Sherif Ramadan, Hideaki Abe, Azusa Hayano, Junichi Yamaura, Tomoaki Onoda, Takeshi Miyake and Miho Inoue-Murayama

Transcription

1 doi: +*.,+.+/jpsa.*+*+*3 Copyright,*++, Japan Poultry Science Association. +,, Sherif Ramadan, Hideaki Abe, Azusa Hayano, Junichi Yamaura, Tomoaki Onoda, Takeshi Miyake and Miho Inoue-Murayama. + + Wildlife Research Center, Kyoto University, Kyoto 0*0 2,*-, Japan, Faculty of Veterinary Medicine, Benha University, Moshtohor +-1-0, Egypt - Japan Racing Pigeon Association, Jonan Combine, Kashiwa,11 **2/, Japan. Graduate School of Agriculture, Kyoto University, Kyoto 0*0 2/*,, Japan Ancient Egyptians used pigeons not only as food in the form of squab but also as a messenger by virtue of their strong homing ability. Pigeons are bred for many purposes like meat in the form of squabs, exhibition as fancy and ornamental, flying and sports like racing competition, and finally for laboratory experiments of cognitive sciences. In this study, a total of +-- pigeon samples of six Egyptian breeds ( n ++* ) and Japanese racing pigeons ( n,-) were surveyed. One sample from each breed was sequenced for mitochondrial COI gene and all samples were genotyped across ++ microsatellites loci. From COI sequence, all the seven studied populations were found to belong to same the species ( Columba livia). By the analysis of ++ microsatellite loci a total of 23 alleles were observed with an average of 2. + alleles per locus. The expected heterozygosities of the six Egyptian breeds and Japanese racing pigeons were *./2* and *. 0-*, respectively. FST showed a relatively high mean of *.,*- which indicated that there is a great di# erentiation among the seven pigeon populations. Zagel breed and Japanese racing pigeons showed the lowest values for both pairwise FST (*. +*2 ) and Nei s genetic distance (*. +/. ). The information from this study would be useful for genetic characterization and provide a foundation for developing sustainable genetic improvement and conservation programs of this agriculturally and commercially important species. Key words: Egyptian breed, genetic diversity, microsatellite, mitochondrial COI, pigeon J. Poult. Sci.,.2: 13 2.,,*++ Introduction form of squab and used pigeon s nitrogen-rich guano or feaces as fertilizers (Jerolmack,,**1). They discovered The importance of maintaining genetic diversity in do- the strong homing ability of pigeons and used them as a mestic livestock is advocated worldwide by the Food and messenger. An Egyptian bas-relief from around +-/* B.C. Agriculture Organization (FAO). Therefore, conserva- depicts a flock of doves being released from their cages to tion of native breeds as a genetic resource is important to fly and then return (Glover and Beaumont, +333). fill unanticipated breeding demands in the future (Tadano The six Egyptian indigenous pigeon breeds used in this et al.,,**1b). Among these species, pigeons are believed study don t belong to feral pigeons. Five of these breeds: to be domesticated as early as -*** B.C. (Glover and Ablaq (Levi, +330), Krezly, Zagel, Safi and Asfer Weraq Beaumont, +333) and today there are over -** breeds of characterize by strong homing and flying abilities and domestic pigeons, all originating from one wild source, the mainly used for certain kind of a very popular flying game rock dove (Bodio, +33* ). Pigeons are bred for many in Egypt, whereas the last one (Romani breed) characterpurposes like meat in the form of squabs, exhibition as ize by heavy body weight and used mainly for meat fancy and ornamental, flying and sports like racing compe- production in form of squabs. During the flying game, tition (Blechman,,**1; Jerolmack,,**1; Hiatt and pigeons stock may often be seen flying in circles over Esposito,,***) and finally for laboratory experiments of rooftops. The breeder trains his birds to fly to nearby cognitive sciences (Watanabe et al., +33/ ). rooftops where they meet another s stock. Birds may Pigeons appeared on Egyptian bas-reliefs from at least become disoriented when their stock meets unknown,1** B.C. Ancient Egyptians used pigeons as food in the others. If the stock is well trained, the pigeons would return with or steal strays from other stock (Jerolmack, Received: October,2,,*+*, Accepted: December,,,*+* Released Online Advance Publication: February,/,,*++,**1). Correspondence: Dr. M. Inoue-Murayama, Wildlife Research Center In Egypt, despite the importance of this species, little is of Kyoto University, Kyoto 0*0 2,*-, Japan. known about its genetic diversity regarding the di# erent ( mmurayama@wrc.kyoto-u.ac.jp) types of uses and local population size. Research work on

2 80 Journal of Poultry Science,.2 (,) the genetic variation of this species is important to charac- from Osaka Museum of Natural History, Osaka, Japan. terize the genetic structure of local populations. This DNA was extracted from feather samples using the serves as an important first step to identify the valuable QIAGEN DNeasy Tissue Kit (QIAGEN, Valencia, CA, genetic characters and resources of the domestic pigeon USA). for conservation against future needs. In the face of Mitochondrial COI Analysis daunting global challenges such as climate change, emerg- A determined region near the / terminus of the COI ing diseases, population growth, and rising consumer was amplified using primers BirdF+ -TTCTCCAACCACdemands, it is likely that maintaining genetic variation is AAAGACATTGGCAC and BirdR+ -ACGTGGGAGATA- quite important for the future (Kayang et al.,,*+*). ATTCCAAATCCTG (Hebert et al.,,**.). The PCR Mitochondrial DNA (mtdna) has been widely em- was performed on a +/ ml reaction mixes including,* ng ployed in phylogenetic studies of various animals because of genomic DNA,, x PCR bu# er, each dntp at.** mm, it evolves much more rapidly than nuclear DNA, resulting each primer at *.. mm and *1/. U of LA-Taq DNA polyin the accumulation of di# erences between closely related merase (TaKaRa, Shiga, Japan). After an initial incubaspecies. In fact, the rapid pace of sequence changes in tion at 3/ for, min, PCR amplification was performed mtdna results in di# erences between populations that for -/ cycles consisting of 3/ for -* sec, /* for -* sec, have only been separated for brief periods of time (Hebert 1. for 0* sec, followed by a final extension of 1. for et al.,,**.). Pigeon mtdna sequence was used to con- +* min. The amplified products were purified using PCR struct a phylogeny for Streptopelia, Columba and related purification kit (Roche, Mannheim, Germany) and the taxa (Johnson et al.,,**+). resultant products were sequenced by using the same Because of their high degree of polymorphism, being primers and the Big Dye Terminator ver. -+. Cycle Senumerous and ubiquitous throughout the genome and quencing Kit (Applied Biosystems, Foster City, CA, codominant inheritance, microsatellite markers are valua- USA) according to the standard protocol, and electroble tools for the studying of genetic diversity between phoresed on an ABI PRISM -+-* xl sequencer (Applied populations and assessing the relationships among closely Biosystems). The MEGA / Software (Kumar et al., related livestock breeds (Tadano et al.,,**1 a). Indeed,,**2) was used for sequences alignment and to infer the pigeon DNA microsatellites were used to clarify the origin phylogenetic relationships based on neighbor-joining (NJ) and genetic relationship between di# erent pigeon lines methods (Saitou and Nei, +321). (Traxler et al.,,***) and to provide a rapid identification Microsatellite Analysis to resolve identification and paternity disputes arising Eleven microsatellite markers ( ClimD +1, ClimT +1, ClimD +0, from racing pigeons (Lee et al.,,**1). In this study, we ClimD -,, ClimT+- and ClimD*+ from Traxler et al.,,***; applied the previous markers for analysis of genetic diver- PG +, PG,, PG., PG0 and PG1 from Lee et al.,,**1) were sity and relationships of pigeon populations in Egypt. used in multiplex PCR reactions employing the QIAGEN Such information would provide a foundation for develop- Multiplex PCR Kit (QIAGEN, Valencia, CA, USA). ing sustainable genetic improvement and conservation PCR was carried out in +* ml reactions containing,* ng of programs aimed at enhancing the flying ability, meat DNA template, *,. mm of each primer, of which the quality, as well as growth and reproduction traits of this agriculturally and commercially valuable species. Materials and Methods Sample Collection and DNA Extraction A total of +-- pigeon samples were obtained from six Egyptian indigenous breeds: Krezly ( n,0), Zagel ( n,+), Safi ( n,+ ), Romani ( n,+ ), Asfer Weraq ( n +, ), and Ablaq ( n +* ) together with Japanese racing pigeons ( n,-; 2 from Japanese Imanishi line and others from Belgian lines). Mitochondrial cytochrome c oxidase subunit I gene ( COI) sequence was analyzed also for one individual each from some wild pigeon species, oriental turtle-dove ( Streptopelia orientalis), emerald dove ( Chalcophaps indica), white-bellied green pigeon ( Treron sieboldii) forward ones were fluorescently labelled ( 0-FAM, NED, and HEX) and, x QIAGEN Multiplex PCR Master Mix. After an initial incubation of 3/ for +/ min, PCR amplification was performed for -/ cycles consisting of 3. for -* sec, // for 3* sec, 1, for 0* sec, followed by a final extension of 0* for -* min. Subsequently, the PCR products were electrophoresed on an ABI -+-* xl DNA Sequencer (Applied Biosystems) and the sizes of the fragments were estimated based on.** HD Rox size marker using the GENEMAPPER software (Applied Biosystems). Data Analysis The genetic diversity of each breed was assessed by calculating the observed number of alleles ( NA) and its mean ( MNA), observed heterozygosity ( HO) and expected and whistling green pigeon ( Treron formosae) for heterozygosity ( HE) by using the program package comparison. Egyptian samples were collected from eight ARLEQUIN version,.*** (Schneider et al.,,***) software. breeders in four provinces (Cairo, Giza, Kaliobia and F-statistics fixation coe$ cient of an individual Zagazig) located in the Nile river delta in the northern part of Egypt, whereas, samples of Japanese racing pigeons were collected from one breeder in Kashiwa city, within a subpopulation ( FIS), fixation coe$ cient of an individual within the total population ( FIT), and fixation coe$ cient of a subpopulation within the total population Chiba, Japan. Samples of wild pigeons were obtained ( F ) per locus, in addition to pairwise F (Weir and ST ST

3 Ramadan et al. : Genetic Diversity of Egyptian Pigeon Breeds 81 Cockerham, +32. ) across the seven studied populations by ++ microsatellite markers. Across all the populations, were calculated using the GENEPOP version -.. program the number of alleles per locus ranged from -( PG0) to +. (Raymond and Rousset, +33/ ). Genetic distances among ( CLimD+0) and the average number of alleles observed the seven populations were evaluated by Nei s genetic was 2+. (Table + ). Across populations, locus PG0 had the distance ( DA; Nei et al., +32- ). A phylogenetic tree was lowest values for both HO (*..,3) and HE (*..,0), whereconstructed based on the DA genetic distance by using the as locus CLimT+1 and locus CLimD*+ had the highest HO neighbor-joining (NJ) method (Saitou and Nei, +321 ). (*. 02* ) and HE (*. 1+* ), respectively (Table + ). The aver- The robustness of tree topologies was evaluated with a age numbers of alleles, expected and observed heterozygbootstrap test of +,*** resampling across loci. These osity in addition to FIS for each population across ++ loci processes were conducted using POPULATIONS version are shown in Table,. Across ++ loci, the lowest value of +,-*.. software (http: //bioinformatics.org/~tryphon/ expected heterozygosity (*..,-) was obtained for the populations/). Ablaq breed, and the highest value (*. 1-, ) was found for Results and Discussion Romani breed. The relatively high diversity obtained for Romani breed may be explained as Romani breed is kept Mitochondrial COI Analysis by fanciers as meat breed in a large area of Egypt, which We obtained 03- base pairs of sequence for one sample leads to breeding from many individuals. This condition each of the six Egyptian breeds, the Japanese racing pi- seems to result in higher degree of diversity than other geons and the four wild pigeon species. After alignment, breeds which are mainly kept for racing and flying. The there were only two substitution sites among the six stud- overall expected heterozygosity of Egyptian indigenous ied Egyptian breeds and the Japanese racing pigeons. pigeons together with Japanese racing pigeons was *./2.. From the NJ phylogenetic tree (Fig. + ) these seven popu- As a measure of deviation from Hardy-Weinberg equiliblations clustered into the same clade with Columba livia rium, the FIS value was calculated and found to range from sequence retrieved from GenBank (accession number GQ.2+0*/). The branching pattern of other species reflected *.,** (Asfer Weraq) to *.*1- (Japanese racing) with mean *.*+1. Table, shows that +, breed-specific alleles their phylogeny. The low sequence divergence within were detected among the seven populations. The number Egyptian breeds together with the Japanese racing pigeons of breed-specific alleles per breed ranged from * (Zagel can be explained as all these breeds belong to same species and Asfer Weraq) to. (Krezly and Romani). ( Columba livia) and the mtdna COI sequence diver- Genetic Di# erentiation Among Populations: Genetic difgence is more suited for the analysis of among species ferentiation was examined by fixation indices F IS, F ST, FIT divergence than within species divergence (Hebert et al., for each locus (Table + ). The fixation coe$ cients of sub-,**.). populations within the total population, measured as FST Microsatellite Analysis value, for the ++ loci varied from *. +/. ( CLimD+1) to Genetic diversity: A total of 23 alleles were detected in the *.-+- ( PG. ), with a relatively high mean *.,*- which six Egyptian breeds together with Japanese racing pigeons indicated that there is a great di# erentiation among the Fig. +. Neighbor-joining tree of mitochondrial COI gene sequence of six Egyptian breeds, Japanese racing pigeons, and four wild pigeon species. The sequence for Columba livia with asterisk was retrieved from GenBank (accession number GQ.2+0*/). The four wild pigeon species act as outgroup.

4 82 Journal of Poultry Science,.2 (,) Table +. Observed ( NA) number of alleles, observed ( HO) and expected ( HE) heterozygosities, and F statistics [fixation coe$ cient of an individual within a subpopulation ( FIS), within the total population ( FIT) and fixation coe$ cient of a subpopulation within the total population ( FST)] across 1 studied populations Locus N H H F F F A O E IS ST IT CLimD+1 CLimT+1 CLimD *40.0 *402* *4/,3 *40+3 *4011 *40+. *4*+, *4**2 *4*1/ *4+/. *4+0* *4,*, *4+.. *4+/. *4,0+ CLimD-, CLimT+- CLimD* , *4.3. *40.. *4010 *4.2+ *400* *41+* *4*/* *4*-, *4*01 *4,.0 *4,+* *4+11 *4,*2 *4+2/ *4,-, PG+ / *4/+. *4/1- *4*2* *4+2, *4,.1 PG, PG. 3 0 *4001 *4/,- *40.+ *4/++ *4**/ *4*1, *4+3- *4-+- *4+23 *4,0. PG0 - *4.,3 *4.,0 *4**. *4+11 *4+2* PG1 0 *4.2* *4/+. *4*+2 *4,,, *4,-0 Mean* *4/1+ *4*3, *4/2. *4*3* *4**2 *4*+/ *4,*- *4*+. *4,*3 *4*+- * The means are given SD for N,H and H and SEfor F,F and F. A O E IS ST IT Table,. Observed ( NA) and mean ( MNA) number of alleles, unique alleles, observed ( HO) and expected ( HE) heterozygosities, and fixation coe$ cient of an individual within a subpopulation ( F ) per breed IS Egyptian pigeon Krezly Zagel Safi Romani Asfer Weraq Ablaq Japanese racing Total average n N SD MNA SD Unique alleles H SD H SD F SE A O E IS ++*..4/ * *4/1* *4++, *4/2* *4+,/ *4*-, *4*-0,0 /1 /4,. *40** *4+/, *40,* *4+/3 *4*,1,+ /-.42 * *4002 *4+*1 *4020 *4*31 *4*+0, *4//2 *4,*- *4/.2 *4+0+ *4*/,,* /2 /4-. *403/ *4++/ *41-, *4++, *4*-, +,,3,40 * *4/*1 *4--3 *4./* *4,0/ *4,** +*,0,4. + *4-3* *4,-3 *4.,- *4+03 *4*+.,- /,.41, *4/2+ *4+03 *40-* *4*33 *4*1-./40 +-4,.4+ +4, +41 *4/1+ *4+*, *4/2. *4++0 *4*+1 *4*-. seven pigeon populations. It is clear that about,*.- of *./+2 (Zagel-Ablaq). The closest pair was thus Zagel and the total genetic variation corresponds to di# erences of Japanese racing pigeons. Similarly, the genetic di# erentipopulations. This FST value is higher than in some other ation (pairwise FST) values were lowest in Zagel-Japanese poultry species. For instance, Shahbazi et al. (,**1) re- racing pair (*. +*2). These results are supported by clusported a FST value of *+/. from five Iranian native chicken tering in the neighbor-joining phylogenetic tree (Fig.,) populations, and Bao et al. (,**2) reported a FST value of where the tree topology showed close relation between *. +01 from Chinese domestic fowls. The global deficit of Zagel breed and Japanese racing pigeons. The close reheterozygote across populations ( FIT) amounted to,*. 3 lation between Zagel breed and Japanese racing pigeons (Table + ). For the coe$ cient F IS, which shows the degree may be explained as they may have a common ancestor. of departure from random mating, positive FIS values The origin of Japanese racing pigeons was said to be indicate deficit of heterozygote, while the negative FIS military messenger pigeons imported from European values indicate an excess of heterozygous genotypes with countries since early +3** s, and the old ancestors of the respect to the expected value. In this study, the relatively Japanese samples used in this study would be from France, high average FIS (*.**2) in addition to five loci ( CLimD +0, Netherlands and Belgium (Komahara, +32* ). The early CLimD *+, PG +, PG 0, and PG1) showing a deficit of heter- use of pigeons as a messenger led to their value as a ozygote might indicate that these loci are under selection commodity and to its further global proliferation. As far (genetic hitchhiking e# ect) with some morphological or as conquerors and traders moved, they brought their productive traits of selective interest. pigeons with them. Even as the invaders left, descendents Genetic Relationship: As shown in Table -, the pairwise of their pigeons stayed behind to be bred for future wars Nei s genetic distance between the seven studied pigeon with new enemies. By the middle of the nineteenth cenpopulations ranged from *+/.. (Zagel-Japanese racing) to tury Belgians had established the modern messenger and

5 Ramadan et al. : Genetic Diversity of Egyptian Pigeon Breeds 83 (below diagonal) esti- Table -. Nei s genetic distance (above diagonal) and pairwise mates for ++ loci between seven pigeon populations F ST Krezly Zagel Safi Romani Asfer Ablaq Japanese racing Asfer Krezly Zagel Safi Romani Ablaq Weraq Japanese racing *4,,0 *4,.3 *4,.1 *4,32 *4-// *4,,3 *4+0* *4-1/ *4,1* *4-3, *4/+2 *4+/. *4,*+ *4,,- *4-*+ *4,./ *4-/- *4-1+ *4+-/ *4+-, *4+13 *4-0/ *4.*/ *4,30 *4,,2 *4,0. *4,.* *4,*. *4-,+ *4-23 *4,// *4-,. *4-., *4,-- *4-,0 *4.3- *4+.* *4+*2 *4,-0 *4+.- *4,0+ *4--* Fig.,. Neighbor-joining tree of six Egyptian breeds and Japanese racing pigeons by ++ microsatellites. The consensus tree was generated with +*** bootstraps over loci and bootstrap values lower than /* are not shown in the diagram. racing pigeon now used throughout the world through the continual crossbreeding of several types of pigeons (Jerolmack,,**1). In conclusion, we confirm the applicability and e$ ciency of microsatellites for assessing genetic variation and divergence in Egyptian native pigeon breeds and populations. Relatively reliable results can be obtained even with a small number of microsatellites, as shown in this and other similar studies (e.g., Vanhala et al., +332). The information from this study would be useful for genetic characterization and provide a foundation for developing sustainable genetic improvement and conservation programs of this agriculturally and commercially valuable species. We suggest that an increase in the e# ective number of populations for breed reproduction will assist in preventing both a decline in genetic diversity and an increase of inbreeding. The further cataloging and genetic characterization of Egyptian pigeon breeds and populations based on highly variable mitochondrial DNA markers (mtdna control region) together with more micro- satellite loci are eagerly anticipated. Acknowledgments We thank the breeders in Egypt and Dr. Takeshi Wada in Osaka Museum of Natural History for providing samples. We are grateful to Ms. Kazumi Kato of Kyoto University for her assistance in genetic analysis. This study was supported financially in part by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) with a Grant-in-aid for Science Research (#,+-+*+/* to MI-M) and the Global Center of Excellence Program Formation of a Strategic Base for Biodiversity and Evolutionary Research: from Genome to Ecosystem. References Bao W, Chen G, Li B, Wu X, Shu J, Wu S, Xu Q and Weigend S. Analysis of genetic diversity and phylogenetic relationships among red jungle fowls and Chinese domestic fowls. Science in China Series C-life Sciences, /+: /0* /02.,**2. Blechman A. Pigeons-The fascinating saga of the world s most revered and reviled bird. University of Queensland Press. St Lucia, Queensland.,**1. Bodio S. Aloft: A Meditation on Pigeons and Pigeon-Flying. Lyons and Burford Publishers. New York. +33*. Glover D and Beaumont M. Racing Pigeons. Crowood Press Ltd. Ramsbury, Marlborough (Great Britain) Hebert PDN, Stoeckle MY, Zemlak TS and Francis CM. Identification of birds through DNA barcodes. Public Library of Science Biology,,: +0/ ,**.. Hiatt S and Esposito DVM. The Pigeon Guide. Silvio Mattacchione and Company. Port Perry, Ontario.,***. Jerolmack C. Animal archeology: Domestic pigeons and the nature-culture dialectic. Qualitative Sociology Review, III: 1. 3/.,**1. Johnson KP, De Kort S, Dinwoodey K, Mateman AC, Cate CT, Lessells CM and Clayton DH. A molecular phylogeny of the dove genera Streptopelia and Columba. Auk, ++2: ,**+. Kayang BB, Youssao I, Inoue E, Naazie A, Abe H, Ito S and Murayama M. Genetic diversity of helmeted guineafowl ( Numida meleagris) based on microsatellite analysis. Journal of Poultry Science,.1: +,* +,..,*+*. Komahara K. How to keep pigeons. Airyudo. Tokyo. +32*. (in Japanese). Kumar S, Nei M, Dudley J and Tamura K. MEGA: A biologist-

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