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Supplementary Information
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This supplement contains additional data and details relating to the
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crosses and experiments described in the Results.
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Progeny viability of unmated triploid daughters of the Screen 2
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diploid male: Progeny viability was quantified using a method
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described by Pultz et al (2000) for directly observing embryonic
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lethality. Females were set individually on single host pupae and
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allowed to lay eggs for several hours; the embryos were then collected
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and monitored for hatching. We collected broods (ranging from 8-49
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embryos) from 25 daughters of the Screen 2 male; all showed a high
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level of embryonic death (unhatched embryos), ranging from 79% to
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100%. When data from individual females are pooled (n = 530
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embryos), the frequency of embryonic death is 93.5% (corresponding to
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6.5% viability). Progeny inviability for control diploid females assayed
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at the same time was 14.3% (86.7% viability; n = 666 embryos); we
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attribute this to trauma occurring during the collection or incubation of
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the embryos. Adjusting for the background of inviability in the controls,
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the expected frequency of unhatched embryos from triploid females in
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this assay is 94.6% [15/16 + 1/16(0.14)]. This predicted value agrees
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with the frequency of embryonic death observed for the progeny of the
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daughters of the Screen 2 diploid male ( =1.069 p= 0.3).
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The progeny of Screen 1 diploid males were triploid females with
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one maternal and two paternal genomes: Three phenotypically wild-
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type diploid males of genotype pm-541/+ I; rdh-5/+ II; st-5219 /+ III
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were isolated in Screen 1. Two of these males were mated with
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genotypically wild-type diploid females. (The third male died before
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mating.) The pm-541/+/+; rdh-5/+/+; st-5219/+/+ daughters generated
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in these crosses produced the high frequency of inviable progeny (due to
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aneuploidy) characteristic of triploid mothers (see Results and Figure 2).
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The ploidy of these females and the genotype of their father were also
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confirmed by the observation that they produced viable male progeny
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exhibiting one or more of the recessive mutations contributed from the
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diploid father: pm-541, rdh-5, and st-5219. If the phenotypically wild-
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type males recovered during Screen 1 had been haploid males (carrying
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only wild-type alleles) rather than diploid males (heterozygous for the
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three recessive markers), they could not have produced daughters
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carrying recessive mutant alleles upon mating with the genotypically
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wild-type females.
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The sex-determining lesion was not transmitted via haploid
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grandsons of the Screen 2 diploid male: Assuming Mendelian
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segregation, one-third of the haploid males produced by triploid S+/S+/S
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–
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2 diploid male were of genotype stDR oy+ /stDR oy+/ st+ oy*; + / + / bl-
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13*. (The asterisk indicates the allele carried by the mutagenized
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grandparent.) An oyster male segregating from a stDR oy+ /stDR oy+/ st+
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oy* triploid female must be haploid. If an oyster male carrying the S-
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allele is mated to an S+/S+ female, all progeny will be of genotype S+/S-
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and develop into biparental diploid males (Figure 4b). Twenty-five st+
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oy* male progeny of these triploid females were individually mated to
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stDR oy+/stDR oy+ females. All crosses resulted in phenotypically wild-
females should carry the S – allele. The triploid daughters of the Screen
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type (st+ oy* /stDR oy+) females and scarlet (stDR oy+) males; no wild-
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type (st+ oy* /stDR oy+) biparental male progeny were observed.
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Similar results were obtained when the bl-13 allele was used to
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identify the haploid males. For example, 13 stDR oy+; bl-13* grandsons
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of the Screen 2 male were also tested individually for transmission of the
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mutant allele. No biparental male progeny were generated in the crosses
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with these males, confirming the results obtained with the st+ oy*
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haploid males. Assuming that the sex-determining mutation is unlinked
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to the bl-13* allele (which was carried by the mutagenized haploid great
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grandfather), that there is no segregation bias and no loss of viability,
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then each stDR oy+; bl-13* haploid male has a 1/3 probability of carrying
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the mutant allele. Mating 13 stDR oy+; bl-13 males corresponds to a
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probability of >99% of testing at least one male with the mutant allele.
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The sex-determining lesion was not transmitted via haploid
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grandsons of the Screen 1 diploid males: We performed a series of
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experiments to look for transmission of a sex-determining mutation via
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the haploid grandsons of the Screen 1 diploid males. Phenotypically
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wild-type males produced by pm-541/+/+; rdh-5/+/+; st-5219/+/+
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daughters are either haploid or diploid; males exhibiting one or more
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mutant phenotypes must be haploid. Haploid male progeny of these
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triploid females were selected and mated individually with appropriately
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marked females so that biparental diploid male progeny could be easily
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differentiated from uniparental male progeny. If the sex-determining
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mutation assorts independently of the three recessive genetic markers
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used in Screen 1, then a haploid male exhibiting either one, two or all
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three of the recessive phenotypes has a 1/3 probability of carrying the
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sex-determining mutation. For each of the two Screen 1 diploid males, at
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least 25 haploid grandsons (singly or doubly mutant) were mated. We
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did not observe transmission of a sex-determining mutation: no
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biparental diploid male progeny were observed in any of these crosses.
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(In addition, some phenotypically wild-type grandsons were also tested
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with the same results.) Testing 25 haploid males corresponds to a
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probabilty of >99.99% of mating at least one male carrying the sex-
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determining mutation, assuming that it is unlinked to the markers used
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and that it does not confer reduced viability in haploid males. If the sex5
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determining mutation is linked to one of the recessive genetic markers,
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then a haploid Screen 1 grandson carrying that marker has less than a 1/3
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chance of carrying the sex-determining mutation since the recessive
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mutations used to identify the haploid grandsons of the Screen 1 males
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were contributed by the triply mutant females that were mated with the
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mutagenized wild-type males (Table 2). For each of the three marker
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loci, at least five males were mated that were mutant at that locus but
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wild-type for the other two loci; this means that for each locus at least
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ten haploid males carrying wild-type alleles of the locus were mated.
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Assuming that the sex-determining mutation is linked to the marker
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locus, this corresponds to a probability of 99.9% [1- (1/2)10] of mating at
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least one haploid male carrying the mutant (sex-determining) allele.
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Thus it is highly unlikely that we failed to see transmission of the sex-
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determining mutations in Screen 1 because of linkage of this mutation to
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the wild-type allele of one of the mutant loci used identify the haploid
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grandsons.
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