# Patent

## Title
Method for gender identification of eagles with probe-based real-time PCR and the sequences used for gender identification of eagles

## Abstract

A method for gender identification of eagles includes: providing a DNA of an eagle; performing a probe-based real-time PCR using the DNA as a template, a universal primer pair P2/P8 as a primer pair and a first probe and a second probe as probes, wherein the 5′ ends of the first probe and the second probe are labeled with a first fluorescent dye and a second fluorescent dye, respectively, and the first probe is a sequence with about 15-38 nucleotides in length of SEQ ID No. 1 and the second probe is a sequence with about 15-44 nucleotides in length of SEQ ID No. 2; and analyzing a result of the PCR, wherein if the result is positive for both the first and the second fluorescent dye, the eagle is a female, and if the result is positive for only the first fluorescent dye, the eagle is a male.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This Application claims priority of Taiwan Patent Application No. 96135735, filed on Sep. 26, 2007, the entirety of which is incorporated by reference herein.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to gender identification of eagles, and in particular relates to gender identification of eagles with probe-based real-time PCR.
2. Description of the Related Art
Monitoring the population sex ratio of eagles is essential to prevent extinction. However, efforts to measure population sex ratios for sexually monomorphic birds, including eagles (Ararajuba 2003;11:65-73), often yield male-biased sex ratios (Proc Biol Sci 2004;271(Suppl 5):S321-3-4, Omis Scand 1987;18:122-8, and Curr Ornithol 1989;6:1-50). Many techniques exist for gender identification of monomorphic birds (Yi Chuan 2005;27:297-301). For instance, the Griffiths procedure, which uses the universal P2/ P8 primers (Mol Ecol 1998;7:1071-5), is a common tool for avian gender identification.
The Griffiths procedure is based on the intron length difference between the chromo-helicase-DNA-binding (CHD)-Z and CHD-W gene amplicons. In general, the gender of birds is identified by the P2/ P8-primed PCR, followed by electrophoresis. The CHD-W gene is unique to females, whereas the CHD-Z gene is found in both sexes (i.e., female, ZW, and male, ZZ). Samples with one band are regarded as males, whereas those with two bands are regarded as females. However, intron lengths between the CHD-Z and CHD-W genes usually vary among species (Mol Ecol 1998;7:1071-5, Auk 1998;115:1074-8, and J Avian Biol 1999;30:116-21.). Additionally, due to the limited length difference of the intron for CHD-Z and CHD-W genes, there is accumulating evidence (J Avian Biol 1999;30:116-21, Mol Cell Probes 2004;18:193-6, J Raptor Res 2005;39:286-95, IBIS 2006;148:167-8 and Curr Sci 2007;92:659-62) that the gender of some avian species cannot be accurately determined by the PCR-based protocol alone. Specifically, the length difference in some eagles is extremely short (approximately 3 to 9-bp). Thus, several solutions have been proposed, such as using re-designed primers of the PCR (J Avian Biol 1999;30:116-21 and BMC Biotechnol 2008;8:12.,13), PCR-restriction fragment of length polymorphism (RFLP) (Mol Cell Probes 2004;18:193-6 and Curr Sci 2007;92:659-62) and random amplified polymorphic DNA (RAPD) fingerprinting (Theriogenology 2007;67:328-33 and Theriogenology 2006;65:1759-68). However, the methods are unable to provide universal primers for high-throughput gender identification for multiple species of eagles.
BRIEF SUMMARY OF THE INVENTION
The invention provides a method for gender identification of eagles with probe-based real-time PCR, comprising: (a) providing a DNA of an eagle; (b) performing a probe-based real-time PCR using the DNA as a template, a universal primer pair P2 and P8 as a primer pair and a first probe and a second probe as probes, wherein the 5′ ends of the first probe and the second probe are labeled with a first fluorescent dye and a second fluorescent dye, respectively, the 3′ ends of the first probe and the second probe are both labeled with quencher dyes and the first fluorescent dye is different from the second fluorescent dye, and wherein a sequence of the first probe is a sequence with about 15-38 nucleotides in length of SEQ ID No. 1 or the complementary sequence thereof and a sequence of the second probe is a sequence with about 15-44 nucleotides in length of SEQ ID No. 2 or the complementary sequence thereof; and (c) analyzing a result of the probe-based real-time PCR, wherein if the result is positive for both the first and the second fluorescent dye, the eagle is a female, and if the result is positive for only the first fluorescent dye, the eagle is a male.
The invention further provides a nucleotide sequence used for gender identification of eagles comprising SEQ ID. No. 3 or the complementary sequence thereof.
The invention also provides a nucleotide sequence used for gender identification of eagles, comprising SEQ ID. No. 4 or the complementary sequence thereof.
A detailed description is given in the following embodiments with reference to the accompanying drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
The present invention can be more fully understood by reading the subsequent detailed description and examples with references made to the accompanying drawings, wherein:
FIGS. 1A-1D shows sequence alignment of the CHD-Z and CHD-W genes amplified by P2/P8 primers in Circaetus gallicus, Gyps indicus, Gyps bengalensis and Spilornis cheela hoya. The sequence for Gvps indicus-CHD-W is indicated by SEQ ID NO:13, the sequence for Gyps bengalensis-CHD-W is indicated by SEQ ID NO:15, the sequence for Spilornis cheela-CHD-W is indicated by SEQ ID NO:9 and the sequence for Circaetus gallicus CHD-W is indicated by SEQ ID NO:11. Moreover, the sequence for Gyps bengalensis-CHD-Z is indicated by SEQ ID NO:14, the sequence for Gyps indicus-CHD-Z is indicated by SEQ ID NO:12, the sequence for Spilornis cheela-CHD-Z is indicated by SEQ ID NO:8 and the sequence for Circaetus gallicus CHD-Z is indicated by SEQ ID NO:10. P8 primer is indicated by SEQ ID NO:6 and P2 primer (antisense) is indicated by SEQ ID NO:26. CHD-W-Specific probe is indicated by SEQ ID NO:27 and CHD-ZW-common probe is indicated by SEQ ID NO:28.
FIGS. 2A-2D shows sequence alignment of the CHD-Z and CHD-W genes amplified by P2/P8 primers in Accipiter nisus, Spizaetus nipalensis, Aquila chrysaetos, Circus spilonotus and Milvus migrans. The sequence for Aquila chrysaetos CHD1W is indicated by SEQ ID NO:20, the sequence for Spizaetus nipalensis CHD1W is indicated by SEQ ID NO:18, the sequence for Circus spilonotus CHD1W is indicated by SEQ ID NO:22, the sequence for Accipiter nisus CHD1W is indicated by SEQ ID NO:16, the sequence for Milvus migrans CHD1W is indicated by SEQ ID NO:24 and the sequence for Spilornis cheela-CHD-W is indicated by SEQ ID NO:9. Moreover, Circus spilonotus CHD1Z is indicated by SEQ ID NO:23, the sequence for the sequence for Accipiter nisus CHD1Z is indicated by SEQ ID NO:17, the sequence for Aquila chrvsaetos CHD1Z is indicated by SEQ ID NO:21, the sequence for Spizaetus nipalensis CHD1Z is indicated by SEQ ID NO:19, the sequence for Milvus migrans CHD1Z is indicated by SEQ ID NO:25 and the sequence for Spilornis cheela-CHD-Z is indicated by SEQ ID NO:8. P8 primer is indicated by SEQ ID NO:6 and P2 primer (antisense) is indicated by SEQ ID NO:26. CHD-W-specific probe is indicated by SEQ ID NO:7 and CHD-ZW-common probe is indicated by SEQ ID NO:28.
FIG. 3 shows a real-time PCR curve for gender identification of Spilornis cheela hoya (S. c. hoya); and
FIG. 4 shows a demonstration of high-throughput gender identification of Spilornis cheela hoya (S. c. hoya) by probes with the sequences of the invention and auto-gender calling by software.



DETAILED DESCRIPTION OF THE INVENTION
The following description is of the best-contemplated mode of carrying out the invention. This description is made for the purpose of illustrating the general principles of the invention and should not be taken in a limiting sense. The scope of the invention is best determined by reference to the appended claims.
The invention provides a method for gender identification of eagles with probe-based real-time PCR. The detailed descriptions and processes are shown in the following.
Probe designing
The eagle species with similar CHD-Z and CHD-W gene sequences are selected. In one embodiment, the eagles may belong to a family of Accipitridae or further belongs to a subfamily of Accipitrinae. For example, the eagle may comprise Aquila chiysaetos, Spizaetus nipalensis, Circus spilonotus, Accipiter nisus, Milvus migrans or Spilornis cheela hoya. 
The CHD-Z and CHD-W sequences of the species selected above are determined by sequencing their P2 SEQ ID NO:5)/P8(SEQ ID NO:6) amplicons, respectively (Mol Cell Probes 2004;18:193-6, Curr Sci 2007;92:659-62, and Zoolog Sci 2003;20:339-44). The sequences of P2/P8 amplicons of CHD-Z and CHD-W of the eagle selected above are based upon sequence alignment. Then SEQ ID. No. 1 or the complementary sequence thereof for the CHD-Z and CHD-W common region, and SEQ ID. No. 2 or the complementary sequence there of for the CHD-W specific region of the eagle selected above are respectively recognized.
SEQ ID. No. 1 is 38 nucleotides in length and SEQ ID. No. 2 is 44 nucleotides in length. A sequence of a first probe for the CHD-ZW common region may be designed in the range of SEQ ID. No. 1 or the complementary sequence thereof, and a sequence of a second probe for the CHD-W specific region may be designed in the range of SEQ ID. No. 2 or the complementary sequence thereof.
In one embodiment, the sequence of the first probe is a sequence with about 15-38 nucleotides in length of SEQ ID No. 1 or the complementary sequence thereof and the sequence of the second probe is a sequence with about 15-44 nucleotides in length of SEQ ID No. 2 or the complementary sequence thereof. In other embodiments, the sequence of the first probe is SEQ ID. No. 3 or the complementary sequence thereof, and the sequence of the second probe is SEQ ID. No. 4 or the complementary sequence thereof. Preferably, the sequence of the first probe may be SEQ ID. No. 3 and the sequence of the second probe may be SEQ ID. No. 4. It is noted that SEQ ID. No. 3 and SEQ ID. No. 4 are a part of SEQ ID. No. 1 and SEQ ID. No. 2, respectively.
The first probe and the second probe may be artificial. Furthermore, the 5′ ends of the first probe and the second probe may be labeled with a first fluorescent dye and a second fluorescent dye, respectively, and the first fluorescent dye may be different from the second fluorescent dye. The 3′ ends of the first probe and the second probe are both labeled with quencher dyes. The first fluorescent dye may comprise 6-carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE) or hexachloro-6-carboxyfluorescein (HEX) and the second fluorescent dye may comprise 6-carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE) or hexachloro-6-carboxyfluorescein (HEX). However, FAM and FITC are not recommended to use at the same time due to the similar emission of color (521 and 519 nm, respectively). The quencher dye may comprise 6-carboxytetramethyl-rhodamine (TAMRA) or dimethylaminoazosulphonic acid (Dabsyl).
DNA Sample of the Eagle
A DNA sample of an eagle used to identify gender is provided. The DNA sample may be extracted from a blood sample or a tissue sample of the eagle.
Probe-based Real Time PCR
A probe-based real time PCR is performed to identify gender of the eagle. The DNA sample mentioned above is used as a template and a universal primer pair P2 (SEQ ID NO:5) and P8 (SEQ ID NO:6) is used as a primer. Moreover, the first probe and the second probe mentioned previously are used as probes. In one embodiment, the sequence first probe is SEQ ID. No. 3and the 5′ end of the first probe is labeled with hexachloro-6-carboxyfluorescein (HEX) and the 3′ end of the first probe is labeled with 6-carboxytetramethyl-rhodamine (TAMRA). In another embodiment, the sequence first probe is SEQ ID. No. 4 and the 5′ end of the second probe is labeled with 6-carboxyfluorescein (FAM) and the 3′ end of the second probe is labeled with 6-carboxytetramethyl-rhodamine (TAMRA). In one embodiment, the eagle may comprise Aquila chrysaetos , Spizaetus nipalensis , Circus spilonotus, Accipiter nisus, Milvus migrans or Spilornis cheela hoya and Spilornis cheela hoya preferably.
After the probe-based real time PCR has been completed, a result of the probe-based real-time PCR is analyzed. If the result is positive for both the first and the second fluorescent dye, the eagle is a female, and if the result is positive for only the first fluorescent dye, the eagle is a male.
EXAMPLE
Identifying Eagles With Similar CHD Sequences and Designing Common Probes for Gender Identification
The CHD-Z and CHD-W gene sequences of Spilornis cheela hoya (S. c. hoya) (DQ885238 (SEQ ID. NO:8), and DQ885237 (SEQ ID. NO:9), respectively) were used to identify other species of eagles with similar sequences by BLAST analysis (Nucleic Acids Res 2004;32:W20-5). The CHD-Z and CHD-W gene sequences of the similar species were compared and aligned using the Biology Workbench 3.2. After alignment inspection, many species listed in the panel of BLAST hits were excluded, because their sequences were too diverse (low score and high E-value in BLAST analysis) to design common probes for gender identification of various species of eagles.
Length Difference Between CHD-Z and CHD-W Genes of the Same Species
The CHD-Z and CHD-W sequences of the species selected above (listed in FIGS. 1A-1D and FIGS. 2A-2D) were determined by sequencing their P2/P8 amplicons (Mol Cell Probes 2004;18:193-6, Curr Sci 2007;92:659-62 and Zoolog Sci 2003;20:339-44). The length difference between the CHD-Z and CHD-W genes amplified by the P2/P8 primers for each species was calculated by subtracting the deleted regions (indicated by the dashed lines between P8 and P2 primer sequences in FIGS. 1A-1D and FIGS. 2A-2D) for the CHD-Z and CHD-W genes in their aligned sequences of the same species. The same criteria were used to calculate the length difference between the CHD-Z and CHD-W genes in other species.
FIGS. 1A-1D shows sequence alignment of the CHD-Z and CHD-W genes amplified by P2/P8 primers in four species of eagles. The sequences start with the P8 primer and end with the P2 primer. The CHD-W-specific and CHD-ZW-common probes are indicated by boxes. The star symbols indicate the conserved region between the CHD-Z and CHD-W genes of all sequences in the tested species. The primer sequences were sometimes not included. The sequence of the S. c. hoya was incomplete and shorter than the others, especially at the end of the P2 primer. All accession numbers of CHD-Z-CHD-W genes are described in the following, such as Circaetus gallicus (C. gallicus) AY313610 (SEQ ID. NO:10)/AY313609 (SEQ ID. NO:11), Gyps indicus (G. indicus) DQ156155 (SEQ ID. NO:12)/DQ156156 (SEQ ID. NO:13), Gyps bengalensis (G. bengalensis) DQ156153 (SEQ ID. NO:14)/DQ156154 (SEQ ID. NO:15), and Spilornis cheela hoya (S. c. hoya) DQ885238 (SEQ ID. NO:8)/DQ885237 SEQ ID. NO:9), respectively. Solid squares after the name of the species indicate the tested species, S. c. hoya. 
FIGS. 2A-2D shows sequence alignment of the CHD-Z and CHD-W genes amplified by the P2/P8 primers in the five species of eagles (A. chrysaetos, S. nipalensis, C. spilonotus, A. nisus, and M. migrans) compared with the S. c. hoya. All symbols are the same as those described in FIGS. 1A-1D. The major difference between the eagles and the S. c. hoya was only one nucleotide, marked with an arrow beside the text of “CHD-W-specific probe”, i.e., G vs. A (A is circled in S. c. hoya). The probe sequences shown in FIGS. 2A-2D are the complementary strand of the sequence described previously. All accession numbers of CHD-W/CHD-Z genes are described in the following, such as Accipiter nisus (A. nisus) AB096151 (SEQ ID. NO:16)/AB096152 (SEQ ID. NO:17), Spizaetus nipalensis (S. nipalensis) AB096149 (SEQ ID. NO:18)/AB096150 (SEQ ID. NO:19), Aquila chrysaetos (A. chrysaetos) AB096147 (SEQ ID. NO:20)/AB096148 (SEQ ID. NO:21), Circus spilonotus (C. Spilonotus) AB096145 (SEQ ID. NO:22)/AB096146 (SEQ ID. NO:23), and Milvus migrans (M. migrans) AB096141 SEQ ID. NO:24)/AB096142 (SEQ ID. NO:25), respectively.
(DQ885238 (SEQ ID. NO:8)/DQ885237 (SEQ ID. NO:9)) were found using BLAST analysis. According to the criteria described above, nine species of eagles (FIGS. 1A-1D and FIGS. 2A-2D) with the highest score and lowest E values for the BLAST analysis were chosen (data not shown). Their sequence information was presented as the GenBank accession nos. of CHD-Z/CHD-W, followed by the length difference between P2/P8-amplified PCR products of the two genes: C. gallicus AY313610 (SEQ ID. NO:10)/AY313609 (SEQ ID. NO:11) (9-bp), G. indicus DQ156155 (SEQ ID. NO:12)/DQ156156 (SEQ ID. NO:13) (6-bp), G. bengalensis DQ156153 (SEQ ID. NO:14)/DQ156154 SEQ ID. NO:15) (5-bp), A. nisus AB096152 (SEQ ID. NO:17)/ AB096151(SEQ ID. NO:16) (4-bp), Spizaetus nipalensis AB096150 (SEQ ID. NO:19)/AB096149 (SEQ ID. NO:18) (8-bp), A. chrysaetos (SEQ ID. NO:21) (SEQ ID. NO:20)4409-64-4-g (3-bp), Circus spilonotus (SEQ ID. NO:23)/AB096145 (SEQ ID. NO:22)(4-bp), Milvus migrans AB096142 (SEQ ID. NO:25)/AB096141 (SEQ ID. NO:24) (2-bp), and S. c. hoya DQ885238 (SEQ ID. NO:8)/DQ885237 (SEQ ID. NO:9) (13-bp). All of the eagles belong to the same order of Falconiformes, family of Accipitridae, and subfamily of Accipitrinae, but to different genera. Therefore, the length difference between the CHD-Z and CHD-W PCR products using the P2/P8 primers ranged from 2-bp to 13-bp for the species (too small to be resolved by a conventional agarose gel).
Given the sequence similarity of the CHD-Z and CHD-W genes in the species of eagles, novel universal probes for gender identification of the birds were able to be designed. The results of the sequence alignment of the CHD-Z and CHD-W genes from the four species (C. gallicus, G. indicus, G. bengalensis vs. S. c. hoya) and the six species (A. nisus, S. nipalensis, A. chrysaetos, C. spilonotus, M migrans vs. S. c. hoya) of eagles are shown FIGS. 1A-1D and FIGS. 2A-2D, respectively). The ends of the alignments were all flanked by the P8 and P2 primers (Mol Ecol 1998;7:1071-5). Both the CHD-W-specific and CHD-ZW-common probes were marked with boxes around them (FIGS. 1A-1D and FIGS. 2A-2D, respectively). The sequences for the probes are the sequences of the complementary strands shown in FIGS. 1A-1D and FIGS. 2A-2D. It is to be noted that C. gallicus, G. indicus, G. bengalensis and S. c. hoya shared the same sequence in the CHD-W-specific region (FIGS. 1A-1D), whereas A. nisus, S. nipalensis, A. chrysaetos, C. spilonotus, and M migrans had only one nucleotide difference (G) in the CHD-W- specific region, as compared to the nucleotide A in the S. c. hoya as well as in the species described in FIGS. 1A-1D (i.e., SEQ ID NO:7, in which the nucleotide at position 17 is G instead of A, FIGS. 2A-2D). In contrast, the CHD-ZW-common region was completely conserved in the nine species of eagles (FIGS. 1A-1D and FIGS. 2A-2D). Consequently, it is determined that the CHD-W-specific and CHD-ZW-common probes combined with the P2/P8 primers were suitable for subsequent tests of real-time PCR-based gender identification.
Samples Collection and DNA Extraction
Thirteen blood samples (Birds 12-24) and two tissue samples (Birds 4966 and 4968) from the S. c. hoya were collected, with the official permission of the Kenting National Park, Taiwan, and Taiwan Endemic Species Research Institute, respectively. Based on anatomical inspection, Bird 4966 and Bird 4968, was identified as male and female, respectively. Blood and tissue DNA was extracted by the QIAamp DNA Blood Mini Kit and DNeasy tissue kit (Qiagen, Valencia, Calif., USA).
Molecular Gender Identification Using Probes of the Invention and P2/P8 Primers
The sequences of probes for the CHD-ZW common and CHD-W-specific regions of the S. c. hoya were 5′-HEX-(SEQ ID NO:3)-TAMRA and, 5′-FAM-(SEQ ID NO:4)-TAMRA, respectively and the sequences were complementary to those shown in FIGS. 1 and 2. The universal primer pair P2 SEQ ID NO:5) and P8 SEQ ID NO:6) were reported previously (Mol Ecol 1998;7:1071-5). The locations of the primers and probes are shown (FIGS. 1 and 2). To perform the probe-based real-time PCR, DNA samples were added to the PCR reaction mixture (10 μL), which contained 1xPCR buffer, 6 mM MgCl2, 0.2 mM dNTPs, 2.5 U Taq enzyme (Invitrogen Inc., Sao Paulo, SP, Brazil), 0.16 μM primers (Gen-Script Corp., Piscataway, NJ, USA), 200 mM probes for the CHD-ZW common or CHD- W-specific regions of the S. c. hoya (Sigma-Proligo Inc., The Woodlands, TX, USA) (sequences as mentioned above), and 10-20 ng DNA. The PCR was performed by the two-step program built into the iQ5 real-time system (Bio-Rad Laboratories, Hercules, Calif., USA), as follows: 95 ° C. for 10 min, 92 ° C. for 15 s, and 60 ° C. for 1 min (total of 50 cycles).
The S. c. hoya, DNA from one male and one female (confirmed by anatomical inspection) were used to delineate the real-time PCR curve by the probes mentioned previously (FIG. 3). FIG. 3 shows a real-time PCR curve for gender identification of eagles. Anatomically confirmed female and male (Bird 4968 and Bird 4655) were used as controls. All tested birds were analyzed in duplicate. W-specific and ZW-common indicated the probes for the CHD-W specific and CHD-ZW-common regions, respectively. And relative fluorescence unit (RFU) indicated relative fluorescence unit. Samples of Bird 4968 and Bird 4655 had a positive fluorescence signal of HEX (indicated by ZW common). Therefore, they were used as the positive control for both the CHD-Z and CHD-W genes, representing the CHD-ZW-common sequence existing in both female and male birds. Conversely, samples of Bird 4968 and Bird 4655 had the presence or absence of FAM fluorescence (indicated by W-specific). Therefore, they served as the positive and negative controls for the CHD-W gene, representing female and male birds, respectively. Moreover, the blank control had very low fluorescence intensities for both FAM and HEX, suggesting that probes of the invention did not detect false positives. Similarly, samples of Birds 12, 14, 15, 17, 19, 20, and 23 were identified as females, whereas other samples listed in this study were ascertained to be males in the real-time PCR curve (data not shown).
In addition to the real-time PCR curve assay (FIG. 3), the data in two dimensions using the axis of allele 1 (HEX-CHD-ZW) and allele 2 (FAM-CHD-W), is shown in FIG. 4. FIG. 4 shows a demonstration of high-throughput gender identification of eagles by mentioned probes and auto-gender calling by software. FIG. 4 is the screen view of a real-time PCR analysis software (iQ5, Bio-rad) after performing the function of “allele discrimination” as indicated by the arrow. All tested birds (n=15) were analyzed in duplicate. Part (A) of FIG. 4 shows the region for internal negative control for the CHD probes. Part (B) of FIG. 4 shows the blank sample for the template-negative control, representing the fluorescence background of the probes (Part (C) and (D)). The CHD-ZW probe was common to both male and female. The CHD-W probe was only specific for females. The anatomically confirmed female (Bird 4968) and male (Bird 4655) controls were included in the test and their fluorescence intensities are shown in Table 1. Allele 1 and heterozygote were regarded as the male and female (as illustrated in Part (A)-(D) of FIG. 4), respectively. Complete data is provided in Table 1. Because birds are always CHD-Z-positive, it is unreasonable to expect to find birds with only the CHD-W gene. Therefore, both regions in FIG. 4 (on the left, Part (A) and (B)) were chosen as the negative controls for the presence and absence of DNA templates, respectively. Samples with the heterozygous alleles representing both CHD-W-positive and CHD-ZW-positive, were predicted to be females (i.e., Birds 12, 14, 15, 17, 19, 20, 23, and 4968). Conversely, samples with allele 1 (CHD-ZW-common probe) alone representing both the CHD-ZW-positive and CHD-W-negative were predicted to be males (i.e., Birds 13, 16, 18, 21, 22, 24, and 4966). Finally, all gender information of the tested samples was immediately displayed in a chart and auto-called in a high-throughput manner (not shown, detailed in Table 1). Moreover, the chart's data set was exported to Microsoft Excel software for statistical analysis (Table 1).







TABLE 1







Summary of gender identification of the S. c. hoya (n = 15) using


probes of the example















RFU1b
RFU2b






(CHD-
(CHD-



Well
ID #a
ZW)
W)
Callc

















A02
Bird
586.77
372.07
Heterozygote




12



A03
Bird
495.69
358.56
Heterozygote




12



A04
Bird
833.72
799.85
Heterozygote




20



A05
Bird
859.60
713.03
Heterozygote




20



B02
Bird
266.27
−18.06
Allele 1




13



B03
Bird
251.43
−20.23
Allele 1




13



B04
Bird
854.44
−17.86
Allele 1




21



B05
Bird
891.52
−10.82
Allele 1




21



C02
Bird
407.40
314.45
Heterozygote




14



C03
Bird
431.69
324.50
Heterozygote




14



C04
Bird
575.08
−17.62
Allele 1




22



C05
Bird
540.09
−15.82
Allele 1




22



D02
Bird
307.87
197.74
Heterozygote




15



D03
Bird
394.99
319.84
Heterozygote




15



D04
Bird
779.47
825.01
Heterozygote




23



D05
Bird
666.31
672.20
Heterozygote




23



E02
Bird
473.88
−25.19
Allele 1




16



E03
Bird
413.67
−17.78
Allele 1




16



E04
Bird
751.58
−9.39
Allele 1




24



E05
Bird
683.89
−11.95
Allele 1




224



E08
Blank
−2.86
−9.52
None



E09
Blank
−5.54
−13.25
None



F02
Bird
357.35
348.55
Heterozygote




17



F03
Bird
520.33
456.77
Heterozygote




17



G02
Bird
381.13
−19.83
Allele 1




18



G03
Bird
390.18
−22.04
Allele 1




18



G04
Bird
268.83
−22.10
Allele 1




4966d



G05
Bird
327.92
−17.89
Allele 1




4966d



G08
Blank
10.16
−18.07
None



G09
Blank
−23.48
−16.48
None



H02
Bird
597.51
319.02
Heterozygote




19



H03
Bird
473.46
398.29
Heterozygote




19



H04
Bird
793.04
657.46
Heterozygote




4966d



H05
Bird
584.12
560.44
Heterozygote




4966d



H08
Blank
−11.18
−15.88
None



H09
Blank
−5.45
−10.83
None







aAll samples were tested in duplicates, e.g., wells A02 and A03 indicate the same sample ID# Bird 12.



bRelative fluorescence unit. RFU1, RFU of allele 1 (CHD-ZW-common probe); RFU2, RFU of allele 2 (CHD-W-specific probe).



cHeterozygote indicated female, i.e., RFU1 (+) and RFU2 (+). None indicated no calling for gender.



dThe female and male controls were confirmed by anatomical inspection.






In Table 1, all heterozygotes had positive signals of both RFU1 and RFU2, i.e., fluorescence for the CHD-ZW and CHD-W probes, respectively. For male birds, all RFU1 signals were positive, whereas all RFU2 were negative. For the blank control, almost all RFU1 and RFU2 signals were negative, except for the well G08. Although the well G08 had a positive RFU1 signal (10.16), the value was very small compared to the values (range, 891.52-251.50 in all female and male samples). Furthermore, no signal was detected in the region of Part (A) of FIG. 4. Therefore, the probes allowed accurate and efficient gender identification of the S. c. hoya in a high-throughput manner.
While the invention has been described by way of example and in terms of the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments. To the contrary, it is intended to cover various modifications and similar arrangements (as would be apparent to those skilled in the art). Therefore, the scope of the appended claims should be accorded the broadest interpretation so as to encompass all such modifications and similar arrangements.

## Claims

1. A method for gender identification of eagles with probe-based real-time PCR, comprising:
(a) providing a DNA of an eagle;
(b) performing a probe-based real-time PCR using the DNA as a template, a universal primer pair P2 and P8 as a primer pair and a first probe and a second probe as probes, wherein the 5′ ends of the first probe and the second probe are labeled with a first fluorescent dye and the second fluorescent dye, respectively, the 3′ ends of the first probe and the second probe are both labeled with quencher dyes and the first fluorescent dye is different from the second fluorescent dye, and wherein a sequence of the first probe is a sequence with about 15-38 nucleotides in length of SEQ ID No. 1 or the complementary sequence thereof and a sequence of the second probe is a sequence with about 15-44 nucleotides in length of SEQ ID No. 2 or the complementary sequence thereof; and
(c) analyzing a result of the probe-based real-time PCR, wherein if the result is positive for both the first and the second fluorescent dye, the eagle is a female, and if the result is positive for only the first fluorescent dye, the eagle is a male.

2. The method as claimed in claim 1, wherein the eagle belongs to a family of Accipitridae.

3. The method as claimed in claim 1, wherein the eagle belongs to a subfamily of Accipitrinae.

4. The method as claimed in claim 1, wherein the eagle comprises Aquila chrysaetos, Spizaetus nipalensis, Circus spilonotus, Accipiter nisus, Milvus migrans or Spilornis cheela hoya.

5. The method as claimed in claim 1, wherein the first fluorescent dye comprises 6-carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE) or hexachloro-6-carboxyfluorescein (HEX).

6. The method as claimed in claim 1, wherein the second fluorescent dye comprises 6-carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC), 2,7-dimethoxy-4, 5-dichloro-6-carboxyfluorescein (JOE) or hexachloro-6-carboxyfluorescein (HEX).

7. The method as claimed in claim 1, wherein the quencher dye comprises 6-carboxytetramethyl-rhodamine (TAMRA) or dimethylaminoazosulphonic acid (Dabsyl).

8. The method as claimed in claim 1, wherein the sequence of the first probe is a sequence with 28 nucleotides in length of SEQ ID No. 1 or the complementary sequence thereof.

9. The method as claimed in claim 1, wherein the sequence of the first probe is a sequence with 24 nucleotides in length of SEQ ID No. 2 or the complementary sequence thereof.

10. The method as claimed in claim 1, wherein the sequence of the first probe is SEQ ID. No. 3 or the complementary sequence thereof.

11. The method as claimed in claim 1, wherein the sequence of the second probe is SEQ ID. No. 4 or the complementary sequence thereof.

12. The method as claimed in claim 1, wherein the sequence of the first probe is SEQ ID. No. 3 and the sequence of the second probe is SEQ No. 4.