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Epistasis is an important feature of the genetic architecture of quantitative traits, but the dynamics of epistatic interactions in natural populations and the relationship between epistasis and pleiotropy remain poorly understood. Here, we studied the effects of epistatic modifiers that segregate in a wild-derived Drosophila melanogaster population on the mutational effects of P-element insertions in Semaphorin-5C (Sema-5c) and Calreticulin (Crc), pleiotropic genes that affect olfactory behaviour and startle behaviour and, in the case of Crc, sleep phenotypes. We introduced Canton-S B (CSB) third chromosomes with or without a P-element insertion at the Crc or Sema-5c locus in multiple wild-derived inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and assessed the effects of epistasis on the olfactory response to benzaldehyde and, for Crc, also on sleep. In each case, we found substantial epistasis and significant variation in the magnitude of epistasis. The predominant direction of epistatic effects was to suppress the mutant phenotype. These observations support a previous study on startle behaviour using the same D. melanogaster chromosome substitution lines, which concluded that suppressing epistasis may buffer the effects of new mutations. However, epistatic effects are not correlated among the different phenotypes. Thus, suppressing epistasis appears to be a pervasive general feature of natural populations to protect against the effects of new mutations, but different epistatic interactions modulate different phenotypes affected by mutations at the same pleiotropic gene.
Predicting functional gene annotations remains a significant challenge, even in well-annotated genomes such as yeast and Drosophila. One promising, high-throughput method for gene annotation is to use correlated gene expression patterns to annotate target genes based on the known function of focal genes. The Drosophila melanogaster transcriptome varies genetically among wild-derived inbred lines, with strong genetic correlations among the transcripts. Here, we leveraged the genetic correlations in gene expression among known seminal fluid protein (SFP) genes and the rest of the genetically varying transcriptome to identify 176 novel candidate SFPs (cSFPs). We independently validated the correlation in gene expression between seven of the cSFPs and a known SFP gene, as well as expression in male reproductive tissues. We argue that this method can be extended to other systems for which information on genetic variation in gene expression is available.
Drosophila melanogaster, like other organisms, move and orient themselves in response to the earth's gravitational force. The ability to sense and respond to gravity is essential for an organism to navigate and thrive in its environment. The genes underlying this behaviour in Drosophila remain elusive. Using 88 recombinant inbred lines, we have identified four quantitative trait loci (QTLs) that contribute to adult gravitaxis (geotaxis) behaviour in Drosophila. Candidate genes of interest were selected from the QTLs of highest significance based on their function in chordotonal organ formation. Quantitative complementation tests with these candidate genes revealed a role for skittles in adult gravitaxis behaviour in D. melanogaster.
A comprehensive understanding of the genetic basis of phenotypic adaptation in nature requires the identification of the functional allelic variation underlying adaptive phenotypes. The manner in which organisms respond to temperature extremes is an adaptation in many species. In the current study, we investigate the role of molecular variation in senescence marker protein-30 (Smp-30) on natural phenotypic variation in cold tolerance in Drosophila melanogaster. Smp-30 encodes a product that is thought to be involved in the regulation of Ca2+ ion homeostasis and has been shown previously to be differentially expressed in response to cold stress. Thus, we sought to assess whether molecular variation in Smp-30 was associated with natural phenotypic variation in cold tolerance in a panel of naturally derived inbred lines from a population in Raleigh, North Carolina. We identified four non-coding polymorphisms that were strongly associated with natural phenotypic variation in cold tolerance. Interestingly, two polymorphisms that were in close proximity to one another (2 bp apart) exhibited opposite phenotypic effects. Consistent with the maintenance of a pair of antagonistically acting polymorphisms, tests of molecular evolution identified a significant excess of maintained variation in this region, suggesting balancing selection is acting to maintain this variation. These results suggest that multiple mutations in non-coding regions can have significant effects on phenotypic variation in adaptive traits within natural populations, and that balancing selection can maintain polymorphisms with opposite effects on phenotypic variation.
Epistasis is an important feature of the genetic architecture of quantitative traits. Previously, we showed that startle-induced locomotor behaviour of Drosophila melanogaster, a critical survival trait, is highly polygenic and exhibits epistasis. Here, we characterize epistatic interactions among homozygous P-element mutations affecting startle-induced locomotion in the Canton-S isogenic background and in 21 wild-derived inbred genetic backgrounds. We find pervasive epistasis for pairwise combinations of homozygous P-element insertional mutations as well as for mutations in wild-derived backgrounds. In all cases, the direction of the epistatic effects is to suppress the mutant phenotypes. The magnitude of the epistatic interactions in wild-derived backgrounds is highly correlated with the magnitude of the main effects of mutations, leading to phenotypic robustness of the startle response in the face of deleterious mutations. There is variation in the magnitude of epistasis among the wild-derived genetic backgrounds, indicating evolutionary potential for enhancing or suppressing effects of single mutations. These results provide a partial glimpse of the complex genetic network underlying the genetic architecture of startle behaviour and provide empirical support for the hypothesis that suppressing epistasis is the mechanism underlying genetic canalization of traits under strong stabilizing selection. Widespread suppressing epistasis will lead to underestimates of the main effects of quantitative trait loci (QTLs) in mapping experiments when not explicitly accounted for. In addition, suppressing epistasis could lead to underestimates of mutational variation for quantitative traits and overestimates of the strength of stabilizing selection, which has implications for maintenance of variation of complex traits by mutation–selection balance.
P-element mutagenesis was used to contaminate M-strain second chromosomes with P elements. The effect of P-element transposition on abdominal and sternopleural bristle scores and on female productivity was deduced by comparing the distributions of these quantitative traits among the contaminated second-chromosome lines with a control population of M-strain second-chromosome lines free of P elements. Estimates of P-element-induced mutational variance, Vm, for these characters are very high, and mutational ‘heritabilities’ (Vm/Ve, the ratio of mutational variance to environmental variance) are of the same order as heritabilities of these traits from natural populations. P-element-induced mutational variance of abdominal bristle score is roughly two orders of magnitude greater than spontaneous and X-ray-induced Vm/Ve for this trait.
Rates of transposition and excision of the Drosophila melanogaster retrotransposon elements mdg3, 297, Doc, roo and copia were estimated directly, by in situ hybridization analysis of their cytological insertion sites in 31 replicates of a highly inbred line that had accumulated spontaneous mutations for approximately 160generations. Estimated transposition rates of Doc, roo and copia were, respectively, 4·2 × 10−5, 3·1 × 10−3 and 1·3 − 10−3; no transpositions of 297 nor mdg3 were observed. Rates of transposition of copia varied significantly among sublines. Excisions were only observed for roo elements, at a rate of 9·0 × 10−6 per element per generation. Copy number averaged over these element families increased 5·9 %; therefore, in these lines the magnitude of the forces opposing transposable element multiplication were weaker than transposition rates.
P element mutagenesis was used to contaminate M strain second chromosomes with P elements. The contaminated lines were compared to uncontaminated control lines for homozygous and heterozygous fitness and its components. Mean homozygous fitness, viability and fertility of chromosome lines contaminated with P elements is decreased relative to the uncontaminated control lines by, respectively, 55, 28 and 40%. Variance among contaminated homozygous lines of total fitness increases by a factor of 1·5, variance of viability by a factor of 5·9, and variance of fertility by a factor of 1·9, compared to variance of these traits among the population of uncontaminated homozygous chromosomes. Estimates of P-element-induced mutational variance among second chromosome lines for homozygous fitness, viability and fertility are, respectively, 2 × 10−2, 5 × 10−2 and 2 × 10−2. This magnitude of mutational effect is equivalent, in terms of incidence of induced recessive lethal chromosomes and D:L ratio, to a dose of approximately 1·0–2·5 × 10−3 m EMS. The distributions of fitness traits among M-derived second chromosome homozygous lines contaminated with P elements are remarkably similar in many regards to distributions of fitness and viability of chromosomal homozygotes derived from natural Drosophila populations. It is possible that a proportion of the fitness variation previously observed (reviewed by Simmons & Crow, 1977) following homozygosis of wild chromosomes was not present in the natural populations, but was generated by P-element transposition during the chromosome extraction procedure. P-element-induced fitness mutations appear to be completely recessive. Implications for models of evolution of transposable elements are discussed.
When males from a strain of Drosophila melanogaster which have multiple copies of the P family of transposable elements integrated into their genome (Bingham, Kid well & Rubin, 1982) (‘P’ strains) are crossed to females of strains lacking those elements (‘M’ strains), the F1 progeny manifest a number of aberrant traits known collectively as hybrid dysgenesis (Kidwell, Kidwell & Sved, 1977). Progeny of the reciprocal cross (P♀ × ♂M) are normal. The dysgenic syndrome is characterized by a high degree of genomic instability caused by enhanced transposition of P elements. Since mutations at visible loci and chromosomal rearrangements are frequently observed in dysgenic hybrids, it was hypothesized that this phenomenon could be used to investigate the effects of transposition on quantitative characters; therefore, the progeny of dysgenic and non-dysgenic crosses were artificially selected for high and low abdominal bristle number. The response to selection, phenotypic variation, and realized heritability were all increased in the dysgenic lines, relative to the non-dysgenic control. It is postulated that these features are the result of P element-induced mutations of loci affecting bristle number, and that future work should lead to the identification of these loci, as well as elucidating the role of transposition in population differentiation for metric traits
The male mating ability and male fertility of 40 third chromosome homozygote lines has been measured. There was significant between-line differentiation for both characters, and comparison with a heterozygous stock indicated inbreeding depression and hence dominance variation for them. The characters showed significant positive correlation both with each other and with other fitness components and total fitness, as measured by Mackay (1985). This pattern of large positive correlations between fitness components is not expected to occur in outbred populations.
Forty-one third chromosomes extracted from a natural population of Drosophila melanogaster were assessed for net fitness and for the quantitative characters viability, net fertility, female productivity, male weight, abdominal bristle number, and sternopleural bristle number. Net homozygous and heterozygous fitness of the third chromosomes was estimated by competition against a marked balancer third chromosome. Average fitness of the homozygous lines relative to wild-type heterozygotes was 0·13, indicating substantial inbreeding depression for net fitness. All significant correlations of quantitative characters with fitness and with each other were high and positive. Homozygous fitness is strongly correlated with net fertility, viability, and female productivity, moderately associated with male weight, and not significantly associated with bristle traits. The combination of metric traits which best predicts homozygous fitness is the simple multiple of viability and female productivity. Heterozygous fitness is not correlated with homozygous fitness; furthermore, the relative contribution of metric traits to fitness in a heterozygous population is likely to be different from that deduced from homozygous lines. These observations are consistent with a model of genetic variation for fitness in natural populations caused by segregation of rare deleterious recessive alleles.
X chromosomes derived from crosses of inbred P and M Drosophila melanogaster strains that had extreme effects on abdominal and/or sternopleural bristle number in males, were further analyzed to determine their effects in females and to map the loci at which the mutations occurred. Seven lines that had on average 3.9 fewer sternopleural bristles than wildtype in males had average homozygous sternopleural bristle effects of −2·2. The bristle effects were partially recessive, with an average degree of dominance of −0·60. Physical mapping of the sternopleural bristle effects of these lines placed them all at approximately 24·7 cM. These mutations are apparently allelic on the basis of a complementation test, and deficiency mapping indicates they occur within chromosomal bands 8A4; 8C6. In situ hybridization analysis of the sites of P element insertions of these lines suggests that mutations probably resulted from excision of P elements at 8C on the original inbred P strain chromosome. Two additional lines, NDC(19) and DP(146), had reduced numbers of sternopleural and abdominal bristles. NDC(19) males had 9·7 fewer abdominal and 8·6 fewer sternopleural bristles than wildtype. The corresponding homozygous abdominal and sternopleural bristle number effects were −5·8 and −3·8, respectively; with the abdominal bristle effect completely recessive and the sternopleural bristle effect nearly additive. DP(146) males had 6·2 fewer abdominal and 4·1 fewer sternopleural bristles than wildtype, with homozygous abdominal bristle effects of −4·3 and sternopleural bristle effects of −2·0. Abdominal bristle effects of this line were partially recessive whereas the sternopleural bristle effects were additive. Physical mapping showed effects on both bristle traits segregated jointly in these two lines, with the NDC(19) mutation closely linked to y and the DP(146) mutation 0·17 cM from it. Complementation tests and deficiency mapping also indicate the mutations in lines NDC(19) and DP(146) are at closely linked but separate loci within chromosomal bands 1B2; 1B4–6 and 1B4–6; 1B10 respectively, with some epistatic effects. In situ hybridization analysis of sites of P element insertion suggest that the NDC(19) mutation, which may be a scute allele, was probably caused by a P element insertion in the IB region; the DP(146) mutation is also associated with an insertion at IB.
In order to assess the relationship between genetic and environmental variability, a large natural population of Drosophila melanogaster was replicated as eight subpopulations, which were subjected to four different patterns of environmental variation. The environmental variable imposed was presence of 15% ethanol in the culture medium. Experimental treatments of the populations were intended to simulate constant environmental conditions, spatial heterogeneity in the environment, and two patterns of temporal environmental variation with different periodicity (long- and short-term temporal variation). Additive genetic and phenotypic variation in sternopleural and abdominal chaeta number, and body weight, were estimated in two successive years, and measurements were taken of the genotype–environment correlation of body weight and sternopleural bristle score with medium type.
Additive genetic variance of sternopleural chaeta number and of body weight was significantly greater in the three populations experiencing environmental heterogeneity than in the control population, but additive genetic variance of abdominal bristle score was not clearly affected by exposing populations to varying environments. Temporal environmental variation was equally, if not more, efficient in promoting the maintenance of genetic variation than spatial heterogeneity, but the cycle length of the temporal variation was of no consequence. Specific genotype–environment interactions were not present, therefore adaptation to heterogeneous environments is by selection of heterozygosity per se, rather than by differential survival of genotypes in the alternate niches.
In Drosophila melanogaster, the P−M and I−R systems of hybrid dysgenesis are associated with high rates of transposition of P and I elements, respectively, in the germlines of dysgenic hybrids formed by crossing females of strains without active elements to males of strains containing them. Transposition rates are not markedly accelerated in the reciprocal, nondysgenic hybrids. Previous attempts to evaluate the extent to which hybrid dysgenesis-mediated P transposition contributes to mutational variance for quantitative characters by comparing the responses to selection of P−M dysgenic and nondysgenic hybrids have given variable results. This experimental design has been extended to include an additional quantitative trait and the I−R hybrid dysgenesis system. The selection responses of lines founded from both dysgenic and nondysgenic crosses showed features that would be expected from the increase in frequency of initially rare genes with major effects on the selected traits. These results differ from those of previous experiments which showed additional selection response only in lines started from dysgenic crosses, and can be explained by the occasional occurrence of large effect transposable element-induced polygenic mutations in both dysgenic and nondysgenic selection lines. High rates of transposition in populations founded from nondysgenic crosses may account for the apparently contradictory results of the earlier selection experiments, and an explanation is proposed for its occurrence.