DOI: 10.5061/dryad.x95x69pfr
title: Data from: Distinct genetic architectures underlie divergent thorax, leg, and wing pigmentation between Drosophila elegans and D. gunungcola

description: Understanding the genetic basis of species differences is a major goal in
 evolutionary biology. Pigmentation divergence between Drosophila species
 often involves genetic changes in pigmentation candidate genes that
 pattern the body and wings, but it remains unclear how these changes
 affect pigmentation evolution in multiple body parts between the same
 diverging species. Drosophila elegans and D. gunungcola show pigmentation
 differences in the thorax, legs, and wings, with D. elegans exhibiting
 male-specific wing spots and D. gunungcola lacking wing spots with
 intensely dark thoraces and legs. Here, we performed QTL mapping to
 identify the genetic architecture of these differences. We find a large
 effect QTL on the X chromosome for all three body parts. QTL on Muller
 Element E were found for thorax pigmentation in both backcrosses but were
 only marginally significant in one backcross for the legs and wings.
 Consistent with this observation, we isolated the effects of the Muller
 Element E QTL by introgressing D. gunungcola alleles into a D. elegans
 genetic background and found that D. gunungcola alleles linked near the
 pigmentation candidate gene ebony caused intense darkening of the thorax,
 minimal darkening of legs, and minimal shrinking of wing spots. D. elegans
 ebony mutants showed changes in pigmentation consistent with Ebony having
 different effects on pigmentation in different tissues. Our results
 suggest that multiple genes have evolved differential effects on
 pigmentation levels in different body regions.
description: <i>Fly stocks </i>
 <i>Drosophila elegans HK (Hong Kong)</i> and
 <i>D. gunungcola SK (Sukarami) </i>species stocks were a gift
 from John True (Stony Brook University). Stock maintenance and food
 recipes are described in Massey <i>et al.</i> (2020). In
 brief, stocks were maintained at 23ºC on a 12 h light-dark cycle. At the
 third instar larval stage of development, adults were transferred onto new
 food, and Fisherbrand filter paper (cat# 09-790-2A) was added to the
 larval vials to facilitate pupariation.
 <i>Generating hybrid progeny</i>
 Male and female <i>D. elegans HK </i>flies will
 reproduce with male and female <i>D. gunungcola SK </i>in the
 laboratory to produce fertile F<sub>1 </sub>hybrid female and
 sterile F<sub>1 </sub>hybrid male offspring (Yeh <i>et
 al</i>., 2006). Creating these F<sub>1 </sub>hybrids in
 populations large enough for genetic analysis, however, is difficult.
 Detailed methods are described in Massey <i>et al</i>. (2020).
 In brief, both <i>D. elegans HK </i>and <i>D. gunungcola
 SK</i> species stocks were expanded to establish populations of more
 than 10,000 flies. Next, virgin males and females from each species were
 placed in heterospecific crosses (<i>D. elegans HK </i>males
 with <i>D. gunungcola SK</i> females and vice versa) in groups
 of ten males and females to generate fertile F<sub>1
 </sub>hybrid female offspring. Dozens of these crosses were set to
 create ~120 F<sub>1 </sub>hybrid female offspring. Since
 F<sub>1 </sub>hybrid males are sterile (Yeh <i>et
 al</i>., 2006; Yeh and True, 2014), the F<sub>1
 </sub>hybrid females were used to generate two backcross populations
 for QTL mapping. Briefly, for the <i>D. elegans HK
 </i>backcross population, ~60 F<sub>1 </sub>hybrid
 females were crossed in the same vial with ~60 <i>D. elegans HK
 </i>males and transferred onto new food every two weeks for ~2.5
 months, resulting in 724 recombinant individuals. For the <i>D.
 gunungcola SK </i>backcross population, ~60 F<sub>1
 </sub>hybrid females were crossed in the same vial with ~60
 <i>D. gunungcola SK </i>males and transferred onto new food
 every two weeks for ~2.5 months, resulting in 241 recombinant
 individuals. <i>Pigmentation
 quantification</i> For thorax pigmentation QTL
 analysis, male recombinants from each backcross population were organized
 into three thorax pigmentation classes. The lightest recombinants showed
 thorax pigmentation intensities similar to <i>D. elegans HK
 </i>and were given a score of 0; recombinants with intermediate
 thorax pigmentation intensities were given a score of 1; and recombinants
 with dark thorax pigmentation intensities similar to <i>D.
 gunungcola SK</i> were given a score of 2 (Figure 3A). To quantify
 the effects of the Muller Element E introgression region on thorax
 pigmentation (Figure 4B,C), individuals were placed thorax-side up on
 Scotch double sided sticky tape on glass microscope slides (Fisherbrand)
 (cat# 12-550-15) and imaged at the same exposure using a Canon EOS Rebel
 T6 camera mounted to a Canon MP-E 65 mm macro lens equipped with a ring
 light. The images were then imported into ImageJ software (version 1.50i)
 (Wayne Rasband, National Institutes of Health, USA;
 http://rsbweb.nih.gov/ij/) and converted to 32 bit grayscale. Using the
 “straight line segment” tool, a line was drawn between the anterior
 scutellar bristles (Figure 4B white dashed box) to measure the mean
 grayscale value of the cuticle. For leg pigmentation
 QTL analysis, methods were identical to the thorax procedures above except
 recombinants were organized into light (0), intermediate (1), and dark (2)
 classes based on the pigmentation intensity of the medial side of their
 right hindleg femur (Figure 3B). This region of the leg was chosen because
 it contains few cuticular bristles that could obscure pigmentation. To
 quantify the effects of the Muller Element E introgression region on leg
 pigmentation (Figure 4B,E), the same methods as above were used except
 right medial hindleg images were captured. In ImageJ, the “polygon
 selections” tool was used to draw a polygon selection around the medial
 side of the right hindleg femur (Figure 4B red transparency selection) to
 measure the mean grayscale value of the cuticle. Data shown in Figure 4E
 were inverted so that higher values indicated darker
 pigmentation. For wing spot pigmentation QTL analysis,
 methods are described in Massey <i>et al</i>., (2020).
 Briefly, right wings from male recombinants from each backcross population
 were imaged, and spots were quantified in ImageJ using the “polygon
 selections” tool to quantify wing spot size relative to wing size (Figure
 4B red transparency selection). The same procedure was used to quantify
 the effects of the Muller Element E introgression region (Figure
 4D). <i>Library preparation, sequencing, and
 genome assembly</i> Detailed methods for
 preparing and sequencing the genomic DNA (gDNA) libraries for the
 <i>D. elegans HK</i> and<i> D. gunungcola SK
 </i>backcross populations and advanced introgression line were
 described in Massey <i>et al</i>., (2020). Briefly, gDNA was
 extracted from male recombinants by homogenizing individuals singly in
 each well of a 96-well plate (Corning, cat# 3879). Each recombinant gDNA
 sample was then barcoded with unique adaptors, pooled into a single
 multiplexed sequencing library, size selected, and sequenced in a single
 lane of Illumina HiSeq by the Janelia Quantitative Genomics Team. Methods
 for assembling the <i>D. elegans HK </i>and <i>D.
 gunungcola SK </i>genomes to facilitate marker generation were
 described in Massey <i>et al</i>. (2020).
 <i>Marker generation with Multiplexed Shotgun
 Genotyping</i> Chromosome ancestry “genotypes”
 for the backcross progeny and introgression line were estimated with two
 Multiplexed Shotgun Genotyping (MSG) (Andolfatto et al., 2011) libraries,
 following methods described in Cande <i>et al</i>., (2012).
 Briefly, reads generated from the Illumina backcross sequencing library
 were mapped to the assembled <i>D. elegans HK </i>and
 <i>D. gunungcola SK </i>parental genomes to estimate
 chromosome ancestry for each backcross individual. We generated 3,425 and
 3,121 markers for the <i>D. elegans HK </i>and <i>D.
 gunungcola SK </i>backcrosses, respectively, for QTL analysis
 [markers, phenotypes, and procedures for QTL mapping are deposited on
 Dryad (doi:10.5061/dryad.gb5mkkwm5)]. PDFs of chromosomal breakpoints for
 each recombinant are available here:
 https://deepblue.lib.umich.edu/data/concern/data_sets/j098zb17n?locale=en. <i>QTL analysis </i> QTL analysis was performed using R/qtl (Broman and Sen, 2009) in R for Mac version 3.3.3. (R Core Team 2018). We imported ancestry data for both backcross populations into R/qtl using a custom script (https://github.com/dstern/read_cross_msg). This script directly imports the conditional probability estimates produced by the Hidden Markov Model (HMM) of MSG (described in detail in Andolfatto <i>et al</i>., 2011). We performed genome scans with a single QTL model using the “scanone” function of R/qtl and Haley-Knott regression (Haley and Knott, 1992) for thorax, leg, and wing pigmentation. For QTL mapping using the <i>D. elegans HK </i>backcross population, we excluded 18 and 20 individuals for thorax and leg pigmentation, respectively, because fly samples were either too poor to image or sequencing reads were too shallow to map. For the <i>D. gunungcola SK</i> backcross population, we excluded 12 and 9 individuals for thorax and leg pigmentation, respectively, for the same reasons. For wing pigmentation QTL analysis, we previously (Massey <i>et al</i>., 2020) reported on an ~400 kb fine-mapped region on the X chromosome explaining the majority of variation for wing spot size. We also reported on QTL underlying variation in wing spot size with spotless recombinants removed from the analysis (Massey <i>et al.</i>, 2020). Here, in Figure 3D, we show QTL underlying this variation to emphasize the role of Muller Element E in spot size divergence. Significance of QTL peaks at α = 0.01 was determined by performing 1000 permutations of the data. <i>Introgression of black body color alleles from </i>D. gunungcola SK<i> into </i>D. elegans HK To isolate individual QTL underlying body color differences between <i>D. elegans HK </i>and <i>D. gunungcola SK</i>, F<sub>1</sub> hybrid females and <i>D. elegans HK </i>backcross recombinants were generated using the methods described above. Next, dark black/brown female recombinants were repeatedly backcrossed with <i>D. elegans HK </i>males for four generations (BC3-BC6). Finally, we generated a single BC6 dark black homozygous introgression line that we then genotyped using MSG (Figure 4A,B). <i>Creating an </i>ebony<i> null allele in </i>Drosophila elegans HK<i> via CRISPR-Cas9 genome editing</i> Using methods described in Bassett <i>et al</i>. 2013, we <i>in vitro</i> transcribed (MEGAscript T7 Transcription Kit, Invitrogen) three single guide RNAs (sgRNAs) (Supplemental File S1) with target sequences designed based on conserved sites between <i>D. elegans HK </i>and <i>D. gunungcola SK</i> in exon 2 of the <i>ebony</i> gene. After transcription, sgRNAs were purified using an RNA Clean and Concentrator 5 kit (Zymo Research), eluted with nuclease-free water, and quantified using a Qubit RNA BR Assay Kit (Thermo Fisher Scientific). Next, mature (&gt;2 weeks old) <i>D. elegans HK </i>males and females were transferred to 60 mm embryo lay cages (GENESEE Part Number: 59-100) on top of 60 mm grape plates (3% agar + 25% grape juice + 0.3% sucrose) at high densities (&gt;300 flies) after brief CO<sub>2</sub> anesthesia. After CO<sub>2</sub> anesthesia, mature, mated <i>D. elegans HK </i>females will often dispel an embryo from their abdomen where it sticks briefly to the anus. Tapping the grape plate + embryo cage down on a hard surface 10 times causes the embryos to stick to the grape agar. Flies were then transferred back into food vials and embryos were lined up on glass cover slips taped to a glass microscope slide. For CRISPR/Cas9 injections into <i>D. elegans HK </i>embryos, Cas9 protein (PNA Bio #CP01), phenol red, and all three sgRNAs were mixed together at 400 ng/µl, 0.05%, and 100 ng/µl final injection concentrations, respectively. All CRISPR injections were performed in-house, using previously described methods (Miller <i>et al</i>, 2002). Finally, we screened for germline mutants based on body pigmentation and confirmed loss of Ebony protein by western blot (Supplemental Figure S1). To the best of our knowledge, these are the first gene editing experiments to succeed in <i>D. elegans</i>. We attempted the same experiments in <i>D. gunungcola SK</i>, but failed to recover any mutants. <i>Western blotting</i> Western blot methods were followed similar to Wittkopp <i>et al</i>. (2002). In brief, for each replicate per genotype, four newly eclosed (within 60 min) male flies were homogenized in 100 µl of 125 mM Tris pH 6.8, 6% SDS and centrifuged for 15 min. The supernatant was then transferred to a new protein low-bind Eppendorf tube with an equal amount of 2X Laemmli sample buffer [4% SDS, 20% glycerol, 120 mM Tris-Cl (pH 6.8)], boiled for 10 min, and stored at -80ºC. Before gel electrophoresis, samples were thawed at room temperature for 30 min, and 20 µl of each sample was loaded into individual wells of an Invitrogen NuPAGE 4-12% Bis-Tris Gel. The gel was run at 175 V for 60 min, washed, and transferred to an Invitrogen iBlot 2 PVDF Mini Stack Kit. The mini stack was run on an iBlot 2 (ThermoFisher, Catalog Number: IB21001) to perform western blotting transfer, and blocked using an Invitrogen Western Breeze anti-rabbit kit (Catalog Number: WB7106). After two washes with diH<sub>2</sub>O, samples were incubated in 1:400 rabbit anti-Ebony (Wittkopp <i>et al</i>., 2002) overnight at 4ºC. Finally, samples were washed using the Western Breeze wash solution, incubated in secondary antibody solution alk-phos. (Catalog Number: WP20007) with conjugated anti-rabbit for 30 min, washed for 2 min with diH<sub>2</sub>O, and prepared for chromogenic detection. <i>Statistics</i> Statistical tests were performed in R for Mac version 3.3.3 (R Core Team 2018). ANOVAs were performed with post hoc Tukey HSD for pairwise comparisons adjusted for multiple comparisons. See “QTL analysis” methods for statistical tests used during QTL mapping.