Dominance and overdominance of mildly deleterious induced mutations for fitness traits in Caenorhabditis elegans

We estimated the average dominance coefficient of mildly deleterious mutations (h, the proportion by which mutations in the heterozygous state reduce fitness components relative to those in the homozygous state) in the nematode Caenorhabditis elegans. From 56 worm lines that carry mutations induced by the point mutagen ethyl methanesulfonate (EMS), we selected 19 lines that are relatively high in fitness and estimated the viabilities, productivities, and relative fitnesses of heterozygotes and homozygotes compared to the ancestral wild type. There was very little effect of homozygous or heterozygous mutations on egg-to-adult viability. For productivity and relative fitness, we found that the average dominance coefficient, h, was approximately 0.1, suggesting that mildly deleterious mutations are on average partially recessive. These estimates were not significantly different from zero (complete recessivity) but were significantly different from 0.5 (additivity). In addition, there was a significant amount of variation in h among lines, and analysis of average dominance coefficients of individual lines suggested that several lines showed overdominance for fitness. Further investigation of two of these lines partially confirmed this finding.     

On the average coefficient of dominance of deleterious spontaneous mutations

T. Mukai and co-workers in the late 1960s and O. Ohnishi in the 1970s carried out a series of experiments to obtain direct estimates of the average coefficient of dominance (h) of minor viability mutations in Drosophila melanogaster. The results of these experiments, although inconsistent, have been interpreted as indicating slight recessivity of deleterious mutations, with h approximately 0.4. Mukai obtained conflicting results depending on the type of heterozygotes used, some estimates suggesting overdominance and others partial dominance. Ohnishi's estimates, based on the ratio of heterozygous to homozygous viability declines, were more consistent, pointing to the above value. However, we have reanalyzed Ohnishi's data, estimating h by the regression method, and obtained a much smaller estimate of approximately 0.1. This significant difference can be due partly to the different weighting implicit in the estimates, but we suggest that this is not the only explanation. We propose as a plausible hypothesis that a putative nonmutational decline in viability occurring in the first half of Ohnishi's experiment (affecting both homozygotes and heterozygotes) has biased upward the estimates from the ratio, while it would not bias the regression estimates. This hypothesis also explains the very high h approximately 0.7 observed in Ohnishi's high-viability chromosomes. By constructing a model of spontaneous mutations using parameters in the literature, we investigate the above possibility. The results indicate that a model of few mutations with moderately large effects and h approximately 0.2 is able to explain the observed estimates and the distributions of homozygous and heterozygous viabilities. Accounting for an expression of mutations in genotypes with the balancer chromosome Cy does not alter the conclusions qualitatively.     

Nature of Deleterious Mutation Load in Drosophila

Much population genetics and evolution theory depends on knowledge of genomic mutation rates and distributions of mutation effects for fitness, but most information comes from a few mutation accumulation experiments in Drosophila in which replicated chromosomes are sheltered from natural selection by a balancer chromosome. I show here that data from these experiments imply the existence of a large class of minor viability mutations with approximately equivalent effects. However, analysis of the distribution of viabilities of chromosomes exposed to EMS mutagenesis reveals a qualitatively different distribution of effects lacking such a minor effects class. A possible explanation for this difference is that transposable element insertions, a common class of spontaneous mutation event in Drosophila, frequently generate minor viability effects. This explanation would imply that current estimates of deleterious mutation rates are not generally applicable in evolutionary models, as transposition rates vary widely. Alternatively, much of the apparent decline in viability under spontaneous mutation accumulation could have been nonmutational, perhaps due to selective improvement of balancer chromosomes. This explanation accords well with the data and implies a spontaneous mutation rate for viability two orders of magnitude lower than previously assumed, with most mutation load attributable to major effects.     

The effect of antagonistic pleiotropy on the estimation of the average coefficient of dominance of deleterious mutations

We investigate the impact of antagonistic pleiotropy on the most widely used methods of estimation of the average coefficient of dominance of deleterious mutations from segregating populations. A proportion of the deleterious mutations affecting a given studied fitness component are assumed to have an advantageous effect on another one, generating overdominance on global fitness. Using diffusion approximations and transition matrix methods, we obtain the distribution of gene frequencies for nonpleiotropic and pleiotropic mutations in populations at the mutation-selection-drift balance. From these distributions we build homozygous and heterozygous chromosomes and assess the behavior of the estimators of dominance. A very small number of deleterious mutations with antagonistic pleiotropy produces substantial increases on the estimate of the average degree of dominance of mutations affecting the fitness component under study. For example, estimates are increased three- to fivefold when 2% of segregating loci are over-dominant for fitness. In contrast, strengthening pleiotropy, where pleiotropic effects are assumed to be also deleterious, has little effect on the estimates of the average degree of dominance, supporting previous results. The antagonistic pleiotropy model considered, applied under mutational parameters described in the literature, produces patterns for the distribution of chromosomal viabilities, levels of genetic variance, and homozygous mutation load generally consistent with those observed empirically for viability in Drosophila melanogaster.     

Mutation accumulation and the effect of copia insertions in Drosophila melanogaster

Repeated efforts to estimate the genomic deleterious mutation rate per generation (U) in Drosophila melanogaster have yielded inconsistent estimates ranging from 0.01 to nearly 1. We carried out a mutation-accumulation experiment with a cryopreserved control population in hopes of resolving some of the uncertainties raised by these estimates. Mutation accumulation (MA) was carried out by brother sister mating of 150 sublines derived from two inbred lines. Fitness was measured under conditions chosen to mimic the ancestral laboratory environment of these genotypes. We monitored the insertions of a transposable element, copia, that proved to accumulate at the unusually high rate of 0.24 per genome per generation in one of our MA lines. Mutational variance in fitness increased at a rate consistent with previous studies, yielding a mutational coefficient of variation greater than 3%. The performance of the cryopreserved control relative to the MA lines was inconsistent, so estimates of mutation rate by the Bateman-Mukai method are suspect. Taken at face value, these data suggest a modest decline in fitness of about 0.3% per generation. The element number of copia was a significant predictor of fitness within generations; on average, insertions caused a 0.76% loss in fitness, although the confidence limits on this estimate are wide.     

Genome‐wide survey of artificial mutations induced by ethyl methanesulfonate and gamma rays in tomato

Genome‐wide mutations induced by ethyl methanesulfonate (EMS) and gamma irradiation in the tomato Micro‐Tom genome were identified by a whole‐genome shotgun sequencing analysis to estimate the spectrum and distribution of whole‐genome DNA mutations and the frequency of deleterious mutations. A total of ~370 Gb of paired‐end reads for four EMS‐induced mutants and three gamma‐ray‐irradiated lines as well as a wild‐type line were obtained by next‐generation sequencing technology. Using bioinformatics analyses, we identified 5920 induced single nucleotide variations and insertion/deletion (indel) mutations. The predominant mutations in the EMS mutants were C/G to T/A transitions, while in the gamma‐ray mutants, C/G to T/A transitions, A/T to T/A transversions, A/T to G/C transitions and deletion mutations were equally common. Biases in the base composition flanking mutations differed between the mutagenesis types. Regarding the effects of the mutations on gene function, >90% of the mutations were located in intergenic regions, and only 0.2% were deleterious. In addition, we detected 1 140 687 spontaneous single nucleotide polymorphisms and indel polymorphisms in wild‐type Micro‐Tom lines. We also found copy number variation, deletions and insertions of chromosomal segments in both the mutant and wild‐type lines. The results provide helpful information not only for mutation research, but also for mutant screening methodology with reverse‐genetic approaches.     

Estimation of Deleterious-Mutation Parameters in Natural Populations

The rate and average effects of spontaneous deleterious mutations are important determinants of the evolution of breeding systems and of the vulnerability of small populations to extinction. Nevertheless, few attempts have been made to estimate the properties of such mutations, and those studies that have been performed have been extremely labor intensive, relying on long-term, laboratory mutation-accumulation experiments. We present an alternative to the latter approach. For populations in which the genetic variance for fitness is a consequence of selection-mutation balance, the mean fitness and genetic variance of fitness in outbred and inbred generations can be expressed as simple functions of the genomic mutation rate, average homozygous effect and average dominance coefficient of new mutations. Using empirical estimates for the mean and genetic variance of fitness, these expressions can then be solved to obtain joint estimates of the deleterious-mutation parameters. We employ computer simulations to evaluate the degree of bias of the estimators and present some general recommendations on the application of the technique. Our procedures provide some hope for obtaining estimates of the properties of deleterious mutations from a wide phylogenetic range of species as well as a mechanism for testing the validity of alternative models for the maintenance of genetic variance for fitness.     

Spontaneous and Ethyl Methanesulfonate-Induced Mutations Controlling Viability in DROSOPHILA MELANOGASTER. III. Heterozygous Effect of Polygenic Mutations

Spontaneous and EMS-induced mutations were accumulated for several generations on the second chromosome of Drosophila melanogaster by keeping this chromosome heterozygous under conditions of minimal natural selection. This article reports studies of heterozygous effects of these mutants.--Both lethal and mildly deleterious mutants have a deleterious heterozygous effect. There was no discernible difference between heterozygotes in which all the mutants were on one chromosome and those where the mutants were distributed over both homologs; thus the coupling-repulsion effect of MUKAI and YAMAZAKI (1964, 1968) is not confirmed. The spontaneous polygenic mutants have a dominance of 0.4 to 0.5, and the same value is found at very low EMS doses. However, the value at higher EMS doses is only about half as high. Since the low doses have a large fraction of spontaneous mutants, the dominance of EMS mutants is less, in the range 0.1 to 0.3, but still larger than for lethals.     

Chromosomal Distribution of Transposable Elements in Drosophila Melanogaster: Test of the Ectopic Recombination Model for Maintenance of Insertion Site Number

Data of insertion site localization and site occupancy frequency of P, hobo, I, copia, mdg1, mdg3, 412, 297, and roo transposable elements (TEs) on the polytene chromosomes of Drosophila melanogaster were extracted from the literature. We show that TE insertion site number per chromosomal division was significantly correlated with the amount of DNA. The insertion site number weighted by DNA content was not correlated with recombination rate for all TEs except hobo, for which a positive correlation was detected. No global tendency emerged in the relationship between TE site occupancy frequency, weighted by DNA content, and recombination rate; a strong negative correlation was, however, found for the 3L arm. A possible dominant deleterious effect of chromosomal rearrangements due to recombination between TE insertions is thus not the main factor explaining the dynamics of TEs, since this hypothesis implies a negative relationship between recombination rate and both TE insertion site number and site occupancy frequency. The alternative hypothesis of selection against deleterious effects of insertional mutations is discussed.     

The rate of mutation and the homozygous and heterozygous mutational effects for competitive viability: a long-term experiment with Drosophila melanogaster

The effect of 250 generations of mutation accumulation (MA) on the second chromosome competitive viability of Drosophila melanogaster was analyzed both in homozygous and heterozygous conditions. We used full-sib MA lines, where selection hampers the accumulation of severely deleterious mutations but is ineffective against mildly deleterious ones. A large control population was simultaneously evaluated. Competitive viability scores, unaffected by the expression of mutations in heterozygosis, were obtained relative to a Cy/L(2) genotype. The rate of decline in mean DeltaM approximately 0.1% was small. However, that of increase in variance DeltaV approximately 0.08 x 10(-3) was similar to the values obtained in previous experiments when severely deleterious mutations were excluded. The corresponding estimates of the mutation rate lambda > or = 0.01 and the average effect of mutations E(s) < or = 0.08 are in good agreement with Bateman-Mukai and minimum distance estimates for noncompetitive viability obtained from the same MA lines after 105 generations. Thus, competitive and noncompetitive viability show similar mutational properties. The regression estimate of the degree of dominance for mild-to-moderate deleterious mutations was approximately 0.3, suggesting that the pertinent value for new unselected mutations should be somewhat smaller.     

EMS-induced polygenic mutation rates for nine quantitative characters in Drosophila melanogaster.

Polygenic mutations were induced by treating Drosophila melanogaster adult males with 2.5 mM EMS. The treated second chromosomes, along with untreated controls, were then made homozygous, and five life history, two behavioral, and two morphological traits were measured. EMS mutagenesis led to reduced performance for life history traits. Changes in means and increments in genetic variance were relatively much higher for life history than for morphological traits, implying large differences in mutational target size. Maximum likelihood was used to estimate mutation rates and parameters of distributions of mutation effects, but parameters were strongly confounded with one another. Several traits showed evidence of leptokurtic distributions of effects and mean effects smaller than a few percent of trait means. Distributions of effects for all traits were strongly asymmetrical, and most mutations were deleterious. Correlations between life history mutation effects were positive. Mutation parameters for one generation of spontaneous mutation were predicted by scaling parameter estimates from the EMS experiment, extrapolated to the whole genome. Predicted mutational coefficients of variation were in good agreement with published estimates. Predicted changes in means were up to 0.14% or 0.6% for life history traits, depending on the model of scaling assumed.     

Sequencing of pooled DNA samples (Pool-Seq) uncovers complex dynamics of transposable element insertions in Drosophila melanogaster

Transposable elements (TEs) are mobile genetic elements that parasitize genomes by semi-autonomously increasing their own copy number within the host genome. While TEs are important for genome evolution, appropriate methods for performing unbiased genome-wide surveys of TE variation in natural populations have been lacking. Here, we describe a novel and cost-effective approach for estimating population frequencies of TE insertions using paired-end Illumina reads from a pooled population sample. Importantly, the method treats insertions present in and absent from the reference genome identically, allowing unbiased TE population frequency estimates. We apply this method to data from a natural Drosophila melanogaster population from Portugal. Consistent with previous reports, we show that low recombining genomic regions harbor more TE insertions and maintain insertions at higher frequencies than do high recombining regions. We conservatively estimate that there are almost twice as many "novel" TE insertion sites as sites known from the reference sequence in our population sample (6,824 novel versus 3,639 reference sites, with on average a 31-fold coverage per insertion site). Different families of transposable elements show large differences in their insertion densities and population frequencies. Our analyses suggest that the history of TE activity significantly contributes to this pattern, with recently active families segregating at lower frequencies than those active in the more distant past. Finally, using our high-resolution TE abundance measurements, we identified 13 candidate positively selected TE insertions based on their high population frequencies and on low Tajima's D values in their neighborhoods.     

Estimates of the genomic mutation rate for detrimental alleles in Drosophila melanogaster

The net rate of mutation to deleterious but nonlethal alleles and the sizes of effects of these mutations are of great significance for many evolutionary questions. Here we describe three replicate experiments in which mutations have been accumulated on chromosome 3 of Drosophila melanogaster by means of single-male backcrosses of heterozygotes for a wild-type third chromosome. Egg-to-adult viability was assayed for nonlethal homozygous chromosomes. The rates of decline in mean and increase in variance (DM and DV, respectively) were estimated. Scaled up to the diploid whole genome, the mean DM for homozygous detrimental mutations over the three experiments was between 0.8 and 1.8%. The corresponding DV estimate was approximately 0.11%. Overall, the results suggest a lower bound estimate of at least 12% for the diploid per genome mutation rate for detrimentals. The upper bound estimates for the mean selection coefficient were between 2 and 10%, depending on the method used. Mutations with selection coefficients of at least a few percent must be the major contributors to the effects detected here and are likely to be caused mostly by transposable element insertions or indels.     

On the rate and linearity of viability declines in Drosophila mutation-accumulation experiments: genomic mutation rates and synergistic epistasis revisited

High rates of deleterious mutations could severely reduce the fitness of populations, even endangering their persistence; these effects would be mitigated if mutations synergize each others' effects. An experiment by Mukai in the 1960s gave evidence that in Drosophila melanogaster, viability-depressing mutations occur at the surprisingly high rate of around one per zygote and that the mutations interact synergistically. A later experiment by Ohnishi seemed to support the high mutation rate, but gave no evidence for synergistic epistasis. Both of these studies, however, were flawed by the lack of suitable controls for assessing viability declines of the mutation-accumulation (MA) lines. By comparing homozygous viability of the MA lines to simultaneously estimated heterozygous viability and using estimates of the dominance of mutations in the experiments, I estimate the viability declines relative to an appropriate control. This approach yields two unexpected conclusions. First, in Ohnishi's experiment as well as in Mukai's, MA lines showed faster-than-linear declines in viability, indicative of synergistic epistasis. Second, while Mukai's estimate of the genomic mutation rate is supported, that from Ohnishi's experiment is an order of magnitude lower. The different results of the experiments most likely resulted from differences in the starting genotypes; even within Mukai's experiment, a subset of MA lines, which I argue probably resulted from a contamination event, showed much slower viability declines than did the majority of lines. Because different genotypes may show very different mutational behavior, only studies using many founding genotypes can determine the average rate and distribution of effects of mutations relevant to natural populations.     

Rapid mutational declines of viability in Drosophila

High rates of mildly deleterious mutation could cause the extinction of small populations, reduce neutral genetic variation and provide an evolutionary advantage for sex. In the first attempts to estimate the rate of mildly deleterious mutation, Mukai and Ohnishi allowed spontaneous mutations to accumulate on D. melanogaster second chromosomes shielded from recombination and selection. Viability of the shielded chromosomes appeared to decline rapidly, implying a deleterious mutation rate on the order of one per zygote per generation. These results have been challenged, however; at issue is whether Mukai and Ohnishi may have confounded viability declines caused by mutation with declines resulting from environmental changes or other extraneous factors. Here, using a method not sensitive to non-mutational viability changes, I reanalyse the previous mutation-accumulation (MA) experiments, and report the results of a new one. I show that in each of four experiments, including Mukai's two experiments, viability declines due to mildly deleterious mutations were rapid. The results give no support for the view that Mukai overestimated the declines. Although there is substantial variation in estimates of genomic mutation rates from the experiments, this variation is probably due to some combination of sampling error, strain differences and differences in assay conditions, rather than to failure to distinguish mutational and non-mutational viability changes.     

Analysis of the estimators of the average coefficient of dominance of deleterious mutations

We investigate the sources of bias that affect the most commonly used methods of estimation of the average degree of dominance (h) of deleterious mutations, focusing on estimates from segregating populations. The main emphasis is on the effect of the finite size of the populations, but other sources of bias are also considered. Using diffusion approximations to the distribution of gene frequencies in finite populations as well as stochastic simulations, we assess the behavior of the estimators obtained from populations at mutation-selection-drift balance under different mutational scenarios and compare averages of h for newly arisen and segregating mutations. Because of genetic drift, the inferences concerning newly arisen mutations based on the mutation-selection balance theory can have substantial upward bias depending upon the distribution of h. In addition, estimates usually refer to h weighted by the homozygous deleterious effect in different ways, so that inferences are complicated when these two variables are negatively correlated. Due to both sources of bias, the widely used regression of heterozygous on homozygous means underestimates the arithmetic mean of h for segregating mutations, in contrast to their repeatedly assumed equality in the literature. We conclude that none of the estimators from segregating populations provides, under general conditions, a useful tool to ascertain the properties of the degree of dominance, either for segregating or for newly arisen deleterious mutations. Direct estimates of the average h from mutation-accumulation experiments are shown to suffer some bias caused by purging selection but, because they do not require assumptions on the causes maintaining segregating variation, they appear to give a more reliable average dominance for newly arisen mutations.     

Environment dependence of mutational parameters for viability in Drosophila melanogaster

The genomic rate of mildly deleterious mutations (U) figures prominently in much evolutionary and ecological theory. In Drosophila melanogaster, estimates of U have varied widely, from <0.1 to nearly 1 per zygote. The source of this variation is unknown, but could include differences in the conditions used for assaying fitness traits. We examined how assay conditions affect estimates of the rates and effects of viability-depressing mutations in two sets of lines with accumulated spontaneous mutations on the second chromosome. In each set, the among-line variance in egg-to-adult viability was significantly greater when viability was assayed using a high parental density than when it was assayed using a low density. In contrast, the proportional decline in viability due to new mutations did not differ between densities. Two other manipulations, lowering the temperature and adding ethanol to the medium, had no significant effects on either the mean decline or among-line variance. Cross-environment genetic correlations in viability were generally close to one, implying that most mutations reduced viability in all environments. Using data from the low-density, lower-bound estimates of U approached the classic, high values of Mukai and Ohnishi; at the high density, U estimates were similar to recently reported low values. The difference in estimated mutation rates, taken at face value, would imply that many mutations affected fitness at low density but not at high density, but this is shown to be incompatible with the observed high cross-environment correlations. Possible reasons for this discrepancy are discussed. Regardless of the interpretation, the results show that assay conditions can have a large effect on estimates of mutational parameters for fitness traits.     

Spontaneous and Ethyl Methanesulfonate-Induced Mutations Controlling Viability in DROSOPHILA MELANOGASTER. II. Homozygous Effect of Polygenic Mutations

Polygenic mutations affecting viability were accumulated on the second chromosome of Drosophila melanogaster by treating flies with EMS in successive generations. The treated chromosomes were later made homozygous and tested for their effects on viability by comparison of the frequency of such homozygotes with that of other genotypes in the same culture. The treated wild-type chromosomes were kept heterozygous in Pm/+ males by mating individual males in successive generations to Cy/Pm females. The number of generations of accumulation was 1 to 30 generations, depending on the concentration of EMS. A similar experiment for spontaneous polygenic mutations was also conducted by accumulating mutations for 40 generations. The lower limit of the spontaneous mutation rate of viability polygenes is estimated to be 0.06 per second chromosome per generation, which is about 12 times as high as the spontaneous recessive lethal mutation rate, 0.005. EMS-induced polygenic mutations increase linearly with the number of treated generations and with the concentration of EMS. The minimum mutation rate of viability polygenes is about 0.017 per 10(-4)m, which is only slightly larger than the lethal rate of 0.013 per 10(-4) m. The maximum estimate of the viability reduction of a single mutant is about 6 to 10 percent of the normal viability. The data are consistent with a constant average effect per mutant at all concentrations, but this is about three times as high as that for spontaneous mutants. It is obvious that one can obtain only a lower limit for the mutation rate, since some mutants may have effects so near to zero that they cannot be detected. The possibility of measuring something other than the lower limit is discussed. The ratio of the load due to detrimental mutants to that caused by lethals, the D/L ratio, is about 0.2 to 0.3 for EMS-induced mutants, as compared to about 0.5 for spontaneous mutants. This is to be expected if EMS treatment produces a large fraction of small deletions and other chromosome rearrangements which are more likely to be lethal.     

Experimental estimate of the abundance and effects of nearly neutral mutations in the RNA virus phi 6

Although the frequency and effects of neutral and nearly neutral mutations are critical to evolutionary patterns and processes governed by genetic drift, the small effects of such mutations make them difficult to study empirically. Here we present the results of a mutation-accumulation experiment designed to assess the frequencies of deleterious mutations with undetectable effects. We promoted the accumulation of spontaneous mutations by subjecting independent lineages of the RNA virus 6 to repeated population bottlenecks of a single individual. We measured fitness following every bottleneck to obtain a complete picture of the timing and effects of the accumulated mutations with detectable effects and sequenced complete genomes to determine the number of mutations that were undetected by the fitness assays. To estimate the effects of the undetected mutations, we implemented a likelihood model developed for quantitative trait locus (QTL) data (Otto and Jones 2000) to estimate the number and effects of the undetected mutations from the measured number and effects of the detected mutations. Using this method we estimated a deleterious mutation rate of U = 0.03 and a gamma effects distribution with mean s=0.093 and coefficient of variation = 0.204. Although our estimates of U and s fall within the range of recent mutation rate and effect estimates in eukaryotes, the fraction of mutations with detectable effects on laboratory fitness (39%) appears to be far higher in 6 than in eukaryotes.     

Accumulation of transposable elements in the genome of Drosophila melanogaster is associated with a decrease in fitness

Replicates of the two isogenic laboratory strains of Drosophila melanogaster, 2b and Harwich, contain different average transposable element (TE) copy numbers in the same genetic background. These lines were used to analyze the correlation between TE copy number and fitness. Assuming a weak deleterious effect of each TE insertion, a decrease in fitness is expected with an increase in genomic TE copy number. Higher rates of ectopic exchanges and, consequently, chromosomal rearrangements resulting in early embryonic death are also predicted from an increase in TE copy number. Therefore egg hatchability is expected to decrease as the genomic TE copy number increases. In 2b, where replicate lines have diverged up by 90 TE copies per haploid genome, a negative correlation between the number of TE insertions and both fitness and egg hatchability were found. Neither correlation was significant for the Harwich replicates, which have only diverged by 30 TE copies. The average deleterious effect of a TE insertion on fitness and its components was estimated as 0.004. Both homozygous and heterozygous TE insertions were shown to have deleterious effects on fitness and its components.