Complex patterns of cis‐regulatory polymorphisms in ebony underlie standing pigmentation variation in Drosophila melanogaster

Abstract

Pigmentation traits in adult Drosophila melanogaster were used in this study to investigate how phenotypic variations in continuous ecological traits can be maintained in a natural population. First, pigmentation variation in the adult female was measured at seven different body positions in 20 strains from the Drosophila melanogaster Genetic Reference Panel (DGRP) originating from a natural population in North Carolina. Next, to assess the contributions of cis-regulatory polymorphisms of the genes involved in the melanin biosynthesis pathway, allele-specific expression levels of four genes were quantified by amplicon sequencing using a 454 GS Junior. Among those genes, ebony was significantly associated with pigmentation intensity of the thoracic segment. Detailed sequence analysis of the gene regulatory regions of this gene indicated that many different functional cis-regulatory alleles are segregating in the population and that variations outside the core enhancer element could potentially play important roles in the regulation of gene expression. In addition, a slight enrichment of distantly associated SNP pairs was observed in the ~10 kb cis-regulatory region of ebony, which suggested the presence of interacting elements scattered across the region. In contrast, sequence analysis in the core cis-regulatory region of tan indicated that SNPs within the region are significantly associated with allele-specific expression level of this gene. Collectively, the data suggest that the underlying genetic differences in the cis-regulatory regions that control intraspecific pigmentation variation can be more complex than those of interspecific pigmentation trait differences, where causal genetic changes are typically confined to modular enhancer elements.

Factors underlying natural variation in body pigmentation of <i>Drosophila melanogaster</i>

“…Table 1 shows the correlation coefficients between pigmentation traits quantified in Miyagi et al (2015) and two stressrelated phenotypes, starvation resistance and chill coma recovery time, from Ayroles et al (2009) and Mackay et al (2012). The results indicated a negative correlation between the % dark-pigmented area of the A4 abdominal segment and starvation resistance, which indicated that darker individuals are less resistant to starvation.…”

“…After the food mixture was sufficiently heated and stirred, ~15 ml was dispensed into a glass vial (diameter 30 mm, height 103 mm), which was then plugged by a cotton ball wrapped in a piece of cotton cloth. Data on the following DGRP strains (Ayroles et al, 2009;Mackay et al, 2012;Miyagi et al, 2015) were used for the association study: Temperature treatment Flies were allowed to lay eggs on the standard corn-meal fly medium in a glass vial at 25 °C for 1−2 days. After adult flies were removed, the vial was kept at 25 °C for 7 days.…”

“…Repeatedly observed clines along latitude (David et al, 1985;Munjal et al, 1997;Parkash et al, 2008a;Telonis-Scott et al, 2011;Bastide et al, 2014) and altitude (Pool and Aquadro, 2007;Parkash et al, 2009Parkash et al, , 2010Bastide et al, 2014) indicate that these traits are involved in adaptation to clinal environmental factors. Furthermore, even within local populations, a considerable degree of variation exists in populations from many regions of the world (Pool and Aquadro, 2007;Parkash et al, 2008b;Rebeiz et al, 2009;Takahashi and TakanoShimizu, 2011;Bastide et al, 2013Bastide et al, , 2014Dembeck et al, 2015;Miyagi et al, 2015), which suggests that these traits are controlled by some balanced forces.…”

“…The major gene causing the intraspecific variation in overall intensity of body pigmentation is known to be ebony (Pool and Aquadro, 2007;Takahashi et al, 2007;Rebeiz et al, 2009;Takahashi and Takano-Shimizu, 2011;Telonis-Scott et al, 2011;Miyagi et al, 2015). Variations in the extent of the dark stripe on the abdominal segments are associated with cis-regulatory SNPs of the bric-à-brac and tan genes True et al, 2005;Bastide et al, 2013;Dembeck et al, 2015).…”

“…In this study, we first analyze the association between body pigmentation and several stress resistance traits by comparing body pigmentation measurements (Miyagi et al, 2015) and stress resistance scores (Ayroles et al, 2009;Mackay et al, 2012) using strains sampled from the DGRP. Next, we quantify changes in the expression levels of genes encoding enzymes in the melanin biosynthesis pathway under different rearing temperatures and assess the degrees of genotype-by-environment interaction in these genes.…”