Fig 1.
Heritable phenotypic variation in tolerance of P-element activity in the female germline.
(A) Crossing scheme for documenting variation in tolerance of P-element activity among RIL offspring, including representative images of atrophied and normal ovaries. (B and C) Phenotypic variation in the proportion of 3-day-old (B) and 21-day-old (C) F1 female offspring of different RILs. RILs are sorted according to the proportion of F1 atrophy observed in their offspring, and error bars indicate the standard error of the estimated proportion. (D) Scatterplot of the arsine-transformed proportion of F1 atrophy observed among 3-day-old and 21-day-old offspring of the same line, after accounting for the effects of experimenter and experimental block. The individual numerical values for panels B, C, and D can be found in S1, S2 and S3 Data, respectively. F1, filial 1; RIL, recombinant inbred line.
Table 1.
The position of the major QTL peak for 3-day-old F1 females, 21-day-old F1 females, and the combined data set are shown. For each analysis, the peak position, Δ2-LOD drop confidence interval, and Bayesian credible interval [44] in dm6 [45] are provided. The percent of phenotypic variation explained by the QTL peak is based on the genotype of each sampled RIL at the LOD peak position. The individual numerical values required to identify LOD peaks and intervals for 3-day-old, 21-day-old, and both F1 females can be found in S4, S5 and S3 Data, respectively.
Fig 2.
QTL mapping of variation in P-element tolerance.
(A) LOD ratio of the observed association between maternal RIL genotype [29] and the adjusted proportion of F1 atrophy phenotype. Higher LOD scores correspond to stronger evidence of linkage, and significant LOD scores are above the threshold (dotted line), which was obtained from 1,000 permutations of the observed data. (B) Two LOD drop confidence interval of the QTL peak based on a combined QTL analysis including both 3-day-old and 21-day-old F1 females. Genes indicated by red letters are potentially affected by polymorphisms in the RIL founders that are in phase with the inferred allelic classes (Fig 3 [46]). Gene models indicated in red are highly expressed in the Drosophila melanogaster ovary [47]. The individual numerical values required to generate LOD plots for 3-day-old and 21-day-old F1 females can be found in S4 and S5 Data, respectively. bru1, bruno; bru2, bruno-2; cM, centimorgan; crok, crooked; F1, filial 1; LOD, logarithm of the odds; P-element; QTL, quantitative trait locus; rho-6, rhomboid 6; RIL, recombinant inbred line.
Fig 3.
Phenotypic classes of founder alleles at the major QTL peak.
The mean adjusted F1 atrophy among 3-day-old females (A) and 21-day-old females (B) is shown for RILs carrying each of the eight founder alleles. Error bars denote standard error. QTL phasing (see Materials and methods) detects two allelic classes for both the 3-day-old and 21-day-old phenotypes: a sensitive allele that increases the odds of F1 ovarian atrophy (pink) and a tolerant allele that decreases the odds of F1 ovarian atrophy (red). The assignment of founder alleles to phenotypic classes across ages is consistent for all founders except A8. (C) In-phase SNPs in Population A RIL founder genomes, indicated by their position in the Drosophila melanogaster reference assembly (dm6, [45]). SNPs are colored according to the function of the affected sequence, and shaded according to whether the founder exhibits the reference (light) or alternate (dark) allele. The individual numerical values required to generate bar plots for panels A and B can be found in S4 and S5 Data, respectively. In-phase polymorphisms represented in panel C are provided in S1 Table. bru1, bruno CDS, coding sequence; dm6, Drosophila melanogaster reference assembly; F1,filial 1; QTL, quantitative trait locus; RIL, recombinant inbred line; SNP, single nucleotide polymorphism.
Fig 4.
Tolerant alleles are associated with enhanced fertility and reduced bruno expression.
Phenotypes of background-matched RILs (A, B, and C) carrying sensitive (A5, pink) and tolerant (A4, red) haplotypes across the QTL peak are compared. (A-B) Incidence of ovarian atrophy (A) and sterility (B) among dysgenic F1 female offspring of crosses between RIL females and Harwich males. Numbers in each bar indicate the sample size. (C) Offspring production of fertile F1 females from B. (D) Ovarian bruno expression relative to rpl32 in each RIL. Error bars in A, B, and D indicate the standard error. The individual numerical values required to generate bar and box plots for panels A, B/C, and D can be found in S6, S7 and S8 Data, respectively. F1, filial 1; F2, filial 2; QTL, quantitative trait locus; RIL, recombinant inbred line.
Fig 5.
Mutational analysis of candidate genes.
(A) Loss-of-function heterozygotes for three candidate genes are compared to their siblings, inheriting the balancer chromosome CyO in order to detect zygotic effects on the incidence of ovarian atrophy among 3–7-day-old dysgenic F1 offspring. (B) Single and double loss-of-function and deficiency heterozygotes for bruno and oskar are compared with their siblings inheriting the balancer chromosomes CyO (bruno) and TM2, TM3, or MKRS (oskar), in order to detect zygotic effects on the incidence of atrophy among 3–7-day-old dysgenic F1 offspring. Offspring from the same cross are represented consecutively on the graph. While bruno alleles act as consistent dominant suppressors in both single and double mutants, oskar deficiencies and mRNA null mutants exhibit only stochastic effects on the atrophy phenotype, suggesting that the mechanism of bruno suppression of F1 atrophy is independent of oskar mRNA function. (C-D) Representative germline development among atrophied and non-atrophied ovaries of brunoQB/+ and CyO/+ dysgenic offspring (B). (C) Atrophied ovaries lack any Vasa-positive germline cells in the ovarioles, including GSCs. Nuclear Vasa external to Hts-1B1 corresponds to the somatic ovarian sheath [35]. (D) Non-atrophied (i.e., morphologically normal) ovaries exhibit the full range of developing oocytes, including GSCs (white arrow). Arrowheads correspond to CBs, the undifferentiated daughters of GSCs. Representative ovaries in both C and D are CyO/+; however, atrophied ovaries (C) are more common among brunoQB/+ (B). Cytological markers for C and D are hu li tai shao (Hts-1B1), which labels somatic follicle cell membranes, the circular fusomes of GSCs and CBs, and the spectrosomes that connect cells in the developing cyst, and Vasa, which labels the cytoplasm of germline cells and nuclei of the ovarian sheath, and DAPI staining of nuclear DNA. The individual numerical values required to generate bar plots in panels A and B can be found in S9 and S10 Data, respectively. CB, cystoblast; CyO, Curly-O; F1, filial 1; GSC, germline stem cell; Hts-1B1, hu li tai shao; ThrRs, Threonyl-tRNA synthetase; TM2, third marked 2; TM3, third marked 3.