Association of polymorphisms in odorant-binding protein genes with variation in olfactory response to benzaldehyde in Drosophila

Abstract

Adaptive evolution of animals depends on behaviors that are essential for their survival and reproduction. The olfactory system of Drosophila melanogaster has emerged as one of the best characterized olfactory systems, which in addition to a family of odorant receptors, contains an approximately equal number of odorant-binding proteins (OBPs), encoded by a multigene family of 51 genes. Despite their abundant expression, little is known about their role in chemosensation, largely due to the lack of available mutations in these genes. We capitalized on naturally occurring mutations (polymorphisms) to gain insights into their functions. We analyzed the sequences of 13 Obp genes in two chromosomal clusters in a population of wild-derived inbred lines, and asked whether polymorphisms in these genes are associated with variation in olfactory responsiveness. Four polymorphisms in 3 Obp genes exceeded the statistical permutation threshold for association with responsiveness to benzaldehyde, suggesting redundancy and/or combinatorial recognition by these OBPs of this odorant. Model predictions of alternative pre-mRNA secondary structures associated with polymorphic sites suggest that alterations in Obp mRNA structure could contribute to phenotypic variation in olfactory behavior.

Figure 1.—

LD in the Obp56 and Obp99 clusters. Boxes below the diagonal reflect R² values for all possible marker combinations and boxes above the diagonal indicate the corresponding P-values. Obp gene structures are denoted at the top by the horizontal line with exons represented by blue boxes, 5′-untranslated regions by orange boxes, and 3′-untranslated regions by white boxes. Introns are represented by the intervening line. The numbers in the circles indicate the number of polymorphisms contained in individual introns, exons, and untranslated regions. Since singletons have been excluded from the LD analysis, the number of polymorphisms for each gene indicated is sometimes lower than the number listed in Table 1.

Figure 2.—

Distribution of mean olfactory response scores for male and female flies of 193 wild-derived inbred lines (a) and lack of correlation between olfactory response scores and locomotion scores (b). Olfactory responses were measured at an odorant concentration of 3.5% (v/v) benzaldehyde. Blue and pink bars indicate scores for males and females, respectively. The correlation between olfactory responses and locomotion scores, shown in b is r = 0.144 for males (blue symbols) and r = 0.020 for females (pink symbols).

Figure 3.—

Associations between polymorphisms in the Obp99 cluster with variation in behavioral responses to benzaldehyde. The Obp99a, b, c, and d gene structures are schematically represented at the top of each graph with blue boxes representing exons, orange boxes 5′-untranslated regions, white boxes 3′-untranslated regions, and the intervening black line introns. The purple horizontal line indicates the significance threshold determined by permutation tests. Arrowheads indicate the locations of SNPs with significant phenotypic associations. The bar graphs show variation in olfactory behavior in response to benzaldehyde associated with four haplotypes corresponding to the two associated markers in Obp99d. Data were analyzed by ANOVA and haplotypes that differ significantly in olfactory behavior in response to benzaldehyde were identified by Tukey's test and are indicated with different letters at the top of the bars.

Figure 4.—

Predicted local stem-loop structures associated with polymorphic markers C75G in Obp99a, C141G in Obp99c, and G67/T78, G67/G78, and A67/G78 in Obp99d. The local stem-loop structures in Obp99d modulate base pairing of their neighbors in an ∼100-nt window around positions 67 and 78. Free energies of local secondary structures for G67/T78 = −132.5 kcal/mol, for G67/G78 = −133.9 kcal/mol, and for A67/G78 = −126.5 kcal/mol.