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Parallel Evolution of Polydactyly Traits in Chinese and European Chickens

  • Zebin Zhang ,

    Contributed equally to this work with: Zebin Zhang, Changsheng Nie, Yaxiong Jia

    Affiliation Department of Animal Genetics and Breeding, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China

  • Changsheng Nie ,

    Contributed equally to this work with: Zebin Zhang, Changsheng Nie, Yaxiong Jia

    Affiliation Department of Animal Genetics and Breeding, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China

  • Yaxiong Jia ,

    Contributed equally to this work with: Zebin Zhang, Changsheng Nie, Yaxiong Jia

    Affiliation Beijing Municipal General Station of Animal Science, Beijing, 100107, China

  • Runshen Jiang,

    Affiliation College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, China

  • Haijian Xia,

    Affiliation Department of Animal Genetics and Breeding, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China

  • Xueze Lv,

    Affiliation Beijing Municipal General Station of Animal Science, Beijing, 100107, China

  • Yu Chen,

    Affiliation Beijing Municipal General Station of Animal Science, Beijing, 100107, China

  • Junying Li,

    Affiliation Department of Animal Genetics and Breeding, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China

  • Xianyao Li,

    Affiliation College of Animal Science, Shandong Agricultural University, Taian, 271018, China

  • Zhonghua Ning,

    Affiliation Department of Animal Genetics and Breeding, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China

  • Guiyun Xu,

    Affiliation Department of Animal Genetics and Breeding, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China

  • Jilan Chen,

    Affiliation Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China

  • Ning Yang,

    Affiliation Department of Animal Genetics and Breeding, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China

  • Lujiang Qu

    quluj@163.com

    Affiliation Department of Animal Genetics and Breeding, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China

Abstract

Polydactyly is one of the most common hereditary congenital limb malformations in chickens and other vertebrates. The zone of polarizing activity regulatory sequence (ZRS) is critical for the development of polydactyly. The causative mutation of polydactyly in the Silkie chicken has been mapped to the ZRS; however, the causative mutations of other chicken breeds are yet to be established. To understand whether the same mutation decides the polydactyly phenotype in other chicken breeds, we detected the single-nucleotide polymorphism in 26 different chicken breeds, specifically, 24 Chinese indigenous breeds and 2 European breeds. The mutation was found to have fully penetrated chickens with polydactyly in China, indicating that it is causative for polydactyly in Chinese indigenous chickens. In comparison, the mutation showed no association with polydactyly in Houdan chickens, which originate from France, Europe. Based on the different morphology of polydactyly in Chinese and European breeds, we assumed that the trait might be attributable to different genetic foundations. Therefore, we subsequently performed genome-wide association analysis (GWAS) to locate the region associated with polydactyly. As a result, a ~0.39 Mb genomic region on GGA2p was identified. The region contains six candidate genes, with the causative mutation found in Chinese indigenous breeds also being located in this region. Our results demonstrate that polydactyly in chickens from China and Europe is caused by two independent mutation events that are closely located in the chicken genome.

Introduction

The chicken (Gallus gallus) is a modern descendant of the dinosaurs and the first non-mammalian amniote [1]. This species is one of the most common and widespread agricultural animals, providing essential proteins for the human food industry. In addition, it is the main laboratory model for human disease and genetics analysis [2, 3].

Polydactyly is one of the most common hereditary congenital limb malformations in humans, chickens, mice, and other vertebrates that have supernumerary fingers and/or toes [47]. Although the number of extra fingers/toes is subject to variability, polydactyly is classified as a rare qualitative trait [8]. High rates of polydactyly are attributed to its autosomal-dominant trait [9]. For instance, polydactyly occurs in 5 to 19 cases of every 10,000 live births in humans [10]. This high incidence in humans has resulted in focused research, with the chick limb becoming a classical model for studying developmental mechanisms and pattern formation during the embryogenesis of vertebrate embryos, due to its size and accessibility [11].

In general, chickens have four toes on each foot, but some breeds have more, such as the Beijing-You, Silkie, Jiningbairi, Dorking, and Houdan. The additional toes in polydactylous breeds of fowl do not represent the restoration of the fifth digit lost from the typical pentadactyl foot of higher vertebrates; rather, it is the result of the development of new toes on the opposite side of the foot [9]. Classic embryonic studies of the chick limb have revealed that the growth and patterning along the proximal/distal (P/D), anterior/posterior (A/P), and dorsal/ventral (D/V) axes are controlled by 3 signaling centers, specifically, the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the limb bud ectoderm [12]. For instance, chick limb A/P patterning is controlled by the secretion of the sonic hedgehog (SHH) protein by a small group of cells in the ZPA, which is located on the posterior mesoderm [13, 14]. The overexpression of SHH throughout the mesoderm of developing chicken limbs causes mutant polydactyly [15]. In comparison, low SHH expression, to the extent that it is not detectable in developing limbs, causes mutant oligozeugodactyly [13]. A cis-regulator of SHH has been identified in human preaxial polydactyly (PPD), and is responsible for the initiation and spatially specific expression of SHH in the ZPA. This regulator is located in intron 5 of the LMBR1 gene, which is located 1 Mb upstream of SHH [16]. The intron 5 sequence of LMBR1, which represents the ZPA regulatory sequence (ZRS), is conserved in humans, mice, pufferfish (fugu), and chickens [1720]. A few point mutations in the ZRS region have been identified as being responsible for human limb malformation, including the presence of a triphalangeal thumb and PPD [2125].

Various genetic mechanisms underlie chicken polydactyly phenotypes. In the Silkie chicken, SNPs in the ZRS region of LMBR1 intron 5 lead to ectopic SHH expression during embryonic development, resulting in polydactyly and oligozeugodactyly [15, 26, 27]. In the Dorking chicken, polydactyly is caused by the up-regulation of FGF4 expression, rather than that of SHH [28]. Through linkage analysis and GWAS, polydactyly has been mapped to genomic regions on GGA2, with several candidate genes potentially responsible for this trait in the Beijing-You chicken [2931]. However, the genetic background for polydactyly has yet to be elucidated in other chicken breeds.

In this study, we discriminated the polydactyly phenotypes of 26 chicken breeds and identified the genetic basis for the traits. Our results are expected to provide new insights about the evolution of polydactyly in chicken breeds, which is expected to contribute towards human research on this topic.

Materials and Methods

Ethics statement

The entire procedure was carried out in strict accordance with the protocol approved by the Animal Welfare Committee of China Agricultural University (Permit Number: XK622).

Sampling and DNA extraction

We selected four polydactylous chicken breeds, including three Chinese indigenous breeds (Beijing-You, Silkie, and Jiningbairi) and one European breed (Houdan) for which the polydactyly trait has not yet been elucidated. We also selected 21 local Chinese breeds with high genetic diversity of each other from 21 provinces [32] and White Leghorn as non-polydactyly comparisons. The sampling datasets are summarized in Table 1. Blood samples were collected from the brachial veins of chickens by standard venipuncture. Genomic DNA was extracted from blood using the standard phenol/chloroform method. DNA quality was controlled using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA).

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Table 1. Genotypes of the causative mutation and their distribution in 26 chicken breeds.

https://doi.org/10.1371/journal.pone.0149010.t001

Polydactyly phenotype identification

For non-polydactyly birds with four digits on each foot, the digits were labeled 1 to 4 from the anterior to posterior position on the foot (Fig 1). Chickens with more than four toes on one or both feet were designated as having the polydactyly phenotype by visual inspection and X-ray digital radiography. Some birds had four toes, but the anterior-most toe had an extra phalanx that was termed the polyphalange, and was considered a variation of polydactyly in the analyses of the present study [9].

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Fig 1. Variation in X-ray digital radiography with respect to Beijing-You foot polydactyly.

A-D show the different foot phenotypes of Beijing-You chicken, E-H show the X-ray digital radiography coordinately with A–D. A & E: non-polydactylous foot with four digits, identified from anterior to posterior (1, 2, 3, 4). B & F: Beijing-You foot with four digits, with a polyphalange in digit “1” making it digit “2” (2, 2, 3, 4). C & G: polydactylous Beijing-You foot with five digits (2, 1, 2, 3, 4). D & H: polydactylous Beijing-You foot with six digits (1, 2, 1, 2, 3, 4).

https://doi.org/10.1371/journal.pone.0149010.g001

Linkage analysis of causative SNP and diagnostic PCR

An SNP (ss161109890) identified in the intronic ZRS region of LMBR1, which is the cis-regulatory element of SHH, has been confirmed to cause Silkie polydactyly [26]. We first analyzed the SNP that has been found in all sampled Silkie chickens by using a diagnostic PCR method. We developed a diagnostic PCR-restriction fragment length polymorphism (PCR-RFLP) method with forward (5ʹ-GCGATTTCCTCTCACCCACA-3ʹ) and reverse (5ʹ-AGCTGAGCAACATGACAGCA-3ʹ) primers, producing a 389-bp fragment. When the genotype is CC, the PCR product is digested into two fragments (245 bp and 144 bp) by the restriction enzyme BsrDI. Some of the PCR products were also sequenced for verification. Furthermore, we analyzed the correlation between this SNP and polydactyly in all chicken breeds.

GWAS for polydactyly in Houdans

All Houdan chickens were wild homozygous types at the causative mutation point, regardless of whether birds had polydactyly or not. To refine the genomic regions responsible for polydactyly in Houdan chickens, we performed genome-wide association studies using the 600 K Affymetrix Axiom Chicken Genotyping Array (Affymetrix, Inc. Santa Clara, CA, USA) by the case-control method, with 43 polydactylous and 43 non-polydactylous Houdan chickens.

Genotyping and quality control

From an initial set of 580,961 SNPs [33], 8,374 SNPs with unknown genomic positions and 56 SNPs with redundant genomic coordinates were disregarded. Subsequently, first-pass quality control and genotype calling from the raw data in the form of CEL files were implemented with Affymetrix Power Tools v1.16.0 (APT) software using the Axiom GT1 algorithm. Only samples with a dish quality control (DQC) of 0.82 or more and a call rate of 98% were included in downstream analyses. An R script supplied by Affymetrix was run to compute SNP QC metrics and filter out individual SNPs falling below the given threshold. All parameters were set to the default values recommended by Affymetrix. After these QC steps, 86 samples and 546,124 SNPs remained. In addition, we excluded 26,656 SNPs on sex chromosomes because current statistical methods are more powerful at detecting the associations between phenotypes and autosomal genotypes. To improve the power of the association analyses, we disregarded a further 171,426 SNPs with minor allele frequency (MAF) < 1% and 441 SNPs that deviated from the Hardy-Weinberg equilibrium (HWE) test P < 10−6 using the PLINK v1.90 package [34]. Finally, a total of 86 samples and 369,020 SNPs were eligible for inclusion in the following GWAS.

Association analysis

Prior to GWAS, we conducted a principal component analysis (PCA) to eliminate spurious associations due to potential cryptic relatedness or hidden population stratification. Considering that clusters of highly correlated SNPs may distort resulting PCs, we first pruned the full SNP set to 24,449 independent SNPs via the—indep-pairwise 25 5 0.2 command (PLINK), and then calculated the top 5 PCs as covariates in the mixed model. To establish proper thresholds for genome-wide suggestive and significant associations, we corrected for multiple testing using the simpleM method [35], which accounts for linkage disequilibrium (LD) relationships among SNPs. Using simpleM, we estimated the effective number of independent tests as Meff = 57,657; thus, the genome-wide suggestive and significant P-values were 1.73 × 10−5 (1.00/57,657) and 8.67 × 10−7 (0.05/57,657), respectively.

We performed univariate tests of association for SNPs that had MAF ≥ 0.05 using an exact mixed model approach implemented in the GEMMA v0.94 software [36]. The centered relatedness matrix was calculated based on these independent SNPs for all cases. Subsequently, each SNP was tested for additive association with each trait by modeling the effects of genotypes and the additional covariates, including the top 5 PCs as fixed effects and genetic relatedness as random effects, because our samples were from highly structured populations with strong family relatedness. The Manhattan plot depicting -log10-transformed observed P-values was generated using the “gap” package in R.

Results

Polydactyly phenotype differs between Chinese and European chicken breeds

Polydactyly, including polyphalange, was detected in 55 out of 236 Beijing-You chickens, 101 out of 110 Silkie chickens, 18 out of 38 Jiningbairi chickens, and 43 out of 86 Houdan chickens (Table 1). The rest of the birds had the non-polydactyly phenotype (Table 1). In the four analyzed chicken breeds, we discovered two main subtypes of polydactyly (Fig 2). Subtype I is the presence of extra toes arising from the second phalanx of the most anterior toe, while subtype II is when extra toe separation originates from the first phalanx. Both subtypes were found in all three breeds, but with different prevalence. For Beijing-You, Silkie, and Jiningbairi, the primary trait was subtype I, while subtype II was the principal polydactyly phenotype in Houdans.

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Fig 2. Principal polydactyly subtypes in Beijing-You, Silkie, and Houdan chickens.

Two subtypes of polydactyly were found in the three chicken breeds, but with difference prevalence. Subtype I: principal subtype of Beijing-You (A) and Silkie (B), in which an extra toe was separated from the second phalanx in the most anterior toe; Subtype II: principal subtype of Houdan (C), with extra toe separation occurring from the first phalanx.

https://doi.org/10.1371/journal.pone.0149010.g002

Causative SNP found in Silkie is also completely associated with polydactyly in Beijing-You and Jiningbairi, but not in Houdan

The reported causative SNP (ss161109890, C > A) in Silkie was also found in Beijing-You, Silkie, and Jiningbairi flocks, but not in Houdan flocks (Table 1 and Fig 3). We analyzed the association between this point mutation and polydactyly in Beijing-You, Silkie, and Jiningbairi. We found that when birds have the polydactyly phenotype (including polyphalange), the genotype on this locus is AA or AC (Fig 3), showing complete association with polydactylous birds (Table 1). The causative mutation was not found in any of the other 306 non-polydactyly chickens, which included the White Leghorn breed and 21 indigenous Chinese breeds, except for 1 heterozygous chicken from the Langya breed, which has been recorded to have a low frequency of polydactyly [37] (Table 1).

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Fig 3. Genotypes of causative SNP loci detected by sequencing and diagnostic PCR.

The genotypes shown on the left side are wild type (CC), mutation heterozygote (AC), and derived homozygote (AA), from top to bottom. The mutation genotypes were fully associated with polydactyly in Chinese chickens, regardless of whether the mutation was homozygotic or heterozygotic. PCR products digested by restriction enzyme BsrDI are presented on the right.

https://doi.org/10.1371/journal.pone.0149010.g003

Relationship of a ~0.39 Mb region to polydactyly in Houdan

To determine the molecular mechanism that contributes to polydactyly in European chickens, we performed GWAS on Houdan, as the representative of European chickens by using the 600 K chicken SNP array. We found that 24 SNPs (Table 2) located at 8.16–8.55 Mb on GGA2 in the current assembly of the chicken genome (galGal4) were significantly associated with polydactyly in Houdan, with the causative mutation in local Chinese breeds also being located in this region (Fig 4). In addition, 16 SNPs that were also clustered on GGA2 may be significantly associated with polydactyly (S1 Table). The 24 SNPs defined a 386.94 kb region (2:8165823–8552766), while the six involved genes were Sonic hedgehog (SHH), ENSGALG00000026913, RING finger protein 32 (RNF32), Limb region 1 protein homolog (LMBR1), Nucleolar protein with MIF4G domain 1 (NOM1), and Motor neuron and pancreas homeobox 1 (MNX1), also known as Homeobox Hb9 (HLXB9) (Table 2).

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Fig 4. Genome-wide scan for polydactyly in Houdan chickens.

Manhattan plot showing the association of all SNPs with the polydactyly trait of Houdan chickens. SNPs were plotted on the x-axis according to their position on each chromosome against their association with these traits on the y-axis (shown as -log10 P-value). The green line and black line indicate the genome-wide suggestive and significant association with P-values of 1.73 × 10−5 (1.00/57,657) and 8.67 × 10−7 (0.05/57,657), respectively.

https://doi.org/10.1371/journal.pone.0149010.g004

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Table 2. Single nucleotide polymorphisms (SNPs) showing significant association with polydactyly in Houdans.

https://doi.org/10.1371/journal.pone.0149010.t002

Parallel evolution of polydactyly traits in Chinese and European chickens

We demonstrated that polydactyly phenotypes in indigenous Chinese breeds and Houdans have different molecular mechanisms. The causative SNP in Silkie had 100% association with polydactyly in all Chinese indigenous chicken breeds, but not in Houdans or other European breeds, such as Salmon Faverolle and Silver Gray Dorking [15]. This result indicates that the polydactyly phenotypes in Chinese and European chickens were caused by different mutation events, providing another example of parallel evolution in vertebrates.

Discussion

The causative SNP in Silkie completely associated with polydactylous Beijing-You and Jiningbairi, indicating that the SNP is also causative for polydactyly in Beijing-You and Jiningbairi. For Silkie, our result supports the previous study on other Silkie flocks [26]. For Beijing-You, our results are more substantial than previous studies, in which the polydactyly loci were mapped in special regions and several candidate association genes were identified [2731]. Our results also identified the causative mutation of polydactylous Silkie was not exist in other nonpolydactyly chickens, suggesting that SNP of polydactyly in Silkie has fully penetrated all other indigenous Chinese chicken breeds.

In this study, the causative SNP in polydactylous Silkie was not detected in our Houdan flocks. Previously, the SNP was confirmed as the causative mutation of polydactyly in Silkie [26]. Furthermore, another study on five polydactylous chicken breeds (Silkie, Salmon Faverolle, Mottled Houdan, Silver Gray Dorking, and White Sultan) found that the same SNP (ss161109890) is highly associated with polydactyly in Silkie and Sultan breeds, but could not be detected in the other three polydactylous breeds [15]. On investigating the origin of these five breeds, we found that the Silkie originates from China, whereas the White Sultan originates from Turkey (Asia) [38]. In addition, Salmon Faverolle and Mottled Houdan originate from France (Europe), while the Silver Gray Dorking originates from Italy (Europe) [38]. Thus, the principal subtype of the polydactyly phenotype differs between European [15] and Chinese breeds, and even between European and Asian breeds. We hypothesize that the molecular mechanism of polydactyly in Chinese and Asian chickens may differ to that of European chickens.

To determine the molecular mechanism that contributes to polydactyly in European chickens, we performed GWAS on Houdan, as the representative European chicken breed. The GWAS results identified 24 SNPs in a ~0.39 Mb region on GGA2, and identified 6 candidate genes: SHH, ENSGALG00000026913, RNF32, LMBR1, NOM1, and MNX1/HLXB9. The RNF32 gene is one of the cis-regulators of SHH in the mouse [39]. This regulatory element directs the initiation and spatial expression of SHH [40]. SHH is one of the proteins of the mammalian signaling pathway family hedgehog [41]. SHH is the best studied ligand of the hedgehog signaling pathway, because of its key role in regulating vertebrate organogenesis [42], such as the growth of digits on limbs [43] and the organization of the brain [44]. We speculate that the SNPs close to or in RNF32 may be critical for regulating the temporal and spatial expression of SHH, which contributes to polydactyly in Houdans.

The LMBR1 gene is one of the cis-regulators of SHH, and is critical for SHH expression [11, 16]. The mutations in LMBR1 may directly cause polydactyly in vertebrates. Thus, a point mutation in the ZRS may directly regulate the expression of SHH, and, consequently, the occurrence of polydactyly or other types of limb malformation [22, 24, 25]. For instance, different SNPs in intron 5 of the LMBR1 gene may lead to triphalangeal thumb-polysyndactyly syndrome in humans [45] and polydactyly in Silkie chickens [26]. Deletions in exon 4, and in portions of intron 3 and 4 of chicken LMBR1, may cause the oligozeugodactyly mutant phenotype [13], which is similar to that of humans affected with acheiropodia [46].

Compared with genes detected in previous studies on chicken polydactyly, the MNX1/HLXB9 gene is a novel candidate gene associated with chicken polydactyly [29, 30, 47]. In humans, the MNX1/HLXB9 gene is hypothesized to be a candidate gene for polydactyly [48, 49]. Mutations in the MNX1/HLXB9 gene may cause neonatal diabetes and Currarino syndrome [50, 51]. Currarino syndrome is a malformation with an autosomal dominant genetic trait similar to polydactyly [5254]. However, the function of MNX1/HLXB9 in the chicken has not been documented yet. Thus, this gene may also affect skeletal development, including the incidence of polydactyly in chickens.

In this study, the causative SNP in Silkie was validated be the causative mutation of all Chinese indigenous chicken breeds, but not of Houdan or the other European chicken breeds, such as Salmon Faverolle and Silver Gray Dorking [15]. This result indicates that the polydactyly phenotypes in Chinese and European chickens are caused by different mutation events, providing another example of parallel evolution in vertebrates. Parallel evolution is ubiquitous in living organisms [55]. For instance, the blue eggshell of Chinese, South American, and European chickens is caused by an EAV-HP insertion in the 5ʹ flanking region of SLCO1B3; however, the retrovirus integration site of American and European chickens differs from that of Chinese chickens [56, 57]. As another example, the black coat color of Asian and European domestic pigs is caused by independent missense mutations in melanocortin 1 receptor (MC1R), which occurred on different haplotypes originating from Asian and European wild boar [58].

In conclusion, we confirm that the genetic basis of chicken polydactyly traits differs between Chinese and European breeds, providing a clear example of parallel evolution. The results of this study are expected to facilitate further studies on limb malformation in chickens, which could be extrapolated to other vertebrates.

Supporting Information

S1 Table. Summary of identified SNP association for polydactyly in Houdan chickens.

https://doi.org/10.1371/journal.pone.0149010.s001

(XLSX)

Acknowledgments

We are grateful for the help of all members of the poultry team of the National Engineering Laboratory for Animal Breeding.

Author Contributions

Conceived and designed the experiments: LQ. Performed the experiments: ZZ HX YJ. Analyzed the data: ZZ CN. Contributed reagents/materials/analysis tools: NY LQ CN RJ HX X. Lv YC JL X. Li ZN GX JC. Wrote the paper: ZZ.

References

  1. 1. LaDeana W, Hillier WMEB. Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature. 2004;432(7018): 695–716. pmid:15592404
  2. 2. Yan Y, Yi G, Sun C, Qu L, Yang N. Genome-wide characterization of insertion and deletion variation in chicken using next generation sequencing. PloS One. 2014;9(8): e104652. pmid:25133774
  3. 3. Dodgson JB, Romanov MN. Use of chicken models for the analysis of human disease. Curr Protoc Hum Genet. 2004;15: 15.
  4. 4. Clark RM, Marker PC, Kingsley DM. A novel candidate gene for mouse and human preaxial polydactyly with altered expression in limbs of Hemimelic extra-toes mutant mice. Genomics. 2000;67(1): 19–27. pmid:10945466
  5. 5. Akhtar S, Grizenkova J, Wenborn A, Hummerich H, Fernandez DMM, Brandner S, et al. Sod1 deficiency reduces incubation time in mouse models of prion disease. PLoS One. 2013;8(1): e54454. pmid:23349894
  6. 6. Riddle RD, Johnson RL, Laufer E, Tabin C. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell. 1993;75(7): 1401–1416. pmid:8269518
  7. 7. Babbs C, Furniss D, Morriss-Kay GM, Wilkie AOM. Polydactyly in the mouse mutant Doublefoot involves altered Gli3 processing and is caused by a large deletion in cis to Indian hedgehog. Mech Develop. 2008;125(5–6): 517–526.
  8. 8. Britton NF. Population genetics and evolution. In: Britton NF, editor. Essential mathematical biology. Springer London; 2003. pp. 117–146.
  9. 9. Warren DC. Inheritance of polydactylism in the fowl. Genetics. 1944;29(3): 217–231. pmid:17247117
  10. 10. Zguricas J, Heus H, Morales-Peralta E, Breedveld G, Kuyt B, Mumcu EF, et al. Clinical and genetic studies on 12 preaxial polydactyly families and refinement of the localisation of the gene responsible to a 1.9 cM region on chromosome 7q36. J Med Genet. 1999;36(1): 32–40. pmid:9950363
  11. 11. Lettice LA, Heaney SJ, Purdie LA, Li L, de Beer P, Oostra BA, et al. A long-range SHH enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum Mol Genet. 2003;12(14): 1725–1735. pmid:12837695
  12. 12. Yang Y, Guillot P, Boyd Y, Lyon MF, McMahon AP. Evidence that preaxial polydactyly in the Doublefoot mutant is due to ectopic Indian Hedgehog signaling. Development. 1998;125(16): 3123–3132. pmid:9671585
  13. 13. Maas SA, Fallon JF. Isolation of the chicken LMBR1 coding sequence and characterization of its role during chick limb development. Dev Dyn. 2004;229(3): 520–528. pmid:14991708
  14. 14. McMahon AP, Ingham PW, Tabin CJ. Developmental roles and clinical significance of hedgehog signaling. Curr Top Dev Biol. 2003;53: 1–114. pmid:12509125
  15. 15. Dorshorst B, Okimoto R, Ashwell C. Genomic regions associated with dermal hyperpigmentation, polydactyly and other morphological traits in the silkie chicken. J Hered. 2010;101(3): 339–350. pmid:20064842
  16. 16. Lettice LA, Horikoshi T, Heaney SJ, van Baren MJ, van der Linde HC, Breedveld GJ, et al. Disruption of a long-range cis-acting regulator for SHH causes preaxial polydactyly. Proc Natl Acad Sci USA. 2002;99(11): 7548–7553. pmid:12032320
  17. 17. Dubchak I, Brudno M, Loots GG, Pachter L, Mayor C, Rubin EM, et al. Active conservation of noncoding sequences revealed by three-way species comparisons. Genome Res. 2000;10(9): 1304–1306. pmid:10984448
  18. 18. Mayor C, Brudno M, Schwartz JR, Poliakov A, Rubin EM, Frazer KA, et al. VISTA: visualizing global DNA sequence alignments of arbitrary length. Bioinformatics. 2000;16(11): 1046–1047. pmid:11159318
  19. 19. Yee SP, Rigby PW. The regulation of myogenin gene expression during the embryonic development of the mouse. Genes Dev. 1993;7(7A): 1277–1289. pmid:8391506
  20. 20. Elgar G, Sandford R, Aparicio S, Macrae A, Venkatesh B, Brenner S. Small is beautiful: comparative genomics with the pufferfish (Fugu rubripes). Trends Genet. 1996;12(4): 145–150. pmid:8901419
  21. 21. Gurnett CA, Bowcock AM, Dietz FR, Morcuende JA, Murray JC, Dobbs MB. Two novel point mutations in the long-range SHH enhancer in three families with triphalangeal thumb and preaxial polydactyly. Am J Med Genet. 2007;143A(1): 27–32. pmid:17152067
  22. 22. Lohan S, Spielmann M, Doelken SC, Flöttmann R, Muhammad F, Baig SM, et al. Microduplications encompassing the Sonic hedgehog limb enhancer ZRS are associated with Haas-type polysyndactyly and Laurin-Sandrow syndrome. Clin Genet. 2014;86(4): 318–325. pmid:24456159
  23. 23. Johnson EJ, Neely DM, Dunn IC, Davey MG. Direct functional consequences of ZRS enhancer mutation combine with secondary long range SHH signalling effects to cause preaxial polydactyly. Dev Biol. 2014;392(2): 209–220. pmid:24907417
  24. 24. VanderMeer JE, Lozano R, Sun M, Xue Y, Daentl D, Jabs EW, et al. A novel ZRS mutation leads to preaxial polydactyly type 2 in a heterozygous form and Werner Mesomelic Syndrome in a homozygous form. Hum Mutat. 2014;35(8): 945–948. pmid:24777739
  25. 25. Girisha KM, Bidchol AM, Kamath PS, Shah KH, Mortier GR, Mundlos S, et al. A novel mutation (g.106737G > T) in zone of polarizing activity regulatory sequence (ZRS) causes variable limb phenotypes in Werner mesomelia. Am J Med Genet. 2014;164(4): 898–906.
  26. 26. Dunn IC, Paton IR, Clelland AK, Sebastian S, Johnson EJ, McTeir L, et al. The chicken polydactyly (Po) locus causes allelic imbalance and ectopic expression of SHH during limb development. Dev Dynam. 2011;240(5): 1163–1172.
  27. 27. Albuisson J, Isidor B, Giraud M, Pichon O, Marsaud T, David A, et al. Identification of two novel mutations in SHH long-range regulator associated with familial pre-axial polydactyly. Clin Genet. 2011;79(4): 371–377. pmid:20569257
  28. 28. Bouldin CM, Harfe BD. Aberrant FGF signaling, independent of ectopic hedgehog signaling, initiates preaxial polydactyly in Dorking chickens. Dev Biol. 2009;334(1): 133–141. pmid:19616534
  29. 29. Sun Y, Liu R, Zhao G, Zheng M, Sun Y, Yu X, et al. Genome-wide linkage analysis and association study identifies loci for polydactyly in chickens. G3 (Bethesda). 2014;4(6): 1167–1172.
  30. 30. Sheng X, Chen Y, Jia Y, Qi X, Feng Y, Huang Z, et al. The identification of loci for polydactyly in chickens using a genome-wide association study. Gene. 2015;568(2): 176–180. pmid:26003297
  31. 31. Yang K, Chen Y, Gu C, Zhou Z, He C, Tang L, et al. Polydactyly mutation is linked with chromosome 2p region in Beijing fatty chicken. Anim Genet. 2013;44(1): 118–119.
  32. 32. Qu L, Li X, Xu G, Chen K, Yang H, Zhang L, et al. Evaluation of genetic diversity in Chinese indigenous chicken breeds using microsatellite markers. Sci China C Life Sci. 2006:49(4): 332–341. pmid:16989278
  33. 33. Kranis A, Gheyas AA, Boschiero C, Turner F, Yu L, Smith S, et al. Development of a high density 600K SNP genotyping array for chicken. BMC Genomics. 2013;14: 59. pmid:23356797
  34. 34. Beasley TM, Erickson S, Allison DB. Rank-based inverse normal transformations are increasingly used, but are they merited? Behav Genet. 2009;39(5): 580–595. pmid:19526352
  35. 35. Gao X, Becker LC, Becker DM, Starmer JD, Province MA. Avoiding the high Bonferroni penalty in genome-wide association studies. Genet Epidemiol. 2010;34(1): 100–105. pmid:19434714
  36. 36. Zhou X, Stephens M. Genome-wide efficient mixed-model analysis for association studies. Nat Genet. 2012;44(7): 821–824. pmid:22706312
  37. 37. China National Commission of Animal Genetic Resources. Animal genetic resources in China poultry. Beijing: China Agriculture Press; 2010.
  38. 38. American Poultry Association. The American standard of perfection. Mendon (MA): American Poultry Association: Inc; 1998.
  39. 39. Sagai T, Amano T, Tamura M, Mizushina Y, Sumiyama K, Shiroishi T. A cluster of three long-range enhancers directs regional SHH expression in the epithelial linings. Development. 2009;136(10): 1665–1674. pmid:19369396
  40. 40. Anderson E, Devenney PS, Hill RE, Lettice LA. Mapping the SHH long-range regulatory domain. Development. 2014;141(20): 3934–3943. pmid:25252942
  41. 41. Marigo V, Roberts DJ, Lee SM, Tsukurov O, Levi T, Gastier JM, et al. Cloning, expression, and chromosomal location of SHH and IHH: two human homologues of the Drosophila segment polarity gene hedgehog. Genomics. 1995;28(1): 44–51. pmid:7590746
  42. 42. Zeller R, López-Ríos J, Zuniga A. Vertebrate limb bud development: moving towards integrative analysis of organogenesis. Nat Rev Genet. 2009;10(12): 845–858. pmid:19920852
  43. 43. Harfe BD, Scherz PJ, Nissim S, Tian H, McMahon AP, Tabin CJ. Evidence for an expansion-based temporal SHH gradient in specifying vertebrate digit identities. Cell. 2004;118(4): 517–528. pmid:15315763
  44. 44. Liu A, Joyner AL, Turnbull DH. Alteration of limb and brain patterning in early mouse embryos by ultrasound-guided injection of SHH-expressing cells. Mech Develop. 1998;75(1): 107–115.
  45. 45. Wang Z, Tian S, Shi Y, Zhou P, Wang Z, Shu R, et al. A single C to T transition in intron 5 of LMBR1 gene is associated with triphalangeal thumb-polysyndactyly syndrome in a Chinese family. Biochem Bioph Res Co. 2007;355(2): 312–317.
  46. 46. Ianakiev P, Van Baren MJ, Daly MJ, Toledo S, Cavalcanti MG, Neto JC, et al. Acheiropodia is caused by a genomic deletion in C7orf2, the human orthologue of the LMBR1 gene. Am J Hum Genet. 2001;68(1): 38–45. pmid:11090342
  47. 47. Huang YQ, Deng XM, Du ZQ, Qiu X, Du X, Chen W, et al. Single nucleotide polymorphisms in the chicken LMBR1 gene are associated with chicken polydactyly. Gene. 2006;374: 10–18. pmid:16650944
  48. 48. Li H, Wang CY, Wang JX, Wu GS, Yu P, Yan XY, et al. Mutation analysis of a large Chinese pedigree with congenital preaxial polydactyly. Eur J Hum Genet. 2009;17(5): 604–610. pmid:19066618
  49. 49. Wang ZQ, Tian SH, Shi YZ, Zhou PT, Wang ZY, Shu RZ, et al. A single C to T transition in intron 5 of LMBR1 gene is associated with triphalangeal thumb-polysyndactyly syndrome in a Chinese family. Biochem Bioph Res Co. 2007;355(2): 312–317.
  50. 50. Merello E, De Marco P, Ravegnani M, Riccipetitoni G, Cama A, Capra V. Novel MNX1 mutations and clinical analysis of familial and sporadic Currarino cases. Eur J Med Genet. 2013;56(12): 648–654. pmid:24095820
  51. 51. Flanagan SE, De Franco E, Lango AH, Zerah M, Abdul-Rasoul MM, Edge JA, et al. Analysis of transcription factors key for mouse pancreatic development establishes NKX2-2 and MNX1 mutations as causes of neonatal diabetes in man. Cell Metab. 2014;19(1): 146–154. pmid:24411943
  52. 52. Ashcraft KW, Holder TM. Hereditary presacral teratoma. J Pediatr Surg. 1974;9(5): 691–697. pmid:4418917
  53. 53. Gopal M, Turnpenny PD, Spicer R. Hereditary sacrococcygeal teratoma—not the same as its sporadic counterpart! Eur J Pediatr Surg. 2007;17(3): 214–216. pmid:17638164
  54. 54. Belloni E, Martucciello G, Verderio D, Ponti E, Seri M, Jasonni V, et al. Involvement of the HLXB9 homeobox gene in Currarino syndrome. Am J Hum Genet. 2000;66(1): 312–319. pmid:10631160
  55. 55. Rubin CJ, Megens HJ, Barrio AM, Maqbool K, Sayyab S, Schwochow D, et al. Strong signatures of selection in the domestic pig genome. Proc Nat Acad Sci USA. 2012;109(48): 19529–19536. pmid:23151514
  56. 56. Wragg D, Mwacharo JM, Alcalde JA, Wang C, Han J- L, Gongora J, et al. (2013) Endogenous Retrovirus EAV-HP Linked to Blue Egg Phenotype in Mapuche Fowl. PLoS ONE 8(8): e71393. pmid:23990950
  57. 57. Wang Z, Qu L, Yao J, Yang X, Li G, Zhang Y, et al. An EAV-HP insertion in 5′ flanking region of SLCO1B3 causes blue eggshell in the chicken. PLoS Genet. 2013;9(1): e1003183. pmid:23359636
  58. 58. Fang M, Larson G, Ribeiro HS, Li N, Andersson L. Contrasting mode of evolution at a coat color locus in wild and domestic pigs. PLoS Genet. 2009;5(1): e1000341. pmid:19148282