Wild-type and Pea-comb chickens. (A) Wild-type male, (B) wild-type female, (C) Pea-comb male and (D) Pea-comb female (Photo by David Gourichon). doi:10.1371/journal.pgen.1000512.g001 

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... in Pea-comb embryos including the scattered cells in the ectoderm. However in Pea-comb embryos, striking ectopic SOX5 expression was observed in mesenchymal cells located just beneath the surface ectoderm where the comb and wattles will develop ( Figure 3A–3J). Differential expression was confirmed with in situ hybridization (Figure 3G–3H) and quantitative real-time PCR (see above). The ectopic expression is transient. Whereas few cells with ectopic expression are visible in the comb region by day E6, they are prominent at E9, and almost completely absent at E12 (Figure 3K–3P). Thus, Pea-comb appears to be a spatiotemporal- specific, cis-acting regulatory SOX5 mutation. A major challenge in current genome biology is to reveal the biological significance of the many Evolutionary Conserved Non- coding Sequences (ECNS). The analysis of the functional significance of ECNS is hindered by a paucity of mutations in such regions which show an association with a phenotype. Here we demonstrate the first spontaneous SOX5 mutation associated with a phenotype, despite the rich abundance of ECNS in the SOX5 region (Figure 2A). SOX5 is under complex regulation and as demonstrated here, mutations affecting its regulation can have very specific effects. It would be surprising if regulatory mutations in this gene do not to some extent contribute to phenotypic diversity present in humans. For instance, the human face shows a bewildering array of diversity. The nearly identical facial appearances of monozygotic twins imply that this diversity is nearly 100% genetically determined, but knowledge concerning the underlying molecular basis of this diversity is restricted to certain craniofacial abnormalities [17]. It is likely that regulatory mutations in developmentally important genes shape this type phenotypic diversity, and SOX5 may very well be one of the genes that contributes. The comb is a sexual ornament that shows strong sexual dimorphism in chickens and the fact that this sexual dimorphism is maintained in Pea-comb birds shows that the Pea-comb tissue maintains the response to the influence of sex hormones (Figure 1). That the comb is under sexual selection is evidenced by red junglefowl females showing mating preferences for males with large combs and reciprocally, males tend to favour females with larger combs [18,19]. The size of the comb is proportionally larger in many breeds of domestic chickens compared to their wild ancestors. In our previous study of a large intercross between White Leghorn chicken (with larger combs) and red junglefowl, we identified a number of Quantitative Trait Loci (QTL) affecting the size of the comb [20]. Interestingly, one of the QTL controlling the size of the female comb overlaps the SOX5 locus, which now becomes an obvious candidate gene for this QTL. However, the confidence interval for the QTL is large, as is usually the case in an F 2 intercross, and the entire SOX5 region needs to be considered in a search for possible causative mutation(s). SOX genes are defined by their high-mobility-group (HMG) domains and are divided into eight groups (A to H) based on protein sequence comparison [14]. SOX5 belongs to the D family of SOX genes, along with SOX6 and SOX13 . SOX5 has been termed an architectural transcription factor [21], as binding to this protein will cause a sharp bend (80–135 degrees) in the bound DNA and may lead to different regulatory regions of a target gene coming into closer proximity. SOX5 has been reported to have a co-operative role in chondrogenesis; during embryonic cartilage formation SOX5 and SOX6 assist SOX9 to activate specific genes [22], and have a repressive role in oligodendrogenesis during neural development [23]. SOX5 is also expressed in the developing neocortex and cranial neural crest during the early stages of development. SOX5 postmitotically regulates migration, axon projection and postmigratory differentiation of certain neocortical neurons [24] but little is known about SOX5 function in neural crest derivatives [25]. With these different roles, the functional consequence of the transient ectopic SOX5 expression in Pea-comb birds is not clear. The comb is composed of layers of epidermis, dermis and central connective tissue, of which collagen and hyaluronan are the major components [26]. The ectopic SOX5 expression is first seen in E7 (st28) mesenchyme (Figure 3). Previous studies with grafts of comb-primordia from different ages at various locations imply that cells giving rise to the comb are already determined by E4 (st24) [27,28] and that the determination resides in the mesenchymal components and not in the ectoderm [27]. These experiments also revealed that the morphology of the comb was under control of the mesenchyme [27,29]. Heterotopic grafts of single-comb primordia to the neck region without beak mesenchyme, lost the serrated single ridge morphology and expanded laterally following the development, resembling that of complex comb types [29] such as the Pea-comb. Hence, changes in the underlying mesenchyme at the time of the ectopic SOX5 expression will not affect the determination and initial stages of the comb development but rather the development of comb shape. Our results indicate that ectopic SOX5 expression changes the modulating properties of the mesenchyme of the nasofacial region beneath the regions of the developing comb and wattles. The serration of a single comb is associated with loosely coherent clusters or points of proliferating mesenchymal cells [30,31]. Such clusters were not observed in the developing Pea-comb mesenchyme and this difference may be due to the ectopic SOX5 expression. Pea-comb is an additional example of a Copy Number Variation (CNV) associated with a phenotype. About 12% of the human genome contains tandem duplications that may show CNV [32] and a number of human diseases have been reported to be associated with CNVs [33,34]. It is important to distinguish CNVs that are due to duplications of single copy sequences ( de novo duplications) and expansions or contractions of already duplicated sequences. We have previously reported three de novo duplications associated with phenotypic traits in domestic animals, Dominant white colour in pigs [35], the Ridge phenotype in Ridgeback dogs [36] and Greying with age in horses [37]. In contrast, Pea-comb and most human diseases associated with CNVs involve expansions or contractions of existing duplications. Pea-comb is however an unusual CNV associated with a phenotype because it involves the amplification of a non-coding region located far from any coding sequence. Pea-comb therefore to some extent resembles the ...

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... 1902 Bateson [1] reported the first examples of Mendelian inheritance in animals based on the genetic studies of four traits in chicken, one of these being the Pea-comb phenotype ( Figure 1). The Pea-comb allele results in reduced comb and wattle size compared to wild-type individuals. Pea-comb shows incomplete dominance and as such the small comb shape can differ slightly between homo- and heterozygous birds. Homozygotes present three longitudinal rows of papillae, whilst heterozygotes can have a well-developed central blade (still of reduced size compared to wild-type) [2]. The wild-type has a single central blade of tissue and is therefore often denoted single comb. Bateson and Punnet [3] reported the first example of an epistatic interaction between genes when they showed that walnut comb is caused by the combined effect of Pea-comb and Rose-comb . Subsequent studies revealed that Pea-comb , besides its effect on comb and wattles, was also associated with a ridge of thickened skin that runs the length of the keel over the breast bone [4]. The Pea-comb mutation may have occurred early during domestication as the phenotype is widespread among both European and Asian breeds of chickens. Furthermore, it has been speculated that a reproduction in the tomb of Rekhmara at Thebes, Egypt, dated to 3,450 years before present depicts a rooster with the characteristic Pea-comb phenotype [5]. Chickens were domesticated from the red junglefowl with some contributions from the grey junglefowl [6], two species adapted to subtropical or tropical environments. Chickens do not sweat, instead they dissipate up to 15 percent of their body heat through the comb and wattles [7], making the Pea-comb phenotype adaptive to cold environments since it reduces heat loss. This phenotype has also been favoured in chickens bred for cock- fighting, as noted by Darwin [8] the smaller ornaments provided smaller targets for injury. In the present study we show that the classical Pea-comb phenotype in chickens is caused by a large expansion of a duplicated sequence in intron 1 of the gene for the SOX5 transcription factor. Pea-comb has previously been assigned to chromosome 1 [9,10]. We refined the localization by linkage analysis using a dense set of genetic markers and a large segregating family. The interval harbouring Pea-comb was defined as 67,831,796–68,456,921 bp on chromosome 1, based on flanking markers showing recombination with Pea-comb (Table 1). This interval contains a single gene, SOX5 , a member of the SRY -related HMG box family of transcription factors. SOX5 is located in a one Mb gene desert that is enriched for Evolutionary Conserved Non-coding Sequences (ECNS; Figure 2A). This is a typical feature of developmentally important genes [11,12]. SOX5 was not an obvious candidate gene for Pea- comb but the comb is composed of extracellular matrix and SOX5 has a well-established role in chondrocyte development and production of extracellular matrix [13]. Mouse SOX5 knockouts die at birth from respiratory distress caused by a cleft secondary palate and narrow thoracic cage [13]. Mouse SOX5/SOX6 double knockouts die in utero with severe skeletal dysplasia, demonstrating that these two genes have critical, redundant roles during development [13,14]. To further refine the localization of Pea-comb we characterized SOX5 haplotype patterns among three breeds of chicken, a French experimental population, the Russian Orlov and the Chinese Hua- Tung. These breeds all carry Pea-comb and, to the best of our knowledge, there has been no exchange of genetic material between them for 100 generations or more. The Orlov and Hua- Tung are not fixed for Pea-comb , allowing recombination to reduce the size of the shared haplotype associated with the mutation. Initial IBD mapping using 12 samples from the three different populations revealed a completely shared haplotype between 67,961,701 bp and 68,061,854 bp (Table 2). SNP genotyping of all Hua-Tung and Orlov individuals available narrowed the shared haplotype further to a 50 kb region spanning positions 67,985,285 bp and 68,035,337 bp (Figure 2A; Table 2). The upstream break-point (67,985,285 bp) was identified using a single Hua-Tung bird. The break was confirmed in two additional individuals from the same population which were homozygous at the six SNPs diagnostic of the Pea-comb haplotype, but heterozy- gous at this break-point. Downstream, the haplotype was broken at 68,035,337 bp in three Orlov birds (Table 2). This critical region is located upstream of the first annotated exon however a comparison with SOX5 from mammalian species indicated that exon 1 is missing from the chicken genome assembly and is expected to be found more than 200 kb upstream of exon 2 (Figure 2A). We confirmed the existence of an upstream exon in chicken by 5 9 RACE analysis. The obtained nucleotide sequence (GenBank accession number FJ548639) showed 90% identity to human SOX5 exon 1, but did not give a match in the chicken genome, implying a gap in the current chicken assembly. Resequencing the 50 kb region associated with Pea-comb from a set of Pea-comb and wild-type birds revealed a limited number of sequence polymorphisms, with fixed differences between genotypes. These potentially causative SNPs were interrogated using a larger set of wild-type birds from the AvianDiv panel [15], however none of the alleles were found to be unique to the Pea-comb haplotype (Table 2). The failure to identify a causative point mutation led to a screen of the Pea-comb region for structural changes using Southern blot analysis. The SOX-85kb_SB probe (Table S1) revealed a dramatic increase in the hybridization signal of a 3.2 kb Bam HI fragment in Pea-comb birds (Figure 2C) whilst other probes from the region gave identical restriction fragment patterns for both alleles. The result implied that Pea-comb is associated with a large tandem array of a duplicated sequence containing a Bam HI restriction site. PCR and sequence analysis revealed that this DNA fragment is also duplicated on wild- type chromosomes which have two copies (Figure 2B), whereas the Pea-comb allele has a large number of copies. Quantification of the copy number of the duplicated fragment using both pulsed field gel electrophoresis (PFGE) and real-time PCR analysis confirmed that a massive amplification of a duplicated sequence is associated with the Pea-comb allele. PFGE analysis using the restriction enzyme Psh A1, which cuts outside the duplicated region, gave a 97 kb restriction fragment in Pea-comb birds in contrast to a predicted 10 kb fragment based on the reference genome sequence from a wild-type bird (Figure S1). The result indicates that the Pea-comb allele contains about 30 copies of the duplicated sequence. Real-time PCR analysis of Pea-comb birds from three breeds confirmed this finding and revealed a 20- to 40-fold sequence amplification (Figure 2D). The real-time PCR analysis did not indicate two clear groupings corresponding to Pea- comb heterozygotes and homozygotes suggesting that the duplication may show further copy number variation among Pea-comb individuals. Interestingly, 100 years ago Bateson and Punnett [16] reported variable expression of the Pea-comb phenotype which may reflect a copy number variation of the duplicated sequence. Although the duplicated sequence is not evolutionary conserved, it is located close to two highly conserved ECNSs (Figure 2A). The distance between these elements is about 10 kb on wild-type chromosomes in contrast to about 100 kb on Pea-comb chromosomes. The duplication includes a sequence repeated in two copies on wild-type chromosomes and each copy contains two partial LINE fragments (Figure 2B). The expansion of this duplication must be the causative mutation because it was the only polymorphism showing complete association with the phenotype. A closer examination of the duplicated sequence shows that it is particularly GC-rich and contains a small CpG island (Figure 2A and 2B). The wild-type chromosome contains two copies of this CpG island whereas the Pea-comb chromosome contains about 30. This could be relevant for the mechanism of action of this intronic mutation. The Pea-comb phenotype is apparent at hatch and must therefore reflect altered gene expression during development. Tissue samples from the comb region were collected from both homozygous Pea-comb and homozygous wild-type birds at embryonic (E) days 6, 7, 8, 9, 12 and 19 for expression analysis. Quantitative RT-PCR analysis only revealed significant differences in SOX5 expression at stage E7 and E8 (which were combined due to the low number of E8 samples). The results for E7 + 8 revealed significant upregulated SOX5 expression in the comb region in Pea-comb birds (t = 2 5.0, p = 0.002; Figure S2A). Expression analysis was also conducted using primers specific for each exon of SOX5 (including the previously un-annotated exon 1 described above), however the results did not indicate any difference between genotypes in regards to differential splicing of SOX5 (Figure S2B). Immunohistochemical staining with a human SOX5 antibody as well as in situ -hybridization with a chicken-specific cRNA probe was carried out to investigate SOX5 expression in both Pea-comb and wild-type embryos during development (Figure 3). Specific immunostaining of nuclei was seen in developing cartilaginous structures including the nasal septum, Meckel’s cartilage and optic sclera (Figure 3A and 3D). Scattered and rare SOX5 positive cells were seen in the surface ectoderm (Figure 3B and 3M). All structures with SOX5 staining in wild-type embryos were ...