAO3) in maintaining this variation. However, these studies have been done mostly in experimental populations. Sporulation is a response to nutrient deprivation. Unlike laboratory strains, natural isolates have likely undergone multiple selections for quick adaptation to varying nutrient conditions. As a result, sporulation efficiency in natural isolates may have different genetic factors contributing to phenotypic variation. Using Saccharomyces cerevisiae strains in the genetically and environmentally diverse SGRP collection, we have identified genetic loci associated with sporulation efficiency variation in a set of sporulation and sporulation-associated genes. Using two independent methods for association mapping and correcting for population structure biases, our analysis identified two linked clusters containing 4 non-synonymous mutations in genes – HOS4, MCK1, SET3, and SPO74. Five regulatory polymorphisms in five genes such as MLS1 and CDC10 were also identified as putative candidates. Our results provide candidate genes contributing to phenotypic variation in the sporulation efficiency of natural isolates of yeast.     

Genetic mapping of variation in dauer larvae development in growing populations of Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the appropriate induction of dauer larvae development within growing populations is likely to be a primary determinant of genotypic fitness. The underlying genetic architecture of natural genetic variation in dauer formation has, however, not been thoroughly investigated. Here, we report extensive natural genetic variation in dauer larvae development within growing populations across multiple wild isolates. Moreover, bin mapping of introgression lines (ILs) derived from the genetically divergent isolates N2 and CB4856 reveals 10 quantitative trait loci (QTLs) affecting dauer formation. Comparison of individual ILs to N2 identifies an additional eight QTLs, and sequential IL analysis reveals six more QTLs. Our results also show that a behavioural, laboratory-derived, mutation controlled by the neuropeptide Y receptor homolog npr-1 can affect dauer larvae development in growing populations. These findings illustrate the complex genetic architecture of variation in dauer larvae formation in C. elegans and may help to understand how the control of variation in dauer larvae development has evolved.     

Mapping determinants of gene expression plasticity by genetical genomics in C. elegans

Recent genetical genomics studies have provided intimate views on gene regulatory networks. Gene expression variations between genetically different individuals have been mapped to the causal regulatory regions, termed expression quantitative trait loci. Whether the environment-induced plastic response of gene expression also shows heritable difference has not yet been studied. Here we show that differential expression induced by temperatures of 16 °C and 24 °C has a strong genetic component in Caenorhabditis elegans recombinant inbred strains derived from a cross between strains CB4856 (Hawaii) and N2 (Bristol). No less than 59% of 308 trans-acting genes showed a significant eQTL-by-environment interaction, here termed plasticity quantitative trait loci. In contrast, only 8% of an estimated 188 cis-acting genes showed such interaction. This indicates that heritable differences in plastic responses of gene expression are largely regulated in trans. This regulation is spread over many different regulators. However, for one group of trans-genes we found prominent evidence for a common master regulator: a transband of 66 coregulated genes appeared at 24 °C. Our results suggest widespread genetic variation of differential expression responses to environmental impacts and demonstrate the potential of genetical genomics for mapping the molecular determinants of phenotypic plasticity.     

A Multiparent Advanced Generation Inter-Cross to Fine-Map Quantitative Traits in Arabidopsis thaliana

Identifying natural allelic variation that underlies quantitative trait variation remains a fundamental problem in genetics. Most studies have employed either simple synthetic populations with restricted allelic variation or performed association mapping on a sample of naturally occurring haplotypes. Both of these approaches have some limitations, therefore alternative resources for the genetic dissection of complex traits continue to be sought. Here we describe one such alternative, the Multiparent Advanced Generation Inter-Cross (MAGIC). This approach is expected to improve the precision with which QTL can be mapped, improving the outlook for QTL cloning. Here, we present the first panel of MAGIC lines developed: a set of 527 recombinant inbred lines (RILs) descended from a heterogeneous stock of 19 intermated accessions of the plant Arabidopsis thaliana. These lines and the 19 founders were genotyped with 1,260 single nucleotide polymorphisms and phenotyped for development-related traits. Analytical methods were developed to fine-map quantitative trait loci (QTL) in the MAGIC lines by reconstructing the genome of each line as a mosaic of the founders. We show by simulation that QTL explaining 10% of the phenotypic variance will be detected in most situations with an average mapping error of about 300 kb, and that if the number of lines were doubled the mapping error would be under 200 kb. We also show how the power to detect a QTL and the mapping accuracy vary, depending on QTL location. We demonstrate the utility of this new mapping population by mapping several known QTL with high precision and by finding novel QTL for germination data and bolting time. Our results provide strong support for similar ongoing efforts to produce MAGIC lines in other organisms.     

Methods for analyzing neuronal structure and activity in Caenorhabditis elegans

The model research animal Caenorhabditis elegans has unique properties making it particularly advantageous for studies of the nervous system. The nervous system is composed of a stereotyped complement of neurons connected in a consistent manner. Here, we describe methods for studying nervous system structure and function. The transparency of the animal makes it possible to visualize and identify neurons in living animals with fluorescent probes. These methods have been recently enhanced for the efficient use of neuron-specific reporter genes. Because of its simple structure, for a number of years, C. elegans has been at the forefront of connectomic studies defining synaptic connectivity by electron microscopy. This field is burgeoning with new, more powerful techniques, and recommended up-to-date methods are here described that encourage the possibility of new work in C. elegans. Fluorescent probes for single synapses and synaptic connections have allowed verification of the EM reconstructions and for experimental approaches to synapse formation. Advances in microscopy and in fluorescent reporters sensitive to Ca²⁺ levels have opened the way to observing activity within single neurons across the entire nervous system.     

Naturally occurring variation in Arabidopsis: an underexploited resource for plant genetics

The definition of gene functions requires the phenotypic characterization of genetic variants. Currently, such functional analysis of Arabidopsis genes is based largely on laboratory-induced mutants that are selected in forward and reverse genetic studies. An alternative complementary source of genetic variation is available: the naturally occurring variation among accessions. The multigenic nature of most of this variation has limited its application until now. However, the use of genetic methods developed to map quantitative trait loci, in combination with the characteristics and resources available for molecular biology in Arabidopsis, allow this variation to be exploited up to the molecular level. Here, we describe the current tools available for the forward genetic analysis of this variation, and review the recent progress in the detection and mapping of loci and the cloning of large-effect genes.     

Mos1-Mediated Transgenesis to Probe Consequences of Single Gene Mutations in Variation-Rich Isolates of Caenorhabditis elegans

Caenorhabditis elegans, especially the N2 isolate, is an invaluable biological model system. Numerous additional natural C. elegans isolates have been shown to have unexpected genotypic and phenotypic variations which has encouraged researchers to use next generation sequencing methodology to develop a more complete picture of genotypic variations among the isolates. To understand the phenotypic effects of a genomic variation (GV) on a single gene, in a variation-rich genetic background, one should analyze that particular GV in a well understood genetic background. In C. elegans, the analysis is usually done in N2, which requires extensive crossing to bring in the GV. This can be a very time consuming procedure thus it is important to establish a fast and efficient approach to test the effect of GVs from different isolates in N2. Here we use a Mos1-mediated single-copy insertion (MosSCI) method for phenotypic assessments of GVs from the variation-rich Hawaiian strain CB4856 in N2. Specifically, we investigate effects of variations identified in the CB4856 strain on tac-1 which is an essential gene that is necessary for mitotic spindle elongation and pronuclear migration. We show the usefulness of the MosSCI method by using EU1004 tac-1(or402) as a control. or402 is a temperature sensitive lethal allele within a well-conserved TACC domain (transforming acidic coiled-coil) that results in a leucine to phenylalanine change at amino acid 229. CB4856 contains a variation that affects the second exon of tac-1 causing a cysteine to tryptophan change at amino acid 94 also within the TACC domain. Using the MosSCI method, we analyze tac-1 from CB4856 in the N2 background and demonstrate that the C94W change, albeit significant, does not cause any obvious decrease in viability. This MosSCI method has proven to be a rapid and efficient way to analyze GVs.     

Phenotypic evolution from genetic polymorphisms in a radial network architecture

Background  

The genetic architecture of a quantitative trait influences the phenotypic response to natural or artificial selection. One of the main objectives of genetic mapping studies is to identify the genetic factors underlying complex traits and understand how they contribute to phenotypic expression. Presently, we are good at identifying and locating individual loci with large effects, but there is a void in describing more complex genetic architectures. Although large networks of connected genes have been reported, there is an almost complete lack of information on how polymorphisms in these networks contribute to phenotypic variation and change. To date, most of our understanding comes from theoretical, model-based studies, and it remains difficult to assess how realistic their conclusions are as they lack empirical support.     

High-volume mapping of maize mutants with simple sequence repeat markers

Many genes in maize (Zea mays L.) are revealed by mutations that cause phenotypic variation from normal. These mutants are valuable resources of genetic information. From among the huge collection of maize mutants, it is ultimately necessary to establish which alleles are of the same genes and which are novel genes. Although any given mutant can be subjected to complementation tests or can be mapped by using conventional techniques, the number of mutants at this time makes these approaches prohibitive to encompass the whole collection. Here we describe procedures to efficiently map large numbers of mutants. Included are methods for generating polymorphic mapping progenies, for simply and rapidly preparing samples to use in polymerase chain reaction (PCR), for tissue pooling and application of simple sequence repeat (SSR), markers, and for stepwise determination of linkage followed by mapping to chromosomal region.     

Bias and Evolution of the Mutationally Accessible Phenotypic Space in a Developmental System

Genetic and developmental architecture may bias the mutationally available phenotypic spectrum. Although such asymmetries in the introduction of variation may influence possible evolutionary trajectories, we lack quantitative characterization of biases in mutationally inducible phenotypic variation, their genotype-dependence, and their underlying molecular and developmental causes. Here we quantify the mutationally accessible phenotypic spectrum of the vulval developmental system using mutation accumulation (MA) lines derived from four wild isolates of the nematodes Caenorhabditis elegans and C. briggsae. The results confirm that on average, spontaneous mutations degrade developmental precision, with MA lines showing a low, yet consistently increased, proportion of developmental defects and variants. This result indicates strong purifying selection acting to maintain an invariant vulval phenotype. Both developmental system and genotype significantly bias the spectrum of mutationally inducible phenotypic variants. First, irrespective of genotype, there is a developmental bias, such that certain phenotypic variants are commonly induced by MA, while others are very rarely or never induced. Second, we found that both the degree and spectrum of mutationally accessible phenotypic variation are genotype-dependent. Overall, C. briggsae MA lines exhibited a two-fold higher decline in precision than the C. elegans MA lines. Moreover, the propensity to generate specific developmental variants depended on the genetic background. We show that such genotype-specific developmental biases are likely due to cryptic quantitative variation in activities of underlying molecular cascades. This analysis allowed us to identify the mutationally most sensitive elements of the vulval developmental system, which may indicate axes of potential evolutionary variation. Consistent with this scenario, we found that evolutionary trends in the vulval system concern the phenotypic characters that are most easily affected by mutation. This study provides an empirical assessment of developmental bias and the evolution of mutationally accessible phenotypes and supports the notion that such bias may influence the directions of evolutionary change.     

Epistasis in a quantitative trait captured by a molecular model of transcription factor interactions

With technological advances in genetic mapping studies more of the genes and polymorphisms that underlie Quantitative Trait Loci (QTL) are now being identified. As the identities of these genes become known there is a growing need for an analysis framework that incorporates the molecular interactions affected by natural polymorphisms. As a step towards such a framework we present a molecular model of genetic variation in sporulation efficiency between natural isolates of the yeast, Saccharomyces cerevisiae. The model is based on the structure of the regulatory pathway that controls sporulation. The model captures the phenotypic variation between strains carrying different combinations of alleles at known QTL. Compared to a standard linear model the molecular model requires fewer free parameters, and has the advantage of generating quantitative hypotheses about the affinity of specific molecular interactions in different genetic backgrounds. Our analyses provide a concrete example of how the thermodynamic properties of protein-protein and protein-DNA interactions naturally give rise to epistasis, the non-linear relationship between genotype and phenotype. As more causative genes and polymorphisms underlying QTL are identified, thermodynamic analyses of quantitative traits may provide a useful framework for unraveling the complex relationship between genotype and phenotype.     

Ectodysplasin signalling genes and phenotypic evolution in sculpins (Cottus)

Despite their deeply conserved function among vertebrates, ectodysplasin (Eda) signalling genes are involved in microevolutionary change in humans and sticklebacks. If such a dual role is common, Eda signalling genes constitute hotspots for morphological evolution. Variation in sculpin (Cottus) skin prickling and body shape resembles patterns caused by variation in Eda signalling in sticklebacks. We mapped Eda signalling genes and performed quantitative trait locus mapping in crosses between Cottus rhenanus and Cottus perifretum. A genomic region containing the Eda receptor (Edar) was strongly associated with prickling and contributed to shape. The expression of Edar in developing prickles and skeletal elements in Cottus was confirmed by in situ hybridization. Coding sequence changes between Edar alleles in C. rhenanus and C. perifretum exceeded sequence differentiation in other vertebrates. However, it is likely that additional genetic elements besides coding changes affect the phenotypic variation. Although the phenotype in a natural hybrid lineage between C. rhenanus and C. perifretum resembles C. perifretum, the respective coding Edar alleles are not fully fixed (88.6%). Hence, our results support an involvement of Eda signalling in microevolutionary changes, but imply that the Edar gene is affected by multiple evolutionary processes that vary among freshwater sculpins.     

Exploiting the Extraordinary Genetic Polymorphism of Ciona for Developmental Genetics with Whole Genome Sequencing

Studies in tunicates such as Ciona have revealed new insights into the evolutionary origins of chordate development. Ciona populations are characterized by high levels of natural genetic variation, between 1 and 5%. This variation has provided abundant material for forward genetic studies. In the current study, we make use of deep sequencing and homozygosity mapping to map spontaneous mutations in outbred populations. With this method we have mapped two spontaneous developmental mutants. In Ciona intestinalis we mapped a short-tail mutation with strong phenotypic similarity to a previously identified mutant in the related species Ciona savignyi. Our bioinformatic approach mapped the mutation to a narrow interval containing a single mutated gene, α-laminin3,4,5, which is the gene previously implicated in C. savignyi. In addition, we mapped a novel genetic mutation disrupting neural tube closure in C. savignyi to a T-type Ca²⁺ channel gene. The high efficiency and unprecedented mapping resolution of our study is a powerful advantage for developmental genetics in Ciona, and may find application in other outbred species.     

solQTL: a tool for QTL analysis,visualization and linking to genomes at SGN database

Background  

A common approach to understanding the genetic basis of complex traits is through identification of associated quantitative trait loci (QTL). Fine mapping QTLs requires several generations of backcrosses and analysis of large populations, which is time-consuming and costly effort. Furthermore, as entire genomes are being sequenced and an increasing amount of genetic and expression data are being generated, a challenge remains: linking phenotypic variation to the underlying genomic variation. To identify candidate genes and understand the molecular basis underlying the phenotypic variation of traits, bioinformatic approaches are needed to exploit information such as genetic map, expression and whole genome sequence data of organisms in biological databases.     

Ten years of life in compost: temporal and spatial variation of North German Caenorhabditis elegans populations

The nematode Caenorhabditis elegans is a central laboratory model system in almost all biological disciplines, yet its natural life history and population biology are largely unexplored. Such information is essential for in‐depth understanding of the nematode's biology because its natural ecology provides the context, in which its traits and the underlying molecular mechanisms evolved. We characterized natural phenotypic and genetic variation among North German C. elegans isolates. We used the unique opportunity to compare samples collected 10 years apart from the same compost heap and additionally included recent samples for this and a second site, collected across a 1.5‐year period. Our analysis revealed significant population genetic differentiation between locations, across the 10‐year time period, but for only one location a trend across the shorter time frame. Significant variation was similarly found for phenotypic traits of likely importance in nature, such as choice behavior and population growth in the presence of pathogens or naturally associated bacteria. Phenotypic variation was significantly influenced by C. elegans genotype, time of isolation, and sampling site. The here studied C. elegans isolates may provide a valuable, genetically variable resource for future dissection of naturally relevant gene functions.     

Two genomic regions together cause dark abdominal pigmentation in Drosophila tenebrosa

Pigmentation is a rapidly evolving trait that is under both natural and sexual selection in many organisms. In the quinaria group of Drosophila, nearly all of the 30 species have an abdomen that is light in color with distinct markings; D. tenebrosa is the exception in that it has a completely melanic abdomen with no visible markings. In this study, we use a combination of quantitative genetic and candidate gene approaches to investigate the genetic basis of abdominal pigmentation in D. tenebrosa. We find that abdominal pigmentation is invariant across wild-caught lines of D. tenebrosa and is not sexually dimorphic. Quantitative genetic mapping utilizing crosses between D. tenebrosa and the light-colored D. suboccidentalis indicates that two genomic regions together underlie abdominal pigmentation, including the X-chromosome and an autosome (Muller Element C/E). Further support for their central importance in pigmentation is that experimental introgression of one phenotype into the other species, in either direction, results in introgression of these two genomic regions. Finally, the expression of the X-linked gene yellow in the pupae exactly foreshadows the adult melanization pattern in the abdomen of both species, suggesting that changes in the regulation of yellow are important for the phenotypic divergence of D. tenebrosa from the rest of the quinaria group. These results contribute to a body of work that demonstrates how changes in expression of highly conserved genes can cause substantial phenotypic differences even between closely related species.