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THE DOMINANCE OF NEWLY ACQUIRED MUTATIONS IN
RHABDITID NEMATODES Jeff Rosenbloom, Kunjal Gandh
i, Joseph Hong, Matt Salomon, Andrew Custer, and
Charles Baer University of Florida department of
Zoology, Gainesville, FL
ABSTRACT
RESULTS
We present a study to further analyze dominance
in C. elegans in the attempt to clarify the
recurring trend of overdominance in previous
studies. Two methods were used, a regression
analysis and by the deviation of the heterozygote
from the homozygotes. A heterozygote cross was
compared to two homozygote crosses, and point
estimates of fitness suggest overdominance with
high variability. Also, we found that under the
regression method, a new mutation was nearly
recessive, and using the comparison of the
heterozygote, overdominance was strongly
represented.
Heterozygote fitness was greater than that of
either homozygote for R12, R3, and Productivity
(Figure 1), although means did not differ
significantly between cross types for any trait.
The point estimates for hr,R12 0.20, hr,R3
0.14 hr,Productivity was inestimable because the
REML estimate of the among-(MA)line variance was
zero. Thus, the point estimates from the
regression suggest that the average new mutation
is partially recessive. This result is
consistent with previous estimates from
spontaneous mutations in C. elegans (Vassilieva
et al. 2000). Conversely, the point estimates
when control lines are included suggest that the
average mutation is substantially overdominant
hM,R12 -15.9, hM,R3 -1.0, hM,Prod 3.1
(Figures 2a, b, and c).
INTRODUCTION
The degree of dominance of new mutations has
important consequences for several areas of
biology, among them the evolution of sexual
reproduction (Chasnov et al. 2000 Kondrashov
1988) and mating system (Charlesworth and Hughes
1996 Houle et al. 1997), the response to
selection and the mean fitness of populations
(Peters et al. 2003 Vassilieva et al. 2000), and
the maintenance of genetic variation
(Charlesworth and Hughes 1996 Zhang et al.
2003). An issue of particular importance is the
prevalence of overdominance, i.e., mutations that
confer higher fitness on heterozygotes than for
either homozygote, because overdominant alleles
will be maintained at high frequency by natural
selection (Peters et al. 2003). From work in
Drosophila and Caenorhabditis elegans - on
average, new mutations are partially recessive
(i.e., 0 et al. 2003 Zhang et al. 2003, Mukai et al.
1972). The coefficient of dominance varies
inversely with the homozygous effect - mutations
of large effect (i.e., lethal or sterile) are
nearly completely recessive, whereas mutations
with small effects tend to be nearly additive.
The degree to which taxa may vary in average
dominance is an open question, and in fact there
are reasons to suspect that dominance
relationships may evolve, e.g., in predominantly
selfing or asexual diploid taxa. As part of a
larger comparative investigation into the
mutational properties of Rhabditid nematodes, we
report an initial investigation into the
dominance properties of new spontaneous mutations
in the N2 strain of C. elegans.
CONCLUSIONS
On average, heterozygotes had higher fitness than
either homozygote, although we cannot rule out a
model in which mutations have equal effects with
a mean effect of zero. The point estimates of
fitness showed overdominance in all 9 lines
(Figures 2a, b, and c). Conversely, point
estimates from the regression method (hR) suggest
that the average new mutation is nearly
recessive. This result emphasizes the importance
of including controls in such studies.
Nevertheless, all other fitness assays with this
set of MA lines have shown that they have lower
fitness than the controls (Baer 2005), so this
result is necessarily suspect. When the controls
are included, we found the dominance coefficient
to be negative, suggesting that the mutations are
overdominant. We have considered various possible
hypotheses to explain our results. We have found
high variance within lines in the pooled data,
and although we expected this based on studies by
Charlesworth and Hughes (1996), Houle et al.
(1997), and Zhang et al. (2000), the high
variability made ruling out complete dominance,
additivity, or recessivity impossible. In
addition, mutations of large deleterious effect
in males would potentially accumulate under our
MA regime, so the MA lines in this study may have
atypically high fitness. Overall, we need
greater numbers of both MA lines and (especially)
replicates within line to be able to conclusively
resolve the issue of variability of dominance of
new mutations.
Figure 2Comparison of among-line variation in
productivity between mutant, heterozygote, and
controls. Data was measured by counting offspring
produced by the F1 generation of the cross during
the a. first two days of reproduction b. the
third day of reproduction c. the total
productivity among all three days. The control
line is represented by the horizontal bar (116.5,
69.24, 185.7 respectively). Bars indicate
standard errors.
METHODS
Caenorhabditis elegans is an androdieocious
hermaphrodite the frequency of males is 0.1
under standard conditions (Baer et al. 2005).
Heat shocking C. elegans during gametogenesis can
raise the frequency of males to about 2-5
(Vassilieva et al. 2000). When males mate with
hermaphrodites, the progeny display a 11 sex
ratio. After the nematodes were heat shocked for
six hours at 30C, we isolated males and mated
them with hermaphrodites at a male hermaphrodite
ratio of 31. After two generations, we
transferred 15 males and 5 hermaphrodites per
line to a 100mm x 15mm Petri dish, from which the
population was frozen and stored until needed.
The nematodes used in this study were taken from
a set of lines that had previously undergone 250
generations of mutation accumulation (MA) by
single progeny descent, details illustrated by
Vassilieva and Lynch (1999). Three crosses were
constructed to analyze the effect of dominance of
accumulated mutations, consisting of two
homozygote crosses and a heterozygote cross. From
each of these crosses, a hermaphroditic offspring
was collected and its fitness was measured. This
study consisted of 9 lines of C. elegans at
generation 250 and their ancestral control. Each
cross was replicated ten times per line. To
ensure that there was no self-fertilization prior
to male fertilization, three worms per line per
replicate were isolated for three days and
allowed to void their sperm. A single spent
hermaphrodite was taken from that subset and
placed onto a plate with three fertile males.
From their offspring, a single, unfertilized
hermaphrodite was isolated and allowed to lay
eggs over a three day span. Plates were stored at
4C for two weeks, which arrested any further
development and reproduction. Productivity was
measured by counting the offspring after staining
individual plates with 0.075 toluidine blue
(Baer et al. 2005). Fecundity for days 1 and 2 (R
12) and day 3 (R3) were analyzed separately and
pooled (Productivity). Coefficients of dominance
(h) were calculated in two ways - hr from the
slope of the regression of heterozygote phenotype
on MA homozygote phenotype (Vassilieva et al.
2000), and hM from the deviation of the
heterozygote from the midpoint of the wildtype
and MA homozygotes. hM 1 is indicative of
overdominance. Components of covariance were
calculated by REML as implemented in SAS v. 9.1
PROC MIXED. The regression slope (hr) was
estimated as the among-line covariance between
heterozygous and homozygous MA phenotype divided
by the among-line component of variance in the MA
phenotype.
LITERATURE CITED
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Figure 1 Productivity of C. elegans. Three
crossed were set up mating mutated homozygote,
control homozygote, and heterozygote in C.
elegans. Productivity was measured by counting
total offspring generated over a three day span,
which was separated into two categories (R12,
R3). Bars indicate standard errors.