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A novel molecular genetic marker for gender determination of pigeons
Theriogenology 65 (2006) 1759–1768 www.journals.elsevierhealth.com/periodicals/the
A novel molecular genetic marker for gender determination of pigeons Yan-Ming Horng b, Chean-Ping Wu a,b, Yng-Chyu Wang b, Mu-Chiou Huang a,* a
Department of Animal Science, National Chung Hsing University, 250 Kao-Kung Road, Taichung, Taiwan b Department of Animal Science, National Chiayi University, 300 University Road, Chiayi, Taiwan
Received 14 June 2005; received in revised form 5 October 2005; accepted 8 October 2005
Abstract The absence of conspicuous sexual dimorphism in pigeons often makes it difficult to determine their sex on the basis of external morphology. We identified a novel female-specific DNA marker in pigeons, presenting the possibility of pigeon gender determination using a PCR-based method. Onehundred and twenty random primers were used for RAPD fingerprinting in order to find any sexspecific fragments in pigeons. One of these primers, OPC-20, produced a female-specific band in the DNA fingerprints. This DNA fragment was isolated from the gel and inserted into a vector for nucleotide sequencing. A novel female-specific 732 bp sequence was obtained. A pair of primers (DoveOPC20F & R) was designed, based on the cloned sequence, for amplifying the female-specific band by PCR for pigeon gender determination. Sex-specific bands in the gel were observed in all females but not in males. The PCR products in the gel were then transferred onto nylon membranes and hybridized with a DIG-labeled probe of the cloned female-specific DNA fragment. Clear hybridization signals were found only in all of the female pigeons; the same result was obtained from dot blot hybridization. This demonstrates that the sex of pigeons can be accurately and rapidly identified by PCR. # 2005 Elsevier Inc. All rights reserved. Keywords: PCR; Gender determination; Pigeons; RAPD; Female-specific DNA marker
* Corresponding author. Tel.: +886 4 22852469; fax: +886 4 22860265. E-mail address: [email protected] (M.-C. Huang). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.10.011
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1. Introduction In many bird species, juveniles exhibit little external sexual differentiation, thus often making it difficult to identify their gender based on outward appearances. The same problem is encountered in most of these birds even when they are fully developed, and it can still be almost impossible to determine their gender based on external morphology [1]. In birds, the male and female sex chromosome roles are reversed from mammals, meaning that the female is heterogametic (ZW) while the male is homogametic (ZZ), and sexing can thus be made by detection of the W chromosome sequences. Initial attempts in this direction were based on screening for W-linked repetitive or anonymous sequences [2]. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) has been successfully applied in genetic studies of animal species identification as well as for gender determination of animals [3–8]. RAPD-PCR is based on amplification of DNA in PCR by oligonucleotide random primers. It is easy to perform, involves low cost, does not require known prior sequence of template, and requires only a small amount of template DNA. This technique has recently elicited much interest in modern biology because of its capability of evaluating DNA variation. If various primers are tested, it may be possible to find a sex-specific fragment in RAPD fingerprints. The absence of conspicuous sexual dimorphism in pigeons often makes it difficult to determine the sex on the basis of external morphology. Here we describe a novel femalespecific DNA marker cloned from pigeons by RAPD fingerprinting. Two primers were designed from the cloned sex-specific sequence for accurate and rapid sexing of pigeons by PCR.
2. Materials and methods 2.1. Animals Blood samples were collected with anticoagulant from the wing vein of adult pigeons (Columba livia). Gender confirmation was done by a surgical invasion that allows for examination of the testes under the guidelines for animal experiments at the National Chung Hsing University. Eighteen females and 18 males were analyzed in this study. 2.2. DNA preparation Eighty microliters of each whole blood sample was suspended in 3 mL lysis buffer (10 mM Tris–HCl, 150 mM NaCl, 10 mM EDTA) and mixed well. Subsequently, 300 mL 10% NH4Cl, 75 mL proteinase K (10 mg/mL), 25 mL collagenase (3.8 IU/mL), and 200 mL 10% w/v SDS were added to the sample and it was incubated at 42 8C for 24 h with agitation. DNA was purified by two extractions with equal volumes of phenol, one followed by phenol/chloroform and one by chloroform. A DNA pellet was precipitated with isopropanol. Excess isopropanol was removed using 70% ethanol. The DNA was vacuum dried and resuspended in double distilled water for PCR use.
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2.3. RAPD fingerprinting The RAPD-PCR was performed following the revised method described by Horng and Huang [6]. One hundred micromole Tris–HCl pH 8.0, 1.5 mM MgCl2, 50 mM KCl, 100 mM of each dNTP, 14 mM of primer (Operon Technologies Inc., CA, USA), 100 ng genomic DNA from pigs, 0.5 U DyNAzyme (Finnzymes Inc., Finland), and double distilled water to a total volume of 15 mL were contained in the reaction mixture. One-hundred and twenty random primers (Operon kits AA, AO, AV, C, D, and E series) were used for RAPD-PCR. The RAPD-PCR amplification was carried out in a GeneAmp PCR system 2400 thermal cycler (Applied Biosystems, CA, USA). The amplification conditions were designed as follows: initial denaturation for 5 min at 94 8C, followed by 45 cycles at 94 8C for 1 min, at 36 8C for 1 min, at 72 8C for 2 min, and a final 5 min extension at 72 8C. The PCR products were detected by electrophoresis in 2.0% agarose gels and stained with ethidium bromide (1.5 mg/ mL). RAPD fingerprints were analyzed using an image analysis system (Bertech Co. Ltd., Taiwan). 2.4. Isolation of sex-specific DNA fragment and sequencing Template DNA isolated from female and male pigeons was amplified by RAPD-PCR with OPC-20 primer. The female-specific fragment (732 bp) was isolated from the agarose gel slice using QIA quick gel extraction kit (Qiagen Inc., Valencia, CA, USA) and inserted into pCRII-TOPO vector using the TOPO cloning Kit (Invitrogen, San Diego, CA, USA). The inserted DNA fragment was then sequenced in an ABI PrismTM Genetic Analyzer 3100 using ABI Prim BigDye terminator cycle sequencing ready reaction kits (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. 2.5. Female-specific primer designing for gender determination A pair of female-specific primers (DoveOPC20F: 50 -ACTTCGCCAC AATTTCAGTG GTTCC-30 ; DoveOPC20R: 50 -ACTTCGCCAC TAAAAATGTA AAAAT-30 ) was designed according to the cloned sex-specific sequence to generate a single band of female-specific fragment (732 bp) for pigeon gender determination. The 18S-F (50 -AGCTCTTTCT CGATTCCGTG-3) and –R (50 -GGGTAGACAC AAGCTGAGCC-30 ) primers were also designed to generate a 256 bp 18S ribosomal RNA gene fragment product for use as an internal control of DNA amplification [9,10]. The PCR program consisted of incubation at 94 8C for 5 min and 35 cycles at 94 for 1 min, 58 8C for 1 min, and 72 8C for 1 min, followed by extra extension at 72 8C for 7 min. The PCR products were run on 2.0% agarose gels, and blotted onto nylon membranes (Schericher & Schull Inc., Dassel, Germany). The blots were hybridized with 50 ng/mL DIG-labeled (PCR DIG Probe Synthesis kit; Roche Applied Science, Mannheim, Germany) female-specific DNA probe. Chemiluminescent detection was carried out according to the standard DIG chemiluminescent detection procedure using CSPD at 0.25 mM final concentration (Roche Diagnostics GmbH, Germany).
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2.6. Dot blot hybridization Dot blot hybridization was also conducted. Ten micrograms of genomic DNA per bird were spotted onto nylon membranes and fixed by baking in an oven at 80 8C for 2 h. Hybridization and chemiluminescent detection were performed as described above.
3. Results 3.1. RAPD fingerprinting for find sex-specific band Six series of random primers, OPAA, OPAO, OPAV, OPC, OPD, and OPE series (Operon Alameda, CA, USA), were used for RAPD fingerprinting to investigate the sex-specific sequence of pigeons. Each series consists of 20 random sequence primers. Among the 120 random primers that were used for testing, there were 94 random primers yielding polymorphic bands in the RAPD fingerprints. Fig. 1 presents the polymorphisms in the RAPD fingerprints amplified with OPAA-03 primer; no sexspecific band was found.
Fig. 1. Polymorphisms in the RAPD fingerprints of male and female pigeons. Genomic DNA samples were amplified with OPAA-03 primer. Several obvious common major bands and some minor individual bands in lanes were observed, but no sex-specific band was found. M is Bio 100 bp ladder marker.
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Fig. 2. RAPD fingerprints of pigeons. The template DNA of both sexes was amplified with OPC-20 primer by RAPD-PCR, which produced a sex-specific band in the DNA fingerprints only in all females. M is 100 bp ET marker.
3.2. Isolation of sex-specific DNA fragment and ligated to vector for sequencing One of these random primers, OPC-20, produced a sex-specific band in the DNA fingerprints only in all females tested (Fig. 2). The female-specific DNA fragment was isolated and inserted into pCRII-TOPO vector (Invitrogen, San Diego, CA, USA) for nucleotide sequencing. A sequence of length 732 bp was obtained (Fig. 3). 3.3. Sex-specific primers designed for sexing A pair of primers, DoveOPC20F & R, was designed based on the cloned femalespecific sequence for amplifying the female-specific band by PCR for pigeon gender determination (Fig. 3). Both female and male pigeon genomic DNA were used as templates and amplified with these two primers by PCR. Sex-specific bands were observed in the gel for females, but not for males (Fig. 4A). The PCR products in the gel were transferred onto nylon membranes and hybridized with DIG-labeled probe prepared from the cloned female-specific DNA fragment (732 bp). Clear hybridization signals could be observed in all of the female pigeons, but none was evident for the males (Fig. 4B).
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Fig. 3. A novel sex-specific DNA sequence of pigeons, cloned from the female-specific band in DNA fingerprints. In order to generate a single band in agarose gel by PCR for easy gender determination of pigeons, a pair of primers (shadows) was designed according to the cloned sequence.
3.4. Dot blot hybridization with the female-specific DNA probe Dot blot hybridization was also conducted. Male and female genomic DNA samples collected from pigeons were spotted onto the nylon membranes and fixed by baking in an oven, and hybridization with the female-specific DNA probe and chemiluminescent detection were performed. The probe hybridized strongly to all female DNA samples, but there was no signal for the samples from any male pigeons (Fig. 5).
4. Discussion Some species of birds, including ratite birds, are considered monomorphic species, meaning that mature female and male birds look very similar in external morphology, body weight, color, and behavior. Hence, they are difficult to identity the sexes on the basis of external appearance. Such birds include 20–30% of the 9019 currently known bird species [11]. The methods often used to identify the sex of monomorphic birds include examining the cloacal duct, endoscopy, hormone determination, and cell gene analysis [11,12]. Molecular sexing is an attractive option since it can potentially provide an accurate and rapid means for sex identification, especially if based on non-invasive techniques [13]. Pigeons have a sensitive and docile temperament. Aside from its use for meat production, it has been bred as a hobby pet. In recent years, pigeons have been widely raised in Taiwan for pigeon racing and meat production. Prior to pairing for breeding, it is important to ensure that representatives of both sexes are present. Because the outward appearances of different sexes of pigeons are similar, sexing is not a simple visual task. In current general practice, pigeon gender determination involves having someone hold the bird while another inserts a light probe into the vent to check for the presence or absence of a phallus. The method works with maturing males, but an immature male’s phallus looks and feels
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Fig. 4. Electropherogram of PCR products amplified from the pigeon genomic DNA with female-specific primers (DoveOPC20F & R) and 18S ribosomal RNA gene primers. Female-specific bands of length 732 bp were represented in the gel for only all females (A). The PCR products in the gel were transferred onto nylon membranes and hybridized with DIG-labeled probe prepared from the cloned female-specific DNA fragment. Clear hybridization signals were observed in all of the female pigeons (B). M is 100 bp ET marker.
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Fig. 5. Pigeon genomic DNA samples were spotted onto the nylon membrane and hybridized with female-specific DIG-labeled probe. Strong signals were found only in all females.
very similar to the female’s clitoris. Another method that is often used is to perform a visual examination according to the morphology or voice of the bird. However, there may be questions as to the accuracy of these methods. In the present study, we describe a novel female-specific DNA sequence cloned from pigeons using RAPD-PCR. A pair of primers was designed according to the cloned sex-specific sequence. We have devised a PCR-based method using the designed primers for the gender determination of pigeons. Operon 10-mer kits contain 10-base oligonucleotide primers for use in pigeon DNA fingerprinting by RAPD-PCR. Operon Technologies presently has 1000 different 10-base primers in stock. These primers are sold in kits of 20 sequences each and are designated ‘‘kit A’’ through ‘‘kit Z’’ and ‘‘kit AA’’ through ‘‘kit AX’’. One-hundred and twenty sequences in total of kits AA, AO, AV, C, D, and E were selected randomly for the present study. The DNA amplification product is generated for each genomic region that happens to be flanked by a pair of 10-base priming sites in the appropriate orientation. Assuming that the priming sites are randomly distributed throughout a genome, different DNA fingerprinting patterns can arise when using a single primer that at the same time amplifies different gene positions of the DNA fragment, when the subject gene differs [14,15]. In other words, genomic DNA from two different individuals often produce different amplification fragment patterns. The particular DNA fragment, which is generated for one sex but not for another, represents a DNA polymorphism and can be used as a gender marker. The marker is inherited in a Mendelian fashion. The band numbers of different amplification products for each reaction depend upon the genomic and primer sequences. The RAPD-PCR was performed on the pigeon genomic DNA of both sexes. Among the 120 random primers, 78% (94/120) yielded polymorphisms in the RAPD fingerprints. No sex-specific bands in the RAPD fingerprints amplified with these primers other than OPC20 were found amongst either females or males. The polymorphisms came from different templates of genomic DNA in different pigeons, and the primer binding sites were also
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different, which led to the production of bands of different length in the RAPD fingerprints. Five common major bands, amplified with OPAA 03 primer, can be observed in the fingerprints of different birds at 500 bp, 650 bp, 700 bp, 1000 bp, and 1250 bp positions (Fig. 1). There were some minor bands in the lanes of individual birds. This demonstrates that although the genetic material was more similar from birds of the same breed, it was entirely homogeneous within breeds. Pigeon template DNA of both sexes was amplified with OPC-20 primer by RAPD-PCR, which produced a sex-specific band in the DNA fingerprints only in all females, but not in males (Fig. 2). The female-specific sequence (732 bp) was sequenced and submitted to the GenBank (Accession No. AY944219) [16]. The sex-specific sequence of female pigeons was then compared with the sequences deposited in the GenBank, by GCG sequence analysis software (Genetic Computer Group). There were no matches to the sequence, showing that the cloned sequence can be considered a novel female-specific sequence of pigeons (Fig. 3). The single female-specific bands (732 bp) amplified by PCR using the DoveOPC20F & R primers were observed only in the females and not in any males (Fig. 4). The same result was obtained by dot blot hybridization (Fig. 5). These results demonstrate that the cloned sequence was a female-specific sequence, and that the DoveOPC20F & R primers can be used for effective PCR-based sexing technique in pigeons. The annealing temperature influences the specificity of the amplification reaction. If the temperature is too high, no annealing occurs at all, but if the temperature is too low, nonspecific annealing might increase dramatically. Sometimes the evaluation of optimum annealing temperature is the most time-consuming part of an optimization strategy [17]. In the present experimental design, we found that the most appropriate annealing temperatures were 36 8C for RAPD-PCR and 58 8C for PCR. In conclusion, we have described a novel sex-specific DNA sequence in pigeons obtained by RAPD fingerprinting. The gender determination of pigeons can be accurately and rapidly performed by PCR with DoveOPC20F and R primers that are designed on the basis of the cloned female-specific sequence of pigeons.
References [1] Fridolfsson AK, Cheng H, Copeland NG, Jenkins NA, Liu HC. Evolution of the avian sex chromosomes from an ancestral pair of autosomes. Proc Natl Acad Sci USA 1998;95:8147–52. [2] Griffiths R, Tiwari B. The isolation of molecular genetic markers for the identification of sex. Proc Natl Acad Sci USA 1993;90:8324–6. [3] Yen NT, Huang MC, Tai C. Genetic variation of RAPD polymorphisms in Taoyuan and Duroc pigs. J Anim Breed Genet 2001;118:111–8. [4] Saifi HW, Bhushan B, Kumar S, Kumar P, Patra BN, Sharma A. Genetic identity between Bhadawari and Murrah breeds of Indian buffaloes (Bubalus bubalis) using RAPD-PCR. Asian-Aus J Anim Sci 2004;17:603–7. [5] Huang MC, Horng YM, Huang HL, Sin YL, Chen MJ. RAPD fingerprinting for the species identification of animals. Asian-Aus J Anim Sci 2003;16:1406–10. [6] Horng YM, Huang MC. Male-specific DNA sequences in pigs. Theriogenology 2003;59:841–8. [7] Huang MC, Lin WC, Horng YM, Rouvier R, Huang CW. Female-specific DNA sequences in geese. Br Poult Sci 2003;44:359–64.
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[8] Horng YM, Chen YT, Wu CP, Jea YS, Huang MC. Cloning of Taiwan water buffalo male-specific DNA sequence for sexing. Theriogenology 2004;62:1536–43. [9] Long EO, Dawid IB. Repeated gene in eukaryotes. Ann Rev Biochem 1980;49:727–64. [10] Clinton M, Haines L, Belloir B, Mcbride D. Sexing chick embryos: a rapid and simple protocol. Br Poult Sci 2001;42:134–8. [11] Jones DM, Samour JH, Knight JA, Finch JM. Sex determination of monomorphic birds by fibreoptic endoscopy. Vet Res 1984;115:596–8. [12] Harrison GJ. Endoscopic examination of avian gonadal tissues. Vet Med Small Anim Clin 1978;73:479–84. [13] Fridolfsson AK, Ellegren H. A simple and universal method for molecular sexing of non-ratite bird. J Avian Biol 1999;30:116–21. [14] Welsh J, Honeycutt RJ, Mcclelland M, Sobral BWS. Parentage determination in maize hybrids using the arbitrarily primed polymerase chain reaction (AP-PCR). Theor Appl Genet 1991;82:473–6. [15] Welsh J, Petersen C, Mcclelland M. Polymorphisms generated by arbitrarily primed PCR in mouse: application to strain identification and genetic mapping. Nucl Acids Res 1991;19:303–6. [16] Horng YM, Wu CP, Wang YC, Huang MC. Columba livia female-specific genomic sequence. NCBI 2005; AY944219. [17] Rolfs A, Schuller I, Finckh U, Rolfs IW. PCR principles and reaction components. In: Rolfs A, Schuller I, Finckh U, Rolfs IW, editors. PCR: clinical diagnostics and research. Berlin: Springer-Verlag; 1992. p. 1–21.
A novel molecular genetic marker for gender determination of pigeons Yan-Ming Horng b, Chean-Ping Wu a,b, Yng-Chyu Wang b, Mu-Chiou Huang a,* a
Department of Animal Science, National Chung Hsing University, 250 Kao-Kung Road, Taichung, Taiwan b Department of Animal Science, National Chiayi University, 300 University Road, Chiayi, Taiwan
Received 14 June 2005; received in revised form 5 October 2005; accepted 8 October 2005
Abstract The absence of conspicuous sexual dimorphism in pigeons often makes it difficult to determine their sex on the basis of external morphology. We identified a novel female-specific DNA marker in pigeons, presenting the possibility of pigeon gender determination using a PCR-based method. Onehundred and twenty random primers were used for RAPD fingerprinting in order to find any sexspecific fragments in pigeons. One of these primers, OPC-20, produced a female-specific band in the DNA fingerprints. This DNA fragment was isolated from the gel and inserted into a vector for nucleotide sequencing. A novel female-specific 732 bp sequence was obtained. A pair of primers (DoveOPC20F & R) was designed, based on the cloned sequence, for amplifying the female-specific band by PCR for pigeon gender determination. Sex-specific bands in the gel were observed in all females but not in males. The PCR products in the gel were then transferred onto nylon membranes and hybridized with a DIG-labeled probe of the cloned female-specific DNA fragment. Clear hybridization signals were found only in all of the female pigeons; the same result was obtained from dot blot hybridization. This demonstrates that the sex of pigeons can be accurately and rapidly identified by PCR. # 2005 Elsevier Inc. All rights reserved. Keywords: PCR; Gender determination; Pigeons; RAPD; Female-specific DNA marker
* Corresponding author. Tel.: +886 4 22852469; fax: +886 4 22860265. E-mail address: [email protected] (M.-C. Huang). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.10.011
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1. Introduction In many bird species, juveniles exhibit little external sexual differentiation, thus often making it difficult to identify their gender based on outward appearances. The same problem is encountered in most of these birds even when they are fully developed, and it can still be almost impossible to determine their gender based on external morphology [1]. In birds, the male and female sex chromosome roles are reversed from mammals, meaning that the female is heterogametic (ZW) while the male is homogametic (ZZ), and sexing can thus be made by detection of the W chromosome sequences. Initial attempts in this direction were based on screening for W-linked repetitive or anonymous sequences [2]. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) has been successfully applied in genetic studies of animal species identification as well as for gender determination of animals [3–8]. RAPD-PCR is based on amplification of DNA in PCR by oligonucleotide random primers. It is easy to perform, involves low cost, does not require known prior sequence of template, and requires only a small amount of template DNA. This technique has recently elicited much interest in modern biology because of its capability of evaluating DNA variation. If various primers are tested, it may be possible to find a sex-specific fragment in RAPD fingerprints. The absence of conspicuous sexual dimorphism in pigeons often makes it difficult to determine the sex on the basis of external morphology. Here we describe a novel femalespecific DNA marker cloned from pigeons by RAPD fingerprinting. Two primers were designed from the cloned sex-specific sequence for accurate and rapid sexing of pigeons by PCR.
2. Materials and methods 2.1. Animals Blood samples were collected with anticoagulant from the wing vein of adult pigeons (Columba livia). Gender confirmation was done by a surgical invasion that allows for examination of the testes under the guidelines for animal experiments at the National Chung Hsing University. Eighteen females and 18 males were analyzed in this study. 2.2. DNA preparation Eighty microliters of each whole blood sample was suspended in 3 mL lysis buffer (10 mM Tris–HCl, 150 mM NaCl, 10 mM EDTA) and mixed well. Subsequently, 300 mL 10% NH4Cl, 75 mL proteinase K (10 mg/mL), 25 mL collagenase (3.8 IU/mL), and 200 mL 10% w/v SDS were added to the sample and it was incubated at 42 8C for 24 h with agitation. DNA was purified by two extractions with equal volumes of phenol, one followed by phenol/chloroform and one by chloroform. A DNA pellet was precipitated with isopropanol. Excess isopropanol was removed using 70% ethanol. The DNA was vacuum dried and resuspended in double distilled water for PCR use.
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2.3. RAPD fingerprinting The RAPD-PCR was performed following the revised method described by Horng and Huang [6]. One hundred micromole Tris–HCl pH 8.0, 1.5 mM MgCl2, 50 mM KCl, 100 mM of each dNTP, 14 mM of primer (Operon Technologies Inc., CA, USA), 100 ng genomic DNA from pigs, 0.5 U DyNAzyme (Finnzymes Inc., Finland), and double distilled water to a total volume of 15 mL were contained in the reaction mixture. One-hundred and twenty random primers (Operon kits AA, AO, AV, C, D, and E series) were used for RAPD-PCR. The RAPD-PCR amplification was carried out in a GeneAmp PCR system 2400 thermal cycler (Applied Biosystems, CA, USA). The amplification conditions were designed as follows: initial denaturation for 5 min at 94 8C, followed by 45 cycles at 94 8C for 1 min, at 36 8C for 1 min, at 72 8C for 2 min, and a final 5 min extension at 72 8C. The PCR products were detected by electrophoresis in 2.0% agarose gels and stained with ethidium bromide (1.5 mg/ mL). RAPD fingerprints were analyzed using an image analysis system (Bertech Co. Ltd., Taiwan). 2.4. Isolation of sex-specific DNA fragment and sequencing Template DNA isolated from female and male pigeons was amplified by RAPD-PCR with OPC-20 primer. The female-specific fragment (732 bp) was isolated from the agarose gel slice using QIA quick gel extraction kit (Qiagen Inc., Valencia, CA, USA) and inserted into pCRII-TOPO vector using the TOPO cloning Kit (Invitrogen, San Diego, CA, USA). The inserted DNA fragment was then sequenced in an ABI PrismTM Genetic Analyzer 3100 using ABI Prim BigDye terminator cycle sequencing ready reaction kits (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. 2.5. Female-specific primer designing for gender determination A pair of female-specific primers (DoveOPC20F: 50 -ACTTCGCCAC AATTTCAGTG GTTCC-30 ; DoveOPC20R: 50 -ACTTCGCCAC TAAAAATGTA AAAAT-30 ) was designed according to the cloned sex-specific sequence to generate a single band of female-specific fragment (732 bp) for pigeon gender determination. The 18S-F (50 -AGCTCTTTCT CGATTCCGTG-3) and –R (50 -GGGTAGACAC AAGCTGAGCC-30 ) primers were also designed to generate a 256 bp 18S ribosomal RNA gene fragment product for use as an internal control of DNA amplification [9,10]. The PCR program consisted of incubation at 94 8C for 5 min and 35 cycles at 94 for 1 min, 58 8C for 1 min, and 72 8C for 1 min, followed by extra extension at 72 8C for 7 min. The PCR products were run on 2.0% agarose gels, and blotted onto nylon membranes (Schericher & Schull Inc., Dassel, Germany). The blots were hybridized with 50 ng/mL DIG-labeled (PCR DIG Probe Synthesis kit; Roche Applied Science, Mannheim, Germany) female-specific DNA probe. Chemiluminescent detection was carried out according to the standard DIG chemiluminescent detection procedure using CSPD at 0.25 mM final concentration (Roche Diagnostics GmbH, Germany).
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2.6. Dot blot hybridization Dot blot hybridization was also conducted. Ten micrograms of genomic DNA per bird were spotted onto nylon membranes and fixed by baking in an oven at 80 8C for 2 h. Hybridization and chemiluminescent detection were performed as described above.
3. Results 3.1. RAPD fingerprinting for find sex-specific band Six series of random primers, OPAA, OPAO, OPAV, OPC, OPD, and OPE series (Operon Alameda, CA, USA), were used for RAPD fingerprinting to investigate the sex-specific sequence of pigeons. Each series consists of 20 random sequence primers. Among the 120 random primers that were used for testing, there were 94 random primers yielding polymorphic bands in the RAPD fingerprints. Fig. 1 presents the polymorphisms in the RAPD fingerprints amplified with OPAA-03 primer; no sexspecific band was found.
Fig. 1. Polymorphisms in the RAPD fingerprints of male and female pigeons. Genomic DNA samples were amplified with OPAA-03 primer. Several obvious common major bands and some minor individual bands in lanes were observed, but no sex-specific band was found. M is Bio 100 bp ladder marker.
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Fig. 2. RAPD fingerprints of pigeons. The template DNA of both sexes was amplified with OPC-20 primer by RAPD-PCR, which produced a sex-specific band in the DNA fingerprints only in all females. M is 100 bp ET marker.
3.2. Isolation of sex-specific DNA fragment and ligated to vector for sequencing One of these random primers, OPC-20, produced a sex-specific band in the DNA fingerprints only in all females tested (Fig. 2). The female-specific DNA fragment was isolated and inserted into pCRII-TOPO vector (Invitrogen, San Diego, CA, USA) for nucleotide sequencing. A sequence of length 732 bp was obtained (Fig. 3). 3.3. Sex-specific primers designed for sexing A pair of primers, DoveOPC20F & R, was designed based on the cloned femalespecific sequence for amplifying the female-specific band by PCR for pigeon gender determination (Fig. 3). Both female and male pigeon genomic DNA were used as templates and amplified with these two primers by PCR. Sex-specific bands were observed in the gel for females, but not for males (Fig. 4A). The PCR products in the gel were transferred onto nylon membranes and hybridized with DIG-labeled probe prepared from the cloned female-specific DNA fragment (732 bp). Clear hybridization signals could be observed in all of the female pigeons, but none was evident for the males (Fig. 4B).
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Fig. 3. A novel sex-specific DNA sequence of pigeons, cloned from the female-specific band in DNA fingerprints. In order to generate a single band in agarose gel by PCR for easy gender determination of pigeons, a pair of primers (shadows) was designed according to the cloned sequence.
3.4. Dot blot hybridization with the female-specific DNA probe Dot blot hybridization was also conducted. Male and female genomic DNA samples collected from pigeons were spotted onto the nylon membranes and fixed by baking in an oven, and hybridization with the female-specific DNA probe and chemiluminescent detection were performed. The probe hybridized strongly to all female DNA samples, but there was no signal for the samples from any male pigeons (Fig. 5).
4. Discussion Some species of birds, including ratite birds, are considered monomorphic species, meaning that mature female and male birds look very similar in external morphology, body weight, color, and behavior. Hence, they are difficult to identity the sexes on the basis of external appearance. Such birds include 20–30% of the 9019 currently known bird species [11]. The methods often used to identify the sex of monomorphic birds include examining the cloacal duct, endoscopy, hormone determination, and cell gene analysis [11,12]. Molecular sexing is an attractive option since it can potentially provide an accurate and rapid means for sex identification, especially if based on non-invasive techniques [13]. Pigeons have a sensitive and docile temperament. Aside from its use for meat production, it has been bred as a hobby pet. In recent years, pigeons have been widely raised in Taiwan for pigeon racing and meat production. Prior to pairing for breeding, it is important to ensure that representatives of both sexes are present. Because the outward appearances of different sexes of pigeons are similar, sexing is not a simple visual task. In current general practice, pigeon gender determination involves having someone hold the bird while another inserts a light probe into the vent to check for the presence or absence of a phallus. The method works with maturing males, but an immature male’s phallus looks and feels
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Fig. 4. Electropherogram of PCR products amplified from the pigeon genomic DNA with female-specific primers (DoveOPC20F & R) and 18S ribosomal RNA gene primers. Female-specific bands of length 732 bp were represented in the gel for only all females (A). The PCR products in the gel were transferred onto nylon membranes and hybridized with DIG-labeled probe prepared from the cloned female-specific DNA fragment. Clear hybridization signals were observed in all of the female pigeons (B). M is 100 bp ET marker.
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Fig. 5. Pigeon genomic DNA samples were spotted onto the nylon membrane and hybridized with female-specific DIG-labeled probe. Strong signals were found only in all females.
very similar to the female’s clitoris. Another method that is often used is to perform a visual examination according to the morphology or voice of the bird. However, there may be questions as to the accuracy of these methods. In the present study, we describe a novel female-specific DNA sequence cloned from pigeons using RAPD-PCR. A pair of primers was designed according to the cloned sex-specific sequence. We have devised a PCR-based method using the designed primers for the gender determination of pigeons. Operon 10-mer kits contain 10-base oligonucleotide primers for use in pigeon DNA fingerprinting by RAPD-PCR. Operon Technologies presently has 1000 different 10-base primers in stock. These primers are sold in kits of 20 sequences each and are designated ‘‘kit A’’ through ‘‘kit Z’’ and ‘‘kit AA’’ through ‘‘kit AX’’. One-hundred and twenty sequences in total of kits AA, AO, AV, C, D, and E were selected randomly for the present study. The DNA amplification product is generated for each genomic region that happens to be flanked by a pair of 10-base priming sites in the appropriate orientation. Assuming that the priming sites are randomly distributed throughout a genome, different DNA fingerprinting patterns can arise when using a single primer that at the same time amplifies different gene positions of the DNA fragment, when the subject gene differs [14,15]. In other words, genomic DNA from two different individuals often produce different amplification fragment patterns. The particular DNA fragment, which is generated for one sex but not for another, represents a DNA polymorphism and can be used as a gender marker. The marker is inherited in a Mendelian fashion. The band numbers of different amplification products for each reaction depend upon the genomic and primer sequences. The RAPD-PCR was performed on the pigeon genomic DNA of both sexes. Among the 120 random primers, 78% (94/120) yielded polymorphisms in the RAPD fingerprints. No sex-specific bands in the RAPD fingerprints amplified with these primers other than OPC20 were found amongst either females or males. The polymorphisms came from different templates of genomic DNA in different pigeons, and the primer binding sites were also
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different, which led to the production of bands of different length in the RAPD fingerprints. Five common major bands, amplified with OPAA 03 primer, can be observed in the fingerprints of different birds at 500 bp, 650 bp, 700 bp, 1000 bp, and 1250 bp positions (Fig. 1). There were some minor bands in the lanes of individual birds. This demonstrates that although the genetic material was more similar from birds of the same breed, it was entirely homogeneous within breeds. Pigeon template DNA of both sexes was amplified with OPC-20 primer by RAPD-PCR, which produced a sex-specific band in the DNA fingerprints only in all females, but not in males (Fig. 2). The female-specific sequence (732 bp) was sequenced and submitted to the GenBank (Accession No. AY944219) [16]. The sex-specific sequence of female pigeons was then compared with the sequences deposited in the GenBank, by GCG sequence analysis software (Genetic Computer Group). There were no matches to the sequence, showing that the cloned sequence can be considered a novel female-specific sequence of pigeons (Fig. 3). The single female-specific bands (732 bp) amplified by PCR using the DoveOPC20F & R primers were observed only in the females and not in any males (Fig. 4). The same result was obtained by dot blot hybridization (Fig. 5). These results demonstrate that the cloned sequence was a female-specific sequence, and that the DoveOPC20F & R primers can be used for effective PCR-based sexing technique in pigeons. The annealing temperature influences the specificity of the amplification reaction. If the temperature is too high, no annealing occurs at all, but if the temperature is too low, nonspecific annealing might increase dramatically. Sometimes the evaluation of optimum annealing temperature is the most time-consuming part of an optimization strategy [17]. In the present experimental design, we found that the most appropriate annealing temperatures were 36 8C for RAPD-PCR and 58 8C for PCR. In conclusion, we have described a novel sex-specific DNA sequence in pigeons obtained by RAPD fingerprinting. The gender determination of pigeons can be accurately and rapidly performed by PCR with DoveOPC20F and R primers that are designed on the basis of the cloned female-specific sequence of pigeons.
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