Fig. 1: The ebony gene is linked to the black pupae phenotype in GUA10.
From: Functional genomics implicates ebony in the black pupae phenotype of tephritid fruit flies
a The bp phenotype is the sex-linked trait used for sex-sorting the mexfly GUA10 strain. Females from GUA10 exhibit black larval anal lobes (al) and pupal cases, developing into darker adults. Mutant adults are noticeable due to their darker wing stripes (wg), abdomen (ab), and ovipositor (ov). b To identify the loci responsible for the bp phenotype, we introgressed the GUA10 bp mutation (bp−(GUA10)) into a common wildtype genetic background. Subsequently, we established and whole-genome sequenced a F4 mapping population where siblings develop either wildtype brown (wt) or mutant black (bp) pupal cases. Illustration credit: Alexander Spengler. c Trait mapping analysis (calculated for 100 Kb windows at 20 Kb sliding intervals across the reference genome) located a large interval within the A. ludens chromosome 2 showing significant genetic differentiation (FST) between F4 black and brown pupae siblings (n = 18 from each group). The horizontal line indicates the top 0.25th percentile threshold for significant intervals. d The gene set within this causal region include the candidates yellow-f2 and ebony from the melanin biosynthesis pathway. e RNA-Seq coverage (calculated from merged BAM files of n = 3 biological replicates) revealed the silencing of ebony expression in black pupae females from GUA10. f The ebony gene is differentially expressed between homozygous black pupae female (F) and heterozygous brown pupae male (M) siblings from GUA10 (genotypes in Fig. 1a), while no difference is detectable for yellow-f2. The expression of the white pupae gene, an MFS transporter required to provide pigment precursors to the pupal cuticle, is shown as a reference. Dots represent biologically independent replicates (n = 3), * = FDR < 0.05 and log2FC > 2, and ns = non-significant. The mean ± SD are shown in a gray dot and error bars, respectively. g Semiquantitative RT-PCR assays further validated the RNA-Seq results (n = 3 biological replicates). Amplifications of Rpl18 are internal controls. L = 100 bp DNA ladder (NEB), gDNA = genomic DNA control, and NTC = non-template negative control.
