Nuclear genetic background influences the phenotype of the Drosophila tko25t mitochondrial protein-synthesis mutant

Abstract

The Drosophila tko25t point mutation in the gene encoding mitoribosomal protein S12 produces a complex phenotype of multiple respiratory chain deficiency, developmental delay, bang-sensitivity, impaired hearing, sugar and antibiotic sensitivity, and impaired male courtship. Its phenotypic severity was previously shown to be alleviated by inbreeding and to vary with mitochondrial genetic background. Here, we show similarly profound effects conferred by nuclear genetic background. We backcrossed tko25t into each of 2 standard nuclear backgrounds, Oregon R and w1118, the latter used as recipient line in many transgenic applications requiring selection for the white minigene marker. In the w1118 background, tko25t flies showed a moderate developmental delay and modest bang-sensitivity. In the Oregon R background, males showed longer developmental delay and more severe bang-sensitivity, and we were initially unable to produce homozygous tko25t females in sufficient numbers to conduct a meaningful analysis. When maintained as a balanced stock over 2 years, tko25t flies in the Oregon R background showed clear phenotypic improvement though were still more severely affected than in the w1118 background. Phenotypic severity did not correlate with the expression level of the tko gene. Analysis of tko25t hybrids between the 2 backgrounds indicated that phenotypic severity was conferred by autosomal, X-chromosomal, and parent-of-origin-dependent determinants. Although some of these effects may be tko25t specific, we recommend that, in order to minimize genetic drift and confounding background effects, the genetic background of nonlethal mutants should be controlled by regular backcrossing, even if stocks are usually maintained over a balancer chromosome.

Keywords: mitochondria; nuclear background; ribosome; semilethality.

Fig. 1.

Backcrossing scheme for tko^25t. At each backcross generation, heterozygous (tko^25t/+) females were identified by single-organism genomic DNA extraction, PCR, and sequencing, after pre-mating. Progeny from wild-type females was discarded. Several parallel lines were established in each backcross, both to the w¹¹¹⁸ and to the Oregon R backgrounds. The FM7-balanced, backcrossed lines were established in parallel.

Fig. 2.

Phenotype of tko^25t in different nuclear backgrounds. a) Proportion of tko^25t males among eclosing progeny from crosses of the type tko^25t/FM7×FM7/Y, conducted at 25°C, after back-crossing of tko^25t into the Oregon R background (OR bkd), to create lines OR1, OR3, and OR5 as indicated, or into the w¹¹¹⁸ background, to create lines w3, w4, and w5. The dotted line indicates the expected proportion of tko^25t/Y progeny (25%). n = 208 (OR1), 295 (OR3), 314 (OR5), 363 (w3), 330 (w4), and 338 (w5) total eclosed adults, from 4 individual vials in each case. In the original, lab-maintained tko^25t stock, the percentage of mutant males in similar crosses without other balancers was always close to the expected 25%, i.e. similar to the findings for tko^25t backcrossed into w¹¹¹⁸. The output of males in each of the 2 background control strains in equivalent crosses was routinely the expected ∼50%. b) Eclosion times (means ± SD) for tko^25t flies generated in multiple crosses using the original tko^25t stock. In the 3 experiments shown (orig 1, orig 2, and orig 3), mothers were tko^25t/FM7 and carrying different autosomal balancers (respectively, CyO, TM3-Sb, and TM3-Ser) with the scored tko^25t progeny free of all balancer markers, some of which are known to interact with tko^25t [see Supplementary Fig. 2 of George et al. (2019), which presents some of the same data]. The number of adult flies of each genotype/sex analyzed is indicated above the applicable column. n = 5 replicate vials in all cases except for line w3, where n = 4 replicate vials. All controls were tko^25t/FM7 heterozygotes in the same background which, in both back-cross backgrounds, gave indistinguishable eclosion times as wild-type males in the same background (Supplementary Fig. 1a). c) Eclosion times (means ± SD) for similar crosses as in a), but carried out at room temperature (21–22°C). The number of adult flies of each genotype/sex analyzed is indicated above the applicable column. n = 5 replicate vials in all cases. d) Bang-sensitivity assay (recovery times) for progeny flies of the indicated genotypes from the crosses in c), assayed at 22°C. n = 35 (OR1), 38 (OR5), 38 (w3), and 29 (w5) tko^25t mutant males, and 119 (OR) and 132 (w¹¹¹⁸) control males. Box plots show 25th and 75th percentiles of each distribution and medians (bold lines). Note that the scatter plots are only indicative, since many data points are coincident or almost completely overlapping on this scale: for full details of source data, see Supplementary Table 1.

Fig. 3.

Phenotype after 2 years in the culture of tko^25t backcrossed into different nuclear backgrounds. a) Developmental delay (days, d, means ± SD) and b) bang-sensitivity recovery times (box-plot nomenclature as in Fig. 2) for tko^25t flies and controls of the indicated back-crossed lines, after 2 years in culture as balanced stocks. Crosses of the type tko^25t/FM7×tko^25t/Y were conducted at 25°C, with controls being tko^25t/FM7 heterozygotes. In a), a repeat experiment is shown for the w3 line (hatched rectangles), due to the large error bars seen for mutant females in the primary experiment. The number of adult flies of each genotype/sex analyzed is indicated above the applicable column. n = 3 replicate bottles in all cases. In b), the same data for tko^25t flies in the w¹¹¹⁸ background are shown at 2 different scales, as indicated by the dotted lines, with the 300-seconds data points omitted, for clarity, in the expanded-scale graph (right-hand panel). n = 70 (OR3 females), 46 (OR3 males), 104 (w3 females), and 85 (w3 males). Note that, as for Fig. 2d, the scatter plots are only indicative, since many data points are coincident or almost completely overlapping on both scales: for full details of source data, see Supplementary Table 2. Control flies (see Fig. 2d) were not bang-sensitive.

Fig. 4.

Expression level of tko in tko^25t flies backcrossed into 2 nuclear backgrounds. tko RNA levels by qRT-PCR (means ± SD), for flies of the indicated sex, background, and culture temperature, normalized a) to values for one isolate of Oregon R wild-type females and b) to mean values for control flies of the same sex and background (Oregon R wild-type or w¹¹¹⁸, as appropriate). *, **, *** denote significantly different data classes (pairwise comparisons between genotypes, same sex, and culture temperature, by Student's t-test, P < 0.05, 0.01, or 0.001, respectively). For a), despite experiment-to-experiment variation, tko expression was generally lower in the w¹¹¹⁸ background in both sexes and at all temperatures tested, as in the example shown. n = 3 technical replicates of each of 3 biological replicates of batches of 5–7 adults, for all genotypes and sexes.

Fig. 5.

Eclosion timing in tko^25t strain-background crosses. Developmental delay (days, d, means ± SD) at 25°C of a) female and b) male tko^25t progeny from the indicated crosses between tko^25t/FM7 females and tko^25t/Y males in the backgrounds as shown (OR—Oregon R, w—w¹¹¹⁸). For ease of comparison, dotted lines indicate values for the non-hybrid crosses, reproduced from Fig. 3a, which were conducted in parallel using the same expanded parental stocks. Data from a) and b) are from the same crosses, but with the sexes shown separately, for clarity. Control flies were tko^25t/FM7 heterozygous females, which show an identical eclosion timing as wild-type males (see Supplementary Fig. 1a), with only the SD shown (means were all set to zero, so as to exclude minor, strain-specific differences in eclosion timing from the analysis). The number of adult flies of each genotype/sex analyzed is indicated above the applicable column. n = 3 replicate bottles in all cases.

Fig. 6.

Bang-sensitivity in tko^25t strain-background crosses. Recovery times (plotted cumulatively, over time) at 25°C for a) female and b) male tko^25t progeny from the indicated crosses between tko^25t/FM7 females and tko^25t/Y males of the indicated parental strain backgrounds (OR—Oregon R background, w—w¹¹¹⁸ background). Control progeny from all crosses were not bang-sensitive. n = 60 (OR × OR), 117 (OR × w), 119 (w × OR), and 118 (w × w) females, and 84 (OR × OR), 74 (OR × w), 56 (w × OR), and 53 (w × w) males.

Fig. 7.

Male courtship effects of tko^25t and w¹¹¹⁸. Percentage of successful copulation (mean ± SEM) of multiple samplings of 6 pairs of flies, at the indicated temperatures. In all assays, male and female flies were of the same genotype and background, and all had red eye color. n—number of batches analyzed for each genotype as indicated. Note that, to exclude the possibility of contamination, the tko locus from the red-eyed control flies back-crossed into the w¹¹¹⁸ background was sequenced and confirmed to be tko⁺.