Low mutation rates of microsatellite loci in Drosophila melanogaster.

Analysis of variation at microsatellite DNA loci is widely used in studies of parentage, linkage and evolutionary history. The utility of microsatellites is primarily due to high levels of allelic diversity, believed to reflect mutation rates orders of magnitude higher than base pair substitutions at single-copy genes. For humans, mice, rats and pigs, microsatellite mutation rates have been estimated at 10(-3)-10(-5). However, a recent study comparing microsatellite variation in humans with non-human primates suggests that microsatellite mutation rates may vary considerably across taxa. We measured mutation rates of 24 microsatellite loci in mutation accumulation lines of Drosophila melanogaster. Surprisingly, only a single mutation was detected after screening 157,680 allele-generations, yielding an estimated average mutation rate per locus of 6.3 x 10(-6), a mutation rate considerably lower than reported for various mammals. We propose that the comparatively low mutation rate is primarily a function of short microsatellite repeat lengths in the D. melanogaster genome.

Naturally occurring variation in bristle number and DNA polymorphisms at the scabrous locus of Drosophila melanogaster.

The association between quantitative genetic variation in bristle number and molecular variation at a candidate neurogenic locus, scabrous, was examined in Drosophila melanogaster. Approximately 32 percent of the genetic variation in abdominal bristle number (21 percent for sternopleural bristle number) among 47 second chromosomes from a natural population was correlated with DNA sequence polymorphisms at this locus. Several polymorphic sites associated with large phenotypic effects occurred at intermediate frequency. Quantitative genetic variation in natural populations caused by alleles that have large effects at a few loci and that segregate at intermediate frequencies conflicts with the classical infinitesimal model of the genetic basis of quantitative variation.

The genetic basis of quantitative variation: numbers of sensory bristles of Drosophila melanogaster as a model system.

The numbers of sensory hairs of Drosophila melanogaster present an ideal model system to elucidate the genetic basis of morphological quantitative variation. Loci affecting bristle number can be identified and their properties studied by accumulating spontaneous mutations, by P element mutagenesis, by mapping factors causing divergence between selection lines and by the association of phenotype variation with molecular variation at candidate neurogenic loci. The consensus emerging from the application of all approaches is that much of the mutational and segregating variation affecting bristle number is attributable to alleles with large phenotype effects at a small number of candidate loci.

Genetics of alcohol consumption in Drosophila melanogaster.

Individual variation in alcohol consumption in human populations is determined by genetic, environmental, social and cultural factors. In contrast to humans, genetic contributions to complex behavioral phenotypes can be readily dissected in Drosophila, where both the genetic background and environment can be controlled and behaviors quantified through simple high-throughput assays. Here, we measured voluntary consumption of ethanol in ∼3000 individuals of each sex from an advanced intercross population derived from 37 lines of the Drosophila melanogaster Genetic Reference Panel. Extreme quantitative trait loci mapping identified 385 differentially segregating allelic variants located in or near 291 genes at P < 10-8 . The effects of single nucleotide polymorphisms associated with voluntary ethanol consumption are sex-specific, as found for other alcohol-related phenotypes. To assess causality, we used RNA interference knockdown or P{MiET1} mutants and their corresponding controls and functionally validated 86% of candidate genes in at least one sex. We constructed a genetic network comprised of 23 genes along with a separate trio and a pair of connected genes. Gene ontology analyses showed enrichment of developmental genes, including development of the nervous system. Furthermore, a network of human orthologs showed enrichment for signal transduction processes, protein metabolism and developmental processes, including nervous system development. Our results show that the genetic architecture that underlies variation in voluntary ethanol consumption is sexually dimorphic and partially overlaps with genetic factors that control variation in feeding behavior and alcohol sensitivity. This integrative genetic architecture is rooted in evolutionarily conserved features that can be extrapolated to human genetic interaction networks.

Positive assortative mating with family size as a function of predicted parental breeding values.

While other investigations have described benefits of positive assortative mating (PAM) for forest tree breeding, the allocation of resources among mates in these studies was either equal or varied, using schemes corresponding only to parental rank (i.e., more resources invested in higher-ranking parents). In this simulation study, family sizes were proportional to predicted midparent BLUP values. The distribution of midparent BLUP values was standardized by a constant, which was varied to study the range of distributions of family size. Redistributing progenies from lower- to higher-ranking families to a point where an equal number of progenies were still selected out of each family to the next generation caused minimal change in group coancestry and inbreeding in the breeding population (BP), while the additive genetic response and variance in the BP were both greatly enhanced. This generated additional genetic gains for forest plantations by selecting more superior genotypes from the BP (compared to PAM with equal family sizes) for production of improved regeneration materials. These conclusions were verified for a range of heritability under a polygenic model and under a mixed-inheritance model with a QTL contributing to the trait variation.

Epistatic partners of neurogenic genes modulate Drosophila olfactory behavior.

The extent to which epistasis affects the genetic architecture of complex traits is difficult to quantify, and identifying variants in natural populations with epistatic interactions is challenging. Previous studies in Drosophila implicated extensive epistasis between variants in genes that affect neural connectivity and contribute to natural variation in olfactory response to benzaldehyde. In this study, we implemented a powerful screen to quantify the extent of epistasis as well as identify candidate interacting variants using 203 inbred wild-derived lines with sequenced genomes of the Drosophila melanogaster Genetic Reference Panel (DGRP). We crossed the DGRP lines to P[GT1]-element insertion mutants in Sema-5c and neuralized (neur), two neurodevelopmental loci which affect olfactory behavior, and to their coisogenic wild-type control. We observed significant variation in olfactory responses to benzaldehyde among F1 genotypes and for the DGRP line by mutant genotype interactions for both loci, showing extensive nonadditive genetic variation. We performed genome-wide association analyses to identify the candidate modifier loci. None of these polymorphisms were in or near the focal genes; therefore, epistasis is the cause of the nonadditive genetic variance. Candidate genes could be placed in interaction networks. Several candidate modifiers are associated with neural development. Analyses of mutants of candidate epistatic partners with neur (merry-go-round (mgr), prospero (pros), CG10098, Alhambra (Alh) and CG12535) and Sema-5c (CG42540 and bruchpilot (brp)) showed aberrant olfactory responses compared with coisogenic controls. Thus, integrating genome-wide analyses of natural variants with mutations at defined genomic locations in a common coisogenic background can unmask specific epistatic modifiers of behavioral phenotypes.

Transposable element-induced response to artificial selection in Drosophila melanogaster.

The P family of transposable elements in Drosophila melanogaster transpose with exceptionally high frequency when males from P strains carrying multiple copies of these elements are crossed to females from M strains that lack P elements, but with substantially lower frequency in the reciprocal cross. Transposition is associated with enhanced mutation rates, caused by insertion and deletion of P elements, and chromosome rearrangements. If P element mutagenesis creates additional variation for quantitative traits, accelerated response to artificial selection of progeny of M female female X P male male strain crosses is expected, compared with that from progeny of P female female X M male male strain crosses.--Divergent artificial selection for number of bristles on the last abdominal tergite was carried out for 16 generations among the progeny of P-strain males (Harwich) and M-strain females (Canton-S) and also of M-strain males (Canton-S) and P-strain females (Harwich). Each cross was replicated four times. Average realized heritability of abdominal bristle score for the crosses in which P transposition was expected was 0.244 +/- 0.017, 1.5 times greater than average heritability estimated from crosses in which transposition was expected to be rare (0.163 +/- 0.010). Phenotypic variance of abdominal bristle score increased by a factor of four in lines selected from M female female X P male male crosses when compared with those selected from P female female X M male male hybrids. Not all quantitative genetic variation induced by P elements is additive. A substantial fraction of nonadditive genetic variation is implicated by chromosomal analysis, which demonstrates deleterious fitness effects of the mutations when homozygous.--Several putative "quantitative" mutations were identified from chromosomes extracted from the selected lines; these will form the basis for further investigation at the molecular level of the genes controlling quantitative inheritance.

Hybrid dysgenesis-induced quantitative variation on the X chromosome of Drosophila melanogaster.

To determine the ability of the P-M hybrid dysgenesis system of Drosophila melanogaster to generate mutations affecting quantitative traits, X chromosome lines were constructed in which replicates of isogenic M and P strain X chromosomes were exposed to a dysgenic cross, a nondysgenic cross, or a control cross, and recovered in common autosomal backgrounds. Mutational heritabilities of abdominal and sternopleural bristle score were in general exceptionally high-of the same magnitude as heritabilities of these traits in natural populations. P strain chromosomes were eight times more mutable than M strain chromosomes, and dysgenic crosses three times more effective than nondysgenic crosses in inducing polygenic variation. However, mutational heritabilities of the bristle traits were appreciable for P strain chromosomes passed through one nondysgenic cross, and for M strain chromosomes backcrossed for seven generations to inbred P strain females, a result consistent with previous observations on mutations affecting quantitative traits arising from nondysgenic crosses. The new variation resulting from one generation of mutagenesis was caused by a few lines with large effects on bristle score, and all mutations reduced bristle number.

Quantitative trait loci for life span in Drosophila melanogaster: interactions with genetic background and larval density.

The genetic architecture of variation in adult life span was examined for a population of recombinant inbred lines, each of which had been crossed to both inbred parental strains from which the lines were derived, after emergence from both high and low larval density. QTL affecting life span were mapped within each sex and larval density treatment by linkage to highly polymorphic roo-transposable element markers, using a composite interval mapping method. We detected a total of six QTL affecting life span; the additive effects and degrees of dominance for all were highly sex- and larval environment-specific. There were significant epistatic interactions between five of the life span QTL, the effects of which also differed according to genetic background, sex, and larval density. Five additional QTL were identified that contributed to differences among lines in their sensitivity to variation in larval density. Further fine-scale mapping is necessary to determine whether candidate genes within the regions to which the QTL map are actually responsible for the observed variation in life span.

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.

Candidate quantitative trait loci and naturally occurring phenotypic variation for bristle number in Drosophila melanogaster: the Delta-Hairless gene region.

Delta (Dl) and Hairless (H) are two chromosome 3 candidate neurogenic loci that might contribute to naturally occurring quantitative variation for sensory bristle number. To evaluate this hypothesis, we assessed quantitative genetic variation in abdominal and sternopleural bristle numbers among homozygous isogenic third chromosomes sampled from nature and substituted into the Samarkand (Sam) inbred chromosome 1 and 2 background; among homozygous lines in which the wild-derived Dl-H gene region was introgressed into the Sam chromosome 3 background; and among Dl-H region introgression lines as heterozygotes against the Sam wild-type strain and derivatives of Sam into which mutant Dl and H alleles had been introgressed. Variation among the Dl-H region introgression lines accounted for 36% (8.3%) of the total chromosome 3 among line variance in abdominal (sternopleural) bristle number and for 53% of the chromosome 3 sex x line variance in abdominal bristle number. Naturally occurring alleles in the Dl-H region failed to complement a Dl mutant allele for female abdominal bristle number and sternopleural bristle number in both sexes, and an H mutant allele for both bristle traits in males and females. These results are consistent with the hypothesis that naturally occurring alleles at Dl and H contribute to quantitative genetic variation in sensory bristle number.

Quantitative genetics of ovariole number in Drosophila melanogaster. II. Mutational variation and genotype-environment interaction.

The rare alleles model of mutation-selection balance (MSB) hypothesis for the maintenance of genetic variation was evaluated for two quantitative traits, ovariole number and body size. Mutational variances (VM) for these traits, estimated from mutation accumulation lines, were 4.75 and 1.97 x 10(-4) times the environmental variance (VE), respectively. The mutation accumulation lines were studied in three environments to test for genotype x environment interaction (GEI) of new mutations; significant mutational GEI was found for both traits. Mutations for ovariole number have a quadratic relationship with competitive fitness, suggesting stabilizing selection for the trait; there is no significant correlation between mutations for body size and competitive fitness. Under MSB, the ratio of segregating genetic variance, VG, to mutational variance, VM, estimates the inverse of the selection coefficient against a heterozygote for a new mutation. Estimates of VG/VM for ovariole number and body size were both approximately 1.1 x 10(4). Thus, MSB can explain the level of variation, if mutations affecting these traits are under very weak selection, which is inconsistent with the empirical observation of stabilizing selection, or if the estimate of VM is biased downward by two orders of magnitude. GEI is a possible alternative explanation.

Deficiency mapping of quantitative trait loci affecting longevity in Drosophila melanogaster.

In a previous study, sex-specific quantitative trait loci (QTL) affecting adult longevity were mapped by linkage to polymorphic roo transposable element markers, in a population of recombinant inbred lines derived from the Oregon and 2b strains of Drosophila melanogaster. Two life span QTL were each located on chromosomes 2 and 3, within sections 33E-46C and 65D-85F on the cytological map, respectively. We used quantitative deficiency complementation mapping to further resolve the locations of life span QTL within these regions. The Oregon and 2b strains were each crossed to 47 deficiencies spanning cytological regions 32F-44E and 64C-76B, and quantitative failure of the QTL alleles to complement the deficiencies was assessed. We initially detected a minimum of five and four QTL in the chromosome 2 and 3 regions, respectively, illustrating that multiple linked factors contribute to each QTL detected by recombination mapping. The QTL locations inferred from deficiency mapping did not generally correspond to those of candidate genes affecting oxidative and thermal stress or glucose metabolism. The chromosome 2 QTL in the 35B-E region was further resolved to a minimum of three tightly linked QTL, containing six genetically defined loci, 24 genes, and predicted genes that are positional candidates corresponding to life span QTL. This region was also associated with quantitative variation in life span in a sample of 10 genotypes collected from nature. Quantitative deficiency complementation is an efficient method for fine-scale QTL mapping in Drosophila and can be further improved by controlling the background genotype of the strains to be tested.

Quantitative trait loci for floral morphology in Arabidopsis thaliana.

A central question in biology is how genes control the expression of quantitative variation. We used statistical methods to estimate genetic variation in eight Arabidopsis thaliana floral characters (fresh flower mass, petal length, petal width, sepal length, sepal width, long stamen length, short stamen length, and pistil length) in a cosmopolitan sample of 15 ecotypes. In addition, we used genome-wide quantitative trait locus (QTL) mapping to evaluate the genetic basis of variation in these same traits in the Landsberg erecta x Columbia recombinant inbred line population. There was significant genetic variation for all traits in both the sample of naturally occurring ecotypes and in the Ler x Col recombinant inbred line population. In addition, broad-sense genetic correlations among the traits were positive and high. A composite interval mapping (CIM) analysis detected 18 significant QTL affecting at least one floral character. Eleven QTL were associated with several floral traits, supporting either pleiotropy or tight linkage as major determinants of flower morphological integration. We propose several candidate genes that may underlie these QTL on the basis of positional information and functional arguments. Genome-wide QTL mapping is a promising tool for the discovery of candidate genes controlling morphological development, the detection of novel phenotypic effects for known genes, and in generating a more complete understanding of the genetic basis of floral development.

The effect of combining alleles into electrophoretic classes on detecting linkage disequilibrium.

When alleles are combined into few detectable classes, linkage correlations are underestimated most of the time. The probability that the linkage correlation will be underestimated is a function of the actual degree of correlation and the evenness of the allelic distribution, but is mainly determined by the distribution of alleles into distinguishable classes. With only two alleles per class this probability will usually be higher than 0.7. Also, the consistency in the sign of the linkage disequilibrium over many populations may escape detection. An increase of sample size by one order of magnitude or more may be required to compensate for the loss in detection power. It follows that the available electrophoretic studies of linkage correlations, although negative in their majority, do not suggest that epistatic interactions and linkage disequilibria are rare in natural populations.

Transposable element-induced response to artificial selection in Drosophila melanogaster: molecular analysis of selection lines.

Artificial selection lines for abdominal bristle score of Drosophila melanogaster established from P-M hybrid dysgenic crosses showed increases in selection response, heritability and phenotypic variance compared to similar lines started from nondysgenic crosses. To determine whether this increased genetic variance could be due to enhanced transposition of P elements following the dysgenic cross, the cytological locations (sites) of P elements were determined by in situ hybridization for the whole genome of samples of 20 individuals from the parental P strain, 20 individuals from each of the eight dysgenic selection lines, and ten individuals from each of the eight nondysgenic selection lines. Variation among and within the selection lines and the parental P strain in P element insertion sites was exceptionally high. A total of 601 sites were identified, but there was no difference in total number of sites per line, mean number of sites per individual, mean copy number per individual, or site frequency between dysgenic and nondysgenic selection lines, or between lines selected for high and low bristle score. Transposition following nondysgenic crosses may explain additional observations of accelerated selection responses in nondysgenic selection lines. It was not possible to deduce which, if any, of the several hundred insertions in the dysgenic selection lines were responsible for their extreme bristle phenotypes.

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.

Effects of single P-element insertions on olfactory behavior in Drosophila melanogaster.

Single P-element (P[lArB]) insertional mutagenesis of an isogenic strain was used to identify autosomal loci affecting odor-guided behavior of Drosophila melanogaster. The avoidance response to benzaldehyde of 379 homozygous P[lArB] element-containing insert lines was evaluated quantitatively. Fourteen smell impaired (smi) lines were identified in which P[lArB] element insertion caused different degrees of hyposmia in one or both sexes. The smi loci map to different cytological locations and probably are novel olfactory genes. Enhancer trap analysis of the smi lines indicates that expression of at least 10 smi genes is controlled by olfactory tissue-specific promoter/enhancer elements.

Effects of P element insertions on quantitative traits in Drosophila melanogaster.

P element mutagenesis was used to construct 94 third chromosome lines of Drosophila melanogaster which contained on average 3.1 stable P element inserts, in an inbred host strain background previously free of P elements. The homozygous and heterozygous effects of the inserts on viability and abdominal and sternopleural bristle number were ascertained by comparing the chromosome lines with inserts to insert-free control lines of the inbred host strain. P elements reduced average homozygous viability by 12.2% per insert and average heterozygous viability by 5.5% per insert, and induced recessive lethal mutations at a rate of 3.8% per insert. Mutational variation for the bristle traits averaged over both sexes was 0.03Ve per homozygous P insert and 0.003Ve per heterozygous P insert, where Ve is the environmental variance. Mutational variation was greater for the sexes considered separately because inserts had large pleiotropic effects on sex dimorphism of bristle characters. The distributions of homozygous effects of inserts on the bristle traits were asymmetrical, with the largest effects in the direction of reducing bristle number; and highly leptokurtic, with most of the increase in variance contributed by a few lines with large effects. The inserts had partially recessive effects on the bristle traits. Insert lines with extreme bristle effects had on average greatly reduced viability.

Heterosis for viability, fecundity, and male fertility in Drosophila melanogaster: comparison of mutational and standing variation.

If genetic variation for fitness traits in natural populations ("standing" variation) is maintained by recurrent mutation, then quantitative-genetic properties of standing variation should resemble those of newly arisen mutations. One well-known property of standing variation for fitness traits is inbreeding depression, with its converse of heterosis or hybrid vigor. We measured heterosis for three fitness traits, pre-adult viability, female fecundity, and male fertility, among a set of inbred Drosophilia melanogaster lines recently derived from the wild, and also among a set of lines that had been allowed to accumulate spontaneous mutations for over 200 generations. The inbred lines but not the mutation-accumulation (MA) lines showed heterosis for pre-adult viability. Both sets of lines showed heterosis for female fecundity, but heterosis for male fertility was weak or absent. Crosses among a subset of the MA lines showed that they were strongly differentiated for male fertility, with the differences inherited in autosomal fashion; the absence of heterosis for male fertility among the MA lines was therefore not caused by an absence of mutations affecting this trait. Crosses among the inbred lines also gave some, albeit equivocal, evidence for male fertility variation. The contrast between the results for female fecundity and those for male fertility suggests that mutations affecting different fitness traits may differ in their average dominance properties, and that such differences may be reflected in properties of standing variation. The strong differentiation among the MA lines in male fertility further suggests that mutations affecting this trait occur at a high rate.