1
A maternal-effect genetic incompatibility in Caenorhabditis elegans
Eyal Ben-David
*,ā
, Alejandro Burga
*,ā
, Leonid Kruglyak
ā
Department of Human Genetics, Department of Biological Chemistry, and Howard Hughes Medical
Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
ā
To whom correspondence should be addressed. Email: ebd@ucla.edu (E.B); aburga@mednet.ucla.edu
(A.B.); lkruglyak@mednet.ucla.edu (L.K.).
* These authors contributed equally to this work.
Selfish genetic elements spread in natural populations and have an important role in genome evolution. We
discovered a selfish element causing a genetic incompatibility between strains of the nematode
Caenorhabditis elegans. The element is made up of sup-35, a maternal-effect toxin that kills developing
embryos, and pha-1, its zygotically expressed antidote. pha-1 has long been considered essential for
pharynx development based on its mutant phenotype, but this phenotype in fact arises from a loss of
suppression of sup-35 toxicity. Inactive copies of the sup-35/pha-1 element show high sequence divergence
from active copies, and phylogenetic reconstruction suggests that they represent ancestral stages in the
evolution of the element. Our results suggest that other essential genes identified by genetic screens may
turn out to be components of selfish elements.
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Introduction
Selfish genetic elements subvert the laws of Mendelian segregation to promote their own transmission
(Dawkins 1976; Doolittle and Sapienza 1980; Orgel and Crick 1980; Werren 2011; Sinkins 2011). In what
is perhaps the most extreme scenario, selfish elements can kill individuals that do not inherit them, leading
to a genetic incompatibility between carriers and non-carriers (Beeman et al. 1992; Werren 1997, 2011;
Hurst and Werren 2001; Lorenzen et al. 2008). Selfish elements are predicted to spread in natural
populations (Hurst and Werren 2001; Werren 2011), and consequently, there is significant interest in using
synthetic forms of such elements to drive population replacement of pathogen vectors in the wild (Chen et
al. 2007; Hammond et al. 2015). However, despite the prominent role of genetic incompatibilities in
genome evolution and their promise in pathogen control, their underlying genetic mechanisms have been
resolved in only a few cases (Werren 2011). Our laboratory previously identified the only known genetic
incompatibility in the nematode Caenorhabditis elegans (Seidel et al. 2008, 2011). The incompatibility is
caused by a selfish element composed of two tightly linked genes: peel-1, a sperm-delivered toxin, and
zeel-1, a zygotically expressed antidote. In crosses between isolates that carry the element and ones that do
not, the peel-1 toxin is delivered by the sperm to all progeny, so that only embryos that inherit the element
and the zeel-1 antidote survive. An analogous element, Maternal-effect dominant embryonic arrest (Medea)
has been previously described in the beetle Tribolium; however, the underlying genes remain unknown
(Beeman et al. 1992; Lorenzen et al. 2008).
Results
A maternal-effect genetic incompatibility in C. elegans
As part of ongoing efforts to study natural genetic variation in C. elegans, we introgressed a genetic marker
located on the right arm of Chr. V from the standard laboratory strain N2 into the strain DL238 by
performing eight rounds of backcrossing and selection. DL238 is a wild strain isolated in the Manuka
Natural Reserve, Hawaii, USA, and is one of the most highly divergent C. elegans isolates identified to
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date (Andersen et al. 2012). To confirm the success of the introgression, we genotyped the resulting strain
at single-nucleotide variants (SNVs) between DL238 and N2 by whole-genome sequencing. As expected,
with the exception of a small region on the right arm of Chr. V where the marker is located, most of the
genome was homozygous for the DL238 alleles (Fig. 1A). However, to our surprise, we observed sequence
reads supporting the N2 allele at many SNVs on Chr. III, including two large regions that were homozygous
for the N2 allele despite the eight rounds of backcrossing (Fig. 1A, Fig. S1). This observation suggested
that N2 variants located on this chromosome were strongly selected during the backcrossing.
To investigate the nature of the selection, we performed a series of crosses between the N2 and DL238
strains and examined their progeny. To avoid effects of the peel-1/zeel-1 element, which is present in N2
and absent in DL238, we performed a cross between DL238 males and a near isogenic line (NIL) that lacks
the peel-1/zeel-1 element in an otherwise N2 background (hereafter, N2 peel-1
-/-
) (Seidel et al. 2011). We
observed low baseline embryonic lethality in the F
1
generation and in the parental strains (0.26% (N = 381)
for F
1
; 0.99% (N = 304) for DL238; 0.4% (N = 242) for N2 peel-1
-/-
), and we did not observe any obvious
abnormal phenotypes in the F
1
that could explain the strong selection. However, when we allowed
heterozygous F
1
hermaphrodites from this cross to self-fertilize, we observed 25.15% (N = 855) embryonic
lethality among the F
2
progeny (Fig. 1B). Similar results were obtained for F1 hermaphrodites from the
reciprocal parental cross (26.1%, N = 398). These results suggested the presence of a novel genetic
incompatibility between N2 and DL238 that causes embryonic lethality in their F
2
progeny.
The observed pattern of embryonic lethality (no lethality in the parents nor in the F
1
; 25% lethality in the
F
2
) is consistent with an interaction between the genotype of the zygote and a maternal or paternal effect
(Fig. 1C) (Seidel et al. 2008). We hypothesized that the incompatibility could stem from a cytoplasmically-
inherited toxin that kills embryos if they lack a zygotically expressed antidote, analogous to the mechanism
of the peel-1/zeel-1 element (Seidel et al. 2008, 2011). To test this model and to discriminate between
maternal and paternal effects, we crossed heterozygous F
1
DL238 x N2 peel-1
ā/ā
males and hermaphrodites
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with DL238 hermaphrodites or males, respectively (Fig. 1B, Fig. S2). We observed 48.59% (N = 389)
lethality when F
1
hermaphrodites were crossed to DL238 males, but only baseline lethality (1.17%; N =
171) in the reciprocal cross of F
1
males to DL238 hermaphrodites. 50% lethality when the F
1
parent is the
mother and no lethality when the F1 parent is the father indicates that the incompatibility is caused by
maternal-effect toxicity that is rescued by a linked zygotic antidote (Fig. S2). We tested whether the new
incompatibility was independent from the paternal-effect peel-1/zeel-1 element by crossing DL238 and N2
worms and selfing the F
1
progeny. We observed 41.37% (N = 389) embryonic lethality among the F
2
progeny, consistent with expectation for Mendelian segregation of two independent incompatibilities
(43.75%) (Fig. 1D).
pha-1 and sup-35 constitute a selfish element that underlies the incompatibility between DL238 and
N2
To identify the genes underlying the maternal-effect incompatibility between N2 and DL238, we sequenced
the genome of DL238 using Illumina short reads and aligned those reads to the N2 reference genome. We
focused our attention on the two regions on Chr. III that were completely homozygous for the N2 allele in
the introgressed strain (Fig. 1A, Fig. S1). Inspection of short read coverage revealed a large ~50 kb region
on the right arm of the chromosome with very poor and sparse alignment to the N2 reference (Chr III:
11,086,500 ā 11,145,000) (Fig. 2A). This region contains ten genes and two pseudogenes in N2. We noticed
that pha-1, annotated as an essential gene in the reference genome, appeared to be completely missing in
DL238 (Fig. 2A) (Schnabel and Schnabel 1990). pha-1 was originally identified as an essential gene
required for differentiation and morphogenesis of the pharynx, the C. elegans feeding organ (Schnabel and
Schnabel 1990). But if pha-1 is essential for embryonic development and missing in DL238, then how are
DL238 worms alive? pha-1 lethality can be fully suppressed by mutations in three other genes: sup-35, sup-
36, and sup-37 (Schnabel et al. 1991). We found no coding variants in sup-36 and sup-37 which reside on
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chromosomes IV and V, respectively (Schnabel et al. 1991) (Fig. S3). However, sup-35, which is located
12.5kb upstream of pha-1, also appeared to be missing or highly divergent in DL238 (Fig. 2A, Fig. S3).
We hypothesized that sup-35 and pha-1 could constitute a selfish element responsible for the observed
incompatibility between the N2 and DL238 isolates. In our model, sup-35 encodes a maternally-deposited
toxin that kills embryos unless they express the zygotic antidote, pha-1 (Fig. 2B). N2 worms carry the sup-
35/pha-1 element, which is missing or inactive in DL238, and F
1
hermaphrodites deposit the sup-35 toxin
in all their oocytes. 25% of their F
2
self-progeny do not inherit the element and are killed because they lack
the antidote pha-1. Consistent with our model, an RNA-sequencing time-course of C. elegans
embryogenesis showed that sup-35 transcripts are maternally provided, whereas pha-1 transcripts are first
detected in the embryo at the 100-cell stage (Hashimshony et al. 2014). To test our model, we first asked
whether sup-35 was necessary for the F
2
embryonic lethality in the N2 x DL238 cross. We crossed DL238
males to N2 peel-1
-/-
hermaphrodites carrying a null sup-35(e2223) allele (Fig. 2B). This sup-35 allele was
reported to fully rescue pha-1 associated embryonic lethality (Schnabel et al. 1991). Embryonic lethality in
the F
2
dropped from 25% to baseline in this cross (1.40%, N = 576), demonstrating that sup-35 activity
underlies the incompatibility between N2 and DL238 (Fig. 2B). We next tested whether expression of pha-
1, the zygotic antidote, was sufficient to rescue the embryonic lethality. We introgressed a pha-1 multicopy
transgene into the DL238 and N2 peel-1
-/-
strains and repeated the cross. As predicted, expression of pha-1
was sufficient to reduce embryonic lethality in the F
2
to baseline (1.49%, N = 268) (Fig. 2B). Moreover,
we reasoned that if the sup-35/pha-1 element underlies the maternal incompatibility, arrested embryos from
an N2 x DL238 cross should phenocopy pha-1 mutant embryos. We collected rare L1-arrested F
2
larvae
from an N2 peel-1
-/-
x DL238 cross and observed major morphological defects in the pharynx of these
individuals, as previously reported for pha-1 mutants (Schnabel and Schnabel 1990; Polley et al. 2014)
(Fig. 2C).
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