RESEARCH ARTICLE Lethality caused by a paternal effect by zygotic interaction. The segregation of em- bryonic lethality opposite the visible marker Widespread Genetic Incompatibility implied that the arrested embryos represented those homozygous for the Hawaii allele of a locus linked in C. Elegans Maintained by to the marker, on the left arm of chromosome I. Because embryonic lethality within the Hawaii Balancing Selection strain itself is very low (less than 1%), we reasoned that F2 lethality reflected an incompatibility Hannah S. Seidel,*† Matthew V. Rockman,*† Leonid Kruglyak* between the Hawaii allele of this locus and an element in the Bristol genome. We also reasoned Natural selection is expected to eliminate genetic incompatibilities from interbreeding populations. that it did not reflect two synthetically lethal alleles We have discovered a globally distributed incompatibility in the primarily selfing species segregatingintheF2 population because such an Caenorhabditis elegans that has been maintained despite its negative consequences for fitness. interaction would affect less than one-quarter of F2 Embryos homozygous for a naturally occurring deletion of the zygotically acting gene zeel-1 embryos (up to 3/16, depending on linkage and arrest if their sperm parent carries an incompatible allele of a second, paternal-effect locus, peel-1. dominance). One-quarter lethality is expected, The two interacting loci are tightly linked, with incompatible alleles occurring in linkage however, if the incompatibility involves an inter- disequilibrium in two common haplotypes. These haplotypes exhibit elevated sequence action between the genotype of the zygote and a divergence, and population genetic analyses of this region indicate that natural selection is maternal or paternal effect. preserving both haplotypes in the population. Our data suggest that long-term maintenance To test this possibility, Hawaii × Bristol F1 of a balanced polymorphism has permitted the incompatibility to persist despite gene flow males and hermaphrodites were separately back- across the rest of the genome. crossed to Hawaii individuals, and lethality was scored among the resulting embryos. We ob- aenorhabditis elegans is a globally dis- study natural genetic variation in C. elegans, served 50% lethality when F1 males were mated tributed species of free-living bacteria- and we genotyped the RILs at 1450 single- to Hawaii hermaphrodites but less than 2% C eating nematode. Although rare males nucleotide polymorphism (SNP) markers (8). lethality in the reciprocal cross (Fig. 1). Thus, on February 9, 2008 contribute at a low rate to outcrossing, C. elegans We noted that a region on the left arm of lethality depends on both paternal and zygotic occurs primarily as inbred self-fertilizing her- chromsome I exhibited a dramatic deficit of genotype, but is independent of maternal cyto- maphrodites (1–4). A wild isolate from Hawaii, Hawaii alleles among the RILs. Of 239 RILs, plasm. (Both Hawaii and F1 hermaphrodites CB4856, has been identified among well-studied only 5 carried the Hawaii allele at the most produced dead embryos, 50% and 25%, respec- isolates as the most divergent at the sequence skewed marker, and simulations of the intercross tively, when mated to F1 males.) In sum, lethal- level from the standard laboratory strain, N2, pedigree indicated that this allele frequency skew ity appears to result from a paternal effect by derived from an isolate from Bristol, England could not have arisen by drift, suggesting that zygotic interaction, whereby embryos homozy- (5–7). As a result of this sequence divergence, selection had acted during construction of the gous for the Hawaii allele of a zygotically act- — the Hawaiian strain is widely used to map RILs (fig. S1). We then crossed Hawaii to a ing locus fail to hatch when the sperm parent www.sciencemag.org mutations induced in the Bristol background. Bristol strain carrying a visible marker located male or hermaphrodite—is a Hawaii × Bristol Genetic incompatibility between Bristol 10 cM from the most skewed RIL marker, and heterozygote. An interaction between a paternal and Hawaii. We generated recombinant inbred we examined F2 progeny produced by self- effect and a zygotically acting gene is surprising lines (RILs) from the 10th generation of an ad- fertilizing F1 hermaphrodites. Surprisingly, ap- because sperm-supplied factors are expected to vanced intercross between Bristol and Hawaii to proximately 25% of F2 progeny arrested as act during fertilization and first cleavage (9), embryos, and embryonic lethality segregated whereas early embryogenesis is primarily con- Lewis-Sigler Institute for Integrative Genomics and Depart- opposite the visible marker (Table 1). F2 lethality trolled by maternally contributed factors, and ment of Ecology and Evolutionary Biology, Princeton was not an effect of the marker: Self-fertilizing F zygotic transcription is not known to occur be- Downloaded from University, Princeton, NJ 08544, USA. 1 hermaphrodites derived from reciprocal crosses fore the four-cell stage (10). *To whom correspondence should be addressed. E-mail: [email protected] (H.S.S.); mrockman@princeton. between Hawaii and wild-type Bristol produced Tight linkage of the zygotically acting and edu (M.V.R.); [email protected] (L.K.) 25% dead embryos, as did F1 hermaphrodites paternal-effect loci. To understand the genetic †These authors contributed equally to this work. mated to F1 males (Fig. 1). basis of the incompatibility, we used the RILs F F F F Hawaii Hawaii F Table 1. F2 lethality segregates opposite visible 1 1 1 1 1 marker bli-3, located 10 cM from the most skewed RIL marker. Genotypes of F2 progeny from selfing x x x Bristol/bli-3 and Hawaii/bli-3 hermaphrodites were scored. Embryonic lethality from selfing Hawaii/bli-3 hermaphrodites was slightly greater than 25% because the visible marker introduces a small percentage of lethality. Bristol/bli-3 Hawaii/bli-3 F2 genotype 24.0%(880) 26.5%(1764) 50.4%(4432) 1.7%(4160) hermaphrodite hermaphrodite bli-3/bli-3 21.7% (128) 21.8% (128) Fig. 1. Paternal effect by zygotic lethality. The percent of embryonic lethality (total) was scored from the crosses shown. Orange and blue indicate Bristol and Hawaii haplotypes, respectively. Pie charts show the bli-3/+ 49.9% (295) 42.0% (246) proportions of embryos that hatched (white) and failed to hatch (black). F individuals were derived from +/+ 24.5% (145) 5.1% (30) 1 reciprocal Bristol × Hawaii crosses. Embryonic lethality from selfing Bristol and Hawaii hermaphrodites, Arrested embryos 3.9% (23) 31.1% (182) reciprocal Bristol × Hawaii, and reciprocal Bristol × F1 crosses was less than 0.8% (n > 240 embryos for each). www.sciencemag.org SCIENCE VOL 319 1 FEBRUARY 2008 589 RESEARCH ARTICLE to map both the zygotically acting locus, zeel-1 (zygotic epistatic embryonic lethal–1), and the paternal-effect locus, peel-1 (paternal effect epistatic embryonic lethal–1). We crossed RILs to Bristol and Hawaii and scored lethality subclone. Arrows among embryos laid by self-fertilizing F1 her- 2,376k maphrodites and Hawaii hermaphrodites mated to F1 males. The pattern of lethality among F2 and backcross embryos was consistent with each RIL carrying either the Bristol alleles of Y39G10AR.5 both zeel-1 and peel-1 or the Hawaii alleles of both. We identified only one genomic interval in which all lines of the former class carried the Bristol haplotype and all lines of the latter class carried the Hawaii haplotype. Thus, both zeel-1 2,366k and peel-1 map to this interval, a 62-kb region on chromosome I (position 2,317,234 to 2,379,249) (Fig. 2A). We confirmed tight linkage between the two loci; they do not segregate independently among backcross progeny (table S1). Incomplete penetrance of the incompatibility. The penetrance of the incompatibility (i.e., the extent of lethality among zeel-1Hawaii homozy- ). We used default parameters and Bristol as the reference sequence. The gotes sired by peel-1 heterozygotes) was com- 34 2,356k plete when oocytes were fertilized by male ) mVista alignment of Hawaii and Bristol sequence across a portion of the sperm but incomplete when they were fertilized B by hermaphrodite sperm. We collected embryos on February 9, 2008 interval ( from self-fertilizing F1 hermaphrodites and from F1 hermaphrodites mated to F1 males and geno- peel-1 / typed surviving progeny at the zeel-1 locus. Among self-progeny, approximately 10% of zeel-1Hawaii in red. A black asterisk indicates the frame shift introduced into the zeel-1 indicate markersdisequilibrium. used ( to genotype wild isolates. All markers are in complete linkage black box surrounds the intervalbp) of deletions elevated (green) divergence and betweenbelow insertions Bristol the (gray) and alignment. in Hawaii. Hawaii Large relative (>50 to the Bristol sequence are shown 8M 10M 11M 12M 13M 14M 15M homozygotes survived to hatching, although most had retarded development and abnormal subclone morphologies (8). In contrast, none survived 2,346k * when fertilized by F1 males. Penetrance of the incompatibility also appeared complete among www.sciencemag.org . Gray bars ), fosmid B embryos from F1 males backcrossed to Hawaii Hawaii Bristol Y39G10AR.5 (zeel-1) hermaphrodites, as these broods lacked the peel-1 deformed larvae characteristic of surviving zeel- peel-1 peel-1 fosmid B 1 homozygotes. and Hawaii The morphological defects of surviving Bristol Hawaii WRM0614dC06 zeel-1Hawaii homozygotes were highly variable zeel-1 zeel-1 zeel-1 and often similar to the terminal phenotype ob- 2,336k Downloaded from ) Colored bars represent Bristol served in arrested embryos, which usually showed subclone A tissue differentiation but no elongation past the ugt-31 twofold stage. Nevertheless, some zeel-1Hawaii subclone homozygotes matured to adulthood and produced progeny. These progeny were entirely wild type, implying that the paternal effect is not caused by Y39G10AR.16 heritable defects such as DNA damage or subclone aneuploidy in zeel-1Hawaii sperm. srbc-64 Globally distributed incompatibility. To determine the distribution of alleles causing the Bristol-Hawaii incompatibility in the global C.