The effect of polymorphisms in the enhancer of split gene complex on bristle number variation in a large wild-caught cohort of Drosophila melanogaster - PubMed (original) (raw)

Comparative Study

The effect of polymorphisms in the enhancer of split gene complex on bristle number variation in a large wild-caught cohort of Drosophila melanogaster

Stuart J Macdonald et al. Genetics. 2005 Dec.

Abstract

The Enhancer of split complex [E(spl)-C] in Drosophila encompasses a variety of functional elements controlling bristle patterning and on the basis of prior work is a strong candidate for harboring alleles having subtle effects on bristle number variation. Here we extend earlier studies identifying associations between complex phenotypes and polymorphisms segregating among inbred laboratory lines of Drosophila and test the influence of E(spl)-C on bristle number variation in a natural cohort. We describe results from an association mapping study using 203 polymorphisms spread throughout the E(spl)-C genotyped in 2000 wild-caught Drosophila melanogaster. Despite power to detect associations accounting for as little as 2% of segregating variation for bristle number, and saturating the region with single-nucleotide polymorphisms (SNPs), we identified no single SNP marker showing a significant (additive over loci) effect after correcting for multiple tests. Using a newly developed test we conservatively identify six regions of the E(spl)-C in which the insertion of transposable elements as a class contributes to variation in bristle number, apparently in a sex- or trait-limited fashion. Finally, we carry out all possible 20,503 two-way tests for epistasis and identify a slight excess of marginally significant interactions, although none survive multiple-testing correction. It may not be straightforward to extend the results of laboratory-based association studies to natural populations.

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Figures

Figure 1.

Figure 1.

Extent of linkage disequilibrium across the Enhancer of split gene complex in Drosophila melanogaster. The pairwise LD among a set of 167 frequent (>5% minor allele frequency) sites in Enhancer of split and 7 frequent sites upstream of the hairy gene, calculated from 2000 diploid individuals, is represented as shaded blocks (dark, high LD; light, low LD). _D_′ is shown below the diagonal (values from 0 to 1 are split into 10 equal bins), and _R_2 is shown above the diagonal (values split into 10 bins defined by 1, formula image). The site positions in the Enhancer of split locus are shown below the plot, with the 12 genes labeled (solid boxes, exons; open boxes, untranslated regions).

Figure 2.

Figure 2.

Decay of linkage disequilibrium with distance in the Drosophila melanogaster Enhancer of split gene complex. Each point is the _R_2-value, calculated from 2000 individuals, between a pair of sites from a set of 167 frequent (>5% minor allele frequency) sites in Enhancer of split. The solid line represents a smoothed curve through the raw data using the ksmooth function in the statistical programming language R (

www.R-project.org

). The dashed line is the expected value of R_2 calculated using the formula 1/(1 + 4_Nc) (H

ill

and R

obertson

1968), where 4_Nc_ = ρ, the population recombination rate per base pair. A value of ρ = 0.0069 was estimated from the genotyping data, assuming the gene conversion parameter, f = 1 (see

materials and methods

and Table 1). The inset plot is an expanded version of the bottom portion of the full plot.

Figure 3.

Figure 3.

Recombination rate across the Enhancer of split complex in Drosophila melanogaster. The population recombination rate, ρ, per base pair was estimated for 10 sets of 49 individuals randomly sampled from a large cohort of 2000 individuals, using 167 frequent sites (>5% minor allele frequency). The program RECSLIDER was used to estimate ρ in a sliding-window framework using a window size of 20 segregating sites and an initial estimate of ρ = 0.01, assuming no gene conversion. The solid line represents the mean value of ρ across the 10 samples for each window, and the dashed lines show the upper and lower 95% confidence intervals on the mean calculated from the 10 population samples. Tickmarks at the top of the plot show the positions of the 167 frequent polymorphisms.

Figure 4.

Figure 4.

Saturation genotyping of frequent sites in the Enhancer of split complex. Resequencing 16 Drosophila melanogaster alleles for the complete Enhancer of split locus revealed 550 frequent polymorphisms, where the minor allele was seen in at least 2/16 alleles, and 167 of these were genotyped in a large cohort for association mapping. The positions of the 167 typed sites are shown in black relative to the structure of the locus (solid boxes, exons; open boxes, untranslated regions). The remaining 383 not typed sites are categorized as exonic (both synonymous and nonsynonymous), UTR (in either the 3′- or 5′-untranslated regions), upstream regulatory (present within an annotated regulatory element or the 10 bp flanking each side of the element), conserved intergenic (present in intergenic, nonregulatory sequence conserved between D. melanogaster and D. pseudoobscura; M

acdonald

and L

ong

2005), or nonconserved intergenic. Each not typed site is color coded to reflect the level of LD with a typed site [green, in strong LD with a genotyped site (χ2-test, P < 0.005) within 3 kb; red, not in LD with a genotyped site]. The values on the right of the plot show, for each category of not typed site (from left to right), the number of ungenotyped sites not in LD with a genotyped site, the number of ungenotyped sites, and the percentage of ungenotyped sites in LD with a genotyped site.

Figure 5.

Figure 5.

Single-marker tests for association between bristle number variation and polymorphisms in the Enhancer of split complex in Drosophila melanogaster. (A) Male additive model, (B) female additive model, (C) male dominance model, (D) female dominance model. (A–D) Each point represents the _P_-value (on a −log10 scale) of a single-marker ANOVA test for association with sternopleural bristle number (SBN, above the _x_-axis) or abdominal bristle number (ABN, below the _x_-axis), under either additive or arbitrary dominance models separately for males and females. (E–F) Sex model: Each point is the _P_-value for the genotype term (E) or the genotype-by-sex term (F) from a single-marker genotype-by-sex interaction ANOVA model with either SBN or ABN. The dashed lines delimit sites showing marginal significance at P ≤ 0.01. Open boxes at the bottom represent the 12 transcription units at Enhancer of split.

Figure 6.

Figure 6.

Cumulative distribution of _F_-ratio statistics under a single-marker additive model for sites in Enhancer of split. The distributions of 195 (for males) or 196 (for females) observed _F_-statistics from single-marker tests of association under an additive model are shown for each of the four sex/trait combinations (SBN, sternopleural bristle number; ABN, abdominal bristle number). The expected _F_-distribution for _F_1,1000 (thick solid line) is also presented for comparison.

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