Linkage disequilibrium mapping of molecular polymorphisms at the scabrous locus associated with naturally occurring variation in bristle number in Drosophila melanogaster

We evaluated the hypothesis that the Drosophila melanogaster second chromosome gene scabrous (sca), a candidate sensory bristle number quantitative trait locus (QTL), contributes to naturally occurring variation in bristle number. Variation in abdominal and sternopleural bristle number was quantified for wild-derived sca alleles in seven genetic backgrounds: as homozygous second chromosomes (C2) in an isogenic background, homozygous lines in which approximately 20 cM including the sca locus had been introgressed into the isogenic background (sca BC), as C2 and sca BC heterozygotes and hemizygotes against a P element insertional sca allele and a P-induced sca deficiency in the same isogenic background, and as sca BC heterozygotes against the wild-type sca allele of isogenic strain. Molecular restriction map variation was determined for a 45 kb region including the sca locus, and single-stranded conformational polymorphism (SSCP) was examined for the third intron and parts of the third and fourth exons. Associations between each of the 27 molecular polymorphisms and bristle number were evaluated within each genotype and on the first principal component score determined from all seven genotypes, separately for each sex and bristle trait. Permutation tests were used to assess the empirical significance thresholds, accounting for multiple, correlated tests, and correlated markers. Three sites in regulatory regions were associated with female-specific variation in abdominal bristle number, one of which was an SSCP site in the region of the gene associated with regulation of sca in embryonic abdominal segments.     

Effects of Single P-Element Insertions on Bristle Number and Viability in Drosophila Melanogaster

Single P-element mutagenesis was used to construct 1094 lines with P[lArB] inserts on all three major chromosomes in an isogenic background previously free of P elements. The effects of insertions on bristle number and on viability were assessed by comparison to 392 control lines. The variance and effects of P-element inserts on bristle number and viability were larger than those inferred from spontaneous mutations. The distributions of effects on bristle number were symmetrical and highly leptokurtic, such that a few inserts with large effects caused most of the increase in variance. The distribution of effects on viability were negatively skewed and platykurtic. On average, the effects of P-element insertions on bristle number were partly recessive and on viability were completely recessive. P-element inserts with large effects on bristle number tended to have reduced viability, but the correlation between the absolute value of the effects on bristle number and on viability was not strong. Fifty P-element inserts tagging quantitative trait loci (QTLs) with large effects on bristle number were mapped cytogenetically. Two P-element-induced scabrous alleles and five extramacrochaetae alleles were generated. Single P-element mutagenesis is a powerful method for identifying QTLs at the level of genetic locus.     

The genetic architecture of selection response. Inferences from fine-scale mapping of bristle number quantitative trait loci in Drosophila melanogaster.

Quantitative trait loci (QTL) affecting responses and correlated responses to selection for abdominal and sternopleural bristle number have been mapped with high resolution to the X and third chromosomes. Advanced intercross recombinant isogenic chromosomes were constructed from high and low selection lines in an unselected inbred background, and QTL were detected using composite interval mapping and high density transposable element marker maps. We mapped a total of 26 bristle number QTL with large effects, which were in or immediately adjacent to intervals previously inferred to contain bristle number QTL on these chromosomes. The QTL contributing to response to selection for high bristle number were not the same as those contributing to response to selection for low bristle number, suggesting that distributions of allelic effects per locus may be asymmetrical. Correlated responses were more often attributable to loose linkage than pleiotropy or close linkage. Bristle number QTL mapping to the same locations have been inferred in studies with different parental strains. Of the 26 QTL, 20 mapped to locations consistent with candidate genes affecting peripheral nervous system development and/or bristle number. This facilitates determining the molecular basis of quantitative variation and allele frequencies by associating molecular variation at the candidate genes with phenotypic variation in bristle number in samples of alleles from nature.     

Association of single-nucleotide polymorphisms at the Delta locus with genotype by environment interaction for sensory bristle number in drosophila Melanogaster

The nature of forces maintaining variation for quantitative traits can only be assessed at the level of individual genes affecting variation in the traits. Identification of single-nucleotide polymorphisms (SNPs) associated with variation in Drosophila sensory bristle number at the Delta (Dl) locus provides us with the opportunity to test a model for the maintenance of variation in bristle number by genotype by environment interaction (GEI). Under this model, genetic variation is maintained at a locus under stabilizing selection if phenotypic values of heterozygotes are more stable than homozygotes across a range of environments, and the mean allelic effect is much smaller than the standard deviation of allelic effects across environments. Homozygotes and heterozygotes for two SNPs at Dl, one affecting sternopleural and the other abdominal bristle number, were reared in five different environments. There was significant GEI for both bristle traits. Neither condition of the model was satisfied for Dl SNPs exhibiting GEI for sternopleural bristle number. Heterozygotes for the abdominal bristle number SNPs were indeed the most stable genotype for two of the three environment pairs exhibiting GEI, but the mean genotypic effect was greater than the standard deviation of effects across environments. Therefore, this mechanism of GEI seems unlikely to be responsible for maintaining the common bristle number polymorphisms at Dl.     

Polygenic Mutation in Drosophila Melanogaster: Genetic Interactions between Selection Lines and Candidate Quantitative Trait Loci

We have investigated genetic interactions between spontaneous mutations affecting abdominal and sternopleural bristle number that have accumulated in 12 long-term selection lines derived from an inbred strain, and mutations at 14 candidate bristle number quantitative trait loci. The quantitative test for complementation was to cross the selection lines to an inbred wild-type strain (the control cross) and to a derivative of the control strain into which the mutant allele at the candidate locus to be tested was substituted (the tester strain). Genetic interactions between spontaneous mutations affecting bristle number and the candidate locus mutations were common, and in several cases the interaction effects were different in males and females. Analyses of variance of the (tester - control) differences among and within groups of replicate lines selected in the same direction for the same trait showed significant group effects for several candidate loci. Genetically, the interactions could be caused by allelism of, and/or epistasis between, spontaneous mutations in the selection lines and the candidate locus mutations. It is possible that much of the response to selection was from new mutations at candidate bristle number quantitative trait loci, and that for some of these loci, mutation rates were high.     

The genetic architecture of Drosophila sensory bristle number

We have mapped quantitative trait loci (QTL) for Drosophila mechanosensory bristle number in six recombinant isogenic line (RIL) mapping populations, each of which was derived from an isogenic chromosome extracted from a line selected for high or low, sternopleural or abdominal bristle number and an isogenic wild-type chromosome. All RILs were evaluated as male and female F(1) progeny of crosses to both the selected and the wild-type parental chromosomes at three developmental temperatures (18 degrees, 25 degrees, and 28 degrees ). QTL for bristle number were mapped separately for each chromosome, trait, and environment by linkage to roo transposable element marker loci, using composite interval mapping. A total of 53 QTL were detected, of which 33 affected sternopleural bristle number, 31 affected abdominal bristle number, and 11 affected both traits. The effects of most QTL were conditional on sex (27%), temperature (14%), or both sex and temperature (30%). Epistatic interactions between QTL were also common. While many QTL mapped to the same location as candidate bristle development loci, several QTL regions did not encompass obvious candidate genes. These features are germane to evolutionary models for the maintenance of genetic variation for quantitative traits, but complicate efforts to understand the molecular genetic basis of variation for complex traits.     

Drosophila bristles and the nature of quantitative genetic variation

Numbers of Drosophila sensory bristles present an ideal model system to elucidate the genetic basis of variation for quantitative traits. Here, we review recent evidence that the genetic architecture of variation for bristle numbers is surprisingly complex. A substantial fraction of the Drosophila genome affects bristle number, indicating pervasive pleiotropy of genes that affect quantitative traits. Further, a large number of loci, often with sex- and environment-specific effects that are also conditional on background genotype, affect natural variation in bristle number. Despite this complexity, an understanding of the molecular basis of natural variation in bristle number is emerging from linkage disequilibrium mapping studies of individual candidate genes that affect the development of sensory bristles. We show that there is naturally segregating genetic variance for environmental plasticity of abdominal and sternopleural bristle number. For abdominal bristle number this variance can be attributed in part to an abnormal abdomen-like phenotype that resembles the phenotype of mutants defective in catecholamine biosynthesis. Dopa decarboxylase (Ddc) encodes the enzyme that catalyses the final step in the synthesis of dopamine, a major Drosophila catecholamine and neurotransmitter. We found that molecular polymorphisms at Ddc are indeed associated with variation in environmental plasticity of abdominal bristle number.     

Polygenic Mutation in Drosophila Melanogaster: The Causal Relationship of Bristle Number to Fitness

The association between sternopleural and abdominal bristle number and fitness in Drosophila melanogaster was determined for sublines of an initially highly inbred strain that were maintained by divergent artificial selection for 150 generations or by random mating for 180 generations. Replicate selection lines had more extreme bristle numbers than those that were maintained without artificial selection at the same census size for approximately the same number of generations. The average fitness, estimated by a single generation of competition against a compound autosome strain, was 0.17 for lines selected for high and low abdominal bristle numbers and 0.19 for lines selected for high and low sternopleural bristle number. The average fitness of unselected lines, 0.46, was significantly higher than that of the selection lines. The fitnesses and the relationships of bristle number to fitness in progeny of all possible crosses of high X high (H X H), high X low (H X L) and low X low (L X L) selection lines were examined to determine whether the observed intermediate optima were caused by direct stabilizing selection on bristle number or by apparent stabilizing selection mediated through deleterious pleiotropic fitness effects of mutations affecting bristle number. Although bristle number was nearly additive for progeny of H X H, H X L and L X L crosses among sternopleural bristle selection lines, their mean fitnesses were not significantly different from each other, or from the mean fitness of the unselected lines, suggesting partly or completely recessive pleiotropic fitness effects cause apparent stabilizing selection. The average fitness of the progeny of H X H abdominal bristle selection lines was not significantly different from the fitness of unselected lines, but the mean fitness of the progeny of L X L crosses was not significantly different from that of the pure low lines. This is consistent with direct selection against low but not high abdominal bristle number, but the interpretation is confounded by variation in average degree of dominance for fitness (on average recessive in the high abdominal bristle selection lines and additive in the low abdominal bristle selection lines). Neither direct stabilizing selection nor pleiotropy, therefore, can account for all the observations.     

Quantitative variation of four morphological traits in Drosophila melanogaster under larval crowding

Effects of three different larval densities (low, intermediate and high) on phenotypic and genetic variation of four morphological traits (thorax and wing length, sternopleural and abdominal bristle number) were studied in Drosophila melanogaster using the isofemale line technique. Phenotypic variation was found to increase at high larval density in all traits examined. Environmental variance for three traits (exception was sternopleural bristle number) and fluctuating asymmetry for both bilateral traits were also increased under high density conditions. For estimates of genetic variability (among isofemale lines variance, heritability and evolvability), no statistically significant differences among density regimes were detected. However, the trends in changes of these estimates across densities indicated a possibility for enhanced genetic variation under larval crowding for all traits except abdominal bristle number. For the latter trait, genetic variation seemed not to be dependent on density regime. Generally, two metric traits (thorax and wing length) were more affected by larval crowding than two meristic ones (sternopleural and abdominal bristle number). The Results are in complete agreement with those previously obtained for D. melanogaster using extreme temperatures as stress-factors.     

High-resolution mapping of quantitative trait loci for sternopleural bristle number in Drosophila melanogaster.

We have mapped quantitative trait loci (QTL) harboring naturally occurring allelic variation for Drosophila bristle number. Lines with high (H) and low (L) sternopleural bristle number were derived by artificial selection from a large base population. Isogenic H and L sublines were extracted from the selection lines, and populations of X and third chromosome H/L recombinant isogenic lines were constructed in the homozygous low line background. The polymorphic cytological locations of roo transposable elements provided a dense molecular marker map with an average intermarker distance of 4.5 cM. Two X chromosome and six chromosome 3 QTL affecting response to selection for sternopleural bristle number and three X chromosome and three chromosome 3 QTL affecting correlated response in abdominal bristle number were detected using a composite interval mapping method. The average effects of bristle number QTL were moderately large, and some had sex-specific effects. Epistasis between QTL affecting sternopleural bristle number was common, and interaction effects were large. Many of the intervals containing bristle number QTL coincided with those mapped in previous studies. However, resolution of bristle number QTL to the level of genetic loci is not trivial, because the genomic regions containing bristle number QTL often did not contain obvious candidate loci, and results of quantitative complementation tests to mutations at candidate loci affecting adult bristle number were ambiguous.     

Both naturally occurring insertions of transposable elements and intermediate frequency polymorphisms at the achaete-scute complex are associated with variation in bristle number in Drosophila melanogaster

A restriction enzyme survey of a 110-kb region including the achaete scute complex (ASC) examined 14 polymorphic molecular markers in a sample of 56 naturally occurring chromosomes. Large insertions as a class were associated with a reduction in both sternopleural and abdominal bristle number, supporting deleterious mutation-selection equilibrium models for the maintenance of quantitative genetic variation. Two polymorphic sites were independently associated with variation in bristle number measured in two genetic backgrounds as assessed by a permutation test. A 6-bp deletion near sc alpha is associated with sternopleural bristle number variation in both sexes and a 3.4-kb insertion between sc beta and sc gamma is associated with abdominal bristle number variation in females. Under an additive genetic model, the small deletion polymorphism near sc alpha accounts for 25% of the total X chromosome genetic variation in sternopleural bristle number, and the 3.4 kb insertion accounts for 22% of the total X chromosome variation in female abdominal bristle number. The observation of common polymorphisms associated with variation in bristle number is more parsimoniously explained by models that incorporate balancing selection or assume variants affecting bristle number are neutral, than mutation-selection equilibrium models.     

Studies on the Scutellar Bristles of DROSOPHILA MELANOGASTER. II. Long-Term Selection for High Bristle Number in the Oregon Rc Strain and Correlated Responses in Abdominal Chaetae

Results are presented of 135 generations of selection for high scutellar bristle number in two lines M and M3 derived from the same original mating of one female with 5 bristles by one male with 4 bristles, the latter being the wild-type canalised phenotype. Results are also given of two relaxed lines per line and of a reselection line M2 derived from the first relaxed line of line M which had regressed almost to base population level. The effect of introducing the sc(1) allele into the M and M3 selected backgrounds was studied at generations 39-44. At the end of selection the effect of an extra dose of sc(+) was also studied in males of all selected backgrounds. The correlated responses in abdominal bristles were followed in all lines.-Considering their common origin, the selection lines differed markedly in pattern of scutellar response and in most other aspects observed, namely correlated responses in abdominals and p.c. scutellars, sex differences, and behaviour on relaxation. Selection limits for scutellar bristles in lines M and M2 were equal to or greater than the most extreme reported in the literature.-The probit span of the canalised 4 bristle class decreased in each selection line as the mean scutellar bristle number increased, and increased again in the relaxed lines as the mean bristle number decreased. In the context of an hypothesis that canalisation at 4 bristle is due to regulation of the scute locus, this result is now interpreted as being due mainly to selection for poor regulators of sc(+), in contrast to a previous interpretation that only the minor gene background was altered by selection, the canalisation (regulation) genotype not being affected.-Introducing the sc(1) allele into the selected backgrounds M and M3 showed a reduced effect on sc(1) flies compared with sc(+) flies, and an interaction of sc(1) and sc(+) with selected background. sc(1) flies had about the same number of bristles in both backgrounds though the mean of sc(+) flies in line M was about 3sigma higher than in line M3. Dominance of sc(+) to sc(1) was reduced slightly in M3. However, the effect of an extra dose of sc(+) at the end of selection was about the same as in unselected in all lines, so the first or dominance level of regulation of the scute locus was not significantly affected by selection, though the second or canalisation level of regulation was.-A large positive correlated response in abdominal bristles occurred in all lines. The response in line M was about twice that in M2 and M3 and was in fact as large as can be obtained from direct selection on abdominals. In line M some genes may have been selected with a proportionately greater effect on abdominals than on scutellars. This is supported by the further observation in line M that the abdominal scores of flies with particular scutellar bristles scores increased as the scutellar mean increased. An attempt was made to apply to these results Rendel's (1962) model of competition between scutellars and abdominals for common bristle-making resources. This could not be done satisfactorily mainly because the assumptions in the model about the similarity of effects in scute and wild-type flies were not met in the present material.     

Joint estimates of quantitative trait locus effect and frequency using synthetic recombinant populations of Drosophila melanogaster

We develop and implement a strategy to map QTL in two synthetic populations of Drosophila melanogaster each initiated with eight inbred founder strains. These recombinant populations allow simultaneous estimates of QTL location, effect, and frequency. Five X-linked QTL influencing bristle number were resolved to intervals of approximately 1.3 cM. We confirm previous observations of bristle number QTL distal to 4A at the tip of the chromosome and identify two novel QTL in 7F-8C, an interval that does not include any classic bristle number candidate genes. If QTL at the tip of the X are biallelic they appear to be intermediate in frequency, although there is evidence that these QTL may reside in multiallelic haplotypes. Conversely, the two QTL mapping to the middle of the X chromosome are likely rare: in each case the minor allele is observed in only 1 of the 16 founders. Assuming additivity and biallelism we estimate that identified QTL contribute 1.0 and 8.7%, respectively, to total phenotypic variation in male abdominal and sternopleural bristle number in nature. Models that seek to explain the maintenance of genetic variation make different predictions about the population frequency of QTL alleles. Thus, mapping QTL in eight-way recombinant populations can distinguish between these models.     

Polygenic Mutation in Drosophila Melanogaster: Estimates from Response to Selection of Inbred Strains

Replicated divergent artificial selection for abdominal and sternopleural bristle number from a highly inbred strain of Drosophila melanogaster resulted in an average divergence after 125 generations of selection of 12.0 abdominal and 8.2 sternopleural bristles from the accumulation of new mutations affecting bristle number. Responses to selection were highly asymmetrical, with greater responses for low abdominal and high sternopleural bristle numbers. Estimates of V(M), the mutational variance arising per generation, based on the infinitesimal model and averaged over the responses to the first 25 generations of selection, were 4.32 X 10(-3) V(E) for abdominal bristle number and 3.66 X 10(-3) V(E) for sternopleural bristle number, where V(E) is the environmental variance. Based on 10 generations of divergent selection within lines from generation 93, V(M) for abdominal bristle number was 6.75 X 10(-3) V(E) and for sternopleural bristle number was 5.31 X 10(-3) V(E). However, estimates of V(M) using the entire 125 generations of response to selection were lower and generally did not fit the infinitesimal model largely because the observed decelerating responses were not compatible with the predicted increasing genetic variance over time. These decelerating responses, periods of response in the opposite direction to artificial selection, and rapid responses to reverse selection all suggest new mutations affecting bristle number on average have deleterious effects on fitness. Commonly observed periods of accelerated responses followed by long periods of stasis suggest a leptokurtic distribution of mutational effects for bristles.     

The tumorous-head-1 locus affects bristle number of the Drosophila melanogaster cuticle.

The tuh-1 maternal effect locus contains two naturally occurring isoalleles, tuh-1h and tuh-1g. Until recently there has been no possibility to distinguish between the tuh-lh and the tuh-1g maternal effects other than evaluating their effect on the Bithorax-Complex (BXC) Abdominal B (Abd-B) mutant tuh-3. However, in this report we identify a bristle phenotype associated with the tuh-1 locus that has very interesting evolutionary implications. Females homozygous for tuh-1h always produce adult offspring with more bristles than females homozygous or heterozygous for tuh-1g. The effect is global. Increased bristle number occurs in the head, the thorax, and the anterior and posterior abdomen. Females totally deficient for the tuh-1 gene produce offspring with high bristle number. Thus, the bristle phenotype results from the absence of the maternally contributed tuh-1g factor. Genetic evidence shows that the bristle phenotype is caused by the tuh-1 locus and that tuh-1h is completely recessive to tuh-1g. The tuh-1 locus is located at the euchromatin-beta-heterochromatin junction near the centromere of the X chromosome and deficiency analysis places the locus between the lethal genes extra organs (eo) and lethal B20 (lB20). The variance in bristle number attributable to the tuh-1 locus in nature is approximately 10.1%, an indication that the bristle phenotype is most likely a neutral, pleiotrophic side effect of tuh-1.     

Genetic screens for factors involved in the notum bristle loss of interspecific hybrids between Drosophila melanogaster and D. simulans

Interspecific cross is a powerful means to uncover hidden within- and between-species variation in populations. One example is a bristle loss phenotype of hybrids between Drosophila melanogaster and D. simulans, although both the pure species have exactly the same pattern of bristle formation on the notum. There exists a large amount of genetic variability in the simulans populations with respect to the number of missing bristles in hybrids, and the variation is largely attributable to simulans X chromosomes. Using nine molecular markers, I screened the simulans X chromosome for genetic factors that were responsible for the differences between a pair of simulans lines with high (H) and low (L) missing bristle numbers. Together with duplication-rescue experiments, a single major quantitative locus was mapped to a 13F-14F region. Importantly, this region accounted for most of the differences between H and L lines in three other independent pairs, suggesting segregation of H and L alleles at the single locus in different populations. Moreover, a deficiency screening uncovered several regions with factors that potentially cause the hybrid bristle loss due to epistatic interactions with the other factors.