Background. Quantitative differences between
individuals stem from a combination of genetic and environmental factors, with
the heritable variation being shaped by evolutionary
forces. Drosophila wing shape has emerged as an attractive system for genetic
dissection of multi-dimensional traits. We utilize several experimental genetic
methods to validation of the contribution of several polymorphisms in the Epidermal growth factor receptor (Egfr) gene to wing shape
and size, that were previously mapped in populations of Drosophila melanogaster
from North Carolina (NC) and California (CA). This re-evaluation utilized
different genetic testcrosses to generate heterozygous individuals with a
variety of genetic backgrounds as well as sampling of new alleles from Kenyan
stocks. Results. Only one variant, in the Egfr promoter,
had replicable effects in all new experiments. However, expanded genotyping of
the initial sample of inbred lines rendered the association non-significant in
the CA population, while it persisted in the NC sample, suggesting population
specific modification of the quantitative trait nucleotide QTN
effect. Conclusion. Dissection of quantitative trait
variation to the nucleotide level can identify sites with replicable effects as
small as one percent of the segregating genetic variation. However, the
testcross approach to validate QTNs is both labor
intensive and time-consuming, and is probably less useful than resampling of large independent sets of outbred
individuals.
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Lack of knowledge about how regulatory regions evolve in relation to their structure–function may limit the utility of comparative sequence analysis in deciphering cis-regulatory sequences. To address this we applied reverse genetics to carry out a functional genetic complementation analysis of a eukaryotic cis-regulatory module—the even-skipped stripe 2 enhancer—from four Drosophila species. The evolution of this enhancer is non-clock-like, with important functional differences between closely related species and functional convergence between distantly related species. Functional divergence is attributable to differences in activation levels rather than spatiotemporal control of gene expression. Our findings have implications for understanding enhancer structure–function, mechanisms of speciation and computational identification of regulatory modules.
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Linkage disequilibrium mapping has been used extensively in medical and evolutionary genetics to map causal polymorphisms within genes associated with disease status or phenotypic variation for a trait. However, the initial findings of most non-human studies have not been replicated in subsequent studies, due in part to false-positives, as well as additional factors that can render true positives un-replicable. These factors may be more severe when the initial study is performed using an experimental population of organisms reared under controlled lab conditions. We demonstrate that despite considerable phenotypic differences for wing-shape between a lab-reared experimental population, and a wild-caught cohort of Drosophila melanogaster, an association between a putative regulatory polymorphism in Egfr and wing-shape can be replicated. These results will be discussed both within the framework of future association mapping studies, and within the context of the evolutionary dynamics of alleles in populations.
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As part of an effort to dissect quantitative trait locus effects to the nucleotide level, association was assessed between 238 single-nucleotide and 20 indel polymorphisms spread over 11 kb of the Drosophila melanogaster Egfr locus and nine relative warp measures of wing shape. One SNP in a conserved potential regulatory site for a GAGA factor in the promoter of alternate first exon 2 approaches conservative experiment-wise significance (P < 0.00003) in the sample of 207 lines for association with the location of the crossveins in the central region of the wing. Several other sites indicate marginal association with one or more other aspects of shape. No strong effects of sex or population of origin were detected with measures of shape, but two different sites were strongly associated with overall wing size in interaction with these fixed factors. Whole-gene sequencing in very large samples, rather than selective genotyping, would appear to be the only strategy likely to be successful for detecting subtle associations in species with high polymorphism and little haplotype structure. However, these features severely limit the ability of linkage disequilibrium mapping in Drosophila to resolve quantitative effects to single nucleotides.
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The Epidermal growth factor receptor is an essential gene with diverse pleiotropic roles in development throughout the animal
kingdom. Analysis of sequence diversity in 10.9 kb covering the complete coding
region and 6.4 kb of potential regulatory regions in a sample of 250 alleles
from three populations of Drosophila
melanogaster suggests that the intensity of different population genetic
forces varies along the locus. A total of 238 independent common SNPs and 20 indel polymorphisms
were detected, with just six common replacements affecting >1475 amino
acids, four of which are in the short alternate first exon.
Sequence diversity is lowest in a 2-kb portion of intron 2, which is also
highly conserved in comparison with D. simulans and D.
pseudoobscura. Linkage disequilibrium decays to background levels within
500 bp of most sites, so haplotypes
are generally restricted to up to 5 polymorphisms. The two North American
samples from
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One objective of quantitative genetics is to identify the nucleotide variants within genes that contribute to phenotypic variation and susceptibility [1]. In an evolutionary context, this means characterizing the molecular polymorphisms that modify the penetrance and expressivity of perturbed traits. A survey of association between 267 SNPs in almost 11 kb of the D. melanogaster Egfr and the degree of eye roughening due to a gain-of-function Egfr(E1) allele crossed into 210 isogenic wild-type lines provides evidence that a handful of synonymous substitutions supply cryptic variation for photoreceptor determination. Ten sites exceed Bonferroni threshold for association in two sets of crosses to different Egfr(E1) backgrounds including a particularly significant cluster of sites in tight linkage disequilibrium toward the 3' end of the coding region. Epistatic interaction of this cluster with one other site enhances the expressivity of this haplotype. Replication of the strongest associations with an independent sample of 302 phenotypically extreme individuals derived from 1000 crosses of Egfr(E1) to freshly trapped males was achieved using modified case-control and transmission-disequilibrium tests. A tendency for the rarer alleles to have more disrupted eye development suggests that mutation-selection balance is a possible mechanism contributing to maintaining cryptic variation for Egfr.
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No abstract, first two sentences:
Developmental geneticists' contribution to the study of the evolution of morphological divergence has proceeded along two lines: comparative analysis of gene expression and quantitative genetics. Recent studies highlight how complementation tests between species can bridge the gap between these approaches.
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Quantitative complementation tests provide a quick test of the hypothesis that a particular gene contributes to segregating phenotypic variation. A set of wild-type alleles is assayed for variation in their ability to complement the degree of dominance of the quantitative effect of a loss of function allele. Analysis of 15 loci known to be involved in wing patterning in Drosophila melanogaster suggests that the genes decapentaplegic, thickveins, EGFR, argos and hedgehog, each of which are involved in secreted growth factor signaling, may contribute to wing shape variation. The phenotype of one deficiency, Df(2R)Px2, which removes blistered/Plexate, is also highly sensitive to the wild-type genetic background and at intermediate expressivity reveals six ectopic veins. These form in the same locations as a projection of the ancestral pattern of dipteran wing veins on- to the D. melanogaster wing. This atavistic phenotype indicates that the wing vein prepatterning mechanism can be conserved in highly derived species, and implies that homoplasic venation patterns may be produced by derepression of vein primordia.
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Two composite multiple regression-interval mapping analyses were performed to identify candidate quantitative trait loci (QTL) affecting components of wing shape in Drosophila melanogaster defined by eight relative warp-based measures. A recombinant inbred line design was used to map QTL for the shape of two intervein regions in the anterior compartment of the wing, using a high resolution map of retrotransposon insertion sites between Oregon-R and Russian 2b. A total of 35 QTL representing up to 23 different loci were identified, many of which are located near components of the epidermal growth factor-Ras signal transduction pathway that regulates vein vs. intervein decision making and vein placement. Over one-half of the loci were detected in both sexes, and just under one-half were detected at two different growth temperatures. Different loci were found to affect aspects of shape in each intervein region, confirming that the shape of the whole wing should be regarded as a compound trait composed of several developmental units. In addition, a reciprocal backcross design was used to map QTL affecting shape in the posterior compartment of the wings of 831 flies, using a molecular map of 16 allele-specific oligohybridization single nucleotide polymorphism (SNP) markers between two divergent inbred lines. A total of 13 QTL were detected and shown to have generally additive effects on separable components of shape, in both sexes. By contrast, 8 QTL that affected wing size in these backcrosses were nearly dominant in their effects. The results confirm at the genetic level that wing shape is regulated independent of wing size and set up the hypothesis that wing shape is regulated in part through the regulation of the length and positioning of wing veins, involving quantitative regulation of the activity of secreted growth factors.