TW201031755A - Gene markers and primers for sex identification of crested serpent eagles and crested goshawks - Google Patents

Abstract

The invention provides DNA markers and primer pairs for sex identification of crested serpent eagles and crested goshawks. By using the technology of Random Amplified Polymorphic DNA (RAPD), two DNA markers BB08 and AT15 were found to be effective in gender diagnosis. Based on the two markers, the primer pairs for sex identification of crested serpent eagles, including CseBB08-5aF/R, CseBB08-5aF/CseBB08-5bR, CseBB08-7F/R and CseAT15F/R, are designed and also provided in this invention; wherein the primer pair CseBB08-5aF/CseBB08-5bR are also effective for sex identification of crested goshawks.

Description

201031755 VI. Description of the invention: [Technical field to which the invention belongs] The sex crown 8 and the crested goshawk [Prior Art] In the conservation and reproduction of rare birds, the correct gender-removal efficiency. The determination of the sex of a singlet bird is not easy to be identified by the appearance; the endoscopic examination of the body's heterogeneous gland is judged, but it is easy to be affected by the age of === (4). Therefore, the genetic marker of the bird has been developed. The soil is different in the size of the interposer on chromosomes 2 and 1. Therefore, the synthase chain reaction (PCR) amplification product, the male will produce a fragment, and the female is steeply produced. 2 pieces. Among the commonly used primer groups, only the primer group 1237l/1272h is enough to distinguish the gender of the big crown, and the other primer groups are not. (Wang, L. c., c. τ. . Chen, Η. Υ. Lee, S. Η. Li, JT Lir, SC Chin, CE Pu, and CH • Wang; Sexing a wider range of avian species based on two CHD1 mtrons with a unified reaction condition. Zoo biology 26: 425-431, 2007). Although the primer group 1237L/1272 can be used to determine the gender of the big crown, the Q7Z) 7-F and C//D7-Z are very similar in size, and a very high concentration of agar colloid (for example, 4%) is required. And longer electrophoresis time to be able to separate the fragments slightly, or to distinguish them with high-resolution 10-12% polyacrylamide gel electrophoresis (PAGE)

_ ( Griffiths, R., MC Double, K. Orr, and RJ Dawson; A DNA test to sex most birds. Mol. Ecol. 7: 1071-1075, 1998; Kahn, N. • W., JS John, and TW Quinn.; Chromosome-specific intron size differences in the avian CHD gene provide an efficient method for sex identification in birds. Auk. 115: 1074-1078., 1998), all of which increase the cost of the trial and the time spent.

Chang et al. (Chang, HW, CA Cheng, DL Gu, CC Chang, SH Su, CH Wen, YC Chou, TC Chou, CT Yao, C. 3 201031755 L. Tsai, and CC Cheng, High-throughput avian molecular SMC Biotechnol. 8: 12, 2008) Using PCR-melting curve analysis (PCR/MCA) technique 'according to C//D7- The PF and C//D7-Z sequences were designed into two new sets of large crowns and gender identification primers (CHD-WF/CHD-WR and CHD-ZF/CHD-ZR), which can make the difference with the size of QiDY-Z fragments. Easy to identify. However, this method requires the use of two sets of primers, and the use of instant polymerase chain reaction (real time PCR) machines to complete gender identification.

In view of this, it is necessary to find a marker DNA sequence that has a greater difference in gender-specific fragments and can quickly analyze and more accurately interpret gender, and then develop a primer set that can be used to effectively and quickly identify the sex of the big crown. SUMMARY OF THE INVENTION 'In order to solve the limitations of the prior art, the sex identification of the big crown can be made more rapid' The purpose of the present invention is to provide a gender-specific nucleotide fragment of the Great Crown, using this twin specificity & can effectively identify the leader of the big crown I gender = further, in order to be able to achieve financial efficiency and more rapid green and miscellaneous methods and j 'to mention the artificial breeding efficiency of the South, and then to reach this Taiwan's rare lunar month Conservation and breeding of wild animals. The animal is provided with a nuclear thief that is used to identify the gender specificity of the S. According to the invention of the invention, the proliferation of the DNA dna t=amphfied pdymQrphie DNA, the technology from the big crown 鹫 ii fingerprint ring, found two gender-specific markers nuϊΐ belt t named face 8 and ati5, respectively (10) and One

; SEQID crown gene · Λ ^ ^: 2. And the discovery of the big '4' - the paste (10) to take the lead of the 糸 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ design. In a further aspect of the invention, a method for sex identification of a large crown is provided. In a specific embodiment of the present invention, according to the sex-specific nucleotide fragments BB08 and AT15 of the present invention, a plurality of sets of primers which can be used to identify the sex of the big crown are designed to include 'CseBB08-5aF/R, CseBB08- 5aF/CseBB08-5bR, CseBB08-7F/R and CseAT15F/R, as listed in Table 1: Table 1 Identification of the gender-specific fragment of the big crown 鹫 gender-specific fragment introduction primer sequence (5'-3') CseBB08-5aF/ RF: CTGCAGCTGTTAATCCTCTCTC (SEQ ID NO: 3) R: TTACACTGACGGATTGCGTGGT (SEQ ID NO: 4) BB08 CseBB08-5aF/ CseBB08-5bR F: CTGCAGCTGTTAATCCTCTCTC (SEQ ID NO: 5) R: TTGGTGTTCCAAGTGGTGTTCC (SEQ ID NO: 6) CseBB08- 7F/RF: CTAGACTAATAGGTTGCGGTCC (SEQ ID NO: 7) R: GGTGGCGATATGTATTCACTGC (SEO ID NO: 8) ATI 5 CseAT15F/RF: GTAACGGATACTCTGACCAGGA (SEQ ID NO: 9) R: GTGCACCAGTGTCTACTAGAGC (SEQ ID NO: 10) According to the present invention At the same time, it is found that the introduction group CseBB08_5aF/CseBB08-5bR can also be used to distinguish the gender of the crested goshawk across species, therefore, another aspect of the present invention is to provide an introduction for identifying the sex of the crested goshawk.

CseBB08-5aF/CseBB08-5bR. In a specific embodiment of the present invention, the sex-specific nuclear acid fragment BB08 and the AT15 designed and developed according to the present invention are used to carry out the genome selection of the large crown, and the following sex-specific nucleotide fragments are found: 1) Using the primer set CseBB08-5aF and CseBB08-5aR to increase the genomic DNA of the large crown ' genomic DNAFig. 2, the female big crown 鹫 found a 553 bp sex-specific nucleotide fragment with a nucleotide sequence such as SEQ ID NO: ll (2) The genomic DNA of the large crown scorpion was amplified by the primer group CseBB08-5aF and CseBB08-5bR. The electrophoresis gel map is shown in Fig. 3. The female gentian cockroach found a 895 bp sex-specific nucleotide fragment with a nucleotide sequence such as SEQ IDN0. .12 ; 5 201031755 (3) The primers CseBB08_7F and CseBB08-7R increase the large-crown genomic DNA 'electrophoresis gel map as shown in Figure 4. Female and male big crown mites have found a 543 bp internal control nucleotide fragment with nucleus The nucleotide sequence, such as XSEQ ID NO: 13; and the female Great Crown, found a sex-specific nucleotide fragment of i93 bp, having a nucleotide sequence such as SEQ ID NO: 14; and (4) increasing the size of CseATbF and CseATHR by the primer group. Genomic DNA' Swimming gum FIG 5, the male and female eagles are found in internal control core of a 734bp fragment bitter acids' are having a core acid sequence as SEQ ID N0.15;

And the female Great Crown was found to have a 147 bp sex-specific nucleotide fragment having a nucleotide sequence such as SEQ ID NO: 16. VIII A Any of the above-described sex-specific nucleotide fragments according to the present invention can be used to discriminate the sex of the Great Crown. In still another aspect of the present invention, a primer set for identifying a Crested Goshawk is identified by using the primer set of the present invention for a pCR reaction of a large crown sample to be identified. According to an embodiment of the present invention, an introduction group can be used

CseBB08-5aF/R > CseBB08-5aF/CseBB08-5bR ^ CseBB08-7F/R or CseAT15F/R identifies the sex of the big crown, with an accuracy rate of loo%. According to an embodiment of the present invention, a primer reaction group CseBB08-5aF/R is used to identify a PCR reaction of the large crown scorpion sample to be identified, and the nucleotide sequence fragment having seqid W): ll is a n-specific sex marker. Fragment. According to an embodiment of the present invention, the primer set CseBB08-5aF/CseBB08-5bR ' is used for PCR reaction of the large crown sample to be identified, and the nucleotide sequence fragment having SEQ ID NO: 12 is a female sex marker fragment. According to an embodiment of the present invention, a PCR reaction is performed on the large crown scorpion sample to be identified by using the primer set CseBB08-7F/R, and the nucleoside 歹ij fragment having SEQm NO: 13 is used as an internal control. The fragment, and the nucleotide sequence fragment having seq id NO: 14 is a hi# sex sex marker fragment. According to an embodiment of the present invention, a primer reaction group CseAT15F/R is used to identify a PCR reaction of a large crown sample to be determined, and a nucleotide sequence fragment having a Q IDNO: l5 6 201031755 is an internal control segment, and The nucleotide sequence fragment having SEQ ID NO: 16 is a female sex marker fragment. In still another aspect of the present invention, in order to provide a method for gender identification of the Crested Goshawk, the primers CseBB08-5aF and CseBB08-5bR of the present invention are used to identify a PCR reaction of the Dendrobium sinensis sample. In a specific embodiment of the present invention, the genomic DNA of the Crested Goshawk genomic DNA is amplified by the primer set CseBB〇8_5aF and CseBB08-5bR as shown in Fig. 6. ^ The female Crested Goshawk found a 895 bp sex-specific nucleotide The fragment, having, has a nucleotide sequence such as SEQ ID NO: 17, which can be used to discriminate the sex of the Crested Goshawk. According to an embodiment of the present invention, a method for sex identification of a Crested Goshawk is identified by using a PCR subgroup CseBB08-5aF and CseBB08-5bR 'for a PCR reaction of a Crested Goshawk sample to be identified to have a seq ID NO· · The nucleotide sequence fragment of 17 is a female sex marker fragment. The invention also provides a PCR kit for identifying the sex identification of the Great Crown or the Crested Goshawk. For those of ordinary skill in the art to which the present invention pertains, it is common to combine the primer set of the present invention with the necessary reagents and apparatus for performing the PCR reaction to prepare a PCR kit for commercial use. [Examples] Example 1 DNA extraction and quantification of p-, genomic DNA extraction 1. Genomic DNA extraction of blood samples with reference to Wu β a/. (Wu, CP, Υ·M. Homg, RT Wang, KT Yang, and The method disclosed in MC Huang, A novel sex-specific DNA marker in columbidae birds. Theriogenology 61 328-333, 20〇7) is modified to extract genomic DNA in a blood sample. The fresh blood sample was first centrifuged at 3500 rpm for 15 minutes to separate the plasma, and the blood cell layer was washed twice with physiological saline. Take 20 μΐ of blood cells, add 3 ml of lysis buffer (containing 〇 mM Tris-Cl, 150 mM NaCl, 10 mM EDTA, pH=8.0) to suspend 'add 60 μM proteinase K (proteinase K) (10mg/ml) (Amresco 'United States) and 2〇〇μ1 l〇〇/〇SDS mixed 7 201031755 evenly, at 55 ° C for more than 12 hours. The aqueous layer was then sequentially extracted with an equal volume of phenol, phenol/chloroform and chloroform (chl〇roform) and then added with 0.8 volumes of 100% isopropanol to precipitate the precipitate. The DNA was washed twice with 1 ml of 70% alcohol. After the DNA is dried, add appropriate amount of deionized water (ddH20) and dissolve at 55 °C. After the DNA is re-dissolved, first check the DNA quality and quantitation. If the DNA quality and content are good, store at -80 °C. r 2 · Tissue sample genomic DNA extraction First, take 0.1 g of tissue into a 1.5 ml microcentrifuge tube, add 500 μΐ of lysis buffer, and then cut the tissue with scissors, then add ❿ 100 μΐ 1〇% SDS and 60 μM proteinase K (10 mg/ml) (Amresco 'USA) were uniformly mixed and allowed to act at 55 ° C for more than 12 hours. The aqueous layer was then extracted with equal volumes of phenol, phenol/chloroform and chloroform, followed by 8 times volume of 1% isopropyl alcohol (isopropanol) precipitation. The precipitated DNA was washed twice with 1 ml of 70% alcohol. After the DNA is dried, add appropriate amount of ddH20 and re-dissolve at 55 °C. After the DNA is dissolved, the DNA quality and quantitation are first detected. If the DNA quality and content are good, it is stored at -8 (TC. 3. DNA quantification by spectrometry Photometer (NanoDrop 1000, Thermo Scientific, USA) Detects the absorbance at 260 and 280 nm (〇D value), and judges the purity of the nucleic acid with OD260/280 (generally 〇D26〇/28〇 ratio should be: [8-2.0 The DNA concentration is estimated by the OD260 absorbance value. After the DNA is determined, the DNA sample is adjusted to ddHsO to adjust the DNA concentration to 10 and 50 ng & and stored at -20 ° C for molecular identification. Selection of sex-specific nucleotide fragments of two crowns - RAPD fingerprint identification This experiment uses the genomic DNA of the big crown and the scorpion scorpion as a template, and the used primer sequence from RAPD 1〇mers kits (Operon)

Biotechnologies, Germany) designed 6 sets of primers (〇pBA, 8 201031755 OPBB, OPBC, OPAN, OPAT and OPAZ) 'Total 120 random primers, the sequence of the primers used is shown in Table 2. .

Table 2

Primer number · Primer sequence (5'-3') Primer number primer sequence (5'-3') Primer number primer sequence Θ-30 OPBA-01 TTCCCCACCC OPBB-01 ACACTGGCTG OPBC-01 CCTTCGGCTC OPBA-02. TGCTCGGCTC OPBB-02 CGGCGAGAAC OPBB-03 TCACGTGGCT OPBC-03 GGCTTGACCT OPBA-04 TCCTAGGCTC OPBB-04 ACCAGGTCAC OPBC-04 CCACGTGCCA OPBA-05 TGCGTTCCAC OPBB-05 GGGCCGAACA OPBC-05 GAGGCGATTG OPBA-06 GGACGACCGT OPBB-06 CTGAAGCTGG OPBC-06 GAAGGCGAGA OPBA-〇7 GGGTCGCATC OPBB-07 GAAGGCTGGG OPBC-07 TGTGCCTGAC OPBA-08 CCACAGCCGA OPBB-08 TCGTCGAAGG OPBC-08 GGTCTTCCCT OPBA-09 GGAACTCCAC OPBB-09 AGGCCGGTCA OPBC-09 GTCATGCGAC OPBA-10 GGACGTTGAG OPBB-10 ACTTGCCTGG OPBC -10 AACGTCGAGG OPBA-11 CCACCTTCAG OPBB-11 TGCGGGTTCC OPBC-11 TTTTGCCCCC OPBA-12. TGTTGGGCAC OPBB-12 TTCGGCCGAC OPBC-12 CCTCCACCAG OPBA-13 AGGGCGAATG OPBB-13 CTTCGGTGTG OPBC-13 CCTGGCACAG OPBA-14, TCGGGAGTGG OPBB-14 GTGGGACCTG OPBC -14 GGTCCGACGA OPBA-15 GAAGACCTGG OPBB-15 AAGTGCCCTG OPBC-15 CCAGACTCCA OPBA-16 CC ACGCATCA OPBB-16 TCGGCACCGT OPBC-16 CTGGTGCTCA OPBA-17 TGTACCCCTG OPBB-17 ACACCGTGCC OPBC-17 CCGTTAGTCC OPBA-18 CTCGGATGTC OPBB-18 CAACCGGTCT OPBC-18 GTGAAGGAGG OPBA-19 CCATCCGTTG OPBB-19 TTGCGGACAG OPBC-19 ACAAGCGCGA OPBA-20 GAGCGCTACC OPBB -20 CCAGGTGTAG OPBC-20 AGCACTGGGG OPAN-01 ACTCCACGTC OPAT-01 CAGTGGTTCC OPAZ-01 TCGGATCCGT OPAN-02 CACCGCAGTT OPAT-02 CAGGTCTAGG OPAZ-02 CCTGAACGGA OPAN-03 AGCCAGGCTG OPAT-03 GACTGGGAGG OPAZ-03 GGCTGTGTGG OPAN-04 GGCGTAAGTC OPAT-04 TTGCCTCGCC OPAZ-04 CCAGCCTCAG OPAN-05 GGGTGCAGTT OPAT-05 ACACCTGCCA OPAZ-05 TCCGCATACC OPAN-06 GGGAACCCGT OPAT-06 CCGTCCCTGA OPAZ-06 CCTTCGGAGG OPAN-07 TCGCTGCGGA OPAT-07 ACTGCGACCA OPAZ-07 CACGAGTCTC OPAN-08 AAGGCTGCTG OPAT-08 TCCTCGTGGG OPAZ -08 TCGCTCGTAG OPAN-09 GGGGGAGATG OPAT-09 CCGTTAGCGT OPAZ-09 CCTTGACCCC OPAN-l〇CTGTGTGCTC OPAT-10 ACCTCCGGTC OPAZ-10 ACTCTGGGGA OPAN-11 GTCCATGCAG OPAT-11 CCAGATCTCC OPAZ-11 TCCAGCGCGT OPAN-12. AACGGCGGTC OPAT-12 CTGCCTAGCC OPAZ -12 GATGGGCCTG OPAN-13 CTTCCAGGAC OP AT-13 CTGGTGGAAG OPAZ-13 CCCGAAGCAA OPAN-14 AGCCGGGTAA OPAT-14 GTGCCGCACT OPAZ-14 CACGGCTTCC OPAN-15 TGATGCCGCT OPAT-15 TGACGCACGG OPAZ-15 TCCGCTAGTC OPAN-16 GTGTCGAGTC OPAT-16 CTCTCCGTAG OPAZ-16 AGGCGAACTG OPAN-17 TCAGCACAGG OPAT- 17 AGCGACTGCT OPAZ-17 CACGCAGATG OPAN-18 TGTCCTGCGT OPAT-18 CCAGCTGTGA OPAZ-18 CCGACGTTGA OPAN-19 ACCACGCCTT OPAT-19 ACCAAGGCAC OPAZ-19 ACACTCTCGG OPAN-20 GAGTCCTCAC OPAT-20 ACATCAGCCC ΟΡΆΖ-20 CATCACCCCT 201031755 The total PCR reaction volume is 20μ Contains 50 ng of DNA, 0.4 μM nucleic acid primer and 10 μΐ 2X Taq DNA Polymerase Master Mix RED (Ampliqon, Denmark). The reaction conditions were as follows: heating at 95 ° C for 10 minutes, heating at 95 ° C for 1 minute, bonding at 36 ° C for 1 minute, 72 ° C for 2 minutes, 45 cycles, and finally stretching at 72 ° C for 10 minutes. After the reaction, 10 μl of the PCR product was electrophoresed on a 2% agar colloid, and stained with EtBr staining solution for 10 minutes. After 20 minutes of decoloration, it was placed on a UV light box and photographed by an image processing system.

The total volume of the PCR reaction was 20 μg containing 50 ng of DNA, 0.4 μΜ of nucleotide primer and 10 μΐ of 2Χ Taq DNA Polymerase Master Mix RED • (Ampliqon, Denmark). The reaction conditions were heating at 95 ° C for 10 minutes, heating at 95 ° C for 1 minute, bonding at 36 ° C for 1 minute, 72 ° C for 2 minutes, 45 cycles, and finally extending at 72 ° C for 10 minutes. After the reaction, 1 〇μι PCR product was electrophoresed on 2% agar colloid, and stained with EtBr dyeing solution for 1 , minutes, and after 20 minutes of decoloration, it was placed on a UV light box and observed with an image processing system. Archive. 2. Recovery of sex-specific nucleotide fragments The agar colloid containing the PCR product of the sex-specific fragment was excised, and the DNA was recovered and constructed on pGEM-T easy vector (pGEM-T Easy φ Vector system, Promega, Madison, In the United States, the HIT DH5a competent cells (HITTM Competent Cells, United Bioinformatica, Calgary, Canada) were transformed. A single white strain was selected on LB solid medium: (Bi〇n〇vas 'US) and confirmed by c〇iony_pCR; the length of the selected PCR product was correct. The bacterial solution with the recombinant plastid will be confirmed to complete the nucleotide sequencing work. The sequenced nucleic acid sequence is based on the National Center for Biotechnology Information (NCBI) BLAST (Basic Local Alignment Search Tool) online program (http://www.ncbi.nlm.nih.gov/BLAST). The appropriate genomic database (genomic databases of species selection chicken Gallus gallus) and nucleotide genomic databases were used to perform 201031755 similar gene sequence alignment of different species. From the polymorphic DNA fingerprinting band of Daguan S, the result of face test of OP臓 is shown in Fig. 1(A), and the labeled nuclear thief fragment is named _8, with rib bp, with nuclear sequence As SEQ ID ΝΟ: 1. The results obtained by the tempo-introduction group 〇pAT15 are shown in Fig. 1(B), and the sex-specific labeled nuclear thief fragment named AT15' has a 1388 bp nucleotide sequence such as SEqIDN〇:2. Using the NCBI negative library (animals (5) / (10) fine / (10) genomic database and nucleotides as a library) to compare with BLAST, did not find complete with bb〇8 或 or ATI 5 sex-specific fragments A sequence of high similarity, showing it as a newly discovered sequence. Example 2 Design of the primer set for sex identification of the big crown After the sequencing of the selected DNA fragment, the appropriate primer was designed with the primer Designer software. According to the sex-specific nucleotide fragment BB〇8, the following three groups of primers were obtained:

CseBB08-5aF/R CseBB08-5aF/ CseBB08-5bR CseBB08-7F/RF: CTGCAGCTGTTAATCCTCTCTC (SEQ ID NO: 3) R: TTACACTGACGGATTGCGTGGT (SEQ ID NO: 4) F: CTGCAGCTGTTAATCCTCTCTC (SEQ ID NO: 5) R: TTGGTGTTCCAAGTGGTGTTCC ( SEQ ID NO: 6) F: CTAGACTAATAGGTTGCGGTCC (SEQ ID NO: 7) R: GGTGGCGATATGTATTCACTGC (SEQ ID NO: 8) According to the sex-specific nucleotide segment AT15, the primer set:

CseAT15F/R F: GTAACGGATACTCTGACCAGGA (SEQ ID NO: 9) R: GTGCACCAGTGTCTACTAGAGC (SEQ ID NO: 10) The PCR reaction was carried out using the above primer set. The primer sequence, bonding temperature, and number of cycles are as wrong! Reference source not found. Table 3 Primer sequence designed by sex identification and PCR conditions Species gender-specific primer name primer sequence (5'—adhesion cycle temperature number 201031755 fragment (°C) (times) Daguanfan BB08 CseBB08-5aF/ RF: CTGCAGCTGTTAATCCTCTCTC R: TTACACTGACGGATTGCGTGGT 67 32 CseBB08-5aF/ CseBB08-5bR F: CTGCAGCTGTTAATCCTCTCTC R: TTGGTGTTCCAAGTGGTGTTCC 62 30 CseBB08-7F/RF: CTAGACTAATAGGTTGCGGTCC R: GGTGGCGATATGTATTCACTGC 66 35 ATI 5 CseAT15F/RF: GTAACGGATACTCTGACCAGGA R: GTGCACCAGTGTCTACTAGAGC 64 30 Crested Goshawk CseBB08-5aF/ Cs6BB08 ~5bR F: CTGCAGCTGTTAATCCTCTCTC R: TTGGTGTTCCAAGTGGTGTTCC 63 30 The total volume of PCR reaction is 10μ1, containing l〇ng genomic DNA, 0.3 μΜ each of ❿ primer and 2X Taq DNA Polymerase Master Mix RED (Ampliqon). PCR reaction with thermal cycle reactor (T-Gradient tiiermocycler, Biometra's Germany), the reaction conditions were 95 ° C heating for 5 minutes, 95 ° C heating for 45 seconds, 61-67 ° C bonding for 30 seconds, 72 ° C extended 1 minute 'clock, for 30- 35 cycles, and finally extended at 72 ° C for 10 minutes. .5% agar colloidal electrophoresis analysis, and then stained with EtBr staining solution for 10 minutes' after 20 minutes of de-staining, placed on a UV light box and photographed with an image processing system to identify the sex. φ Example 3 Poultry sex Identification 1. Test animal DNA samples, blood samples or tissue samples were taken from Taipei City. The zoo (TaiPei z〇〇) has 12 large crowns and 3 crested goshawks, and - the special biological research and nine conservation center ( The Endemic Species Research Institute has 29 large crowns and 5 crested goshawks. Among them, the big crown 鹫 2 〇 only uses endoscopy to detect sex, while the big crown 鹫 i and the phoenix goshawk 2 are by other methods (such as The autopsy or the sensation of the egg has been deceived. In the case of internal vision, the sex of the big crown 鹫 ' 丨 4 4 4 4 4 4 已知 已知 已知 已知 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 9 9 9 。 9 Second, PCR sex identification 12 201031755 Taking the blood of the test animal as a sample, genomic DNA extraction, using the genomic DNA of the yellow carp, the crested goshawk and the grasshopper as a template, using the primer set and method obtained in the second example to carry out the temperature gradient (gradient) The PCRt>pcR reaction has a total volume of 10 μg containing 10 ng of genomic DNA, primers of each 〇.3 μΜ and 2X Taq DNA Polymerase Master Mix RED (Ampliqon, Denmark). The PCR reaction was carried out in a thermal cycle reactor (T-Gradient thermocycler, Biome A, Germany) under the reaction conditions of 95 C for 5 minutes, 95 ° C for 45 seconds, and 61, -63, 65 and 67 °C temperature gradient bonding. 30 seconds, 72 ° C extended for 1 minute, 30-35 cycles, and finally 72t: extended for 1 minute. The product obtained by the reaction was subjected to 1.5% agar colloidal electrophoresis analysis, and then stained with EtBr staining solution for 10 minutes, and after 20 minutes of defection, it was placed on a UV light box and photographed by an image processing system. (1) Sex identification of the big crown ··· CseBB08-5aF/R The genomic DNA analysis of the genomic DNA of the large scorpion genomic DNA of the CseBB08_5aF and CseBB08-5aR primers was performed as shown in Fig. 2;鹫 does not amplify any fragment, and the female cockroach can amplify a 553 bp fragment with a nucleotide sequence such as SEQ IDN〇:u, which is a female sex marker fragment of the big crown, and the sex identification accuracy is 100%. (41/41).

(2) Gender identification of the big crown: CseBB〇8_5aF and CseBB〇8_5bR 鹫 The genomic DNA of the large crown was amplified by the CseBB08_5aF and CseBB08-5bR primers. The electrophoresis analysis results of the PCR products are shown in Fig. 3, and the male crown was not expanded. Any fragment is obtained, and the female large crown can be amplified into a 895 bp fragment having a nucleotide sequence such as SEQ ID NO: 12, which is a female sex marker fragment of the large crown. -- The accuracy of gender identification is also 100% (41/41).

(3) Gender identification of the big crown: CseBB08-7F and CseBB08-7R = cseBB08_7F and CseBB08_7R introduced the genome of the large crown, the electrophoresis analysis results of PCR products are shown in Figure 4, the female crown can be amplified, 193 and Two fragments of 543 bp, while the male cockroach cockroach amplified 543 bp tablets. The 543 bp fragment which appears in both female and male large crowns, and has a nucleotide sequence such as SEQ ID NO: 13, which can be used as an intrinsic control fragment; only / out 13 201031755 is now 193 bp in female large crown A fragment having a nucleotide sequence such as SEQ ID N: 14 can be used as a female sex marker fragment of the Great Crown. The accuracy of gender identification was 100% (41 Ml only). (4) Sex identification of the big crown :: CseAT15F/R The CseAT 15F and CseAT 15R primers were used to increase the amplitude of the genomic PCR product. The results of electrophoresis analysis are shown in Figure 5. The female crown can be amplified by 147 and 734 bp. Fragments, while the male cockroach cockroach amplifies the I" bp fragment, which is a 147 bp fragment that appears in both female and male large crowns, with a nucleotide sequence such as SEQ ID NO: 15 'as an intrinsic control fragment; The 734 3卩 fragment that appears in the female large crown has a nucleotide sequence such as 〇〇)): 16,

It can be used as a female sex marker fragment of the Great Crown. The accuracy of gender identification was 1〇〇〇/〇 (41/41). (5) Sex identification of the Crested Goshawk The genomic DNA was extracted from the blood of the Crested Goshawk, and the PCR reaction was carried out with the introduction group CseBB08-7F/R. The reaction condition was 95. (: heating for 5 minutes' 95 C heating for 45 seconds, 63 °C for 30 seconds, 72 °C for 1 minute, for 2 30 cycles, and finally for 72t: for 10 minutes. The reaction product was subjected to 1.5% agar colloidal electrophoresis analysis. As shown in Fig. 6, the male eagle eagle does not amplify any fragments, while the female eagle eagle can amplify a 895 bp fragment, and the nucleus sequence is SEQ ID N〇: 16, which can be used as a phoenix. The female sex score of the goshawk is 1. The accuracy rate of gender identification is 1〇〇% (8/8). [Simple description of the figure] Again = the nuclear thief fragment of the big crown s sex and sex, Figure 1 (A) for the big crown stunned, and the DNA was obtained by RAPD test on the 〇pbb〇8 of the machine introduction group, the representative nucleotide fragment was 1241 bp; the round 1 (8) was the big crown 鹫 gene ^, 2, and the CseB was 5aF. And (10) Na Mi's introduction group increased the Daguan acid tablets and paragraph ^3 swimming, its (four) sexual rank nuclear ί 14 201031755 Figure 3 is the CseBB08-5aF and CseBB08-5bR bow scorpion group increase the large crown stimuli genomic DNA The electrophoresis gel map, in which the female large crown has a gender-specific nucleotide fragment of 895 bp. Figure 4 shows the increase in the CseBB08-7F and CseBB08-7R primers. The electrophoresis gel of the genomic DNA of the genomic DNA of the female genus is 193 bp with a gender-specific fragment and contains 543 bp of the intrinsic control fragment.

Figure 5 shows the electrophoresis map of the genomic DNA of the large crown carp genomic DNA with the CseAT 15F and CseAT 15R primers. The female large crown has a sex-specific nucleotide fragment of 734 bp and contains an intrinsic control fragment of 147 bp. Figure 6 is an electrophoresis gel of genomic DNA of Crested Goshawk eagle with CseBB08-5aF and CseBB08-5bR primers. The female Crested eagle has a sex-specific nucleotide fragment of 895 bp. Eight ‘, 【Main component symbol description】 None

15 201031755 Sequence Listing <110> National Taiwan University; Taipei City Zoo <120> Identification of DNA markers and primers for the gender of the Great Crown and the Crested Goshawk <140><141><160>19

<210>1 <211>1241 <212>DNA <213>Great Crown <400> 1

TCGTCGAAGG

GGCATACAGC

ATCAAAAGCA

GCAGGCCAAT

TCAATACAAA

AGTATTTACA

GTACAAGACT

AGGAGTGTCG

CAGCTAATAT

ACAGATTGGA

ATGTTGGGTT

ATAAGGCACA

TCTGATATCA

TACGTCATTC

GTTGCGGTCC

GTAGTTAATA

CATGTTTTGT

GAATATTCCA

CAATGTTGTA

CATCCCATTC

TGTGGAATAT

AGTGGAACAC

GTGAGGAGGT

TATGTTGATT

GTATCCACAG

AGGAAATAGT

CGTTACCTAC

CTTAGCATAG

GAAACACATT

AGGCAGATGA

TTTTGATCTC

ACAAAGCACG

TCTCCTTCCA

CACTCTGGGT

GAGCTATGGC

CGCGATACCA

GGTATTTGTC

ACAGAGGATG

TCAGGGGTTA

CACCACGCAA

TTTTGGGAGA

ACTGGAGGTG

GCTGTTTAAC

AAGTTTTAGT

ATTCTTTCAA

CCAATCTATC

CACTTGGAAC

CCGTATTGAT

AGCATACTTG

CAGTGAATAC

CCACCGGCAA

TCAGAAAGAA

TATAATTCTG

GTCAAAAGAT

GTTAGTTAAA

ATGGAACTGT

ATGAGCCAAC

ATAATACATA

TTGATAACCT

TTGTGCTTTT

ATAACACCAT

AAAGATACCT

TAAAATAAGA

TTATATGAAC

TCCGTCAGTG

AATACCATCT

TTGTAATATG

AAAAAAACTG

ACTGGTTAGC

TC7VAGCCATT

CCTAGCCCTT

ACCAAGTGTT

GAATGAGTTG

GTGGGTTTAG

ATATCGCCAC

TTGATTTTGT

TAGCAATGAG

TAATACAGCT

CTGCAGCTGT

GTACAGGCTA

ATCCAGGGCA

CTGTGATAGG

TGCCATTGCT

GATGTGGGGT

TCAGACTGAA

TCCAATGCTA

GAAATACAAG

GAAGTTGGGG

CTTCATGCCT

TAAACAATTC

GGAAAAATTT

ATCATAGGGT

CTTTAGTAAG

TTTTTTAATT

AGCTTGGGGA

GAGCCCAATC

CCTTGGTCAC

TTCTAGTGCT

CAAAAAACAG

CCCTTCGACG

AATGCTCCTT

GAACATTAAT

ACTGGGGTTG

TAATCCTCTC

ACCAAATCTA

TGTGCCTGTA

AGAGGAAAAC

CCAGCTCCTT

AATTGCCCAC

CTTCAATTTG

TCTCCTCTAG

AACTCGAGAA

TACAGAACCA

AGACTAATAG

CATTTCCCCT

CAATCCTCAC

GTTTGATTAT

ATGTGAACTC

GTTCCTTCAA

TGGTAGGCAT

AAGGGCTCCA

TTTGTATTTG

TCAATTGTGT

TCCTGACACT

A 50 100 150 200 250 300 350 400 450 500 550 600 651 700 750 800 850 900 950 1000 1050 1100 1150 1200 1241 <210>2 <211>1388 <212>DNA <213> Great Crown < 400>2 1 201031755

TGACGCACGG GTACGGGAAT TGCGAGAAAA AGCAATCGGA AAAGACGATT 50 CTTCTTGGAA AAATGCCGCT CCAGTTTCCC GTGAGCAGTC CCCCAGACGC 100 AGTAGAAGGG CTGATCTCAT TTCTGATCCT CTTGAAGGGA CTTGTGATTC 150 ACGTTTGCGA AAAGTGAGTA ACGGATACTC TGACCAGGAT TAGAGGGGCC 200 CTGCCTCCAG CCAGGTGGAG GAAAGGGACA ACCGGGTCTA CTGGACAGTG 250 TGGATTCGAT GGCCTGGCAC ATCACACTGT CCTGGTTTCA GCTGGGATAG 300 AGTTAATTGT CTTCCTAGTA GCTGGTATAG TGCTATGTTT TGAGTTCAGT 350 ATGAGAAGAA TGTTGATAAC ACTGATGTTT TCAGTTGTTG CTAAGGAATG 400 TTTAGTCTAA AGTCAAGGAT TTTTCAGCTT CTCATGCCCA GCCAGCAAGA 450 AAGCTGGAGG GGCACAAGAA GTTGGCACAG GACAGAGCCA GGGCAGCAGA 500 CCCAAAGAGG CCAACGGGGT ATTCCATACC ATGTGATGTC ATGTCTTGTA 550 TATACACTGG GGGGAGTGGG GGTGGGGGGA TTGCCGCTCA GGGACTAACT 600 GGACATCGGT CGGCGGGTAG TGAGCAATTG CATTGTGCAT CACTTGCATA 650 TTCCAATCCT TTTATTATTA CTGTTGTCAT TTTATTAGTG TTACCATTAT 700 CATTATTAGT TTCTTCCTTT CTGTACTGTT AAACCGTTCC TATCTCAACC 750 CACGAGTTTT ACTTCTTTTC CCGATTCTCT CCCCCATCCC ACTGGGTGGC 800 GGGGAAGTGA GTGAGCAGCT GCGAGGTGCT TAGTTGCTGG CTGGGGTTAA 85 0 ACCATGACAC AGACCCACAG GAATATAAGG CTCTAGTAGA CACTGGTGCA 900 CAATGTACCC TAATGCCATC AAGTTATAAA GGGGCAGAAC CCATCTGTAT 950 CTCTGGTGTG ACAGGGGGAT CCCAAGAGCT AACTGTATTG GAAGCTGAAG 1000 TGAGTCTAAC TGGGAATGAG TGGCAGAAAC ACCCCATTGT GACTGGCCCA 1050 GAGGCCCCAT GCATCCTTGG CATAGACTAC CTCAGGAGAG GGTATTTTAA 1100 GGACCCAAAG GGGTACTGTT GGGCCTTTGG TATAGCTGCA TTGGAGACGG 1150 AGGAGATTGA ACAGCTGTCC GCCTTGCCTG GCCTCTCTGA GGACCCTTCA 1200 GTTGTGGGGT TGCTGAGGGT TGAAGAACAA CAGGTGCCAA TTGCTACCAC 1250 AATGGTGCAC TGGTGGCAAT ATCGCACCAA CTGAGACTCC CTGATTCCCA 1300 TCCACAAGTT GATTCGCCAA CTGGAGAGCC AAGGAGTGAT CAGCAAGACT 1350 CACTCACCCT TTAATAGTCT CATATGGCCC GTGCGTCA 1388 <210>3 <211>22 <212>DNA <213> Synthetic <400> 3 22 5, CTGCAGCTGT TAATCCTCTC TC 3, <210>4 <211>22 <212> DNA <213> Synthetic <400>4 5, TTACACTGAC GGATTGCGTG GT 3' 22

<210 5 <211>22 <212>DNA 2 201031755 <213> Synthetic <400> 5 55 CTGCAGCTGT TAATCCTCTC TC 35 22 <210 6 <211>22 <212>DNA <213>; Synthetic <400> 6 5, TTGGTGTTCC AAGTGGTGTT CC 3' 22

<210>7 <211>22 <212>DNA<213> Synthetic <400> 7 5' CTAGACTAAT AGGTTGCGGT CC 3' 22 <210>8 <211>22 <212>DNA <;213> Synthetic <400>8 5,: GGTGGCGATA TGTATTCACT GC 3, 22

<210>9 <211>22 <212>DNA<213> Synthetic <400> 9 5, GTAACGGATA CTCTGACCAG GA 3, 22 <210> 10 <211>22 <212>DNA <;213>Synthesis<400> 10 5, GTGCACCAGT GTCTACTAGA GC 3' 22 3 201031755 <21011 <211>553 <212>DNA <213>

<400>5

CTGCAGCTGT

GTACAGGCTA

ATCCAGGGCA

CTGTGATAGG

TGCCATTGCT

GATGTGGGGT

TCAGACTGAA

TCCAATGCTA

GAAATACAAG

GAAGTTGGGG

CTTCATGCCT

TAA

TAATCCTCTC

ACCAAATCTA

TGTGCCTGTA

AGAGGAAAAC

CCAGCTCCTT

AATTGCCCAC

CTTCAATTTG

TCTCCTCTAG

AACTCGAGAA

TACAGAACCA

AGACTAATAG

TCAATACAAA

AGTATTTACA

GTACAAGACT

AGGAGTGTCG

CAGCTAATAT

ACAGATTGGA

ATGTTGGGTT

ATAAGGCACA

TCTGATATCA

TACGTCATTC

GTTGCGGTCC

AGGCAGATGA

TTTTGATCTC

ACAAAGCACG

TCTCCTTCCA

CACTCTGGGT

GAGCTATGGC

CGCGATACCA

GGTATTTGTC

ACAGAGGATG

TCAGGGGTTA

CACCACGCAA

GTTAGTTAAA

ATGGAACTGT

ATGAGCCAAC

ATAATACATA

TTGATAACCT

TTGTGCTTTT

ATAACACCAT

AAAGATACCT

TAAAATAAGA

TTATATGAAC

TCCGTCAGTG 50 100 150 200 250 300 350 400 450 500 550 553 <210> 12 <211>895 <212>DNA <213> Great Crown

<400>12

CTGCAGCTGT

GTACAGGCTA

ATCCAGGGCA

CTGTGATAGG

TGCCATTGCT

GATGTGGGGT

TCAGACTGAA

TCCAATGCTA

GAAATACAAG

GAAGTTGGGG

CTTCATGCCT

TAAACAATTC

GGAAAAATTT

ATCATAGGGT

CTTTAGTAAG ΤΤΤΤΤΤΆΑΤΤ

AGCTTGGGGA

GAGCCCAATC

TAATCCTCTC

ACCAAATCTA

TGTGCCTGTA

AGAGGAAAAC

CCAGCTCCTT

AATTGCCCAC

CTTCAATTTG

TCTCCTCTAG

AACTCGAGAA

TACAGAACCA

AGACTAATAG

CATTTCCCCT

CAATCCTCAC

GTTTGATTAT

ATGTGAACTC

GTTCCTTCAA

TGGTAGGCAT

AAGGGCTCCA

TCAATACAAA

AGTATTTACA

GTACAAGACT

AGGAGTGTCG

CAGCTAATAT

ACAGATTGGA

ATGTTGGGTT

ATAAGGCACA

TCTGATATCA

TACGTCATTC

GTTGCGGTCC

GTAGTTAATA

CATGTTTTGT

GAATATTCCA

CAATGTTGTA

CATCCCATTC

TGTGGAATAT

AGTGGAACAC

AGGCAGATGA

TTTTGATCTC

ACAAAGCACG

TCTCCTTCCA

CACTCTGGGT

GAGCTATGGC

CGCGATACCA

GGTATTTGTC

ACAGAGGATG

TCAGGGGTTA

CACCACGCAA

TTTTGGGAGA

ACTGGAGGTG

GCTGTTTAAC

AAGTTTTAGT

ATTCTTTCAA

CCAATCTATC

CACTTGGAAC

GTTAGTTAAA

ATGGAACTGT

ATGAGCCAAC

ATAATACATA

TTGATAACCT

TTGTGCTTTT

ATAACACCAT

AAAGATACCT

TAAAATAAGA

TTATATGAAC

TCCGTCAGTG

AATACCATCT

TTGTAATATG

AAAAAAACTG

ACTGGTTAGC

TCAAGCCATT

CCTAGCCCTT

ACCAA 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 895 <21013 <211> 543 <212>DNA <213> Great Crown 4 201031755 <400> 13 CTAGACTAAT AGGTTGCGGT CCCACCACGC AATCCGTCAG TGTAAACAAT 50 TCCATTTCCC CTGTAGTTAA TATTTTGGGA GAAATACCAT CTGGAAAAAT 100 TTCAATCCTC ACCATGTTTT GTACTGGAGG TGTTGTAATA TGATCATAGG 150 GTGTTTGATT ATGAATATTC CAGCTGTTTA ACAAAAAAAC TGCTTTAGTA 200 AGATGTGAAC TCCAATGTTG TAAAGTTTTA GTACTGGTTA GCTTTTTTAA 250 TTGTTCCTTC AACATCCCAT TCATTCTTTC AATCAAGCCA TTAGCTTGGG 300 GATGGTAGGC ATTGTGGAAT ATCCAATCTA TCCCTAGCCC TTGAGCCCAA 350 TCAAGGGCTC CAAGTGGAAC ACCACTTGGA ACACCAAGTG TTCCTTGGTC 400 ACTTTGTATT TGGTGAGGAG GTCCGTATTG ATGAATGAGT TGTTCTAGTG 450 CTTCAATTGT GTTATGTTGA TTAGCATACT TGGTGGGTTT AGCAAAAAAC 500 AGTCCTGACA CTGTATCCAC AGCAGTGAAT ACATATCGCC ACC 543

<210> 14 <211> 193 <212> DNA <213> Great Crown<400> 14 GGTGGCGATA TGTATTCACT GCTGTGGATA CAGTATCAAG ACTGTTTTTT 50 GCTAAACCCA CCAAATATAC TAATCAGCAT AGCACAATTG AAGCATTAGA 100 ACAACTCATT CTCTCCCAAA ATATTAACTA CAGGGGAAAT GGAATTGTTT 150 ACACCAACGG ATTGTGTGGT GGGACCGCAA CCTATTAGTC TAG 193 <210> 15 <211>734 <212>DNA<213> Great Crown<400> 15 GTAACGGATA CTCTGACCAG GATTAGAGGG GCCCTGCCTC CAGCCAGGTG 50 GAGGAAAGGG ACAACCGGGT CTACTGGACA GTGTGGATTC GATGGCCTGG 100 CACATCACAC TGTCCTGGTT TCAGCTGGGA TAGAGTTAAT TGTCTTCCTA 150 GTAGCTGGTA TAGTGCTATG TTTTGAGTTC AGTATGAGAA GAATGTTGAT 200 AACACTGATG TTTTCAGTTG TTGCTAAGGA ATGTTTAGTC TAAAGTCAAG 250 GATTTTTCAG CTTCTCATGC CCAGCCAGCA AGAAAGCTGG AGGGGCACAA 300 GAAGTTGGCA CAGGACAGAG CCAGGGCAGC AGACCCAAAG AGGCCAACGG 350 GGTATTCCAT ACCATGTGAT GTCATGTCTT GTATATACAC TGGGGGGAGT 400 GGGGGTGGGG GGATTGCCGC TCAGGGACTA ACTGGACATC GGTCGGCGGG 450 TAGTGAGCAA TTGCATTGTG CATCACTTGC ATATTCCAAT CCTTTTATTA 500 TTACTGTTGT CATTTTATT A GTGTTACCAT TATCATTATT AGTTTCTTCC 550 TTTCTGTACT GTTAAACCGT TCCTATCTCA ACCCACGAGT TTTACTTCTT 600 TTCCCGATTC TCTCCCCCAT CCCACTGGGT GGCGGGGAAG TGAGTGAGCA 650 GCTGCGAGGT GCTTAGTTGC TGGCTGGGGT TAAACCATGA CACAGACCCA 700 CAGGAATATA AGGCTCTAGT AGACACTGGT GCAC 734 5 201031755 < 210 > 16 < 211 > 147 < 212 > DNA < 213 > Large Crown <400> 16 GTGCACCAGT GTCTACTAGA GCCTTATATT CCTGTGGGTC TGATGTGCCA 50 GGTCATCGAA TCCACACTGT CCAGTAGACT CGGTTGTCCC TTTCCTCCAC 100 CTGGCTGGAG GCAGGGCCCC TCTAATCCTG GTCAGAGTAC CCGTTAC 147 <210> 17 <211>895 <212>DNA <213> Crested Goshawk ❹ <; 400 >. 17 CTGCAGCTGT TAATCCTCTC TCAATACAAA AGGCAGATGA GTTAGTTAAA 50 GTACAGGCTA ACCAAATCTA AGTATTTACA TTTTGATCTC ATGGAATTGT 100 ATCCAGGGCA TGTGCCTGTA GTACAAGACT ACAAAGCATG ATGAACCAAC 150 CTATGATAGG AGAGGAGAAC AGGAGTGTCG TCTCCTTCCA ATAATACATA 200 TGCCATTGCT CCAGCTCCTT CAGCTAATAT CACTCTGGGT TTGATAACCT 250 GATGTGGGGT AATTGCCCAC ACAGATTGGA GAGCTATGGC TTGTGCTTTT 300 TCAGACTGAA CTTCAATCTG ATGTTGGGTT TGCGACACCA ATAACACCAT 350 TCCAATGCTA TCTCCTCTAG ATAAGGCACA GGTATTTGTC AAAGATACCT 400 GAAATACAAG AACTCGAGAA TCTGATATCA ACAGAGGATG TAAAATAAGA 450 «GGAGAAGTTG GGGTACAGAA CCATGTGTCA TTCTCAGGGG GTATTATATG 500 CACCTTCACG CCTAGACTAA TAGGTTGCGG TCCCACCACG CAATCCGTCA 550 GTGTAAACAA TTCCATTTCC CCTGTAGTTA ATATTTTGGG AGAAATACCA 600 TCTGGAAGAA TTTCAATCCT CACCATGTTT TGTACTGGAG GTGTAATACG 650 ATCATAGGGT GTTTGATTAT GAACATTCTG CCTGTTTAAC AAAAAAACTG 700 CTTTAGTAAG ATGTGAACTC CAATGTTGTA AAGTTTTAGT ACTGGTTGGC 750 TTTTTTAATT GTTCCTTCAA CATCCCATTC ATTCTTTCAA TCAAGCCATT 800 ATCTTGGGGA TGGTAGGCAT TGTGGAATAT CCAATCTATC CCTAGCCCTT 850 A GAGCCCAATC AAGGGCTCCA AGTGGAACAC CACTTGGAAC ACCAA 895 6

Claims (1)

201031755 VII. Patent application scope: h A heterozygous (4) fragment, which is a nucleotide sequence having SEQ ID: 1 or SEQ ID NO: 2. The nuclear section of the scorpion, which is designed to design the w subgroup of the Daqi gender identification. 3. A method for sex identification of a large crown, which uses a nucleotide fragment of SEQ ID NO: 1 according to claim j to design a large crown and sex identification group to perform PCR selection of sex marker fragments. A method for sex identification of a large crown, which uses a nucleotide sequence fragment having SEQ ID NO: 2 in claim 1 to design a large crown 鹫 sex identification group to perform PCR-selection of sex-labeled fragments. 5. A primer set for identifying the sex of a large crown, which is: (1) CseBB08-5aF: CTGCAGCTGTTAATCCTCTCTC (SEQIDNO: 3), and CseBB08-5aR: TTACACTGACGGATTGCGTGGT (SEQ ID NO: 4); (2) CseBB08-5aF: CTGCAGCTGTTAATCCTCTCTC (SEQ ID NO: 5), and CseBB08-5bR: TTGGTGTTCCAAGTGGTGTTCC (SEQ ID NO: 6); (3) CseBB08-7F: CTAGACTAATAGGTTGCGGTCC (SEQ ID NO: 7), and CseBB08-7R: GGTGGCGATATGTATTCACTGC (SEQ ID NO: 8); or (4) CseAT15F: GTAACGGATACTCTGACCAGGA (SEQ ID NO: 9), and CseATl5R: GTGCACCAGTGTCTACTAGAGC (SEQ ID NO: 10) ° 6. According to claim 5, the introduction of the large-crown sex group, wherein the primer group is CseBB08-5aF: CTGCAGCTGTTAATCCTCTCTC (SEQ ID NO: 3) ' and CseBB08-5aR: TTACACTGACGGATTGCGTGGT (SEQ ID NO: 4), a female sex-sequence fragment having a large crown, having the nucleotide sequence of SEQ ID NO: . 16 201031755 7. According to the request 5, the introduction of the big crown 鹫 sex introduction group, wherein the introduction group is CseBB08-5aF: CTGCAGCTGTTAATCCTCTCTC (SEQIDNO: 5), and CseBB08-5bR: TTGGTGTTCCAAGTGGTGTTCC (SEQ ID NO: 6) » A female sex marker fragment of the Great Crown, having the nucleotide sequence of SEQ ID NO: 12. 8. According to claim 5, the introduction of the large-crown sex group, wherein the primer group is CseBB08-7F: CTAGACTAATAGGTTGCGGTCC (SEQIDNO: 7), and CseBB08-7R: GGTGGCGATATGTATTCACTGC (SEQ ID NO: 8) > A female sex marker fragment of the Great Crown, having the nucleotide sequence of SEQ ID NO: 14; and an intrinsically control fragment having the nucleotide sequence of SEQ ID NO: 13. 9. According to claim 5, the introduction of the big crown 鹫 sex introduction group, wherein the introduction group is CseAT15F: GTAACGGATACTCTGACCAGGA (SEQ ID NO: 9), and CseAT15R: GTGCACCAGTGTCTACTAGAGC (SEQ Π) NO: 10), . A female sex marker fragment of 鹫, which is the nucleotide sequence having SEQ ID NO: 16; and an intrinsically control fragment having the nucleotide sequence of SEQ Π) n〇:15. 10. A sex-specific marker fragment for identifying the sex of a large crown, which is a group consisting of a nucleotide sequence fragment having SEQ ID NOill, SEQ ID NO: 12, SEQ K) NaU and SEQ ID NO: 16. One of them. U. An intrinsic control fragment for identifying the sex of a large crown, which is a fragment of a nucleotide sequence having SEQ ID NO: 13 or SEQ ID NO: 15. 12. A method for sex identification of a large crown, which uses a primer set according to claim 5, 6, 7, 8, or 9 to identify the pCR response of the large crown sample to be identified. μ 13· According to the claim 12, the method for identifying the sex of the big crown is to identify the nucleoside having the SEQ ID: 11 by using the primer set according to claim 6 for the pCR reaction of the large crown sample to be identified. The acid sequence fragment is a female sex marker fragment. 17 201031755 : Item 12 of the Great Crown® gender identification method, which uses the PCR reaction for the large crown scorpion sample of the mosquitoes according to the request (4) 丨丨 子 ί ^ ^ g g g 具有 具有 具有 具有 具有 具有 具有 具有 具有 具有 具有 具有The fragment is a female sex 15.= request for the large crown miscellaneous identification method of the item 12, which is carried out by taking the response according to the request (4), and taking the identification of the large crown miscellaneous to take the reaction to have seq id n. The fragment of the nuclear thief of 13 is an intrinsic control fragment, and the nucleotide sequence fragment having the SEQ ID N〇: 14 is female. 16 According to the request 12, the method for identifying the sex of the large crown is used according to the request. • 引 引 primer group 'PCR reaction for the large crown 待 sample to be identified, with the nucleotide sequence fragment of SEQ ID NO: 15 as an intrinsic control fragment, and the nuclear bitter with SEQ ID NO: The acid sequence fragment is a female sex marker fragment. • 17. A primer set used to identify the sex of the Crested Goshawk, which is: • CSeBB〇8_5aF: CTGCAGCTGTTAATCCTCTCTC (SEQIDN0..5), and CseBB〇8-5bR: TTGGTGTTCCAAGTGGTGTTCC (SEQ ro NO: 6). 18. According to claim 17, the indicator set of the Crested Goshawk genus can be amplified, wherein a female sex-sex fragment of a Crested Goshawk can be amplified, which has the SEQ ID νο:1θ7 reference nucleotide sequence. 19. A sex-specific marker fragment for identifying the sex of a Crested Goshawk, which is a fragment of a nucleotide sequence having SEQ ID NO: 17. 20. A method for sex identification of a Crested Goshawk, which is identified by performing a PCR reaction on a large crown sample to be identified according to the introductory group of claim 17 or 18'. m 21. According to the method of gender identification of the Crested Goshawk of the 20th item, the PCR reaction of the Crested Goshawk sample to be identified is identified by the primer set according to claim 17 to have SEQ ID NO: 17 The nucleotide sequence is a female sex marker fragment. 18 201031755 22. A PCR kit for the sex identification of the Great Crown, which contains the primers according to claim 5, 6, 7, 8, or 9, and the reagents and tools required for the PCr reaction. 23· A pcR kit for the identification of the Crested Goshawk Sex, which contains the primer set according to claim 17 and the reagents and instruments required for performing the PCR reaction. 19