Validation of Spilornis cheela hoya TaqMan probes for potential gender identification of many Accipitridae species

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Theriogenology 73 (2010) 404–411 www.theriojournal.com

Validation of Spilornis cheela hoya TaqMan probes for potential gender identification of many Accipitridae species T.-C. Chou a,1, C.-T. Yao b,c,1, S.-H. Su d,1, Y.-C. Hung d, W.-S. Chen d, C.-C. Cheng d, C.-N. Tseng d, H.-M. Wang e, Y.-C. Chou f, S.S.-L. Li g, D.-L. Gu d, H.-W. Chang d,h,i,* a

Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei, Taiwan b Taiwan Endemic Species Research Institute, Nantao, Taiwan c Institute of Biodiversity, National Cheng Kung University, Tainan, Taiwan d Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan e Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, Kaohsiung, Taiwan f Institute of Biotechnology, Chung Hwa College of Medical Technology, Tainan, Taiwan g Department of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan h Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan i Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Received 9 April 2009; received in revised form 15 September 2009; accepted 19 September 2009

Abstract The objective of this study was to test the hypothesis that genders of Accipitridae species, with the same or similar sequences to our previously proposed Spilornis cheela hoya (S. c. hoya) chromo-helicase-DNA binding protein (CHD)-W-specific and CHD-ZWcommon TaqMan probes, can be successfully determined. Eight species of Accipitridae with known genders were collected. After PCR, TA cloning, sequencing, and alignment analyses, sequence length differences of Griffiths P2/P8 PCR amplicons between CHD-Z and CHD-W genes ranged from 2 to 19 bp for these Accipitridae species, and they were unsolved in 3% agarose gel. Using our previous proposed S. c. hoya TaqMan probes, the genders of Circaetus gallicus, completely homologous to the sequences for these CHD probes, were successfully identified. With one nucleotide difference to S. c. hoya CHD-W-specific probe, gender identification of Accipiter gularis, Accipiter soloensis, Accipiter trivirgatus, Accipiter virgatus, and Butastur indicus were validated. With two nucleotide differences in the CHD-W-specific probe and one nucleotide difference in the CHD-ZW-common probe, Pernis ptilorhyncus also performed well for gender identification. In conclusion, the S. c. hoya CHD probes, coupled with the Griffiths P2/ P8 primers, were validated to provide accurate and high-throughput gender identification for many Accipitridae species. # 2010 Elsevier Inc. All rights reserved. Keywords: Accipitridae species; CHD gene; Eagle; Gender identification; Sex-specific probe; TaqMan

1. 1. Introduction * Corresponding author. Tel.: +886 7 312 1101x2691; fax: +886 312 5339. E-mail address: [email protected] (H.W. Chang). 1 Equal contribution. 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.09.024

Accipitridae is the family of hawks, eagles, kites, harriers, and Old World vultures [1,2]. All Accipitridae species are protected under the Convention on International Trade in Endangered Species (CITES) [3]. As

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top predators, birds of prey are sensitive indicators of environmental quality [2]. Monitoring of the sex ratio of the Accipitridae species became an attractive method to evaluate their population stability. However, the length difference of intron for CHD-Z and CHD-W genes in many Accipitridae species is extremely short (approximately 3 to 9 bp) [4–10], making it difficult to determine gender of these species by the traditional Griffiths procedure [11]. This problem is resolved by methods of redesigned primers [7,9], PCR-restriction fragment length polymorphism (RFLP) [5,6], microsatellite DNA markers [12,13], random amplified polymorphic DNA (RAPD) [14,15], single-strand conformation polymorphism (SSCP) analysis [16], amplified fragment length polymorphism (AFLP) [17], melting curve assay (MCA) [10], and TaqMan probe [8]. Except for the electrophoresis-free methods for MCA and TaqMan probe (96 or 384 wells available), these methods were currently unsuitable for highthroughput avian gender identification because they required extra time for multiple sample loadings and for electrophoresis after PCR amplification.

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In our previous study [8], we reported that CHD-Wspecific and CHD-ZW-common TaqMan probes provided high-throughput gender identification for Accipitridae species such as Spilornis cheela hoya (S. c. hoya). Most likely, our method [8] may be implemented for gender identification of other Accipitridae species based on the sequence alignments of CHD gene. For example, the sequence of the CHD-W-specific probe designed for S. c. hoya was completely conserved in Circaetus gallicus (C. gallicus), Gyps indicus (G. indicus), and Gyps bengalensis (G. bengalensis), and it was only one nucleotide different from those of Accipiter nisus (A. nisus), Spizaetus nipalensis (S. nipalensis), Aquila chrysaetos (A. chrysaetos), Circus spilonotus (C. spilonotus), and Milvus migrans (M. migrans) as described [8]. For the CHD-ZW-common probe, all these species were completely conserved. However, this method has been developed for and tested only in the S. c. hoya species [8]. The potential use of these CHD probes [8] with regard to other species of Accipitridae still requires proof. The objective of the current study was to test the hypothesis that our proposed CHD-W-specific and

Table 1 Summary of gender identification of eight species of Accipitridae using S. c. hoya TaqMan probes. Wells

A3 A5 B3 B5 C3 C5 D3 D5 E3 E5 G3 G5 F3 F5 B7 C7 G8 H8 B9 D9 E9 F9 G9 H9 a b c d

Species

A. virgatus A. gularis B. indicus A. soloensis P. ptilorhyncus A. trivirgatus C. gallicus S. c. hoya

ID No.a

Bd-2225 Bd-3108 Bd-1505 Bd-1506 Bd-3264 Bd-4857 Bd-2466 Bd-3088 Bd-3861 Bd-434 Bd-6223 Bd-6224 Bd-cgB4 Bd-cgB2 Bd-498 Bd-475 Blank Blank Blank Blank Blank Blank Blank Blank

Genderb

< , < , < , < , < , < , < , < ,

RFU 1 c

RFU 2 c

(CHD-W)

(CHD-ZW)

6.99 732.47 9.80 678.64 12.72 662.09 13.04 725.54 9.49 363.23 14.93 537.09 17.51 1299.66 29.10 267.99 7.56 4.19 4.87 5.37 8.13 7.73 10.27 44.35

127.82 159.00 110.82 144.12 87.95 156.80 113.51 110.18 126.30 98.46 66.10 75.03 75.75 172.43 93.39 59.44 9.04 7.17 11.92 2.25 12.16 10.05 15.24 13.23

Calld

References

Allele 2 Heterozygote Allele 2 Heterozygote Allele 2 Heterozygote Allele 2 Heterozygote Allele 2 Heterozygote Allele 2 Heterozygote Allele 2 Heterozygote Allele 2 Heterozygote None None None None None None None None

This study

[5] [8]

The ID No. is the original banded number for each sample. The female and male controls were confirmed by anatomic inspection. RFU, relative fluorescence unit; RFU 1, RFU of Allele 1 (CHD-ZW-common probe); RFU 2, RFU of Allele 2 (CHD-W-specific probe). ‘‘ Heterozygote’’ indicates female, that is, RFU 1 (+) and RFU 2 (+). ‘‘None’’ indicates there is no calling for gender.

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CHD-ZW-common TaqMan probes for S. c. hoya [8] can be applied to determine the gender of Accipitridae species with the same or similar sequences to these probes. 2. Materials and methods

buffer AW1 by centrifugation (6000  g, 1 min), and washed with 500 mL buffer AW2 by centrifugation (20,000  g, 3 min). After being moved to a new collection tube for incubation with 70 mL buffer AE (room temperature, 1 min), the DNA-containing solution was eluted by centrifugation (6000  g, 1 min) and stored at 20 8C before use.

2.1. Sample collection and DNA extraction Muscle tissue samples (Table 1) for male-female paired Accipiter virgatus (A. virgatus; ID No. Bd 2225 and No. Bd 3108), Accipiter gularis (A. gularis; ID No. Bd-1505 and No. Bd-1506), Butastur indicus (B. indicus; ID No. Bd-3264 and No. Bd-4857), Accipiter soloensis (A. soloensis; ID No. Bd-2466 and No. Bd-3088), Pernis ptilorhyncus (P. ptilorhyncus; ID No. Bd-3861 and No. Bd-434), Accipiter trivirgatus (A. trivirgatus; ID No. Bd6223 and No. Bd-6224), and Spilornis cheela hoya (S. c. hoya; ID No. Bd-498 and No. Bd-475) were collected in the process of specimen preparation from the carcasses of injured birds after futile medical care at the Taiwan Endemic Species Research Institute (TESRI), a governmental wildlife shelter. The causes of death of these specimens were mainly due to collisions. These known genders were identified based on anatomic inspection (postmortem). Male-female paired Circaetus gallicus (C. gallicus; ID No. Bd-cgB4 and No. Bd-cgB2) DNAs were a gift from Dr. Paola Sacchi [5]. Tissue DNAs were extracted by DNeasy tissue kit (Protocol for Animal Tissues; Qiagen, Valencia, CA, USA), according to its instruction manual. In brief, 20 mL proteinase K and 180 mL buffer ATL were added to the 25-mg tissue samples, mixed thoroughly by vortexing, and incubated at 56 8C overnight. Then, 200 mL buffer AL was added to the sample with vortexing, followed by the addition of 200 mL ethanol (96% to 100%) with vortexing. The mixture was pipetted into the spin column with centrifugation (6000  g, 1 min). This column was placed in a new collection tube, washed with 500 mL

2.2. Primary molecular gender identification by P2/P8 primers The universal P2/P8 primer pair [11] for avian molecular sexing was used. The modified PCR reaction mixture, modified PCR program, and gel electrophoresis were performed as described previously [8]. 2.3. TA cloning and nucleotide sequence analysis for CHD-Z and CHD-W genes Except for S. c. hoya [10] and C. gallicus [5], the P2/ P8 PCR products for other Accipitridae species listed in this study were gel purified, cloned, and sequenced as described previously [10]. 2.4. Sequence alignment to calculate the length difference between CHD-Z and CHD-W genes of the same species and compare the sequence similarity of S. c. hoya CHD-ZW-common and CHD-Wspecific TaqMan probes The CHD-Z and CHD-W gene sequences of the species were aligned using Biology Workbench 3.2 (http://workbench.sdsc.edu/). The length difference between the CHD-Z and CHD-W genes amplified by P2/P8 primers for each species was calculated as described previously [8] by subtracting the deleted regions (indicated by dashed lines between P8 and P2 primer sequences in Fig. 1) for CHD-Z and CHD-W genes in their aligned sequences of the same species.

Fig. 1. Sequence alignment of CHD-Z and CHD-W genes amplified by P2/P8 primers in seven species of Accipitridae (A. gularis, A. soloensis, A. trivirgatus, A. virgatus, B. indicus, P. ptilorhyncus, and C. gallicus) compared with S. c. hoya. The sequences start with the P8 primer and end with the P2 primer; CHD-W-specific and CHD-ZW-common probes are indicated by boxes. P2 primer sequences in CHD genes were sometimes not included such as for A. soloensis, A. trivirgatus, B. indicus, P. ptilorhyncus, and S. c. hoya. The star symbols indicate the conserved region between CHD-Z and CHD-W genes of all sequences in tested species. All accession numbers are described in Section 3.

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Fig. 2. Real-time PCR curve for gender identification of eight different species of Accipitridae using TaqMan probes. All the species listed here (A to H) are anatomically confirmed female and male pairs as described in Section 2. ‘‘W’’ and ‘‘ZW’’ indicated the positive signals of TaqMan probes for CHD-Wspecific and CHD-ZW-common regions, respectively. ‘‘ZW’’ alone and ‘‘W/ZW’’ represent the male and female birds, respectively. RFU, relative fluorescence unit.

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2.5. Secondary molecular gender identification using TaqMan probes with P2/P8 primers All experimental conditions for secondary molecular gender identification in this study were the same as described previously [8]. In brief, the sequences of CHDZW-common and CHD-W-specific TaqMan probes for S. c. hoya were as follows: 50 -HEX-CCCTTCACTTCCATTAAAGCTGATCTGG-TAMRA and 50 -FAM-TGTGCCATGTGTGAAAACCACCCA-TAMRA, respectively. These sequences were complementary to those shown in Fig. 1. The TaqMan-based real-time PCR reaction contained 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.7 U Taq enzyme (Invitrogen Inc., Sao Paulo, SP, Brazil), 0.16 mM primers (GenScript Corp., Piscataway, NJ, USA), 100 nM TaqMan probes (Sigma-Proligo Inc., The Woodlands, TX, USA), and 10 to 20 ng DNA. A two-step PCR program was performed as follows: 95 8C for 10 min, 92 8C for 15 sec, and 60 8C for 1 min (total of 50 cycles). 2.6. Gender calling from TaqMan based on real-time PCR The criteria for gender calling of Accipitridae species using S. c. hoya TaqMan probes has been

described previously [8]. In brief, the female was positive for the intensities of both FAM (Relative Fluorescence Unit [RFU] 1 or Allele 1) and HEX (RFU 2 or Allele 2) for CHD-W-specific and CHD-ZWcommon probes, respectively, whereas the male was only positive for the intensities of HEX (Allele 2) for CHD-ZW-common probe (Fig. 2). For auto-calling of the genders (Fig. 3 and Table 1), the information was determined using the ‘‘Allele Discrimination’’ function of the real-time PCR software iQ5 (Bio-Rad Laboratories, Hercules, CA, USA). Accordingly, the female was the heterozygote with Alleles 1 and 2, whereas the male was the homozygote with Allele 2.

3. Results 3.1. P2/P8 primers-based molecular gender identification of eight species of Accipitridae To test the feasibility of the P2/P8 primers in gender identification of eight species as described in Section 2.1, we performed a regular PCR reaction using this primer pair followed by gel electrophoresis. Among these species, CHD-W and CHD-Z gene products were unsolved in 3% agarose gel (Fig. 4).

Fig. 3. High-throughput auto-gender callings of eight different species of Accipitridae. It is simultaneously analyzed by the allele discrimination function of the real-time PCR analysis software (iQ5; Bio-Rad). (A, B) The signals of the CHD-ZW probes were positive to both males and females. The signals of the CHD-W probes were only positive to females. All the gender callings of these tested birds (e.g., Well Nos. A3, A5, B3, B5, C3, C5, D3, D5, E3, E5, G3, G5, F3, F5, B7, and C7) are anatomically confirmed to the same result, and their fluorescence intensities are shown in Table 1. (C) The blank (G8, H8, B9, D9, E9, F9, G9, and H9) indicates the negative control in the absence of template; it represents the fluorescence background of CHD-W and CHD-ZW probes. (D) The region for internal negative control for CHD probes applied to avian gender identification.

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3.2. Cloning and sequencing for P2/P8 PCR products of six species of Accipitridae After cloning as described in Section 2.3, the CHDW and CHD-Z gene sequences (Fig. 1) for all Accipitridae species in this study except for S. c. hoya [10] and C. gallicus [5] were newly submitted to GenBank and are described in Section 3.3. 3.3. Comparison of the length differences for CHD-Z and CHD-W sequences between S. c. hoya and other non–S. c. hoya Accipitridae species using the alignment tool The sequence information of the PCR products of CHD genes for tested Accipitridae species (in Fig. 4) is presented as GenBank accession numbers of CHD-Z/ CHD-W genes, followed by the length difference between P2/P8-amplified PCR products of these two genes: A. gularis [FJ896038/FJ896027] (3 bp), A. soloensis [FJ896036/FJ896037] (6 bp), A. trivirgatus [FJ896034/FJ896035] (2 bp), A. virgatus [FJ896032/ FJ896033] (4 bp), B. indicus [FJ896030/FJ896031] (2 bp), P. ptilorhyncus [FJ896028/FJ896029] (19 bp), S. c. hoya [DQ885238/DQ885237] (13 bp) [10], and C. gallicus [AY313610/AY313609] (9 bp) [5]. Therefore, these short-length differences of P2/P8 PCR amplicons between CHD-Z and CHD-W genes demonstrated why 3% agarose gel was unable to resolve them, as described in Section 3.1. 3.4. Comparison of the sequence similarity for CHD-ZW-common and CHD-W-specific TaqMan probes between S. c. hoya and other non–S. c. hoya Accipitridae species using the alignment tool In these tested Accipitridae species, their homolog regions to both CHD-W-specific and CHD-ZW-common probes reported in S. c. hoya [8] were marked with boxes around them, along with the completely conserved P2/P8 primers in all tested species (Fig. 1). Both C. gallicus and S. c. hoya shared the same sequence in the CHD-Wspecific and CHD-ZW-common regions [8]. It was noteworthy that A. gularis, A. soloensis, A. trivirgatus, A. virgatus, and B. indicus had only one nucleotide difference in the CHD-W-specific region for S. c. hoya, as indicated in Arrow 1 of Fig. 1, whereas P. ptilorhyncus had two nucleotide differences compared with S. c. hoya as indicated in Arrows 1 and 2 of Fig. 1. Except for one nucleotide mismatch at the 50 end of P. ptilorhyncus (Arrow 3 of Fig. 1), the CHD-ZW-common region was completely conserved in these species of Accipitridae.

Fig. 4. Gel view for molecular gender identification of eight species of Accipitridae by PCR amplification of CHD genes using primers P2 and P8. The genus names and their known genders are provided as indicated. The sample names are mentioned in Section 2.1. Electrophoresis was performed with 3% agarose gel and stained with ethidium bromide. M = 100-bp marker; B = blank.

3.5. Validation of the suitability of S. c. hoya CHDW-specific and CHD-ZW-common probes for non–S. c. hoya Accipitridae species: Real-time PCR curve Using the S. c. hoya TaqMan probes [8], all the Accipitridae species listed in Fig. 2A–H show the same performance for the gender identification; that is, ‘‘ZW’’ is positive for male and female, ‘‘W’’ is positive for female only, and the blank controls had very low (indicated with arrows) or undetectable fluorescence intensities for both ‘‘ZW’’ and ‘‘W.’’ 3.6. Validation of S. c. hoya CHD-W-specific and CHD-ZW-common probes for non–S. c. hoya Accipitridae species: Auto-calling of the genders A two-dimensional plot for gender calling is provided in Fig. 3 using the axes of Allele 1 (FAM; CHD-W-specific probe) and Allele 2 (HEX; CHD-ZWcommon probe). Because birds are always CHD-Zpositive, both regions in Fig. 3C and D were confirmed to be the negative controls for the absence and presence of DNA templates, respectively. Under building software of the real-time PCR machine (as described in Section 2.6), all gender information on the tested samples was immediately auto-called in a highthroughput manner (Table 1). All females of eight different Accipitridae species are the type of heterozygotes that have positive signals of both CHD-W (RFU 1) and CHD-ZW (RFU 2) probes; that is, wells A5, B5, C5, D5, E5, G5, F5, and C7 (Table 1 and Fig. 3B). All males had positive RFU 2 signals but negative RFU 1 signals; that is, wells A3,

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B3, C3, D3, E3, G3, F3, and B7 (in Fig. 3A and the call of Allele 2 in Table 1). All blanks had negative RFU 1 and RFU 2 signals. Two independent experiments had the same gender calling for these tested species. 4. Discussion In this study, six pairs of the novel CHD-Z and CHD-W sequences for six Accipitridae species (such as A. gularis, A. soloensis, A. trivirgatus, A. virgatus, B. indicus, and P. ptilorhyncus) were reported and submitted to GenBank. After alignment to S. c. hoya and C. gallicus, their sequences for CHD-Z and CHD-W genes revealed a high similarity, especially at the regions for S. c. hoya CHD-W-specific and CHD-ZWcommon probes. Similarly, Gyps indicus, Gyps bengalensis, Accipiter nisus, Spizaetus nipalensis, Aquila chrysaetos, Circus spilonotus, and Milvus migrans were also conserved in the regions for these S. c. hoya probes, as described previously [8]. All of these 15 species of birds of prey belong to the same order Falconiformes, family Accipitridae, and subfamily Accipitrinae, but to different genera. Based on these findings, we inferred that the identification of the common gender determination probes for many Accipitridae species was possible. This was the first step to prove our proposed hypothesis that the genders of Accipitridae species with the same or similar sequences to our previously proposed S. c. hoya TaqMan probes can be successfully determined. Under the genomic DNA-based experiments in this study, the gender identification of six Accipitridae species using the S. c. hoya probes [8] was demonstrated successfully compared with their known anatomic confirmed genders. Therefore, our proposed hypothesis was validated. We found the nucleotide mismatches to the regions for CHD-W-specific and CHD-ZW-common S. c. hoya probes [8] are as follows: C. gallicus (0 vs. 0, respectively), A. gularis, A. soloensis, A. trivirgatus, A. virgatus, and B. indicus (1 vs. 0), and P. ptilorhyncus (2 vs. 1). It is possible that the corresponding nucleotides ‘‘T’’ and ‘‘G’’ of CHD-W-specific probe (complementary nucleotides ‘‘A’’ [Arrow 1] and ‘‘C’’ [Arrow 2] in Fig. 1) is base-wobbling to ‘‘G’’ and ‘‘T,’’ respectively). As the Accipitridae is a diverse avian family, comprising up to 14 subfamilies, 65 genera, and 231 species [18,19], the suitability of S. c. hoya CHDW-specific and CHD-ZW-common TaqMan probes [8] cannot be used for evaluation of other non-Accipitrinae subfamilies until their CHD genes have been sequenced and submitted to GenBank.

In the Taxbrowser database of the National Center for Biotechnology Information (NCBI; http:// www.ncbi.nlm.nih.gov/Taxonomy), we found on July 26, 2009, that the family Accipitridae (http://www. ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id =56259) contains 63 different genera with 2989 nucleotide records (http://www.ncbi.nlm.nih.gov/ sites/entrez?db=nucleotide&cmd=Search&dopt=Doc Sum&term=txid56259%5BOrganism%3Aexp%5D), but it only contains 36 records related to CHD genes (when the input keywords are ‘‘txid56259[Organism:exp] chromo-helicase-DNA’’). Most of these records are the pair of CHD-Z/CHD-W genes for each species. These records do not currently include the Accession Nos. FJ896027 to FJ896038 reported in this study, and they will soon be released. Accordingly, the CHD-Z/ CHD-W genes have still not been sequenced for most of the Accipitridae species. Once they are sequenced, their suitability for the S. c. hoya probes [8] can be retested. Although the Taxbrowser of NCBI is helpful, it cannot directly provide the sequence similarity to S. c. hoya probes [8] from the search records. In contrast, the BLAST database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) [20] of NCBI is a common tool to search for the homologous sequences to the input such as the sequences for the S. c. hoya probes. It is convenient to find all submitted CHD-Z and CHD-W sequences (including the new submission) related to S. c. hoya probes [8] based on the BLAST analysis. For example, one new record such as Gyps fulvus (EU430640) was found to be a complete match to the nucleotide sequence of the S. c. hoya CHD-W-specific probes [8] in BLAST analysis. Some records for CHD-W and CHD-Z genes, such as Gyps fulvus (EU430640 vs. EU430641) and Accipiter gentilis (AB096143 vs. AB096144), completely matched the S. c. hoya CHD-ZW-common probes [8] in BLAST analysis. Alternatively, the resulting mismatched nucleotide(s) of our designed probes can be modified to completely match the target sequences from other species of interest. Moreover, we found that our proposed S. c. hoya TaqMan probes [8] may not be limited to the application to the family Accipitridae. For example, the sequence record for CHD-W gene of Bartramia longicauda (EU784668), belonging to the family Scolopacidae, is very similar to the sequence of the S. c. hoya CHD-Wspecific TaqMan probe, that is, Identities = 20/21 (95%), TGTGCCA(T/C)GTGTGAAAACCACCCA, where C is the mismatched nucleotide compared with that of S. c. hoya. Bartramia longicauda (EU784668 and EU784667 for CHD-W and CHD-Z genes, respectively)

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was a complete match to the sequence for S. c. hoya CHD-ZW-common TaqMan probe. In conclusion, our proposed CHD-W-specific and CHD-ZW-common probes [8], coupled with the P2/P8 primers [11], provided accurate and high-throughput gender identification for many Accipitridae species. It also has the potential for application to gender identification for other species with similar sequences of S. c. hoya CHD-W-specific and CHD-ZW-common probes. Acknowledgments This work was supported in part by the National Science Council in Taiwan under grants NSC97-2622B-037-002-CC2, NSC96-2622-B-037-003-CC3, and KMU-EM-98-1.4. References [1] Nanda I, Karl E, Volobouev V, Griffin DK, Schartl M, Schmid M. Extensive gross genomic rearrangements between chicken and Old World vultures (Falconiformes: Accipitridae). Cytogenet Genome Res 2006;112:286–95. [2] Lerner HR, Mindell DP. Phylogeny of eagles, Old World vultures, and other Accipitridae based on nuclear and mitochondrial DNA. Mol Phylogenet Evol 2005;37:327–46. [3] CITES Secretariat. Update to Appendices I, II, III. Convention on International Trade in Endangered Species of Wild Fauna and Flora, CITES, 2003. [4] De volo SB, Reynolds RT, Topinka JR, May B, Antolin MF. Population genetics and genotyping for mark-recapture studies of Northern Goshawks (Accipiter gentilis) on the Kaibab plateau, Arizona. J Raptor Res 2005;39:286–95. [5] Sacchi P, Soglia D, Maione S, Meneguz G, Campora M, Rasero R. A non-invasive test for sex identification in short-toed Eagle (Circaetus gallicus). Mol Cell Probes 2004;18:193–6. [6] Reddy A, Prakash V, Shivaji S. A rapid, non-invasive, PCRbased method for identification of sex of the endangered Old

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