An amino acid polymorphism in the couch potato gene forms the basis for climatic adaptation in Drosophila melanogaster

Abstract

Diapause is the classic adaptation to seasonality in arthropods, and its expression can result in extreme lifespan extension as well as enhanced resistance to environmental challenges. Little is known about the underlying evolutionary genetic architecture of diapause in any organism. Drosophila melanogaster exhibits a reproductive diapause that is variable within and among populations; the incidence of diapause increases with more temperate climates and has significant pleiotropic effects on a number of life history traits. Using quantitative trait mapping, we identified the RNA-binding protein encoding gene couch potato (cpo) as a major genetic locus determining diapause phenotype in D. melanogaster and independently confirmed this ability to impact diapause expression through genetic complementation mapping. By sequencing this gene in samples from natural populations we demonstrated through linkage association that variation for the diapause phenotype is caused by a single Lys/Ile substitution in one of the six cpo transcripts. Complementation analyses confirmed that the identified amino acid variants are functionally distinct with respect to diapause expression, and the polymorphism also shows geographic variation that closely mirrors the known latitudinal cline in diapause incidence. Our results suggest that a naturally occurring amino acid polymorphism results in the variable expression of a diapause syndrome that is associated with the seasonal persistence of this model organism in temperate habitats.

Fig. 1.

Likelihood plot for the QTL analysis of reproductive diapause. The likelihood ratio, as calculated by multiple-interval mapping for ordinal traits (21) and implemented in QTL Cartographer v.2.5 (Category Trait Mapping, Forward Model), is plotted as a function of location in cM units on chromosome 3. The positions of the markers used in the analysis are indicated by arrows, and they correspond to cytological positions 61C, 64E, 65D, 68C, 75F, 83B, 85D, 87E, 89A, 90A, 90D, 92E, 94D, 98B, and 99A. The two markers that flank the identified QTL are indicated.

Fig. 2.

SNP genotypes across the right arm of the third chromosome and diapause incidence in the 20 RILs that were recombinant in the interval to which diapause mapped. The cytological position of each SNP marker is given (Left, top) and ranged from band 83B to 99A. Four SNPs were placed in the cpo gene and are listed as cpo1–4. The variation in diapause phenotype clearly maps only to the interval between SNP markers cpo3 and cpo4; this corresponds to the 3′ end of the cpo locus containing all of the coding sequence in exon 5.

Fig. 3.

The association between diapause phenotype and each of 192 identified polymorphisms in the 3.5-kb region encompassing cpo exon 5. Each point represents the transformed P value resulting from a nominal logistic regression of nucleotide state on diapause incidence in the 35 extracted third chromosomes from the DPF population. The dashed line indicates significance threshold based on Bonferroni adjustment for multiple testing. The four sites (two synonymous and two nonsynonymous substitutions) that are significantly associated with diapause phenotype span 322 bp and are in significant linkage disequilibrium with one another (D ranges from 0.162 to 0.238, P < 0.0001 for each). The amino acid polymorphism at residue 462 is present in only the smallest cpo transcript (cpo-RH).

Fig. 4.

Variation in allele frequency for the five assayed cpo SNPs as a function of latitude of the populations. (A) Latitudinal clines for four reference SNPs in exons 1 (diamonds), 5 (circles), and 6 (5′ SNP, triangles; 3′ SNP, squares). The SNP in the 3′ end of exon 6 exhibits a significant cline in frequency (F_1,10 = 11.82, P < 0.007, R² = 0.568), whereas patterns of geographic allele frequency variation are homogeneous for the other three markers. (B) The SNP corresponding to the polymorphism at amino acid residue 356 exhibits an allele frequency cline (F_1,10 = 82.97, P < 0.00001, R² = 0.92), and it is the derived allele (Val) that increases positively with latitude. This polymorphism is in strong linkage disequilibrium with the amino acid polymorphism at residue 462 (D = 0.216, χ² = 37.99, P < 0.0001). This suggests that the cpo^462Ile/cpo^462Lys polymorphism also varies significantly with geography. This is further supported by direct sequencing of cpo alleles in third chromosomes from the northernmost and southernmost populations sampled. The frequency of the derived cpo^462Lys allele was 0.15 in the southern (n = 23 sequences) and 0.61 in the northern (n = 24 sequences) population (data not shown).

Fig. 5.

Complementation analyses of 24 cpo alleles (12 cpo^462Ile, 12 cpo^462Lys) derived from the DPF population. (A) Diapause incidence in F₁ progeny from crosses to the cpo hypomorphic allele cpo^BG02810 and the wild-type revertant (cpo^P3) created by the precise excision. Patterns of diapause expression exhibited codominance and stepwise effects of allelic substitution. Genotypes possessing two high-diapause alleles (cpo^BG02810 or cpo^462Lys) exhibited a strong diapause phenotype; the substitution of a low-diapause allele (cpo^P3 or cpo^462Ile) resulted in intermediate diapause expression, and substituting a second low-diapause allele resulted in a low-diapause phenotype. In both genetic backgrounds, the cpo^462Lys allele is associated with a higher diapause expression than the cpo^462Ile allele. (B) Patterns of diapause incidence in F₁ progeny from crosses to the cpo deletion (cpo^3.42) and duplication (cpo^3.20) were qualitatively identical. The observation that the duplication of the cpo allele from the high-diapause parental strain (stock 6326) results in a reduction in diapause incidence again indicates that diapause phenotype may be governed by cpo expression.