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British Poultry Science Volume 52, Number 2 (April 2011), pp. 173 176 Feather keratin gene polymorphism (F-KER) in domestic pigeons A. DYBUS AND E. HAASE 1 West Pomeranian University of Technology, Laboratory of Molecular Cytogenetics, Doktora Judyma 10, 71-460 Szczecin, Poland, and 1 Christian-Albrechts-Universität, 24118 Kiel, Germany Abstract 1. A 525-bp fragment of a feather keratin (F-KER) gene was amplified. Four PCR products were sequenced. 2. Two substitutions in the amplified region were observed, one of them in the coding region of the gene (cysteine to glycine substitution in the protein). A new ACRS-PCR test for AvaI enzyme was designed. A total of 344 domestic pigeons (Columba livia var. domestica) were genotyped. 3. There were significant differences in the frequencies of alleles and genotypes between homing and non-homing pigeons. The frequency of the rare F-KER G allele was higher in the group of homing pigeons. 4. The effects of the detected polymorphism on flying performance should be verified in biomechanical studies. INTRODUCTION Bird feathers can serve sensory, ornamental, insulating, thermoregulatory and locomotory functions. Feathers have evolved, especially with respect to flight, to be robust, light and sufficiently resilient to withstand mechanical functioning until replaced during a moult (Cameron et al., 2003). The principal constituent of feathers, ß-keratin, is a single family of closely-related proteins with molecular weight around 105 kda (Woodin, 1954; Harrap and Woods, 1964), which accounts for approximately 90% of the feather rachis (Fraser et al., 1971). The amino acid sequences of feather proteins from different species is rather conservative and exhibit a high degree of homology (O Donnell, 1973; O Donnell and Inglis, 1974; Busch and Brush, 1979; Bonser and Purslow, 1995). In domestic pigeons, where care and shelter are provided by the breeders, some mutations concerning the structure of contour and flight feathers have been described. The mutants silky (L), frayed (F), frizzy (fz), scraggy (sc), and porcupine (p) result in a loss of the ability to fly and the feathers readily becoming water-soaked (Hollander, 1983; Sell, 1994). Under natural selection, these mutants would be rapidly eliminated. Their molecular-genetic basis is unknown. In this paper we report the detection of DNA polymorphisms in a domestic pigeon s feather keratin gene which do not obviously alter the structure of the feathers or the bird s flying ability. Also, we analysed the genotypes/alleles frequencies in homing pigeons and a collection of other breeds summarised as non-homing pigeons. MATERIALS AND METHODS A total of 344 domestic pigeons (two unrelated groups of homing pigeons: 141 individuals from Natural Antwerp Breeding Station, Belgium and 100 pigeons from two colonies kept at the universities of Szczecin and Kiel) and 103 specimens from other breeds, for simplicity termed non-homing pigeons, were genotyped. The non-homing group was composed of Carrier (n ¼ 6), Bagdad of Nuremberg (n ¼ 4), Correspondence to: A. Dybus, West Pomeranian University of Technology, Laboratory of Molecular Cytogenetics, Doktora Judyma 10, 71-460 Szczecin, Poland. E-mail: Andrzej.Dybus@zut.edu.pl Accepted for publication 6th October 2010. ISSN 0007 1668(print)/ISSN 1466 1799 (online)/11/020173 4 ß 2011 British Poultry Science Ltd DOI: 10.1080/00071668.2010.549666
174 A. DYBUS AND E. HAASE Polish Barb (n ¼ 4), Cauchois (n ¼ 5), Danzig Highflier (n ¼ 8), King (n ¼ 14), Polish Helmet (n ¼ 3), Maltese (n ¼ 4), Hungarian (n ¼ 4), Polish Musian (n ¼ 2), Vienna Kiebitz (n ¼ 6), German Magpie (n ¼ 5), German Long-Beaked Tumbler (n ¼ 10), German Show Homer (n ¼ 6), Fantail (n ¼ 4), Polish Short-Beaked (n ¼ 1), Stralsunder Highflier (n ¼ 1), Strasser (n ¼ 14), Weisskopf (n ¼ 1) and Budapest Highflier (n ¼ 1) kept at 7 lofts owned by local breeders (Szczecin city). All pigeons were genotyped using ACRS- PCR (amplification-created restriction site polymerase chain reaction) and restriction enzyme analysis. The DNA was isolated from 5-mL blood samples using MasterPure TM kit (Epicentre Technologies). In the first step of this study, PCR primers were designed to produce 525 base pairs amplicon using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/ primer3/primer3_www.cgi) and AB017906 DNA sequence (Takahashi et al., 2003): Forward 5 0 -TCTTTGGCTCCCTCGTAATG-3 0 Reverse 5 0 -GAAGGGCAAAACAGAGATGG-3 0 The PCR mixture contained 60 ng of genomic DNA, 10 pmol of each primer, 1xPCR buffer, 15 mm MgCl 2, 200 mm dntp and 04 units Taq-polymerase in a total volume of 15 ml. The following cycles were applied: denaturation at 94 C/5 min, followed by 33 cycles at 95 C/ 30 s, primer annealing at 58 C/40 s, PCR products synthesis at 72 C/40 s and final synthesis at 72 C/5 min. Four PCR products from two non-homing pigeons (Strasser and King) and also two homing pigeons (the best racing cock (A.D.) and a purebred Janssen hen (E.H.)) were sequenced in an ABI Prism Sequencer (Perkin-Elmer) and analysed using Chromas software. The sequencing was performed at the Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland. The second pair of primers (forward primer with nucleotide modification) was designed to create the restriction motif for AvaI enzyme in the 146 bp amplification product and enable genotyping of the T/G substitution (detected in previous sequentioning) using an ACRS-PCR test: Forward 5 0 -GGAGTTCCCATCAACTCTCG-3 0 ðc instead of complementary GÞ Reverse 5 0 -GATCCTCTGTCCAGCACCAT-3 0 The following cycles were applied: initial denaturation, 94 C/5 min, followed by 35 cycles: denaturation, 94 C/30 s, primer annealing, 58 C/30 s, PCR products synthesis, 72 C/30 s and final synthesis, 72 C/5 min. The amplified DNA was digested with 4 units of AvaI enzyme. Figure. The results of F-KER/AvaI genotyping The digestion products were separated through 3% agarose gels (PRONA) in 1xTBE and 05 mm ethidium bromide. The chi-square ( 2 ) test was used for comparison of genotypes and alleles frequencies in homing and non-homing pigeons. RESULTS The following DNA fragments were observed due to the F-KER/AvaI polymorphism: 129 and 17 bp for the F-KER GG, 146, 129 and 17 bp for the F-KER GT and 146 bp (no digestion) for F-KER TT genotype (Figure). Analysis of sequencing results (Chromas software v.2.31) indicated two polymorphic sites in the analysed amplicons (heterozygotic genotype in pure-breed Janssen hen). One of them is located in the 5 0 region and the second in the 83rd codon (TGC to GGC) of the F-KER gene. The result of this mutation is a cysteine to glycine (C to G) substitution in the feather keratin protein. The frequency of the allele F-KER G in all studied pigeons was relatively low (0176), but in the homing pigeons the frequency of this rare F-KER G variant was three-fold higher than in nonhoming pigeons (P <0001). Statistically significant differences in genotype frequencies between homing and non-homing pigeons were observed ( 2 ¼ 2414). The frequency of F-KER TT was significantly higher in the group of non-homing than in homing pigeons. In the case of F-KER GT, there was a non-significant trend towards a higher frequency in the homing pigeons. Frequncies of F-KER genotypes in two groups of homing pigeons were similar ( 2 ¼ 423) (Table). DISCUSSION Approximately 25 33% of the keratin protein has a ß-conformation (Fraser et al., 1971) and the
F-KER GENE POLYMORPHISM IN PIGEONS 175 Table. Frequencies of genotypes and alleles in analysed groups of pigeons Group of pigeons Genotype Alleles F-KER GG F-KER GT F-KER TT F-KER G F-KER T Homing 0046 0348 0606** 0220** 0780** (n ¼ 241) (n ¼ 11) (n ¼ 84) (n ¼ 146) Natural Antwerp 0028 0390 0582 0223 0777 (n ¼ 141) (n ¼ 4) (n ¼ 55) (n ¼ 82) Szczecin/Kiel 0070 0290 0640 0215 0785 (n ¼ 100) (n ¼ 7) (n ¼ 29) (n ¼ 64) Non-homing 0019 0107 0874** 0073** 0927** (n ¼ 103) (n ¼ 2) (n ¼ 11) (n ¼ 90) Total 0038 0276 0686 0176 0824 (n ¼ 344) (n ¼ 13) (n ¼ 95) (n ¼ 236) **Homing versus non-homing pigeons significantly different at P 001. regular secondary structure is concentrated in the central part of the molecule (rich in hydrophobic residues) (Suzuki, 1973; Arai et al., 1986). The rest of the keratins form an irregular matrix around the ß-sheet region (Fraser et al., 1971). The X-ray diffraction pattern of the feather rachis can be interpreted in terms of a helical arrangement of molecules and possible conformations have been suggested (Bear and Rugo, 1951; Busch and Brush, 1979; Gregg and Rogers 1986). These models suggest the ß-keratin associates as a two-stranded rope of crystallites that probably act mechanically as a high-modulus, essentially linearly elastic element (Fraser and MacRae, 1980). Within the rachis, subtle changes in molecular orientation and the presence of a laminate structure have been identified qualitatively but not quantitatively in geese (Earland et al., 1962) and peacocks (Busson et al., 1999). Recent studies indicate that avian feather keratin filaments have a helical structure with 4 repeating units per turn (Fraser and Parry, 2008). Mechanical properties of pigeon primary feathers have been studied by Purslow and Vincent (1978) and Corning and Biewener (1998). According to the first two authors, the shape and size of the outer wall of the shaft account for the majority of bending behaviour. Bonser and Purslow (1995) assessed the longitudinal Young s modulus of feather keratin in 8 avian species (including the pigeon) and concluded that the flexural stiffness of the whole rachis is principally controlled by its crosssectional morphology rather than by the material properties of the keratin. In a comparison of the primary structure of the main component (B-4) of feather keratins, Arai et al. (1986) reported a similarity of 85% (82 from 95 positions) among chickens, ducks and pigeons. The variation in amino acid sequence was higher in the C-terminal region than in the N-terminal region, the latter containing the majority of cysteine-residues which seem to be important for the formation of feather structure. Only one of the several cysteine-residues of the duck was replaced in the other two species. In our pigeons, we found an intraspecific replacement of a cysteine-residue by glycine. Because cysteine usually forms disulphidebridges, its loss could affect the structure of the keratine molecule and thereby alter the structure of the plumage. However, we have so far not detected any visible or tactile differences between feathers of the genotype Cys/Cys, Cys/Gly and Gly/Gly. It is interesting to note that the F-KER G has a three-fold higher frequency in the homing-pigeons compared to the nonhoming group. Homing pigeons are strongly selected for rapid return from distant release sites which means selection for speed and endurance. Feathers are their tools ; and it seems unlikely that a genetic variant could spread if it was disadvantageous for flying. This view is further supported by the existence of the rare F-KER G variant in a pure bred Janssen hen at Kiel. The Janssen strain, an inbred line of racing pigeons from the Janssen brothers at Arendonk, Belgium, is highly demanded by breeders from all over the world. In numerous cases, introduction of this strain successfully improved the racing performance of pigeon colonies in different continents (Schaerlaeckens, 2001). The rare F-KER G allele was found in various breeds of non-homing pigeons irrespective of their flying ability, for example, in Cauchois, Carrier, Maltese, Show Homers and Fantails. A more clear-cut idea on the effects of the detected mutation on flying performance may result from biomechanical studies on feathers of the three genotypes that are in progress. REFERENCES ARAI, K.M., TAKAHASHI, R., YOKOTE, Y.& AKAHANE, K. (1986) The primary structure of feather keratins from duck (Anas
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