Natural variation of ebony gene controlling thoracic pigmentation in Drosophila melanogaster

Abstract

We identified the causal genetic variation for the difference in the thoracic trident pigmentation intensity between two wild-derived strains of Drosophila melanogaster. It was found to be the difference in expression level of ebony, which codes for an enzyme in the melanin-synthesis pathway and has pleiotropic effects on vision and behavior.

Figure 1.—

Thoracic trident pigmentation pattern and its association with the third chromosome. (A) Thorax segment of Mel6 (G59) originated from Benin, West Africa. (B) Thorax segment of TW1 (G23) originated from Taiwan. Mel6 (G59) and TW1 (G23) were subjected to 23 and 59 generations of sib mating, respectively, to construct nearly isogenic lines. (C) Trident phenotypes of the whole-chromosome substitution lines between Mel6 and TW1. Chromosome origins and the trident phenotypes of the lines are indicated in the left and right columns, respectively. These lines were generated using balancer chromosomes by a similar scheme used in Hollocher et al. (1997). Pigmentation intensity was examined under the microscope.

Figure 2.—

Genetic mapping of the thoracic trident pigmentation phenotype using 73 homozygous recombinant lines constructed between TW1 and Mel6. (A) Genotypes of the pigmented (n = 19) and nonpigmented (n = 54) phenotype lines at 17 restriction fragment length polymorphism (RFLP) markers (a–q) on the third chromosome: a, Gr61d; b, gry; c, Gr64A; d, lama; e, Gr65c; f, Dhpr; g, Gr68d; h, Or74a; i, desat2; j, pnr; k, Rh2; l, Gr93f; m, RpS3; n, ssh; o, Pr; p, Gr98a; q, Ptx. Horizontal bars indicate genotypes of the recombinant lines. The dotted square indicates the genotypes at Gr93f marker that had almost complete association with the phenotype (r² = 0.93). Note that one exceptional line that showed nonpigmented phenotype but had TW1 genotype at Gr93f is represented as MT10-64 in B. RFLP markers were developed by direct sequencing PCR fragments of ∼600–900 bp from Mel6 and TW1. Further information on RFLP markers can be provided upon request. Primers were designed by Primer3 software (Rozen and Skaletsky 2000) from the publicly available genome sequence (Adams et al. 2000). DNA was extracted from one male each from Mel6 and TW1 using the GeneElute Mammalian Genomic DNA kit (Sigma-Aldrich, St. Louis). PCR products were obtained from these genomic samples by the AmpliTaq Gold PCR kit (Applied Biosystems, Foster City, CA) and were purified using MultiScreen-FB filter plates (Millipore, Billerica, MA). These purified products were directly sequenced on both strands using the BigDye terminator cycle sequencing kit version 3 (Applied Biosystems). The third chromosome recombinant lines were constructed from F₂ backcrossed individuals in the Mel6 homozygous background. The line was backcrossed twice (including the F₂ backcross) to Mel6 and had two generations of recombination opportunity. The lines were made homozygous using balancers in the last step. Note that these third chromosome recombinants possess the X and the second chromosomes from Mel6. (B) Genotypes at eight additional RFLP markers in six recombinant lines from A that had their breakpoints between markers Rh2 (k) and Gr93f (l). T indicates TW1 genotype and “-” indicates Mel6 genotype. Two critical lines, MT10-69 and MT10-64, indicate that the ∼310-kb region between markers r-l and tin is responsible for the phenotype.

Figure 3.—

Recombination breakpoints of the three most informative recombinants between Mel6 and TW1. The recombinants were screened from a laboratory population that experienced over 20 generations of recombination. These third chromosome recombinants had the X and the second chromosomes from Mel6. Horizontal arrows indicate annotated ORFs with their transcriptional directions. Vertical arrowheads indicate positions of the markers genotyped. Open and solid bars represent chromosome segments from Mel6 and TW1, respectively. Shaded bars indicate regions where recombinant breakpoints reside. The markers were genotyped by directly sequencing PCR fragments of ∼600–900 bp. The thoracic tridents of the three recombinants were all pigmented. Note that the critical interval of ∼75kb between CG7000 and CG5892 includes the candidate gene ebony.

Figure 4.—

Thoracic trident pigmentation phenotype obtained from complementation test with ebony and ebony RNAi knockdown experiment. (A) Dorsal view of the thorax segment of the Mel6/e¹ heterozygote. (B) Dorsal view of the thorax segment of the TW1/e¹ heterozygote. The flies in A and B were reared at 25°. The results were almost identical when e^AFA, Df(3R)e-H4, and Df(3R)e-R1 were used as ebony mutants (data not shown). (C) Dorsal view of the thorax segment of the wild-type control, w¹¹¹⁸; P{w^+mc = Actin5C-GAL4}/+. (D) Dorsal view of the thorax segment of the RNAi knockdown individual, w¹¹¹⁸; P{UAS-ebony-IR}/+; P{w^+mc = Actin5C-GAL4}/+. The insert, P{UAS-ebony-IR} contains two fragments of 500 bp ebony cDNA as an inverted repeat (IR). These lines were crossed to w¹¹¹⁸; P{w^+mc = Act5C–GAL4}17bFO1/+ strain to induce IR RNA in the whole body. w¹¹¹⁸; P{w^+mc = Act5C–GAL4}17bFO1/+ was obtained by backcrossing y¹ w*; P{w^+mc = Act5C–GAL4}17bFO1/TM6B, Tb¹ to w¹¹¹⁸ strain for 10 generations. Adult flies carrying one copy of the UAS-IR construct and one copy of the GAL4 driver were investigated for the thorax pigmentation. The flies in C and D were reared at 20°.