Genome features of "Dark-fly", a Drosophila line reared long-term in a dark environment

Abstract

Organisms are remarkably adapted to diverse environments by specialized metabolisms, morphology, or behaviors. To address the molecular mechanisms underlying environmental adaptation, we have utilized a Drosophila melanogaster line, termed "Dark-fly", which has been maintained in constant dark conditions for 57 years (1400 generations). We found that Dark-fly exhibited higher fecundity in dark than in light conditions, indicating that Dark-fly possesses some traits advantageous in darkness. Using next-generation sequencing technology, we determined the whole genome sequence of Dark-fly and identified approximately 220,000 single nucleotide polymorphisms (SNPs) and 4,700 insertions or deletions (InDels) in the Dark-fly genome compared to the genome of the Oregon-R-S strain, a control strain. 1.8% of SNPs were classified as non-synonymous SNPs (nsSNPs: i.e., they alter the amino acid sequence of gene products). Among them, we detected 28 nonsense mutations (i.e., they produce a stop codon in the protein sequence) in the Dark-fly genome. These included genes encoding an olfactory receptor and a light receptor. We also searched runs of homozygosity (ROH) regions as putative regions selected during the population history, and found 21 ROH regions in the Dark-fly genome. We identified 241 genes carrying nsSNPs or InDels in the ROH regions. These include a cluster of alpha-esterase genes that are involved in detoxification processes. Furthermore, analysis of structural variants in the Dark-fly genome showed the deletion of a gene related to fatty acid metabolism. Our results revealed unique features of the Dark-fly genome and provided a list of potential candidate genes involved in environmental adaptation.

Figure 1. History of Dark-fly.

In 1954, a fly population derived from one pair of Oregon-R-S flies was divided into 6 populations. Three of them (aL, bL and cL populations) were reared in normal light-dark cycling conditions and the remaining three populations (dD, eD, and fD populations) were reared in constant dark conditions. Unfortunately, all of the L lines were lost by 2002. The dD and eD lines were lost in 1965 and 1967, and only the fD line has been maintained until now. In 2008, we started to rear the fD line and designated it “Dark-fly”. We have maintained Dark-fly in a minimum medium as done before (black lines), and in a standard cornmeal medium (white lines) in parallel. The population size of Dark-fly has not been controlled but has usually been about 100 flies each in several culture vials.

Figure 2. Fecundity of Dark-fly and Oregon-R-S.

(A) Three-day fecundity (offspring/female) of Dark-fly and Oregon-R-S in LL, LD and DD conditions are shown by box plots. Boxes and median lines represent inter-quartile range and median values of data, and vertical lines represent minimum and maximum values of data within 1.5-fold of the inter-quartile range. Circles indicate values of outliers. * indicates FDR-adjusted p-value<0.05, Welch t-test. n = 10 (total 100 females). (B) Lifetime fecundity (offspring/female) of Dark-fly and Oregon-R-S in LD and DD conditions are shown by box plots in a similar manner to (A). ** indicates p-value<0.01, Welch t-test. n = 10 (total 100 females).

Figure 3. Survival curves of Dark-fly and Oregon-R-S.

The viability of male flies (A) and female flies (B) reared together is plotted versus time (days). Dark-fly (red lines) and Oregon-R-S (blue lines) were reared under LD (dotted lines) and DD (solid lines) conditions. The viability of virgin females (C) was also measured in a similar manner. n = 92–100 flies. Oregon-R-S virgin females showed longer longevity than the mated ones, whereas Dark-fly virgin females showed shorter longevity than the mated ones.

Figure 4. Homozygosity and ROH regions.

Mean homozygosity of SNPs in a sliding window (200-kb window at 100-kb steps) was plotted versus the location on 2L (A), 2R (B), 3L (C), 3R (D) and X (E) chromosomes. The Oregon-R-S genome (blue lines) displayed higher homozygosity than the Dark-fly genome (red lines) in most of the regions. Thick horizontal bars represent ROH regions identified by PLINK software for Oregon-R-S (blue bars) and Dark-fly (red bars) and are plotted above the graph without homozygosity values.

Figure 5. Alignment of read sequences around CG4594 gene.

A view of Integrated Genomics Viewer around the CG4594 gene. The numerous small gray bars represent reads of genome sequencing. A region of about 500 bases in the CG4594 gene (red thick bar) was not covered by any read sequences of the Dark-fly genome (upper), but was fully covered by the sequences of the Oregon-R-S genome (lower). Numbers on a horizontal line indicate nucleotide position on chromosome 2L. Numbers on vertical alignment indicate read depth.