Sex-biased gene expression at homomorphic sexchromosomes in emus and its implication forsex chromosome evolutionBeatriz Vicoso, Vera B. Kaiser, and Doris Bachtrog1

Department of Integrative Biology, University of California, Berkeley, CA 94720

Edited by Kathryn V. Anderson, Sloan-Kettering Institute, New York, NY, and approved March 8, 2013 (received for review October 1, 2012)

Sex chromosomes originate from autosomes. The accumulation ofsexually antagonistic mutations on protosex chromosomes selectsfor a loss of recombination and sets in motion the evolutionaryprocesses generating heteromorphic sex chromosomes. Recombi-nation suppression and differentiation are generally viewed as thedefault path of sex chromosome evolution, and the occurrence ofold, homomorphic sex chromosomes, such as those of ratite birds,has remained a mystery. Here, we analyze the genome and tran-scriptome of emu (Dromaius novaehollandiae) and confirm thatmost genes on the sex chromosome are shared between the Z andW. Surprisingly, however, levels of gene expression are generallysex-biased for all sex-linked genes relative to autosomes, includingthose in the pseudoautosomal region, and the male-bias increasesafter gonad formation. This expression bias suggests that the emusex chromosomes have become masculinized, even in the absenceof ZW differentiation. Thus, birds may have taken different evolu-tionary solutions to minimize the deleterious effects imposed bysexually antagonistic mutations: some lineages eliminate recombi-nation along the protosex chromosomes to physically restrict sexu-ally antagonistic alleles to one sex, whereas ratites evolved sex-biased expression to confine the product of a sexually antagonisticallele to the sex it benefits. This difference in conflict resolutionmayexplain the preservation of recombining, homomorphic sex chromo-somes in other lineages and illustrates the importance of sexuallyantagonistic mutations driving the evolution of sex chromosomes.

sexual antagonism | female heterogamety | dmrt1

Sex chromosomes are derived from ordinary autosomes. Re-striction of recombination and differentiation of sex chromo-

somes is often thought to be the ultimate fate of a pair of protosexchromosomes that acquired a sex-determining function (1–3). Theevolutionary fuel driving the loss of recombination between na-scent sex chromosomes is provided by sexually antagonisticmutations, that is, mutations that benefit one sex but are harmfulto the other (4). If a sexually antagonistic mutation arises in thepopulation on the recombining, nondifferentiated part of a pro-tosex chromosome, selection for increased linkage of the sexuallyantagonistic allele to the sex determination locus can cause thismutation to be transmitted more often through the sex it benefits(5). Loss of recombination between the sex chromosomes allowsthe proto-X and proto-Y (or proto-Z and proto-W) to evolve in-dependently, and also sets the stage for the population processescausing degeneration of the nonrecombining sex chromosome (6).Restriction of recombination across the entire length of the sexchromosomes and continuous gene decay on the Y results inheteromorphism between the X and Y (or Z and W). Sex chro-mosomes that show low levels of differentiation are generallyconsidered to be evolutionarily young and only at the initial stagesof this path; given time, they too are expected to differentiate.However, some clades, such a birds or snakes, contain old, ho-

mologous sex chromosomes that have progressed to heteromor-phism in some species but not others (7–10). The reasons for a lackof differentiation in some lineages are unclear, but several sce-narios are possible. Clades might differ in the amount of sexuallyantagonistic selection operating in the genome. In particular,

species experiencing more conflict between the sexes might besubject to higher rates of sexually antagonistic mutations and arethus expected to more rapidly eliminate recombination betweentheir protosex chromosomes. Species experiencing lower levels ofsexual antagonism, in contrast, lack the selective agent for re-combination suppression and may maintain homomorphic sexchromosomes.Why different clades of birds or snakes would differin the amount of sexual conflict, however, is unclear.Recombination suppression is only one strategy for conflict res-

olution at loci undergoing opposite selection in males and females.In particular, the evolution of sex-biased gene expression is an al-ternative solution to eliminate the deleterious effects of a sexuallyantagonistic allele in the sex to which it is harmful (11–13). Thus,sexually antagonistic alleles might have accumulated at a similar ratein species with homo- and heteromorphic sex chromosomes, butdifferent taxa may have used different strategies to resolve sexualconflict. Species could evolve close linkage between sexually antag-onistic mutations and the sex determining region, eventually leadingto the evolution of heteromorphic sex chromosomes. Alternatively,sex-biased expression might evolve at sexually antagonistic allelesalong the protosex chromosomes, thus eliminating the selectivepressure to abolish recombination on the nascent sex chromo-somes (14). This evolutionary path would result in species withold, homomorphic sex chromosomes, but an excess of sex-biasedexpression at the recombining portion of their sex chromosomes.Birds all have homologous sex chromosomes that formed about

120 Mya (15), similar in age to the mammalian sex chromosomes(about 165 My old, ref. 16). However, whereas all mammals andmost bird lineages have highly differentiated sex chromosomes, insome groups of birds, such as ratites, the sex chromosomes remainhomomorphic (17). The reason for this difference is unclear.Previous mapping studies have shown that the emu Z and W arelargely homologous, containing only a small, differentiated seg-ment (18–21). Here, we analyze male and female genomic se-quences and transcriptomes of emus at different developmentaltime stages, in an attempt to identify the evolutionary forces causingthese differences.

Results and DiscussionLevels of Differentiation Vary Along the Emu Sex Chromosomes. Map-ping studies have suggested that the emu Z and W chromosomesare largely homomorphic, with only a small, differentiated segmenton the short arm of the Z chromosome (18–21). Genomic readcoverage information and SNP patterns from male and female

Author contributions: B.V. and D.B. designed research; B.V. and V.B.K. performed re-search; D.B. contributed new reagents/analytic tools; B.V. and V.B.K. analyzed data;and B.V., V.B.K., and D.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The RNA-seq and DNA-seq reads reported in this study have been de-posited in the National Center for Biotechnology Information Short Reads Archive, www.ncbi.nlm.nih.gov/sra (accession nos. SRP019802 and SRP019803).1To whom correspondence should be addressed. E-mail: dbachtrog@berkeley.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1217027110/-/DCSupplemental.

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samples can be used to identify regions of sex chromosomes thatare either fully or partly differentiated, or still recombining andhomologous between the Z and W chromosome. Autosomalregions should show similar SNP densities in males and femalesand similar genomic read coverage in both sexes (i.e., a similarnumber of genomic reads obtained from male and female samplesshould map to autosomal fragments, normalized by the totalnumber of reads per sample). Fully differentiated regions (whichno longer have a W-linked homolog) are always hemizygous andthus lack SNPs in females but should have normal SNP densities inmale samples. Such regions will also show only half as many ge-nomic reads in females, compared with males. Partially differen-tiated regions will show an increased level of heterozygosity infemales because, if W-derived reads still map to the Z, divergencebetween the W and the Z will be confounded with polymorphism.Such regions should also show reduced genomic coverage infemales because only highly similar reads between the Z and the Wwill contribute to coverage (depending on the exact mappingparameters). Last, pseudoautosomal regions (PARs) are expectedto have similar SNP densities and genomic coverage in males andfemales; i.e., they should be indistinguishable from the autosomes.To identify differentiated regions along the emu sex chromo-

somes, we obtained DNA-seq and RNA-seq reads from male andfemale emu samples, which we assembled into genomic scaffoldsand transcript sequences, respectively. We mapped these emugene sequences along the chicken Z chromosome and used thegene content of the genomic scaffolds to estimate their location onthe chicken Z (22) (Fig. 1). Although physical or genetic maps areunavailable for emus, the low rate of rearrangements observedbetween bird genomes, and reptiles in general, suggests that thelocation of the genes along the chicken genome may be a goodproxy for their location on emu chromosomes (Fig. S1). For ex-ample, a large number of microsyntenic blocks are shared amongchicken and lizard (23), and the gene content between the chickenand flycatcher Z chromosome is well conserved (24). Therefore,although a few rearrangements have probably occurred since theemu–chicken split, synteny at small scales can serve to group emugenes. Repeating our analysis of emu expression, but mapping ofemu genes along Anolis chromosome 2 (which is homologous tothe bird Z chromosome) to identify differentiated and pseudoau-tosomal regions, does not affect our conclusions (Figs. S1–S3).

We mapped the female and male genomic reads separately tothe genomic scaffolds and estimated male and female coverage ofeach scaffold from the resulting SAM alignment, after removal ofreads with mismatches. The male and female SNP density of eachgene was estimated by mapping RNA-seq reads back to the genesequences with Bowtie and calling SNPs when two alleles werepresent at high frequency at a particular site. The genomic readcoverage and levels of polymorphism in male and female emuwere plotted along the chicken Z chromosome, using a slidingwindow of 10 scaffolds or genes (Fig. 1). Using this approach, weobserved several candidate regions for differentiation on the emuZ; lists of genes found in each region are provided in Dataset S1.Specifically, we identify three candidate regions along the chickenZ that appear fully differentiated in emus: genomic read coveragefor these regions is similar to autosomal regions in males, whereasfemales show only half the read coverage; further, few SNPs wereobserved within each female sample whereas the male samplesshowed levels of heterozygosity similar to other regions of the Z.These three regions (26471499–34336577, 50347741–55266322,and 62989377–69684220) presumably correspond to a single an-cestral sex-determining region on the bird sex chromosomes,termed stratum 0, that has been broken into several smallersegments on the chicken Z by chromosomal rearrangements. Anancestral involvement of this genomic segment in sex determi-nation in all birds is further corroborated by the location of dmrt1[doublesex and mab-3 related transcription factor 1; presumablythe ancestral sex-determining gene in birds (25)] within thatsegment, their adjacent location along Anolis chromosome 2 (Fig.2), and a lack of W-homologous genes within this presumablyancient sex-linked region in chicken (Fig. 2 and see Dmrt1 andEvolutionary Strata in Birds). Another region, which we call emustratum 1 (34336577–39235675), also showed reduced genomiccoverage in the female sample, but highly increased heterozy-gosity in females relative to males (Fig. 1). This pattern suggeststhat this Z-linked region stopped recombining with the W rela-tively recently, i.e., sufficiently long ago for divergence betweenthe Z and the W to build up (and thus genomic read coverage isreduced in females due to the more stringent mapping of ge-nomic reads and higher divergence in intergenic and intronsequences), but recently enough for sequence similarity to remainhigh between the chromosomes (and detected as high SNP density

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Fig. 1. Evolutionary strata on the emu sex chromosomes. (A) Female and male coverage along the Z chromosome (dots represent individual scaffolds, thelines a sliding window of 10 scaffolds). (B) Female/male SNP densities for emu transcripts, ordered along the chicken Z chromosome, using a sliding window of10 genes. Putative differentiated regions are colored in yellow (Str0: stratum 0 and Str1: stratum 1), and pseudoautosomal regions are in green (PAR). The redvertical line indicates the location of dmrt1.

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between Z and W orthologs in females). The remaining regionsalong the sex chromosomes showed similar levels of genomiccoverage and diversity in the male and female sample and likelycorrespond to the nondifferentiated, recombining portion of thesex chromosomes. We classified the regions adjacent to the Z-Wdifferentiated regions as pseudoautosomal; here, genomic readcoverage and SNP densities are indistinguishable from autosomalvalues (see Tables S1–S4 for comparisons of SNP densities).Mapping of emu genes to Anolis resulted in a very similar set ofgenes being classified as differentiated or pseudoautosomal, andthe boundaries between the differentiated and pseudoautosomalsegments often correspond to boundaries of large syntenic blocksbetween chicken and Anolis (Fig. 2).The pseudoautosomal region (PAR) makes up most of the emu

sex chromosomes (about 60% of genes according to our classifi-cation, versus 25% for stratum 0 and 5% for stratum 1), consistentwith the observation that the emu Z and W are of similar size andappear largely undifferentiated under the microscope.

Male-Biased Expression in Pseudoautosomal and Differentiated Regions.In chicken, the sex chromosomes are fully differentiated; i.e., theW is completely degenerated, and the Z is hemizygous in females(26–28). Transcripts derived from Z–linked genes in chicken arefound, on average, at higher levels in males compared withfemales, supporting the notion that birds lack global mechanismsof dosage compensation (the equalizing of expression from the Zrelative to the autosomes in the two sexes) (29). In the emu, mostof the Z and W chromosome is pseudoautosomal; therefore, ex-pression of most sex-linked genes in females relative to males(F/M) is expected to be similar to that of the autosomes, and onlygenes located in the differentiated region should show lowerexpression in females. Surprisingly, however, we find that geneslocated on the Z showed an overall reduction in F/M expression atboth 15 d and 42 d (Fig. 3A); i.e., expression from the Z was 1.07(15-d embryos) and 1.33 (42-d embryos) times as high in malescompared with females. If degeneration of the W and lack ofdosage compensation were driving this pattern in emu, a decreased

female over male expression ratio should be largely confined to thedifferentiated regions (that is, stratum 0 and possibly emu stratum1). Consistent with a lack of dosage compensation, both the 15-dand the 42-d samples showed reduced F/M expression at the fullydifferentiated region stratum 0. However, whereas the F/M ex-pression ratio is indeed lowest at stratum 0, the pseudoautosomalregion also shows evidence for reduced F/M expression in the 42-dsample (Fig. 3 B andC and Fig. S4), and we find a larger fraction ofsex-biased genes in the PAR relative to autosomes (Tables S5–S8).Thus, gene expression from the emu Z is sex-biased, both in thedifferentiated region as well as in the PAR.Because genes in the PAR are diploid in both sexes, this pattern

cannot result from simple gene dose differences. Instead, it sug-gests that male-biased genes accumulate on sex chromosomes,even in homologous regions that still recombine between the Zand the W. In particular, whereas a lack of dosage compensationshould be detectable at any developmental stage, sex-biased ex-pression of genes (other than the sex determination genes) shouldbe more pronounced in older embryos, after the full developmentof gonads (30). Consistent with more sex-specific or sexually an-tagonistic selection operating at later stages in development, thevariance in F/M expression among autosomal genes increasedfrom day 15–42 (P value < 2.2e−16, F test), as did the fraction ofsex-biased genes (7–11% using a twofold cutoff; Tables S5–S8).Further, only the 42-d sample shows male-biased expression onthe PAR whereas, at 15 d, the F/M ratio is more similar to that ofthe autosomes (Fig. 3C). Specifically, the distribution of expres-sion ratios of genes on the PAR is bimodal in the 42-d sample, witha second population of more strongly male-biased genes (peakingat M/F ∼1.35; Fig. 3C). Such a bimodal distribution is not found atautosomal genes, or genes within stratum 0, but is also absentat PAR-linked genes at the younger, sexually undifferentiated15-d-old embryos. This pattern demonstrates that the excess ofmale-biased expression at PAR genes at 42 d is not caused by theinclusion of Z-linked genes that are differentiated on the W(which should be detected in both temporal samples) but insteadis consistent with sex-specific expression of male-beneficial genes

BA

Fig. 2. (A) Synteny and evolutionary strata on the bird Z. Dot plot between Anolis chromosome 2 and the chicken Z chromosome. Dashed lines indicate theposition of Z-linked genes in chicken that also have W-linked homologs; these genes have been used to infer the location of three evolutionary strata on thechicken Z (old strata I–III). Regions in yellow are fully differentiated on the emu Z, and the orange region is a more recently differentiated region. The yellowregion supposedly corresponds to the ancestral sex-determining segment shared by all birds but contains no remaining W homologs in chicken and can thusnot be dated based on Z-W divergence. We refer to this ancestral sex-linked region as stratum 0. The red line gives the location of dmrt1, the ancestral sex-determining gene in birds. (B) Schematic comparison of the chicken and emu Z chromosomes, with the emu differentiated (in yellow) and pseudoautosomalregions (green) colored on the chicken Z. DMRT1 has been mapped to the differentiated region of the emu Z chromosome using in situ hybridization.

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Fig. 3. Patterns of gene expression in emus. Genes were assigned to different chromosomes according to their location in the chicken genome. (A) Log2(female/male FPKM) for the different emu chromosomes at 15 and 42 d. (B) Log2(female/male FPKM) along the Z chromosome using a sliding window of 10genes at 15 d and 42 d. (C) Log2(male FPKM/female FPKM) at day 15 and 42 on the autosomes, the differentiated regions (stratum 0 and stratum 1), and thepseudoautosomal region (PAR) of the Z chromosome. The dashed lines indicate the median values for each sample.

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after sexual differentiation. Overall, male-biased expression at42 d seems to be mostly due to a reduction in expression from thePAR in females, rather than an increase in males (Fig. S5). Thisfinding is consistent with a scenario whereby the sex to whichexpression of a sexually antagonistic allele is detrimental reducesthe deleterious consequences of such mutations through tran-scriptional down-regulation. The differences in F/M expressionbetween early and late embryonic development are significantlygreater in the PAR than those observed for the autosomes (P =8.509e−09; Fig. S6) whereas the difference in F/M between theautosomes and stratum 0/stratum1 is not significant (but follows thesame trend). In summary, we observe a distinct shift toward male-biased expression in the pseudoautosomal region during de-velopment, in contrast to the autosomes (where average sex biasdoes not change) or in stratum 0 (where genes are always male-biased, presumably due to a lack of dosage compensation). Stra-tum 1 contains only 26 genes but probably constitutes a mixture ofgenes where the W-linked ortholog is either expressed ordegenerated. This mixture in genes could contribute to the ap-parent (but not significant) shift in the distribution of expressionratios in that region (Fig. 3C and Fig. S4).Interestingly, we also observe a slight, yet significant, shift in

the distribution of F/M expression ratios at PAR-linked genes atthe 15-d sample toward female-biased expression (Fig. 3C andFig. S4). This pattern suggests that female-biased genes accumu-late in the PAR during early development, at the onset of sexualdifferentiation (day 15) whereas, at later stages, male-biased ex-pression is more abundant (day 42). A shift in sex-biased geneexpression over the life cycle has also been observed in chickengonads, with the relative proportion of male-biased genes in-creasing over development (30).

Homomorphic vs. Heteromorphic Sex Chromosomes, and Sex-BiasedExpression. Why is the pseudoautosomal region different com-pared with the autosomes in emu, and how does this differencerelate to sex chromosome evolution? Whereas pseudoautosomalregions are not confined to a single sex, even partial linkage toa sex determination locus can select for sexually antagonisticalleles to accumulate (14, 31, 32). For example, an excess of sex-ually antagonistic genes mapped to the pseudoautosomal region inthe dioecious plant Silene latifolia (13, 33). Sexual conflict resultingfrom the accumulation of sexually antagonistic mutations withinthe PAR can be resolved either by restricting recombination be-tween the sex chromosomes (i.e., restricting a sexually antagonisticmutation to a particular sex) or by down-regulating the expressionof genes in the sex that they harm. In emu, male(female)-beneficialalleles might have accumulated at pseudoautosomal genes, andtheir deleterious effects to females (males) may have beenresolved by down-regulating them in the sex they harm. Thisdownregulation in turn would eliminate selective pressure for re-duced recombination between the Z and W; additionally, most ofthe genes on the emu W chromosome are not subject to delete-rious effects caused by loss of recombination. Sex-biased expres-sion is viewed as the resolution of sexual antagonism at themolecular level, and our results suggest that the evolution of sex-biased expression could be a general mechanism to resolve con-flicts occurring at sex chromosomes and could explain the stabilityof homomorphic, recombining sex chromosomes in some clades.The maintenance of homomorphic, nonrecombining sex chromo-somes could also result from rare recombination in sex-reversedZY male or XY females, as has occurred in tree frogs (34), butdoes not explain the lack of a restriction of recombination ob-served in ratites.Why would different strategies to resolve sexual antagonism be

adopted in different clades? As mentioned in the introduction,one factor likely to be paramount in driving divergence at nascentsex chromosomes is the extent of sexually antagonistic selection onmales and females. Sexual selection is thought to be more preva-lent in males than in females, as females (who usually invest morein the progeny) are more selective when choosing mates. Male-heterogametic clades, which can fully link male-beneficial/female-

deleterious mutations to males by abolishing recombination on theY, may therefore be more prone to suppressing recombinationbetween the sex chromosomes; ZW species can achieve only partiallinkage of these mutations to males by abolishing recombinationand may obtain greater benefits from reducing their expression infemales. The fact that both snakes and birds, the two major ZWclades that have been studied in great detail, show ancestral non-degenerated W chromosomes provides some support for this idea,but the generality of such an association needs to be confirmed ona larger scale. The great diversity of mating systems that occurswithin birds can also lead to drastic differences in the occurrence ofsexual antagonism: very little opportunity for sexual selection isexpected in monogamous species with low frequencies of extra-bond fertilization, whereas the opposite is true in polygamous/promiscuous species. Consistent with sexual antagonism differingbetween lineages, mating systems correlate with the extent of bodysex-dimorphism in birds (35). It is therefore possible that differ-ences in the strength of sexual selection may lead to different op-timal strategies to resolve sexual antagonism in different lineagesof birds. Whereas our current knowledge of the degeneration ofthe bird W chromosome is limited to a few species, the applicationof next-generation sequencing technologies to a variety of specieswith different mating systems will provide a unique opportunity toinvestigate the importance of sexual antagonism in the degenerationofW chromosomes and to disentangle it from other forces drivingthe evolution of sex chromosomes.

Dmrt1 and Evolutionary Strata in Birds. Comparative analysis be-tween Anolis and chicken, combined with patterns of differenti-ation along the emu Z, allows us to reconstruct, to some extend,the early evolutionary history of differentiation and strata for-mation on the bird Z chromosome. In chicken, dmrt1 has beenidentified as the master-switch sex-determining gene (25); dmrt1is present on the Z, but absent on theW, and is believed to triggersexual development in a dose-dependent fashion. dmrt1 has alsobeen mapped to the differentiated region on the Z chromosomeof ratite birds and is absent on the ratite W (21), further sup-porting its role as the master sex-determining switch gene inall birds.In chicken, a limited number of W-linked genes with homologs

on the Z have been identified and are remnants of the ancestralgenes present on the autosome that formed the sex chromosome.The level of differentiation between Z and W homologs, how-ever, is not uniform along the Z; instead, different gene pairsshow discontinuous divergence levels, which are thought torepresent different time points at which recombination ceasedbetween the Z and W (36, 37). Genes with similar sequence di-vergence are grouped together to form “evolutionary strata,”and, in chicken, at least three such strata can be identified (36,37). If dmrt1 is indeed the ancestral sex-determining gene thatinitiated sex chromosome evolution in the ancestor of all birds, itshould be part of the oldest evolutionary stratum. However, thethree evolutionary strata detected on the chicken Z are allreported to have formed after the ratite–chicken split (37), anddmrt1 is actually not found within the oldest stratum of chicken.Instead, it is located in the middle of stratum II, a region thatceased recombination only 50–70 Mya, long after the split ofchicken and ratites. The placement of dmrt1 within a more recentaddition to the nonrecombining region of the chicken sex chro-mosomes is not consistent with its putative role as the ancestralsex-determining gene shared by all birds; it is also counter tomapping studies showing Z-linkage of dmrt1 in ratites (whichlack the three strata identified in chicken). The boundaries ofstratum II, however, are defined only by a few Z-W gametologs,and a large region within that stratum (ranging from roughly17 Mb to 39 Mb) completely lacks any W genes (36, 37); dmrt1 islocated right in the middle of this region (at roughly 26 Mb onthe chicken Z; Fig. 2). Thus, together these observations suggestthat there may be an undetected, even older evolutionary stra-tum present on all bird sex chromosomes that contains the dmrt1gene, but is lacking any W homologs (and thus could not be

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detected based on ZW divergence). Indeed, our genomic cov-erage analysis in emus suggests that part of the ancestral, dif-ferentiated fragment (stratum 0) on the bird sex chromosomescorresponds to position 26–33 Mb along the chicken Z, the re-gion that contains the dmrt1 gene (Fig. 2). Thus, stratum 0corresponds to the very initial differentiated fragment on the birdsex chromosomes and contains the master-switch sex-determininggene of birds. Additional genomic analysis of the sex chromo-somes of other birds, and in particular other ratites, should al-low us to further refine the evolutionary history of the avian sexchromosomes.

Materials and MethodsSample Preparation. Fertilized emu eggs were incubated at 37 °C for 15and 42 d, and embryo heads (for 15-d embryos) and brains (42-d embryos)from sexed embryos were used for RNA extraction. DNA was extractedfrom the sexed 42 d embryos. Paired-end RNA-seq libraries (with a 200-bpfragment size) and DNA-seq libraries (with an 800-bp fragment size) weremade following Illumina’s standard protocol and sequenced on the Illu-mina platform. The number of reads obtained for each sample is listed inTable S9.

Genomic Assembly and Read Coverage Estimation. Female and male genomicreads were trimmed, pooled, and assembled using SOAPdenovo (http://soap.genomics.org.cn/soapdenovo.html). Female and male reads were mappedseparately back to the genome assembly using Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml), and SoapCov (http://soap.genomics.org.cn/soapaligner.html) was used to estimate the female and male cover-age for each scaffold larger than 1 kb.

Mapping of Emu Genomic Scaffolds to the Chicken Z. We mapped all knownchicken gene sequences (www.ensembl.org) to the emu genomic assemblyusing blat (38) with a translated query and a translated database using a re-ciprocal best hit approach. Emu scaffolds were assigned chicken chromo-some coordinates based on gene content.

Processing of the RNA-seq Reads and Transcriptome Assembly. All 43.9 millionRNA-seq reads were trimmed, pooled, and assembled with SOAPdenovo-Trans(http://soap.genomics.org.cn/SOAPdenovo-Trans.html), and only transcriptsof at least 400 bp were kept for further analysis. Chicken protein sequenceswere downloaded from the Ensembl ftp server, and emu transcripts weremapped against the chicken protein sequences using blastx (39), and furtherscaffolded according to their position along the chicken protein sequences.Our resulting transcriptome consists of 10,102 genes, with a median lengthof 1,653 bp.

SNP Analysis. RNA-seq reads from each individual sample were mappedseparately to the assembled transcriptome using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml) with default parameters. SNPs were called(within each sample) when two alleles were present at a frequency higherthan 0.3, considering only sites covered by at least nine reads.

Identification of Pseudoautosomal and Differentiated Regions. The femalecoverage for different genomic scaffolds along the Z chromosome is bimodal(Fig. S7) and was used to find a cutoff value to define differentiatedregions. Sequential windows (window size of 10 scaffolds) with Log2(Fe-male coverage) >1.2 were considered to be part of the PAR. Regions be-tween these PAR segments were considered to be part of stratum 0 if theyhad female:male SNP density below 4, or stratum 1 if they had increasedfemale SNP density (Table S10). A similar classification of genes wasobtained if emu genes were mapped against the zebra finch Z chromosome(Fig. S8).

Expression Analysis. RNA-seq reads from each individual were mappedseparately to the assembled transcriptome using Bowtie2, and Cufflinks(http://cufflinks.cbcb.umd.edu/) was used to estimate expression levels[in fragments per kilobase of transcript per million mapped reads (FPKM)].

ACKNOWLEDGMENTS. This work was funded by National Institutes ofHealth Grants R01GM076007 and R01GM093182 and a Packard Fellowship(to D.B.).

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