The Rose-comb Mutation in Chickens Constitutes a Structural Rearrangement Causing Both Altered Comb Morphology and Defective Sperm Motility

Rose-comb, a classical monogenic trait of chickens, is characterized by a drastically altered comb morphology compared to the single-combed wild-type. Here we show that Rose-comb is caused by a 7.4 Mb inversion on chromosome 7 and that a second Rose-comb allele arose by unequal crossing over between a Rose-comb and wild-type chromosome. The comb phenotype is caused by the relocalization of the MNR2 homeodomain protein gene leading to transient ectopic expression of MNR2 during comb development. We also provide a molecular explanation for the first example of epistatic interaction reported by Bateson and Punnett 104 years ago, namely that walnut-comb is caused by the combined effects of the Rose-comb and Pea-comb alleles. Transient ectopic expression of MNR2 and SOX5 (causing the Pea-comb phenotype) occurs in the same population of mesenchymal cells and with at least partially overlapping expression in individual cells in the comb primordium. Rose-comb has pleiotropic effects, as homozygosity in males has been associated with poor sperm motility. We postulate that this is caused by the disruption of the CCDC108 gene located at one of the inversion breakpoints. CCDC108 is a poorly characterized protein, but it contains a MSP (major sperm protein) domain and is expressed in testis. The study illustrates several characteristic features of the genetic diversity present in domestic animals, including the evolution of alleles by two or more consecutive mutations and the fact that structural changes have contributed to fast phenotypic evolution.


Introduction
Rose-comb (Figure 1) was one of the autosomal dominant traits William Bateson used in his seminal paper describing Mendelian inheritance in animals for the first time [1].This mutation probably occurred early in the process of chicken domestication, as it is widespread among chicken populations originating in both Asia and Europe, separated for hundreds of years.A few years after the first description of the mode of inheritance of Rose-comb, Bateson and Punnet [2] reported the first case of epistatic interaction between genes as they demonstrated that individuals carrying both the Rose-comb and Pea-comb alleles exhibit the walnutcomb phenotype (Figure 1).Rose-comb has been described in many breeds and shows extensive phenotypic variability (Figure S1).Most attention has been paid to variation in surface characteristics and texture, angle and number of posterior spikes [3][4][5].Thus, Rose-comb variability indicates that comb morphogenesis is influenced by several genes and represents an excellent model to study interactions between developmental genes.
Numerous reports have documented reduced male fertility associated with the Rose-comb allele [6][7][8].Gradually it was elucidated that defective sperm motility in roosters homozygous for Rose-comb is the cause of the observed poor fertility and duration of fertility [9][10][11][12][13][14][15].This reduced motility is thought to result in sperm from a homozygous Rose-comb rooster (RR) being outcompeted by sperm from roosters carrying the wild-type allele (Rr and rr) in promiscuous mating or heterospermic insemination experiments [16,17].Heterozygous Rose-comb roosters show normal fertility and transmit their Rose-comb and wild-type alleles to equal number of progeny.Fertility in the hen has been shown to be unaffected by her genotype at the R locus [18].The unaffected fertility of the heterozygous rooster, combined with the effects of sperm competition, has historically confounded breeders' attempts to establish flocks that breed true for Rose-comb, resulting in an equilibrium of allele frequencies that gives rise to about 15% single-combed chicks in each generation [19].
In the present study, we show that Rose-comb is caused by a large structural rearrangement that leads to transient ectopic expression of an important transcription factor in the chicken, the Mnx-class homeodomain protein MNR2 [20].This resembles our previous discovery that the Pea-comb mutation constitutes a copy number expansion in intron 1 of SOX5 leading to transient ectopic expression of SOX5 [21].We also postulate that the sperm motility defect observed in males homozygous for Rose-comb is due to the same structural rearrangement disrupting the CCDC108 gene.

Results
Linkage mapping reveals that Rose-comb is associated with suppressed recombination Linkage mapping was performed using a pedigree consisting of two F 0 males heterozygous for Rose-comb, sixteen F 0 single-combed females, and 383 F 1 progeny segregating for Rose-comb (Rose-comb 50.7%, single-comb 49.3%).Already in 1940 Rose-comb was mapped to chicken linkage group I [22] and in a recent study Rose-comb was assigned to chicken chromosome 7 (GGA7) [23].Our linkage data, presented in Table 1, confirmed the assignment to GGA7 and revealed suppression of recombination in Rose-comb heterozygotes as no recombination event was detected over a 7 Mb region, from position 16.1 Mb to 23.4 Mb on GGA7, despite the chicken consensus linkage map indicating a distance of 50 cM across this region [24].This suppression of recombination associated with Rose-comb was confirmed in a second pedigree, a Chinese Silkie x White Plymouth Rock intercross (Text S1).

Detection and characterization of a large inversion associated with Rose-comb
The observed suppression of recombination suggested that Rosecomb might be associated with an inversion.This hypothesis was strongly supported by a SNP screen using an Illumina 60K SNP array [25] of Rose-comb homozygotes from four Chinese chicken breeds, showing complete homozygosity for all SNPs in the interval 16,424,096 bp to 23,854,241 bp despite this region showing normal levels of heterozygosity in wild-type birds (Figure S2).
We searched for an inversion associated with Rose-comb using whole-genome resequencing of a 3.9 kb mate-pair library because this approach should precisely predict the location of any inversion breakpoints.The library was prepared from a pool of eight Rosecombed males from the Le Mans breed, all presumed to be homozygous Rose-comb.The library was sequenced to 16coverage.Bioinformatic analysis of the data revealed aberrant mate pairs consistent with an inversion (Figure 2).Most aberrant reads (n = 22) indicated a 7.38 Mb inversion with breakpoints located    Rose-combed individuals included in the pool (Figure 2).PCR analysis of genomic DNA from Rose-combed individuals confirmed the inversion breakpoints at nucleotide positions 16,499,781 bp and 23,881,384-23,881,392 bp (Figure 3).A 628 bp gap is predicted around 16.50 Mb in the chicken galGal3 assembly.However, sequencing across the gap in the reference sequence bird (red junglefowl female from the UCD-001 line) revealed that the gap and an additional 87 bp that together constitute the chr7:16,499,808-16,500,522 bp region must be an assembly artefact; the correct sequence of this region has been submitted to GenBank with the accession number JN942757.
The presence of the inversion had an almost perfect association with the Rose-comb phenotype in a PCR-based screen of a large number of individuals from different breeds.No unambiguously single-or Pea-combed individual (n = 679) carried any of the breakpoints, as expected, and almost all Rose-or walnut-combed chickens (n = 872) carried both the 16.50 Mb breakpoint and the 23.89 Mb breakpoint (Table 2).However, some individuals from five breeds, that had an unambiguous Rose-comb, (n = 45) showed an aberrant pattern, scoring positive for the 16.50 Mb breakpoint but not for the 23.89 Mb breakpoint (Table 2).Therefore we postulated that these birds might carry a second Rose-comb allele, R2, that evolved from the original Rose-comb allele (R1) by a second rearrangement.Such an event would also be consistent with the presence of the aberrant mate-pair reads connecting the 16.50 Mb and 23.79 Mb regions (Figure 2).PCR amplification confirmed the existence of R2, and the results showed that the allele must have originated by a recombination event between the wild-type allele at position 16.50 Mb and the R1 allele at position 23.79 Mb in the inverted region (Multimedia S1).The consequence of this recombination event is that R2 does not carry the entire inversion but instead has two duplicated segments, one 91 kb fragment (23,790,881,384 bp) that represents a remaining fragment of the inverted region together with a small duplicated fragment of 198 bp (16,499,583-16,499,781 bp) that is present on both sides Figure 2. Candidate structural variants identified from whole-genome resequencing.Mate-pair information was used to plot structural variants in the region of interest (chr7:12-30 Mb) for sequenced pools of (A) single-combed White Leghorn, (B) Rose-combed Chinese Silkie (C) Rosecombed Le Mans.Structural variants were defined as 1.5 kb windows where at least 25% of the mate pairs had mapping distances exceeding ten standard deviations above the average insert size and those .= 25% that were mapped within 1 kb of each other.Y-axis indicates the size of candidate structural variants in log 10 base pairs.X-axis indicates the genomic coordinates of the pairs supporting structural variants.Red colour indicates mates that map to different strands, indicative of inversion.Blue colour indicates mates that map to the same strand, indicative of a deletion or duplication.The structural variants uniquely observed in the two Rose-combed pools included an inversion candidate, stretching between approximately 16.50-23.88Mb.In the Le Mans pool an additional inversion candidate was also observed between 16.50-23.79Mb, supported by three read pairs.This is depicted at the magnified region at bottom of (C).Candidate structural changes shared by all genotypes may represent errors in the draft chicken assembly.doi:10.1371/journal.pgen.1002775.g002 of the 91 kb duplication (Figure 3).Genotyping the eight resequenced Le Mans males revealed an allele composition of 4 R1, 9 R2, and 3 r, explaining why a large number of mate pairs over the breakpoint located at 16.50 Mb was found, as that is present in both R1 and R2, while few reads spanning the breakpoint at 23.89 Mb were found, as that is only present in R1.The genome assembly is rich in gaps around 23.79 Mb, making it difficult to map reads that span the breakpoint only found in R2, explaining the low number of mapped reads spanning that breakpoint.
FISH analysis using four different BAC clones was employed to confirm the existence of two distinct Rose-comb alleles (Figure 4; Figure S3).The BAC clones CH261-95H11 and CH261-5G3 span the inversion breakpoints at 16.50 Mb and 23.88 Mb, respectively.BAC clones TAM32-24B23 and BW27C3 targeted regions within the inversion.A staining of a metaphase spread from an R1r heterozygote confirmed the presence of a large inversion on chromosome 7.The FISH staining of an R2r heterozygote metaphase spread was consistent with an altered organization, as the BACs CH261-95H11, TAM32-24B23 and BW27C3 showed indistinguishable staining for both R2 and r chromosomes, with only two aberrant patterns obtained, one for BAC clone CH261-5G3 that confirmed the translocated duplication of a segment from the 23.88 MB region to the 16.50 MB region, and the other a slight spatial separation of BAC clone CH261-95H11, consistent with the insertion of the translocated duplication (Figure 4).
Previous studies of the Rose-comb phenotype have established that it involves both an altered comb morphology, showing dominant inheritance, and reduced male fertility, showing   The proximal breakpoint at 16.50 Mb is located in the 59UTR of FKBP7 (FK506 binding protein 7) gene, 72 bp upstream of its start codon.Only 9 bp separate the 59UTRs of FKBP7 and PLEKHA3 (Pleckstrin homology domain containing, family Aphosphoinositide binding specific-member 3), placing the breakpoint 42 bp from the 59UTR, and 150 bp from the start codon of PLEKHA3 (Figure 3B, Multimedia S1).This rearrangement may alter regulation of PLEKHA3 and FKBP7 in both R1 and R2 alleles.
The second proximal breakpoint, unique to R2, where an inverted duplicated 23.88-23.79Mb region is joined with a duplicated 198 bp fragment at 16.50 Mb results in the duplication of a part of the ABCB6 (ATP-binding cassette, sub-family B (MDR/TAP), member 6) gene.ABCB6 has not been adequately annotated in the chicken genome, but this translocated duplication does not involve the first few exons, judging from annotated Expressed Sequence Tags (ESTs).Additionally, the region in which it is located is riddled with gaps in the genome assembly, causing the 39 region of the EST range of the gene to appear truncated.The breakpoint seems to be close to the 39 end of what may be exon 5 or 6, duplicating the very end of that exon and the remainder of the gene.This exon fragment copy is in its novel genomic context situated only 8 bp from the duplicated first exon of PLEKHA3.Whilst this could result in a hybrid transcript, the fact that an intact copy of ABCB6 is present on R1, R2 and r chromosomes, as well as the presence of the duplicated segment being unique to R2, make it an unlikely candidate for the Rosecomb phenotype.
The distal breakpoint at 23.88 Mb is located in intron 3 of CCDC108 (coiled-coil domain containing 108).This breakpoint disrupts CCDC108 and transfers the first three exons to the proximal breakpoint present in both R1 and R2.However, due to Birds labelled with an asterisk were initially assumed to have a single-comb, but genotyping revealed that they carry the R1 allele, a thorough examination of the comb phenotype revealed aberrant comb shape.Further information is given in Figure S1.doi:10.1371/journal.pgen.1002775.t002 the intact nature of CCDC108 in R2 it was excluded as causative for the altered comb morphology.The neighbouring gene MNR2 (MNR2 homeodomain protein), located only 3 kb from the inside of the distal inversion breakpoint, is also transferred to the near vicinity of the proximal breakpoint in R1 and R2.The translocation of the transcription factor MNR2 to a novel genomic context was considered the best candidate for causing altered comb morphology in Rose-comb as it belongs to the Mnx-class of homeodomain proteins which act as transcriptional repressors and specifiers of cell identity [26].Furthermore, hyaluronan (HA), a major component of the extracellular matrix and the comb in chickens, shows strong accumulation around early MNR2expressing neurons [27].
Expression analysis using comb tissue from wild-type, Rose-combed, Pea-combed, and walnut-combed embryos Expression analysis of comb tissue from early embryos (singlecombed wild-type and R1R1 homozygotes) by RT-PCR revealed that PLEKHA3 and FKBP7 were expressed in both wild-type and homozygous Rose-comb tissue, whereas CCDC108 and MNR2 were expressed in Rose-combed but not in wild-type embryos (Figure S4).The ectopic MNR2 expression was restricted to days E6-E12 of embryonic development.This suggested that the Rose-comb phenotype might be due to ectopic expression of the MNR2 homeodomain protein as a copy of MNR2 is translocated close to the 16.50 Mb breakpoint in both R1 and R2.To further explore this possibility, as well as shed light on the interaction between Rose-comb and Pea-comb causing the walnut-comb phenotype, we performed immunohistochemical staining using an anti-chicken MNR2 antibody and an anti-human SOX5 antibody (previously used in our characterization of Pea-comb [21]) in single-combed wild-type, Rose-combed, Pea-combed and walnut-combed embryos (Figure 5).This analysis revealed transient ectopic expression of MNR2 in Rose-combed embryos, consistent with the results of the RT-PCR analysis.Striking MNR2 expression was observed in a layer of mesenchymal cells located in the area where the comb is developing at day E6.5 but not at E9 (Figure 5C and 5D).This pattern of transient ectopic expression resembles that previously reported for SOX5 in Pea-combed embryos, where expression is weak at day E5, strong at E9 (Figure 5E and 5F) and not present at day E12 [21].
Walnut-combed embryos showed transient ectopic expression of both MNR2 and SOX5 as expected from their genotype (Figure 5G-5H).The ectopic expression of MNR2 and SOX5 overlapped only partially, with peak expression occurring first for MNR2.The data revealed ectopic expression of MNR2 and SOX5 in the same cell type as well as MNR2-SOX5 coexpression within individual cells (Figure 5I).Furthermore, MNR2 expression was observed at E9 together with SOX5 (Figure 5H) but this was not the case in the absence of SOX5 expression (Figure 5D) suggesting a potential positive interaction between the Pea-comb allele of SOX5 and the Rose-comb allele of MNR2.Interestingly, at day E6 MNR2 and SOX5 also showed ectopic expression in Rosecombed and Pea-combed birds, respectively, in mesenchyme present in the region where the wattles develop (Figure 5J-5L).The ectopic expression of MNR2, with its transcriptional repression activities, is likely to change the identity of the mesenchyme underlying both the comb and wattles.Seminal work on comb primordium development has shown that the comb shape is directly dependent on instructive signals derived from the underlying mesenchyme/dermal structures [28][29][30].This suggests that the comb and wattle tissue share a common developmental pathway and that the structural variants underlying Rose-comb and Pea-comb activate the expression of MNR2 and SOX5, which both modulate or intercept this pathway.However, wattle phenotype is not altered by the Rose-comb mutation as it is by the Pea-comb mutation (Figure 1).

59RACE analysis of transcripts initiated from the vicinity of the inversion breakpoints
59RACE analysis was performed using tissue from early embryonic comb and from adult testis for the three genes located close to the two R1 inversion breakpoints.Results are summarized in Figure S5.The samples were from single-combed wild-type birds and R1R1 homozygotes.A full length PLEKHA3 transcript (denoted PLEKHA3a in Figure S5) was expressed in both tissues and in both genotypes.A PLEKHA3-CCDC108 hybrid transcript (PLEKHA3b) corresponding to exons 1 and 3 from CCDC108 and exons 2-8 from PLEKHA3 was found in Rose-comb testis (Figure S6).The FKBP7 transcripts showed no difference between genotypes but different splice forms were expressed in comb and testis.Full-length CCDC108 transcripts were only found in wildtype testis (Figures S5 and S6), whereas two very similar hybrid transcripts lacking the first three exons of CCDC108 were expressed in both R1R1 testis and comb tissue (Figures S5 and S6).

Analysis of male fertility in Rose-comb homozygotes
Rose-comb is associated with reduced male fertility in homozygotes due to low sperm motility [12,17,18].However, as R1 is the more common Rose-comb allele (Table 2) we wanted to investigate if the fertility effect is also associated with the R2 allele discovered in the present study.A preliminary study to address this question was carried out by mating single-combed and Rose-combed roosters of several genotypes (R1R1, R2R2, R1r and rr) to single-combed and Rose-combed hens.The data were consistent with reduced male fertility in R1R1 males, as expected, but there were no signs of reduced fertility in R2R2 homozygotes (Text S2; Table S1).Thus, the deleterious effect on male fertility appears to be restricted to the R1 allele, suggesting that the lesion causing this phenotypic effect is located at the 23.88 Mb breakpoint (Figure 3).The obvious candidate for this effect is the disruption of the CCDC108 transcript that leads to the expression of a truncated transcript in testis (Figures S5 and S6).

Discussion
The present study illustrates several striking features of the genetic diversity present in domestic animals.Firstly, the R2 allele exemplifies the evolution of alleles by two or more consecutive mutations.Other examples include the Dominant white allele in pigs which involves a 450 kb duplication encompassing the entire KIT gene combined with a splice mutation in one of the duplicated copies [31] and black spotting in pigs which is determined by the combined effects of two mutations in MC1R, a missense mutation associated with black colour and a somatically unstable two basepair insertion [32].Secondly, it represents a new example of how structural rearrangements have contributed to rapid phenotypic evolution observed in domestic animals [33,34].The majority of the structural changes reported to be associated with phenotypic effects, like the effects of R1 and R2 on comb morphology, constitute cis-acting regulatory mutations.The altered configurations of regulatory elements on the rearranged chromosome lead to altered gene expression patterns.It appears plausible that structural rearrangements similar to those that affect comb There are several mechanisms by which genomic rearrangements are thought to occur, involving either double strand break repair via primarily non-homologous mechanisms or homology mediated replication and recombination based processes [35,36].At the R1 and R2 16.50 Mb breakpoint there is only a single base pair sequence overlap, while at the R1 23.88 Mb breakpoint there is a 7 bp overlap with one mismatch.Although it is not possible to determine the exact mechanism by which the original inversion occurred, it is likely that microhomology at the 23.88 Mb breakpoint was involved in the generation of the R1 allele.The generation of the R2 allele occurred by recombination between a wild-type chromosome and an R1 chromosome (Multimedia S1).On the wild-type chromosome this event occurred 198 bp upstream of the 16.50 Mb breakpoint, on the R1 chromosome the recombination event occurred 91 kb into the inversion from the 16.50 Mb breakpoint.This resulted in the duplication of the 91 kb portion of the R1 chromosome, including the R1 proximal breakpoint and the 199 bp fragment of the wild-type chromosome, effectively inserting the duplicated sequence at 16.50 Mb into a wild-type chromosome.Analysis of the R2 recombination event breakpoint shows 2 bp of sequence homology, again suggesting that sequence microhomology was likely involved.That both events introduced breakpoints around 16.50 Mb, spaced only 198 bp apart, suggests that some characteristic of this region may predispose it to such events.
More than half of the loci identified in human genome-wide association analyses do not overlap coding regions [37], implying that they reflect regulatory polymorphisms.The present study illustrates how challenging it can be to reveal the molecular mechanism underlying even a simple monogenic trait in a model organism such as the chicken.We were able to demonstrate transient ectopic expression of MNR2 during a narrow period of embryonic development because immunohistochemistry provided the spatial resolution allowing the detection of ectopic expression in a subset of cells within the affected tissue (Figure 5).
We postulate that the well-established association between the Rose-comb phenotype and reduced sperm motility is restricted to the R1 allele and that it is caused by the disruption of the CCDC108 transcript.The predicted CCDC108 protein sequence in chickens shows 49% amino acid identity with human CCDC108 (HomoloGene:28093; www.ncbi.nlm.nih.gov).CCDC108 has an unknown function, according to the UNIPROT database (www.uniprot.org/uniprot/Q6ZU64),it is a single pass membrane protein composed of 1925 amino acids, containing one MSP (major sperm protein) domain.The MSP domain is present in major sperm proteins and in sperm specific proteins (SSPs) found in various nematodes as well as in the mammalian Motile sperm domain-containing proteins-1, -2 and -3 (MOSPD1-3).All MSP, SSP and MOSPD proteins are small, with a size range of 107-518 amino acids, and thus much smaller than CCDC108.Mouse Ccdc108 is expressed in testis and shows differential expression during progression of spermatogenesis [38]; expression is absent in juvenile mice but is turned on when male mice reach sexual maturity (www.ncbi.nlm.nih.gov;GEO profiles GDS606/ 164004_at/Ccdc108/Mus musculus).Furthermore, by using reciprocal best-hit protein BLAST searches, putative CCDC108 orthologs can be found in many organisms, including in deep branches on the tree of life.One such orthologous protein has been annotated in Chlamydomonas algae as an axonemal protein named Flagellar Associated Protein 65 (FAP65) [39].FAP65 expression is strongly induced after deflagellation, when cells regenerate their flagella.This sequence homology suggests that CCDC108 is part of the sperm flagellum and thus that a disruption of CCDC108 function may lead to sperm motility defects as observed in Rose-comb R1R1 homozygotes.This study establishes CCDC108 as a candidate gene for sperm motility disorders in humans.
The present study strongly suggests that Rose-comb and Peacomb are caused by transient ectopic expression of two potent transcription factors, MNR2 and SOX5, respectively.However, at present we cannot exclude the possibility that altered expression levels of FKBP7 or PLEKHA3, located on either side of the proximal breakpoint, may have some impact on the comb phenotype.However the ectopic MNR2 expression in the developing embryo is by far the most striking molecular consequence of the Rose-comb inversion.The exact downstream mechanisms leading to the altered comb morphology remain undetermined.Comb tissue is composed of layers of epidermis, dermis and central connective tissue comprising primarily collagen and hyaluronan (HA) [40].The previous report that there is a strong accumulation of HA around early MNR2-expressing neurons [27] may be relevant for the Rose-comb phenotype, but perhaps more importantly, MNR2 acts as a repressor and specifier of cell identity [26].SOX5 also has an established functional role that makes sense in relation to the altered comb morphology observed in Pea-combed and walnut-combed birds.SOX5 contributes to chondrogenesis, together with SOX6 and SOX9 it activates specific genes during embryonic cartilage formation [41], and has a repressive role in oligodendrogenesis during neural development [42].A fascinating observation is that both the Rosecomb mutations and the Pea-comb mutation give rise to ectopic expression in the area of the developing comb, leading to altered comb morphology in both mutants, as well as in the wattle area, which only leads to altered morphology in birds carrying Pea-comb (Figure 1).The fact that the transient ectopic expression of MNR2 and SOX5 apparently occurs in the same population of mesenchymal cells, and with at least partially overlapping expression in individual cells in the comb primordium, provides a reasonable explanation of why the combined effect of the two mutations leads to the formation of the walnut-comb.Thus, 104 years after Bateson and Punnett [2] reported the first example of epistatic interaction between genes, we can now provide a molecular explanation for their seminal observation.

Ethics statement
All animal work has been conducted according to relevant national and international guidelines.

Animals
Linkage mapping was carried out using a pedigree consisting of two heterozygous R1r male parentals, each mated with eight homozygous rr females, resulting in 383 progeny segregating for Rose-comb.The Rose-combed roosters were from an INRA (French National Institute for Agricultural Research) resource population, with the R1 allele having been derived from the French breed Charollaise.The wild-type single-combed hens were from another INRA resource population line.
DNA samples from various chicken breeds were genotyped for the R1, R2 and wild-type alleles.These included samples collected as part of the AvianDiv project [43]

Linkage analysis
Linkage analysis was performed by genotyping two microsatellites and nine SNPs from chromosome 7 using standard procedures.Custom TaqMan SNP Genotyping Assays (Applied Biosystems) were designed by ABI, other primers were designed with the Primer3Plus webtool (http://www.bioinformatics.nl/cgibin/primer3plus/primer3plus.cgi).See Table S2 for primer and probe sequence information.Linkage analysis was performed with the Crimap software (version 2.4) [44].

Whole-genome resequencing
DNA from eight Rose-combed males from the Le Mans breed, all presumed to be homozygous for Rose-comb, were pooled.Whole genome resequencing data from a pool of Rose-combed Silkie chickens, and another pool of single-combed White Leghorns were obtained at later time points and included in this study to verify the results obtained from the Le Mans pool.A sequencing library was generated for the Le Mans sample using a Mate-pair SOLiD3 protocol and sequenced on SOLiD v.3 (Life Technologies, Carlsbad, USA).The White Leghorn library was generated using a Mate-pair SOLiD3+ protocol and sequenced on SOLiD 3+.The Silkie Library was generated using a Mate-pair SOLiD5500 protocol and sequenced on SOLiD5500XL.The Le Mans, White Leghorn and Silkie reads (2650 bp mate-pair reads) were mapped to the chicken genome (WUGSC 2.1/galGal3) reference assembly using the software CoronaLite v0.4r2, Bioscope v1.0.1 and LifeScope v2.0, respectively, with average insert sizes estimated as approximately 3.9, 3.1 and 2.6 kb and average read depth approximately 16, 106 and 206 over the chicken genome.Mapping distances between mate-pairs were used to detect structural variations in relation to the reference assembly.All library kits, alignment software and massively parallel sequencing equipment were used according to the manufacturer's instructions (Life Technologies, Carlsbad, USA).

Fluorescent in situ hybridisation (FISH)
Heterozygous Rose-combed embryos (R1r and R2r) were produced from parental stock maintained at INRA.The R1 allele originated from Belgian Barbu d'Anvers and the R2 allele from French Alsacienne.BAC clones were chosen considering their position in the chicken genome sequence (Table S3).BW27C3 comes from the Wageningen library [45].TAM32-24B23 was ordered from TAMU (Texas A&M BAC Libraries, USA).CH261-5G3 and CH261-95H11 were ordered from the Children's Hospital Oakland Research Institute in Oakland (CHORI), California, USA.BAC clones were grown in LB medium with 12.5 mg/ml chloramphenicol according the instructions of the providers.The DNA was extracted using the Qiagen plasmid midi kit.
FISH was carried out on metaphase spreads obtained from fibroblast cultures derived from 7 days old embryos, arrested with 0.05 mg/ml colcemid (Sigma) and fixed by standard procedures.The FISH protocol is derived from Yerle et al. [46].Two-colour FISH was performed by labelling 100 ng of each BAC clone with alexa fluorochromes (ChromaTide Alexa Fluor 488-5-dUTP, Molecular probes; ChromaTide Alexa Fluor 568-5-dUTP, Molecular Probes) by random priming using the Bioprim Kit (Invitrogen).The probes were purified using spin column G50 Illustra (Amersham Biosciences).Probes were ethanol precipitated together and hybridised to the metaphase slides for 17 h at 37uC in the Hybridizer (Dako) after denaturation for 8 min at 72uC.
Chromosomes were counterstained with DAPI in antifade solution (Vectashield with DAPI, Vector).The hybridised metaphases were screened with a Zeiss fluorescence microscope.A minimum of twenty spreads was analysed for each experiment.Spot-bearing metaphases were captured and analysed with a cooled CCD camera using Cytovision software (Applied Imaging, Leica-Microsystem).Images were formatted, resized and arranged for publication using Adobe Photoshop and Adobe Illustrator.

PCR analysis of rearrangement breakpoints
A set of five PCR primers that together will amplify a series of specific bands over each of the five breakpoints was designed for genotyping the R1, R2 and r alleles.Primer and protocol information are in Table S4.Gel image for the six possible genotypes is presented in Figure S7.

RT-PCR analysis of tissue samples
Comb tissue was collected from homozygous (R1R1) and heterozygous (R1r) Rose-combed birds as well as homozygous (rr) single-combed wild-type birds.Comb tissue was sampled at embryonic (E) days 6, 7, 8, 9, 10, 11, 12, 15 and 19.Testis tissue was sampled from adult roosters at day 200.Samples from three birds of each type were collected and stored in RNAlater (Ambion).RNA was extracted using RNeasy Mini kit (Qiagen).cDNA was synthesized with 1 mg of RNA using oligo(T) primer.Primers spanning introns were used in RT-PCR.The 59 and 39 RACE were performed using GeneRacer Kit (Invitrogen).

Immunohistochemistry
Homozygous Rose-combed Alsacienne, single-combed INRA resource population, homozygous Pea-combed Cheptel and heterozygous walnut-combed Alsacienne x Cheptel embryos were used.Heads from staged embryos were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for one hour at 4uC.Fixed heads were incubated overnight in 30% sucrose in PBS at 4uC, embedded in OCT freezing medium (Tissue-Tek, Sakura), frozen and sectioned in a cryostat.Cross sections, 10 mm thick, were collected on glass slides (Super Frost Plus, Menzel-Gla ¨ser).The sections were rehydrated in PBS for 5 min and then blocked for one hour in PBS containing 1% fetal calf serum, 0.1% Triton-X and 0.02% Thimerosal.The antibodies MNR2 (Developmental studies hybridoma bank, 81.5C10) and SOX5 (Abcam, a_6226041) were diluted 1:250 and 1:1000 respectively in blocking solution and incubated on the slides overnight at 4uC.The secondary antibodies (Invitrogen) were incubated at room temperature for two hours at a 1:1000 dilution in blocking solution.Samples were analysed using a Zeiss Axioplan2 microscope equipped with Axiovision software.Images were formatted, resized, enhanced and arranged for publication using Axiovision and Adobe Photoshop.

URL
Information on the chicken genome sequence is available at http://www.genome.ucsc.edu.

Accession numbers
The sequence data presented in this paper have been submitted to GenBank with accession numbers JN942757-JN942760, JN880446, JN880447, JQ004983, and JQ004984.Text S2 Test matings to assess male fertility.(PDF)

Figure 3 .
Figure 3. Organization of wild-type and Rose-comb chromosomes and description of inversion and duplication breakpoints.(A) Constitution of the two Rose-comb alleles, R1 and R2, in relation to the organization of the wild-type (r) chromosome 7 in chickens.Sequence orientation in relation to the wild-type chromosome is indicated by arrows.Duplicated sequence in R2 (chr7:23,790,414-23,881,384 bp) is in reverse orientation, apart from 198 bps (chr7:16,499,583-16,499,781 bp) flanking the inverted segment.Breakpoint locations are indicated by arrows (a-e).Breakpoints for the R1 inversion are at 16,499,781 and 23,881,384-23,881,392 bp in the wild-type sequence.Additional breakpoints for the R2 duplication are at 16,499,583 and 23,790,414 bp.(B) Organisation of genes in the five different chromosomal configurations associated with Rosecomb.Breakpoint locations are indicated with red arrows.mRNAs with accession numbers XM_422054.2,NM_204929.1,CR353563.1 and AJ719903.1,as well as EST sequences CD218766.1,BG713529.1 and DR426188.1 were used to define the genes illustrated.The copy of ABCB6 that occurs at the second proximal breakpoint unique to the R2 chromosome, is 59 truncated from the duplication event, and appears 39 truncated due to a gap in the assembly.An intact full length copy of this gene is expected to occur at its native chromosomal position (around 23.79 Mb) on R1, R2 and r chromosomes.doi:10.1371/journal.pgen.1002775.g003 inheritance.The presence of two different Rose-comb alleles facilitates the elucidation of the causal relationship between the observed chromosomal rearrangement and these two different aspects of the Rose-comb phenotype.No obvious phenotypic differences in comb morphology were observed amongst birds carrying R1 and R2, matched for breed and genetic background (FigureS1).Thus the critical genetic lesion causing the Rose-comb morphology must be located at the 16.50 Mb breakpoint including the 91 kb segment transferred from the 23.79-23.88Mb region, because this is the only alteration present in both R1 and R2.

Figure 5 .
Figure 5. Immunohistochemical labelling of MNR2 and SOX5 in various comb tissues.Wild-type single-comb (a, b), Rose-comb (c, d), Peacomb (e, f) and walnut-comb (g, h) sections from embryonic day (E) 6.5 (a, c, e, g) and 9 (b, d, f, h) were labelled against MNR2 and SOX5.Nuclei are visualized by DAPI.Boxed regions are shown magnified as single colour.Arrows in (g) and (h) indicate double labelled cells whereas arrowheads indicate single labelled cells.(i) E5.5 walnut comb.The two framed regions are shown magnified and arrows indicate double labelled cells.(j-l) The prospective wattle region for wild-type single-comb, Pea-comb and Rose-comb.(m) Schematic view of an E6.5 head where boxes indicate the regions of the comb depicted in (a-h) and wattles depicted in (j-l).Scale bar equals 100 mm in (h) and is valid for (a-h) and 100 mm in (l) valid for (i-l).doi:10.1371/journal.pgen.1002775.g005 , from resource populations at INRA, and 31 different breeds of Chinese chickens collected by Institute of Poultry Science, Chinese Academy of Agricultural Sciences.Blood samples from privately owned Icelandic chickens were obtained at eight different locations in the South-West of Iceland by a veterinarian with permission from owners.Genomic DNA from the reference red junglefowl bird was kindly provided by Dr. J.B. Dodgson.

Figure S1
Figure S1 Phenotypic variability of Rose-comb in Icelandic chickens (A-W), Alsacienne (X), and INRA resource population

Table
Comb morphology is a trait that shows considerable variability among domestic chickens.The Rose-comb mutation causes a drastically altered shape of the comb, whereas the Pea-comb mutation leads to a considerable reduction in the size of the comb.The combined effect of Rose-comb and Pea-comb is a comb shaped like a walnut, and the phenotype is consequently named walnut-comb.Both Pea-comb and Rose-comb are caused by structural changes in the genome leading to altered expression of important transcription factors.In a previous study we showed that Pea-comb is caused by misexpression of SOX5 during the development of the comb.In this study we report that Rose-comb is caused by a large inversion on chicken chromosome 7.The inversion moves the MNR2 gene to a new genomic location.This leads to misexpression of MNR2 during comb development, similar to the defect causing Pea-comb.Roosters that are homozygous for the Rose-comb inversion show poor sperm motility, and our results suggest that this is caused by the disruption of the CCDC108 gene that is located at one of the inversion breakpoints.CCDC108 is well conserved between chickens and humans, and this study establishes CCDC108 as a candidate gene for sperm motility disorders in humans.approximately at 16.50 Mb and 23.88 Mb.However, three aberrant mate pairs connected the 16.50 Mb region with a region at 23.79 Mb, not consistent with a single inversion in all eight . Two-point linkage analysis of the Rose-comb locus using 11 markers on chicken chromosome 7.C7C19.4420.00112.9 18,865,430 SNP C7C19.5590.00113.8 21,118,705 SNP C7C107.36 0.00 112.9 23,443,405 SNP C7C15.2220.365.8 33,000,168 SNP 1 SNP markers are defined in Table S2.doi:10.1371/journal.pgen.1002775.t001AuthorSummary

Table 2 .
Genotyping of the Rose-comb locus in various chicken breeds.