Associations between sperm competition and natural variation in male reproductive genes on the third chromosome of Drosophila melanogaster

Abstract

We applied association analysis to elucidate the genetic basis for variation in phenotypes affecting postcopulatory sexual selection in a natural population of Drosophila melanogaster. We scored 96 third chromosome substitution lines for nine phenotypes affecting sperm competitive ability and genotyped them at 72 polymorphisms in 13 male reproductive genes. Significant heterogeneity among lines (P < 0.01) was detected for all phenotypes except male-induced refractoriness (P = 0.053). We identified 24 associations (8 single-marker associations, 12 three-marker haplotype associations, and 4 cases of epistasis revealed by single-marker interactions). Fewer than 9 of these associations are likely to be false positives. Several associations were consistent with previous findings [Acp70A with the male's influence on the female's refractoriness to remating (refractory), Esterase-6 with a male's remating probability (remating) and a measure of female offspring production (fecundity)], but many are novel associations with uncharacterized seminal fluid proteins. Four genes showed evidence for pleiotropic effects [CG6168 with a measure of sperm competition (P2') and refractory, CG14560 with a defensive measure of sperm competition (P1') and a measure of female fecundity, Acp62F with P2' and a measure of female fecundity, and Esterase-6 with remating and a measure of female fecundity]. Our findings provide evidence that pleiotropy and epistasis are important factors in the genetic architecture of male reproductive success and show that haplotype analyses can identify associations missed in the single-marker approach.

Figure 1.—

Candidate genes and scored polymorphisms. The approximate location of each gene is shown relative to its cytological position on the third chromosome. Typed polymorphisms are given as asterisks. Protein-coding sequences are shown as open boxes and 5′- and 3′-untranslated regions are shaded gray. Introns and upstream and downstream regions are depicted as solid lines.

Figure 2.—

Variation across lines for sperm competition phenotypes. Rank-order line means (or line medians for P2′) are shown with standard errors (or Q1–Q3 plots for P2′). The test statistics and P-values are shown (see text). The grand mean across lines is shown by an open circle on the y-axis. The x-axis always corresponds to the rank-order lines.

Figure 3.—

Linkage disequilibrium across the 72 markers. Highly significant linkage disequilibrium is shown with solid circles (P < 0.01), and significant linkage disequilibrium is shown with open circles (P < 0.05). Significance tests that could not be completed are marked with a “+” (see text).

Figure 4.—

Examples of three-marker haplotype associations. The mean value of each haplotype is shown with the standard errors for Acp70A with refractory (A), CG14560 with P1′ (B), Esterase-6 with remating (C), CG14560 with fecundity-defense (D), Acp62F with P2′ (E), and Esterase-6 with fecundity-V2 (F). Association between haplotypes at Esterase-6 and fecundity-V2 is driven by a single line with low phenotypic value (see text). Plot of P1′ is back calculated from ASP1′.

Figure 5.—

Epistasis at male reproductive genes affecting sperm competitive ability: interaction plots from two-way ANOVA for markers in Acp76A and CG6168 associating with P1′ (A), in CG6168 and BG642167 associating with fecundity-defense (B), in Acp76A and Acp70A associating with P1′ (C), and in Acp62F and CG14560 associating with P2′ (D). Adjusted r² for each model is shown. Plot of P1′ is back calculated from ASP1′.

Figure 6.—

Association between haplotypes at Acp70A and male-induced female refractoriness to remating. (A) The gene region of Acp70A with the two exons (E1 and E2), the two regions of high LD shaded (5′-LD and coding-LD regions identified by Cirera and Aguadé 1997), and the 210-bp promoter region from Styger-Schmucki (1992). The LD regions are open ended, indicating that they may extend further as suggested by Cirera and Aguadé (1997). The asterisks indicate the locations of the three scored markers. (B and C) Associations between male-induced female refractoriness and two-marker haplotypes at Acp70Asnp902:1412 (B) and the serine:alanine amino acid polymorphism (C).