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Biol Lett. Dec 22, 2005; 1(4): 492–495.
Published online Aug 19, 2005. doi:  10.1098/rsbl.2005.0364
PMCID: PMC1626383

Deleterious genomic mutation rate for viability in Drosophila melanogaster using concomitant sibling controls

Abstract

New deleterious mutations may reduce health and fitness and are involved in the evolution and maintenance of numerous biological processes. Hence, it is important to estimate the deleterious genomic mutation rate (U) in representative higher organisms. However, these estimated rates vary widely, mainly because of inadequate experimental controls. Here we describe an experimental design (the Binscy assay) with concomitant sibling controls and estimate U for viability in Drosophila melanogaster to be 0.31. This estimate, like most published studies, focuses on viability mutations and the overall deleterious genomic mutation rate would therefore be higher.

Keywords: mutation rate, mutation accumulation, concomitant sibling controls, Drosophila melanogaster

1. Introduction

Health and fitness may be reduced each generation by newly arising deleterious mutations (Drake et al. 1998; Lynch et al. 1999). These genetic changes are also involved in the evolution and maintenance of sexual reproduction and recombination, aging, diploidy, inbreeding avoidance, degeneration of Y chromosomes, genetic control of DNA-element transpositions, DNA repair, and the risk of extinction of endangered species (Charlesworth & Charlesworth 1998; Lynch et al. 1999). With relaxed selection, the increased accumulation of deleterious mutations is a particular threat to the genetic well-being of future humans (Crow 1997).

Hence, it is important to estimate the deleterious genomic mutation rate (U) in representative higher organisms. This rate seems to be very high in humans, with indirect estimations of up to three new deleterious mutations per individual (Eyre-Walker & Keightley 1999). However, estimated rates in other higher organisms vary widely. For example, U estimates in the model organism Drosophila melanogaster range from 0.01–1, with extensive ongoing debates on the correct value (Drake et al. 1998; Lynch et al. 1999; Fry 2001; Keightley & Eyre-Walker 2001; Kondrashov 2001; Garcia-Dorado & Caballero 2002; Rice 2002; Garcia-Dorado et al. 2004). One of the main reasons for difficulties in determining U in D. melanogaster is inadequate experimental controls (Kondrashov 1998; Houle & Nuzhdin 2004). In the standard methods of measuring the accumulation of deleterious mutations over time in D. melanogaster, the comparative controls have in many cases been unreliable. Examples of these controls include the original base stock, the most fit lines at the end of the experiment and base stocks frozen at the beginning of the experiment. Problems have arisen in controls because of contamination with other flies, low recovery of frozen stocks, and genetic changes in the base stock over time (Kondrashov 1998).

What is needed is an experimental design where deleterious mutations both do and do not accumulate in morphologically distinct siblings that have similar genetic backgrounds and are raised in identical environments (Kondrashov 1998). In this study, we will describe such an experimental design, the Binscy assay.

2. Material and methods

(a) The Binscy assay

In the synthesis of the Binscy assay stocks (figure 1), we modified a mating scheme of Muller (Muller & Oster 1963). Binscy is a balancer X chromosome with the B (Bar eyes, dominant) and y (yellow body, recessive) mutations, plus multiple inversions that eliminate recombination of the X chromosome in females. We confirmed the balancing ability of the Binscy X chromosome, as described by Lindsley & Zimm (1992), by observing no recombinants among 622 progeny from w m f/Binscy females, whereas 206 recombinants were recovered among 584 progeny from w m f/+++ females. C(1;YS)oc ptg is a compound X and short arm of the Y chromosome, with the oc (ocelliless, missing ocelli, homozygous female sterile) and ptg (pentagon, thoracic trident dark) recessive mutations; and RYL is the long arm of the Y chromosome in the shape of a ring (Lindsley & Zimm 1992). The Binscy/RYL males are sterile because of the missing male fertility factors in the short arm of the Y chromosome, and C(1;YS)oc ptg/C(1;YS)oc ptg females are sterile because of the homozygous oc mutation (Lindsley & Zimm 1992). Each generation, one Binscy/C(1;YS)oc ptg female was mated with one C(1;YS)oc ptg/RYL male per vial at 25 °C on standard cornmeal and agar medium. This mating scheme is designed to allow new deleterious mutations to accumulate over generations on the Binscy balancer X chromosome in Binscy/C(1;YS)oc ptg females. Since C(1;YS)oc ptg homozygous females and Binscy/RYL males from this mating scheme are sterile, new deleterious mutations that occur on the X chromosome of C(1;YS)oc ptg/RYL males are in a hemizygous state and are eliminated by selection. To the extent that deleterious mutations might temporarily be present on C(1;YS)oc ptg, our estimate of U will be reduced. Conversely, deleterious mutations that occur on the Binscy X chromosome in Binscy/C(1;YS)oc ptg females are buffered from selection because mutations are maintained as heterozygotes against wild-type alleles on the C(1;YS)oc ptg chromosome. Hence, with time deleterious mutations will accumulate on the Binscy chromosome, but are not expected to remain on the C(1;YS)oc ptg chromosome. We also tested for hybrid dysgenesis in our crosses (see methods in Boussy et al. 1998). None was observed.

Figure 1
The mating scheme for the accumulation of deleterious mutations in the Binscy X chromosome of Drosophila melanogaster. In every generation (G0, G1, G2 … etc.), one single female and one single male are randomly selected to mate for each ...

A total of 108, 225 and 225 lines were initiated in the three runs, and 80, 141 and 71 lines completed each run, respectively. Run A was for 16 generations, whereas Runs B and C were each for 25 generations. Viability was measured for each line and each generation by counting the ratio of Binscy/RYL males to total males (Binscy/RYL and C(1;YS)oc ptg/RYL males). Any line that gave three generations or more with no Binscy/RYL males was considered to contain a new Binscy lethal mutation and was discarded. Only those lines that completed all generations were included in the final determination of viability.

(b) Method to estimate deleterious genomic mutation rate

The genomic mutation rate can be calculated using a method proposed by Bateman (1959) and Mukai (1964). The accumulation of deleterious mutations will lead to a predicted steady reduction in mean viability and an increase in variance between lines. The rate of decline in mean viability (ΔM) and the rate of increase in variance of viability between lines (ΔV) can be estimated by regression analysis.

If one assumes that spontaneous mutations are distributed on the X chromosome according to a Poisson distribution, ΔM and ΔV can be expressed as

ΔM=MsUx,
(2.1)
ΔV=(Ms2+Vs)Ux,
(2.2)

where Ms and Vs are the mean and the variance of s (the effect of a mutation on viability), respectively, and Ux is the mean number of deleterious mutations in the X chromosome in one generation.

Rearranging equations (2.1) and (2.2) gives

Ux=(1+k)ΔM2ΔV,
(2.3)
Ms=11+kΔVΔM,
(2.4)
Vs=[1(1k)2(1+k)2](ΔV2ΔM)2,
(2.5)

where k=Vs/Ms2. Let

Uxl=Ux1+k=ΔM2ΔV,
(2.6)
Msu=Ms(1+k)=ΔVΔM,
(2.7)
Vsu=Vs1(1k)2(1+k)2=(ΔV2ΔM)2,
(2.8)

then Uxl is a lower bound of Ux, Msu is an upper bound of Ms and Vsu is an upper bound of Vs.

The Binscy assay estimates the haploid genomic mutation rate using the X chromosome. Since an X chromosome contains around 15.97% of the genes in the haploid genome in D. melanogaster1, the lower bound of the diploid genomic mutation rate that we report here (Ul) is about two times Uxl divided by 15.97%.

(c) Statistical analysis

A bootstrap resampling method (Charlesworth et al. 2004) was employed to get interval estimations of Ul, Msu and Vsu, using the Minitab software package. From the observed viability data of each of the three experimental runs, the same number of lines as in that run were sampled with replacement 300 times. For each bootstrap sample, ΔM, ΔV, Ul, Msu and Vsu were calculated by the method mentioned above. Confidence intervals of these parameters then can be obtained from those bootstrap samples.

3. Results and discussion

The numbers of Binscy X chromosomes with new lethal mutations and the total numbers of Binscy X chromosomes assayed in three experimental runs are recorded in table 1. If any line had no Binscy/RYL males for three generations or more, a new lethal mutation in a Binscy X chromosome was counted. The total number of Binscy X chromosomes assayed is the summation of the number of lines screened in every generation. There were 47 lethal mutations among 8446 X chromosomes screened in all three runs. Therefore, the estimated spontaneous lethal mutation rate for the Binscy X chromosome in females is 0.0056. This rate is similar to some previous estimations (Woodruff et al. 1983, 1984).

Table 1
Lethal mutation rates (with standard errors) in three runs of mutation accumulation experiments.

As predicted, the mean viability of Binscy/RYL males decreased and the variance increased for each of the three experimental runs (figure 2). The rate of decline in the mean viability (ΔM) and the rate of increase in the variance of viability between lines (ΔV) were determined from the slopes of the curves, which can be estimated by linear regression. Using the method described above, our estimations of mutational parameters are given in table 2. Weighting each run by the inverse of its bootstrap variance (Charlesworth et al. 2004), the average genomic mutation rate for viability (Ul) is 0.31 and the average effect of a deleterious mutation (Msu) is 0.14.

Figure 2
Changes in the mean viability and variance over time for deleterious mutations in three runs (Run A has 80 lines, Run B has 141 lines and Run C has 71 lines) of the Binscy assay of Drosophila melanogaster.
Table 2
Deleterious mutation rate for the X chromosome (Uxl) and the diploid genome (Ul) in three runs.

The value of Ul (Ul=0.31) is a lower bound of the deleterious genomic mutation rate (U). U equals to Ul only when the mutational effect (s) is constant. The relation between Ul and U depends on the coefficient of variance of s (i.e. k), which is determined by the distribution of the parameter s. For example, if s follows an exponential distribution, k=1, U=2Ul and Ms=0.5Msu (Drake et al. 1998). Hence, our estimation of the deleterious genomic mutation rate is at least 0.31 and could be higher.

This estimation of the deleterious genomic mutation rate in D. melanogaster is lower than the estimations of Mukai (0.7–0.8) (Mukai 1964; Mukai et al. 1974), but higher than some recent estimations (0.04–0.12) (Fry et al. 1999; Garcia-Dorado et al. 1999; Charlesworth et al. 2004). The average effect of a mutation (Msu) of 0.14 is substantially higher than reported by Mukai (0.02–0.03) (Mukai 1964; Mukai et al. 1974), but is more in accordance with other recent reports (0.08–0.25, Garcia-Dorado et al. 2004; 0.10, Charlesworth et al. 2004). In addition, the rate of decline in mean viability of about 4% (Msu of 0.14×Ul of 0.31) is larger than previously reported. Although the reason for this large value is unknown, it may be related to the deleterious mutations recovered in this study being on the X chromosome.

The observed deleterious mutation rate of 0.31 is below 1, which is predicted to be the minimum level at which sex may have evolved to eliminate deleterious mutations through recombination leading to epistatic selection (Kondrashov 1988, 1998). The Binscy assay allows us to avoid one key criticism of other estimation techniques by providing concomitant sibling controls, but there are other complications that affect estimates in general, such as the occurrence of epistatic interactions and mutations affecting processes other than viability, that will make our rate an underestimate of the overall deleterious genomic mutation rate.

Acknowledgments

We thank Priti Azad, Brian Charlesworth, Sheng Gu, Gillian Millburn and Elizabeth Morgan for their valuable advice. This study was sponsored by NASA grants NAG 2-1427 and NCC 2-1355.

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