Marta L. Wayne

Combining mapping and arraying: An approach to candidate gene identification

A combination of quantitative trait locus (QTL) mapping and microarray analysis was developed and used to identify 34 candidate genes for ovariole number, a quantitative trait, in Drosophila melanogaster. Ovariole number is related to evolutionary fitness, which has been extensively studied, but for which few a priori candidate genes exist. A set of recombinant inbred lines were assayed for ovariole number, and QTL analyses for this trait identified 5,286 positional candidate loci. Forty deletions spanning the QTL were employed to further refine the map position of genes contributing to variation in this trait between parental lines, with six deficiencies showing significant effects and reducing the number of positional candidates to 548. Parental lines were then assayed for expression differences by using Affymetrix microarray technology, and ANOVA was used to identify differentially expressed genes in these deletions. Thirty-four genes were identified that showed evidence for differential expression between the parental lines, one of which was significant even after a conservative Bonferroni correction. The list of potential candidates includes 5 genes for which previous annotations did not exist, and therefore would have been unlikely choices for follow-up from mapping studies alone. The use of microarray technology in this context allows an efficient, objective, quantitative evaluation of genes in the QTL and has the potential to reduce the overall effort needed in identifying genes causally associated with quantitative traits of interest.

Regulatory Divergence in Drosophila melanogaster and D. simulans, a Genomewide Analysis of Allele-Specific Expression

Species-specific regulation of gene expression contributes to the development and maintenance of reproductive isolation and to species differences in ecologically important traits. A better understanding of the evolutionary forces that shape regulatory variation and divergence can be developed by comparing expression differences among species and interspecific hybrids. Once expression differences are identified, the underlying genetics of regulatory variation or divergence can be explored. With the goal of associating cis and/or trans components of regulatory divergence with differences in gene expression, overall and allele-specific expression levels were assayed genomewide in female adult heads of Drosophila melanogaster, D. simulans, and their F1 hybrids. A greater proportion of cis differences than trans differences were identified for genes expressed in heads and, in accordance with previous studies, cis differences also explained a larger number of species differences in overall expression level. Regulatory divergence was found to be prevalent among genes associated with defense, olfaction, and among genes downstream of the Drosophila sex determination hierarchy. In addition, two genes, with critical roles in sex determination and micro RNA processing, Sxl and loqs, were identified as misexpressed in hybrid female heads, potentially contributing to hybrid incompatibility.

Sex-Specific Splicing in Drosophila: Widespread Occurrence, Tissue Specificity and Evolutionary Conservation

Many genes in eukaryotic genomes produce multiple transcripts through a variety of molecular mechanisms including alternative splicing. Alternatively spliced transcripts often encode functionally distinct proteins, indicating that gene regulation at this level makes an important contribution to organismal complexity. The multilevel splicing cascade that regulates sex determination and sex-specific development in Drosophila is a classical example of the role of alternative splicing in cell differentiation. Recent evidence suggests that a large proportion of genes in the Drosophila genome may be spliced in a sex-biased fashion, raising the possibility that alternative splicing may play a more general role in sexually dimorphic development and physiology. However, the prevalence of sex-specific splicing and the extent to which it is shared among genotypes are not fully understood. Genetic variation in the splicing of key components of the sex determination pathway is known to influence the expression of downstream target genes, suggesting that alternative splicing at other loci may also vary in functionally important ways. In this study, we used exon-specific microarrays to examine 417 multitranscript genes for evidence of sex-specific and genotype-specific splicing in 80 different genotypes of Drosophila melanogaster. Most of these loci showed sex-biased splicing, whereas genotype-specific splicing was rare. One hundred thirty-five genes showed different alternative transcript use in males vs. females. Real-time PCR analysis of 6 genes chosen to represent a broad range of biological functions showed that most sex-biased splicing occurs in the gonads. However, somatic tissues, particularly adult heads, also show evidence of sex-specific splicing. Comparison of splicing patterns at orthologous loci in seven Drosophila species shows that sexual biases in alternative exon representation are highly conserved, indicating that sex-specific splicing is an ancient feature of Drosophila biology. To investigate potential mechanisms of sex-biased splicing, we used real-time PCR to examine the expression of six known regulators of alternative splicing in males vs. females. We found that all six loci are themselves spliced sex specifically in gonads and heads, suggesting that regulatory hierarchies based on alternative splicing may be an important feature of sexual differentiation.

Simpler mode of inheritance of transcriptional variation in male Drosophila melanogaster.

Sexual selection drives faster evolution in males. The X chromosome is potentially an important target for sexual selection, because hemizygosity in males permits accumulation of alleles, causing tradeoffs in fitness between sexes. Hemizygosity of the X could cause fundamentally different modes of inheritance between the sexes, with more additive variation in males and more nonadditive variation in females. Indeed, we find that genetic variation for the transcriptome is primarily additive in males but nonadditive in females. As expected, these differences are more pronounced on the X chromosome than the autosomes, but autosomal loci are also affected, possibly because of X-linked transcription factors. These differences may be of evolutionary significance because additive variation responds quickly to selection, whereas nonadditive genetic variation does not. Thus, hemizygosity of the X may underlie much of the faster male evolution of the transcriptome and potentially other phenotypes. Consistent with this prediction, genes that are additive in males and nonadditive in females are overrepresented among genes responding to selection for increased mating speed.

Environmental stress and reproduction in Drosophila melanogaster: starvation resistance, ovariole numbers and early age egg production.

BackgroundThe Y model of resource allocation predicts a tradeoff between reproduction and survival. Environmental stress could affect a tradeoff between reproduction and survival, but the physiological mechanisms underlying environmental mediation of the tradeoff are largely unknown. One example is the tradeoff between starvation resistance and early fecundity. One goal of the present study was to determine if reduced early age fecundity was indeed a robust indirect response to selection for starvation resistance, by investigation of a set of D. melanogaster starvation selected lines which had not previously been characterized for age specific egg production. Another goal of the present study was to investigate a possible relationship between ovariole number and starvation resistance. Ovariole number is correlated with maximum daily fecundity in outbred D. melanogaster. Thus, one might expect that a negative genetic correlation between starvation resistance and early fecundity would be accompanied by a decrease in ovariole number.ResultsSelection for early age female starvation resistance favored survival under food deprivation conditions apparently at the expense of early age egg production. The total number of eggs produced by females from selected and control lines was approximately the same for the first 26 days of life, but the timing of egg production differed such that selected females produced fewer eggs early in adult life. Females from lines selected for female starvation resistance exhibited a greater number of ovarioles than did unselected lines. Moreover, maternal starvation resulted in progeny with a greater number of ovarioles in both selected and unselected lines.ConclusionReduced early age egg production is a robust response to laboratory selection for starvation survival. Ovariole numbers increased in response to selection for female starvation resistance indicating that ovariole number does not account for reduced early age egg production. Further, ovariole number increased in a parallel response to maternal starvation, suggesting an evolutionary association between maternal environment and the reproductive system of female progeny.

QUANTITATIVE GENETICS OF OVARIOLE NUMBER IN DROSOPHILA MELANOGASTER. I. SEGREGATING VARIATION AND FITNESS

The number of ovarioles of the Drosophila melanogaster ovary is a trait thought to be associated with female fecundity, and therefore is expected to be under strong natural selection. This hypothesis may be tested by examining patterns of genetic and environmental variation for ovariole number in natural populations, and by determining the association between ovariole number and fitness in isogenic lines derived from a natural population. We measured ovariole number, and competitive fitness and its components, for 48 homozygous chromosome 3 substitution lines in a standard inbred background; and body size in a sample of 15 chromosome 3 substitution lines. We found significant segregating genetic variation for ovariole number, with a broad‐sense heritability (H2) of 0.403 and correspondingly high coefficients of genetic variation (CVC = 20.8) and residual variation (CVR = 25.3). Estimates of quantitative genetic parameters for body size (H2 = 0.191, CVG = 2.15, and CVR = 3.87) are similar to those previously reported for this trait. Although the isogenic chromosome 3 substitution lines varied significantly for components of fitness, there was no significant linear or quadratic association of ovariole number and body size with fitness. There was, however, highly significant sex × genotype interaction for fitness among these lines. This special case of genotype × environment interaction for fitness may contribute to the maintenance of genetic variation for fitness in natural populations.

Tracking adaptive evolutionary events in genomic sequences

As more gene and genomic sequences from an increasing assortment of species become available, new pictures of evolution are emerging. Improved methods can pinpoint where positive and negative selection act in individual codons in specific genes on specific branches of phylogenetic trees. Positive selection appears to be important in the interaction between genotype, protein structure, function, and organismal phenotype.

Genetic architecture of two fitness-related traits in Drosophila melanogaster: ovariole number and thorax length

In Drosophila melanogaster, ovariole number and thorax length are morphological characters thought to be associated with fitness. Maximum daily egg production in females is positively correlated with ovariole number, while thorax length is correlated with male reproductive success and female fecundity. Though both traits are related to fitness, ovariole number is likely to be under stabilizing selection, while thorax length appears to be under directional selection. Current research has focused on examining the sources of variation for ovariole number in relation to fitness, with a view towards elucidating how segregating variation is maintained in natural populations. Here, we utilize a diallel design to explore the genetic architecture of ovariole number and thorax length in nine isogenic lines derived from a natural population. The full diallel design allows the estimation of general combining ability (GCA), specific combining ability (SCA), and also describes variation due to reciprocal effects (RGCA and RSCA). Ovariole number and thorax length differed with respect to their genetic architecture, reflective of the independent selective forces acting on the traits. For ovariole number, GCA accounted for the majority (67.3%) of variation segregating between the lines, with no evidence of reciprocal effects or inbreeding depression; SCA accounted for a small percentage (3.9%) of the variance, suggesting dominance variation; no reciprocal effects were observed. In contrast, for thorax length, the majority of the non-error variance was accounted for by SCA (17.9%), with only one third as much variance (6.2%) due to GCA. Interestingly, RSCA (nuclear–extranuclear interactions) accounted for slightly more variation (7.5%) than GCA in these data. Thus, genetic variation for thorax length is largely in accord with predictions for a fitness trait under directional selection: little additive genetic variation and substantial dominance variation (including a suggestion of inbreeding depression); while the mechanisms underlying the maintenance of variation for ovariole number are more complex.

Quantitative genetics of natural variation of behavior in Drosophila melanogaster: the possible role of the social environment on creating persistent patterns of group activity.

Abstract Using a set of nine effectively isogenic lines collected from nature in 1998, we observed unperturbed behaviors of mixed‐sex groups of Drosophila melanogaster. We repeatedly scanned replicated groups of genetically identical individuals, five females and five males, and recorded the behavior of each individual (i.e., walking, feeding, grooming, flying, courting, mating, fighting, or resting). From these behaviors, we made a composite variable of activity for our quantitative genetic analysis. Genotypes differed in activity, explaining 14.41% of the variation in activity; 8.60% of the variation was explained by a significant genotype 3 sex interaction, which signifies genetic variation for sexual dimorphism in behavior. Phenotypic plasticity explained 11.13% of the variation in activity. Different genotypes and sexes within genotypes had different rank orders of the component behaviors that contribute to activity. We found no effect of common rearing environment. Instead, differences between replicate groups within genotype accounted for 19.47% variation in activity, and activity was significantly repeatable across scans. This emergent group behavior is likely caused by differences between groups of interacting individuals, even though individuals were genetically identical across groups. Thus, emergent group behavior explained almost as much variation in activity as the combined sources of genetic variation (23.01%), and this is an additional level on which selection could operate: individuals and groups. We discuss how differences among groups could change patterns of additive genetic variation available for evolution. Furthermore, because the behavior of an individual is influenced by conspecifics, genotype interactions between individuals could contribute to indirect selection. Finally, if we consider activity as a syndrome governing all component behaviors with strong genetic correlations among behaviors within an individual, then these component behaviors cannot evolve independently. These results suggest that reductionist approaches of molecular behavior genetics may be incomplete and/or misleading when considering similar phenotypes at the population level or when trying to understand how behaviours evolve

Intersection tests for single marker QTL analysis can be more powerful than two marker QTL analysis

BackgroundIt has been reported in the quantitative trait locus (QTL) literature that when testing for QTL location and effect, the statistical power supporting methodologies based on two markers and their estimated genetic map is higher than for the genetic map independent methodologies known as single marker analyses. Close examination of these reports reveals that the two marker approaches are more powerful than single marker analyses only in certain cases.Simulation studies are a commonly used tool to determine the behavior of test statistics under known conditions. We conducted a simulation study to assess the general behavior of an intersection test and a two marker test under a variety of conditions. The study was designed to reveal whether two marker tests are always more powerful than intersection tests, or whether there are cases when an intersection test may outperform the two marker approach.We present a reanalysis of a data set from a QTL study of ovariole number in Drosophila melanogaster.ResultsOur simulation study results show that there are situations where the single marker intersection test equals or outperforms the two marker test. The intersection test and the two marker test identify overlapping regions in the reanalysis of the Drosophila melanogaster data. The region identified is consistent with a regression based interval mapping analysis.ConclusionWe find that the intersection test is appropriate for analysis of QTL data. This approach has the advantage of simplicity and for certain situations supplies equivalent or more powerful results than a comparable two marker test.