TWI288780B - Sex specific sequences in identifying birds or mammals and method thereof - Google Patents

Abstract

The present invention provides sex specific sequences in identifying birds or mammals and a method thereof. The invented method comprises: separately collecting specimen from female and male of a particular kind of bird or mammal; separately amplifying the nucleic acids contained in the collected specimen of female and male, in which the primer pair used is a random primer pair; and comparing the nucleic acids-containing amplified products in the specimen collected from female and male samples to determine the sex specific sequences. The present invention further provides a method for synthesizing an oligonucleotide primer pair capable of amplifying the sex specific sequences.

Description

1288780 玖, INSTRUCTION DESCRIPTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a sex-specific sequence and method for identifying avian or mammalian animals. [Prior Art] Gender identification of birds and mammalian animals is important in both economic and research development. Some poultry livestock are not easy to identify before sex or during embryonic or even life, but many economic activities, such as the proportion of males and females in birds and mammals when buying or selling, often need to identify the animal before sexual maturity. gender. The current state of the art is directly distinguished by appearance recognition, karyotype, Barr body or male specific antibody test. However, the use of appearance identification method to distinguish between male and female, often difficult to judge or misjudged; in addition, the karyotype, Barr body or male specific antibody test resolution, the technical level is complex and costly, and the time spent long. From the above, there is still a lack of a simple and rapid identification method for the sex identification of economic animals such as poultry and livestock. SUMMARY OF THE INVENTION A proprietary name is a sex-specific sequence that means having or lacking a specific sequence on a sex chromosome of a bird or mammal, depending on the sex of the bird or mammal. . The proper term "amplification method" means a test or method for increasing the nucleotide sequence. For example, the ''amplification method'" includes the polymerase chain reaction (pCR) assay, the ligase chain reaction (LCR) assay. The proper term "sample" means a specimen of any biological substance containing nucleic acid. For example, the "sample" includes biological samples such as whole blood, serum, urine, semen, etc. 1288780 The present invention relates to a method for identifying the sex of a bird or mammal. The present invention utilizes novel oligonucleotides. The primer pair is contacted with a bird or a mammalian specimen. 'Amplification by an amplification method detects the presence or absence of the amplification product' to distinguish the sex of the bird and the mammal. The present invention finds that 'in different birds or mammals There are a sex-specific sequence on the sex chromosome, which can be used to distinguish birds or mammals of different genders. Therefore, the present invention provides a method for identifying different sex birds or 嗔0 animals, which is via a special nucleus nucleotide primer pair. The presence or absence of the amplified product is used to determine the sex of the bird or mammal. The method of the invention comprises a method for determining a sex-specific sequence of a bird or mammal, comprising the following steps: • Female from the bird or mammal And the male individuals collect the samples separately; the nucleotides contained in the samples collected from the female and male individuals are separately The increase, wherein the pair of primers used is a pair of primers; the products of the amplified nucleotides contained in the samples collected by the female and the male individuals determine the sex-specific sequence. Preferably, the amplification method is aggregation. The enzyme chain reaction test or the ligase reaction test. More preferably, the amplification method is a polymerase chain reaction test. In another aspect, the present invention is further related to a method for identifying the sex of a black or mammal, #include the following steps : collecting a specimen of a black or mammal, and designing (4) a specific (four) sub-pair according to the sex-specific sequence obtained by the method for obtaining a gender-specific sequence as described above; and increasing the sex-specific primer pair of the nucleic acid disk contained in the money collection; (4) The existence of the amplification product to determine the sex of the bird or mammal. Preferably, the gender-specific primer is used for the amplification test, and 1288780 uses an internal control group primer pair.

More preferably, the internal control group is paired with Seq π ·· 1/SEq ID NO: 2 〇 More preferably, the sample of the amplification test is whole blood, serum, urine or semen. A method for identifying a sex-specific sequence of a bird or a mammal obtained by the steps of: collecting a test from a female or a male of the bird or a mammal; and collecting the sample collected from the self-engraving and male individual The nucleic acid-containing nucleic acid was contacted with the primers; the sex-specific sequences were obtained by comparing the products of the nucleic acids contained in the samples collected by the male and the male individuals. Preferably, SEQ ID NO: 3 〇 Preferably, the animal is a buffalo, and the sex-specific sequence is that the mammal is a yellow cow, the sex thereof.

SEQ ID Ν0··6. J Preferably, the mammal is a pig, basin ID N0:9

Preferably, the fn.n VIII-specific sequence is SEI. The avian is a series with a gender-specific ID of N0:12. Preferably, the avian is a scorpion, a snail, and the sex is SEQ ID Ν 0:15. Preferably, the avian is SEQ ID NO: 16 « ^ specific order

It is preferably listed as SEQ column. 'The bird is a crane, ID N0:21 〇 its gender-specific order

Listed as SEQ 1288780, preferably ID N0:24 〇 The black-eyed animal is a duck

Preferably, the sex-specific sequence is SEQ, the urchin, SEQ ID. The substance is a scorpion, and the sex-specific sequence thereof is, on the other hand, the present invention; the individual combination of the te μ ^ sex-specific sequence, the genus of the genus of the genus The sex increase is different from the sex-specific sequence of the genus or the squirting animal. Preferably, the mammal is a buffalo having the oligo SEQ ID NO: 4 / SEQ ID Ν 0: 5. Preferably, the spray animal is a yellow cattle having an oligonucleotide primer pair of SEQ ID NO: 7 / SEQ ID Ν 0: 8.

Preferably, the oligo-nucleotide primer pair is SEC, and the oligo-nucleotide primer pair is SEQ. Preferably, the mammal is porcine ID N0: l〇/SEQ ID Ν0:11 〇 Preferably, the avian is a crane ID N0:13/SEQ ID Ν0:14. Preferably, the avian is a Muscovy duck having an oligonucleotide primer pair of SEQ ID NO: 17/SEQ ID Ν 0:18. Preferably, the avian is a Muscovy duck having a nucleotide primer pair of SEQ ID NO: 19/SEQ ID NO: 20. Preferably, the avian is a goose with an oligonucleotide primer pair of seq ID NO: 22 / SEQ ID NO: 23. Preferably, the avian is a duck whose oligonucleotide primer pair is seq ID NO: 25 / SEQ ID NO: 26. Preferably, the avian is an ostrich having an oligonucleotide primer pair of SEQ ID NO: 28/SEQ ID ·. 1288780 The identification of sex-specific sequences in birds or mammals by the present invention and the methods disclosed herein provide an easy way to identify the sex of birds and mammals. [Embodiment] The present invention uses a DNA fingerprinting technique to find a gender-specific genetic marker from a polymorphic DNA fingerprinting zone. The DNA sequence of the animal is then selected and sequenced, and compared with the sequence that has been found in the world, and found to be new sequences not found by the predecessors. And based on the selected sex-specific DNA (sex-specif ic DNA), the primer pair is designed by itself, and the designed primer pair is used for amplification reaction and electrophoresis analysis, and the DNA molecular distribution on the electrophoresis film is performed. , can accurately determine the sex of the animal. The following examples are intended to illustrate the invention. The examples are not intended to limit the scope of the invention in any way, but are intended to indicate how to practice the materials and methods of the invention. Feng Shi Example 1: Method of 噼 彳饨 柹庠 1. 1. 1. 1. 1. 1. 1. 1. Φ Φ 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动 自动

1. 2 DNA extraction The collected blood was extracted from genomic DNA by the method recommended by Shiau and Huang (1997). After genomic DNA, the DNA was diluted to 5 〇ng/“i for sterilized deionized water for amplification experiments. 1 · 3 amplification test 10 1288780 The amplification test was based on the recommendations of Sambrook et al. (1989). The method was modified. The concentration of each reactant in the amplification test was as follows: l mM Tris-HCl ρ Η 8 · 0, 1 · 5 mM MgCl 2 , 50 mM KC1, each 100 Μ dATP, dCTP, dGTP and dTTP, each 0 · 14 " individual primers, lOOng of genomic DNA, 0·5U Dynazyme 11 polymerization (Finnzymes Oy), and then sterilized deionized water 2 from 1, so that the reaction volume in each tube is 25 / / 1. The primer pairs used are the primer pairs (〇peron, USA). The amplification test performed by RAPD-PCR (random amplified polymorphic DNA PCR) is: 941 denaturation for 5 minutes, and then each amplification reaction is carried out. 45 temperature cycles (denaturation at 94 °C for 1 minute, 36 for primer binding for 1 minute and 72 °C for 2 minutes), followed by extension at 72 °C for 7 minutes to completely double the DNA. 1.4 Separation of DNA products Recycling a small amount of PCR product (each 20 // 1), After entering the appropriate indicator, electrophoresis was carried out at a voltage of 50 volts on a 2.0% agarose gel. After electrophoresis, the gel was stained with ethidium bromide and observed under a UV light source. The genomic DNA of the animal is amplified by a specific fragment which can distinguish different sexes, and the gel containing the specific DM fragment is excised, recovered and purified, and the purified DNA fragment is again subjected to PCR by the original primer. Preparation of competent cells 30 ml of E. coli bacteria cultured at 37 ° C with shaking to D6 (10) = 〇 · 4~0.6, centrifuged at 6,000 rpm for 5 minutes at 4 ° C, remove the supernatant, add 15 ml The cells were resuspended in ice-cold 50 mM CaCl2, and ice-bathed for 1 minute. Then centrifuge at 6,000 rpm for 5 minutes at 4 ° C, remove the supernatant, add 15 ml of 1288780 ice-cold 50 mM CaCl 2 solution, and ice bath for 10 minutes. After the above steps were carried out and centrifuged under the same conditions, the cells were resuspended in 1 ml of 50 mM CaCl 2 , and then added to one tenth of the total volume of DMSO, and then stored at -70 ° C for use. (transformation) _ The product obtained according to the method of 1.4, refer to Inv Itrogen operating manual, taking 2//1 product of secondary PCR, with 1//1 pd)NA3.1/V5-His-TOPO (Cat·No·K4800-01) carrier or pCRII-TOPO (Cat· No . K4600-01) Carrier, (Invitrogen, CA, USA) mixed. The method of operation is as follows: According to the TOPO ΤΑ Cloning 8 Kit (Invitrogen®) manual, _ sequentially added the PCR product confirmed by electrophoresis and 〇. 5 //1 pCRii— - Τ0Ρ0® vector, the total volume of the reaction was adjusted to 5// 1, at room temperature for 5 minutes, and then moved to ice for later use for the next transformation. Add about 50 ^ 4 / g of recombinant plastid DNA or DNA after the adhesion reaction, add 1.5 to the method of the competent cell 1 〇〇 # 1, ice bath for 1 hour, heat shock in a 42 ° C water bath Add 〇· 9 ml LB culture solution, water bath at 37 ° C for 1 hour, take 100~200 //1 bacterial solution, uniformly apply selective medium to curved glass, and incubate for 3 to 16 hours after 3 7 C. can. The colonies obtained by the transformation reaction were initially screened by the Cracking method (Lin, 1991), and the possible colonies were picked and cultured, and then separated by a QNAprep Spin Miniprep kit (Cat· No. 27106, QIAGEN). DNA, the resulting DNA was adjusted to a concentration of 0.5/zg/〆1. The M13 forward primer/reverse primer' and the Prism Big Dye Terminator Cycle sequencing reaction set (BigDye V· 3.1; Perkin-Elmer/AB, Lot. No. 0303013), in the Applied Biosystem 3100 DNA Auto Sequencer ( The DNA sequence isolated by 12 1288780 sequencing was performed on Perkin-Elmer Cetus Corp., Forster City, CA, USA. 1. Design of 6 primers and amplification response of sex-specific sequences According to the sequenced DNA sequences, primer pairs designed to identify the sex of different animals were designed. Animal genomic DNA (50 ng / / 1) was taken and subjected to an amplification reaction test using the following primer pairs which can be used to identify different animal sexes. At the same time, 18S-F/18S-R (Hedges et al., 1990) was simultaneously subjected to an amplification test based on the 18 S rRNA sequence design primer as an internal control group for the amplification test. 18S~F(SEQ ID NO:1): 5,-AGCTCTTTCT CGATTCCGTG-3; 18S-R(SEQ ID NO:2): 5,-GGGTAGACAC AAGCTGAGCC-3 18S-F/18S-RCSEQ ID N0:1/SEQ ID N0: 2) The amplification conditions of the primer pair are as follows: denaturation at 94 ° C for 5 minutes, and then each amplification reaction is carried out for 30 temperature cycles (denaturation at 94 ° C for 1 minute, bonding at 58 ° C for 45 seconds, and 72 The temperature was extended for 1 minute), and the DNA was completely double-stranded by extension at 72 ° C for 7 minutes. Example 2: buffalo sex-specific sequence 2. 1 Taiwan buffalo (and the sex-specific sequence according to the method described in Example 1.1.1 to 1.4, the first image shown in the RAPD-PCR gel And found on the DNA fragment distribution map of male Taiwan buffalo, with a gender-specific sequence, as indicated by the arrow. Purify the sex-specific sequence and insert the sequence into pcDNA3·1/V5 according to the method of 1.4. -His-Τ0Ρ0 vector, forming a plastid DNA (Fig. 2). The plastid DNA was transformed, the plastid DNA was extracted, and the sex-specific sequence of SEQ ID NO: 3 of Taiwan 12 1388780 buffalo was sequenced. SEQ ID NO: 3, an oligonucleotide primer pair designed to increase the sex-specific sequence of Taiwan buffalo

BuSex0PC16F (SEQ ID NO: 4): 5'-CACTCCAGTC TAACACAGTC AGTAG-3’

BuSex0PC16R (SEQ ID NO: 5): 5'-CTCCAGAGAC TGCATGCTAT GGTGA-3' The above primer was used to increase the genomic DNA (50 ng/zl) of Taiwan buffalo. The amplification conditions were as follows: denaturation at 94 °C for 5 minutes. Then each amplification reaction was carried out for 35 temperature cycles (denaturation at 94 °C for 1 minute, bonding at 66 °C for 45 seconds, and elongation at 72 °C for 1 minute and 30 seconds), and then extending the DNA at 72 °C for 7 minutes to complete the DNA. Become a double share. The electrophoresis analysis was carried out, and the results are shown in the third figure. From the results of the third figure, the first column is the molecular marker of l〇〇bp, the second column is the increase result of the female Taiwan buffalo, and the seventh column is the increase result of the male Taiwan buffalo. From the electrophoresis gel map, the oligonucleotide primer of the present invention can be found in BuSexOPC16F/BuSexOPC16R (SEQ ID NO: 4/SEQ ID NO: 5), indicated by the arrow at 315 bp, and found in male Taiwan buffalo genomic DNA, which can be increased in specificity. A gender-specific sequence, in which each genomic DNA can be increased by 256 bp in the 18rRNA control group (arrows indicate 18S), so BuSexOPC16F/ BuSexOPC16R (SEQ ID NO: 4/SEQ ID NO: 5) does provide rapid Accurate gender identification method. 2. 2 different breeds of cattle are identified by different types of bulls such as buffalo (B), yellow cattle (Y), Dutch cattle (H), Angus cattle (A) and Haifu cattle (HF). Body, at the same time with BuSexOPC16F/ 1288780 31^6 and 0? (: 161? (5 £ (3 10 grab: 4/5 £ (3 10 1^0: 5) primer pair, the amplification test, the fourth map As shown in the fourth figure, it is found that BuSex0PC16F/BuSex0PC16R (SEQIDN0:4/SEQIDN0:5:^l subpairs can be increased in the male cattle of each species as the 315 bp fragment indicated by the arrow, so BuSex0PC16F/ BuSex0PC16R ( SEQ ID NO: 4/SEQ ID NO: 5) The primer pair can be applied to the sex identification of different breeds of cattle. Example 3: Yellow cattle sex-specific sequence 3. 1 Yellow cattle (10,000 6 ^ sex-specific sequence according to The method described in the first embodiment of the first embodiment is the RAPD-PCR electrophoresis gel image shown in the fifth figure, and is found on the DNA fragment distribution map of the male yellow cattle, and has a gender-specific sequence, such as The arrow is labeled. Purify the sex-specific sequence and insert the sequence into the pCRI I-Τ0Ρ0 vector according to the method of 1.4 to form a plastid DNA (sixth Fig.) After the plastid DNA has been transformed, the plastid DNA is extracted and the sex-specific sequence of the yellow cattle is sequenced, as listed in SEQ ID NO: 6. According to SEQ ID NO: 6, designed to increase the sex specificity of the yellow cattle. The oligonucleotide primer pair of the sequence is YCaOPAG06F (SEQ ID NO: 7): 5, -TCCTCCTGCA ATGTGTGAGA CCTG-3' YCaOPAG06R (SEQ ID NO: 8): 5, -CAAGATTCCA AGGCTGCAAC AGCG-3' The genomic DNA (50 ng/zl) of the yellow cattle was subjected to an amplification test. The amplification test conditions were as follows: denaturation at 94 ° C for 5 minutes, and then each amplification reaction was carried out for 25 temperature cycles (denaturation at 94 ° C for 1 minute, introduction at 65 ° C) For 1 minute, and 1 hour at 72 ° C, and then extended by 72 ° C for 7 minutes, the DNA was completely double-stranded. The electrophoresis analysis was carried out, and the results are shown in Fig. 1288780 of the seventh figure. 1 is a molecular marker of lOObp, the second column is the result of the increase of the female yellow cattle, and the seventh column is the increase result of the male yellow cattle. The oligonucleotide primer of the present invention can be found by the electrophoresis gel map to the YCaOPAG06F/YCaOPAG06R (SEQ ID NO: 7 / SEQ ID NO: 8), which can be increased by about 1.5 kb at the arrow. The sex-specific sequence in the genomic DNA of male cattle, and each of the genomic DNA can increase the 256 bp 18SrRNA internal control group fragment, which can prove that the sex-specific primer of the present invention pairs SEQ ID NO: 7/SEQ ID N0·· 8, can specifically increase the sex-specific sequence in the genomic DNA of male cattle, quickly and accurately provide a gender identification method. 3.2 Identification of different breeds of cattle The number of males of different breeds such as yellow cattle (Y), Dutch cattle (H), Angus cattle (A), Haifu cattle (HF) and Taiwan buffalo (B) The specimens were tested for the growth of the sex-specific sequence, and the results are shown in the eighth panel. The genomic DNA of the Dutch cattle, Angus cattle and Heifer cattle may also be increased by about 1. 5 kb in size and specificity. Sex fragments, so the YCaOPAG06F/ YCaOPAGO 6R primer pair can be applied to the sex identification of Dutch cattle, Angus cattle and Haifu cattle. Example 4: Sex-specific sequence of pigs 4.1 克克夏 (Ym pig sex-specific sequence According to the method described in Example I 1.1 to 1.4, the RAPD-PCR electrophoresis gel map shown in the ninth figure was obtained and found In the map of DNA fragment distribution 16 1288780, there is a sex-specific sequence, as indicated by the arrow. Purify the sex-specific sequence and embed the sequence into pCRI Ι-Τ0Ρ0 according to the operation method of 1.4. The vector forms a plastid DNA (Fig. 10). After transformation of the plastid DNA, the plastid DNA is extracted and the sex-specific sequence of the gram porcine pig is sequenced SEQ ID NO: 9. According to SEQ ID NO: 9. The pair of oligonucleotide primers designed to discriminate the sex-specific sequence of the summer pig is PigSexAV18F (SEQ ID NO: 10): 5, -TTGCTCACGG TAGATAACAA GAGAG-3' PigSexAV18R (SEQ ID NO: 11): 5,- TTGCTCACGG ACCAGGTAGG GAATG-3' Take the above primers to increase the genomic DNA (50ng///1) of the Pankesi pig. The amplification test conditions are as follows: β·94°C denaturation for 5 minutes, and then each amplification reaction is carried out for 30 times. Temperature cycle (denaturation at 94 ° C for 1 minute, 62 ° C for primer bonding After 1 minute, and extended at 72 ° C for 1 minute), the DNA was completely double-stranded by extension at 72 ° C for 7 minutes. The electrophoresis analysis was carried out, and the results are shown in Figure 11. The first column of Figure 11 is 100 bp molecular markers, 2-3 columns for male Panke summer pigs, and columns 4-5 for female Panke summer pigs. The arrow indicates that male Panke summer pigs can increase the sex-specific sequence of about lkb. By the oligonucleotide primer of the present invention, PigSexAV18F/ PigSexAV18R (SEQ ID NO: 10/SEQ ID NO: 11) can specifically increase the sex-specific sequence in the genomic DNA of male Pankeshen pigs, and provide it quickly and accurately. Sex identification method 4.2 Identification of pigs of different breeds Different types of boars ($) such as Pankexia (B), Taoyuan pig (T), Lanyu small ear pig (S) and Meishan pig (M) 17 1288780 And the sow (early) number of samples were amplified by the PigSexAV18F/ PigSexAV18R (SEQ ID NO: 10/SEQ ID NO: 11) primer pair, and the results were shown in the tenth. Figure 1. The image of PigSexAV18F/ PigSexAV18R (SEQ ID NO: 10/SEQ) can be seen from Figure 11. ID NO: 11) The pair of primers, indicated by the arrow, shows that the genomic DNA of male pigs such as Taoyuan pig, Lanyu small ear pig and Meishan pig can increase the specific fragment of about lkb, so it can be applied to pigs of different varieties. Only gender identification. Example 5 · Pigeon J/v/a) Sexual dissimilarity All According to the method described in Example 1.1 to 1.4, the RAPD-PCR electrophoresis map shown in Fig. 12 is obtained and found in the hen On the DNA fragment map, there is a gender-specific sequence, as indicated by the arrow. The sex-specific sequence was purified and inserted into the pCRI Ι-Τ0Ρ0 vector according to the procedure of 1.4 to form a plastid DNA (Fig. 13). After the plastid DNA was transformed, the plastid DNA was extracted, and the sex-specific sequence of the hen was sequenced as SEQ ID N0··12. According to SEQ ID Ν0··12, the oligonucleotide primer pair designed to increase the sex-specific sequence of the hen is Dove0PC20F (SEQ ID NO: 1 3): 5, -ACTTCGCCAC AATTTCAGTG GTTCC -3,

Dove0PC20R (SEQ ID NO: 1 4): 5'- ACTTCGCCAC TAAAAATGTA AAAAT-3' Take the above primer to increase the genomic DNA (50 ng / / zl) with the hen, the amplification test conditions are as follows: denaturation at 94 ° C for 5 minutes Then, each amplification reaction was carried out for 35 temperature cycles (denaturation at 94 ° C for 1 minute, binding at 15 ° C for 1 minute, and extension at 72 ° C for 1 minute), and then extension of 72 minutes to 12,88780 minutes for DNA. Completely become a double share. The electrophoresis analysis was carried out, and the results are shown in Fig. 14. From the arrow indicated in Figure 14, it was found that the oligonucleotide primer of the present invention can increase the dot size of about 750 bp in the genomic DNA of the female pigeon by DoveOPC20F / Dove0PC20R (SEQ ID NO: 13 / SEQ ID NO: 14). The sex-specific sequence fragment provides a rapid and accurate method for gender identification. The real side of your J six: Muscovy duck (force a wide a) of the ten generations of another 1 J specific f-sequence according to the method described in Example I 1.1 to 1.4, respectively, the RAPD-PCR shown in the fifteenth and sixteenth Electrophoresis gel images, and on the DNA fragment distribution map of the female muscovy duck, a sequence with gender specificity was found, such as the arrow mark. The sex-specific sequence was purified, and two sex-specific fragments were inserted into the pCRI Ι-Τ0Ρ0 vector according to the method of 1.4 to form two plastid DNAs (Figs. 17 and 18). After the plastid DNA is transformed, the plastid DNA is extracted, and then two specific fragments are sequenced, and the sequences SEQ ID NO: 15 and SEQ ID NO: 16 are obtained, respectively. According to SEQ ID NO: 15, an oligonucleotide primer pair designed to increase the sex-specific sequence of Muscovy duck is MduckA012F (SEQ ID NO: 17): 5'-TGTTGTTCCC TGACAGGGCT G-3,

MduckA012R (SEQ ID NO: 18): 5, -ACAGGGGGTG CTAAGAAGC-3, an oligonucleotide primer pair designed to increase the sex-specific sequence of Muscovy duck according to SEQ ID NO:

MduckAV08F (SEQ ID NO: 19): 5, -AAGCGGTAGA ACGGAGAGAG TC-3 ’

MduckAV08R (SEQ ID NO: 20): 1288780 5'-AACTGCCTCT CGCTGGTAAG-3 ' Take the above-mentioned primer pair and Muscovy duck genomic DNA (50ng//zl) for the amplification test, MduckA012F / MduckA012 R primer for the amplification test conditions The following conditions were: denaturation at 94 ° C for 5 minutes, and then each amplification reaction was carried out for 30 temperature cycles (denaturation at 94 ° C for 1 minute, bonding at 55 ° C for 1 minute, and extension at 72 ° C for 1 minute), followed by 72 ° C is extended for 7 minutes to make the DNA completely double-stranded. After electrophoresis analysis, the results of the nineteenth graph were obtained, and the arrow indicated a section of about 700 bp. The amplification test conditions of the MduckAV08F / MduckAV08R primer pair were as follows: denaturation at 94 ° C for 5 minutes, and then each amplification reaction was carried out for 30 temperature cycles (denaturation at 94 ° C for 1 minute, bonding at 59 ° C for 1 minute, and 72 ° C). The extension was extended for 1 minute, and the DNA was completely double-stranded by extension at 72 ° C for 7 minutes. The results of the electrophoresis analysis are shown in Fig. 20, and a fragment of about 150 bp is displayed at the arrow mark. From the results of the nineteenth and twentyth figures, the oligonucleotide primer of the present invention pairs MduckA012F/MduckA012R (SEQ ID NO: 17/SEQ ID NOH8) and MduckAV08F / MduckAV08R (SEQ ID NO: 19/SEQ ID N0: 20), the female genomic DNA can be increased by a fragment of about 700 bp and 1500 bp, respectively, which can specifically identify the sex-specific sequence in the genomic DNA of the female muscovy duck, thus providing a rapid and accurate method for gender identification.

The implementation of the combination of J7: I Iesar), the specificity of the sequence, J 7. 1 the sex-specific sequence of the goose according to the method described in Example 1.1.1 to 1.4, as shown in the twenty-first figure The RAPD-PCR electrophoresis gel map was found on the DNA fragment distribution map of Chinese goose and has a gender-specific sequence, such as the arrow mark. Purify the sex-specific sequence, and insert the sequence into 1288780, # body dna pCRI I - T0P0 vector according to the method of 1.4, to form a plastid DNA (22). After the negative reaction, the sample is extracted. Plasmin DNA, and sequenced Chinese goose sex-specific & sequence SEQ ID N0··21. According to SEQ ID NO: 21, the oligonucleotide primer pair designed to increase the sex-specific sequence of Chinese goose is CGSex-F (SEQ ID NO: 22): 5, -GGGAAGACAGACCTAACAGAC-3' CGSex-R (SEQ ID NO:23): 5,-AGTCACTTGTATTGTTCAGCC-3” Take the above primers to increase the genomic DNA of Chinese goose (50ng/ // 1). The increase test conditions are as follows: denaturation at 94 °C for 5 minutes, then each increase The reaction was carried out for 30 temperature cycles (denaturation at 94 ° C for 1 minute, adhesion at 63 ° C for 1 minute, and extension at 72 ° C for 2 minutes), and then extension at 72 ° C for 7 minutes to completely double the DNA. For the analysis, the results are shown in Figure 23. The first column of the twenty-third figure is the lOObp molecular marker, the 2-4th column is the female Chinese goose, and the fifth column is the male Chinese goose. It is pointed out that the female Chinese goose can increase the sex-specific sequence of about lkb. Therefore, the oligonucleotide primer of the present invention can specifically increase the female CGSex-F/CGSex_R (SEQ ID NO: 22/SEQ ID NO: 23). The sex-specific sequence in the genomic DNA of Chinese goose provides a rapid and accurate method for gender identification. The goose of the same species was identified by taking the samples of different species of goose, such as Chinese goose, Landaise goose and white roman goose, with CGSex-F/CGSex-R (SEQ ID NO: 22/SEQ ID NO: 23). The growth response of the sex-specific sequences of different species of goose is carried out. The results are shown in the twenty-third figure. Among them, columns 8-13 are white roman geese (21 2188780 8-10 are female, first 1 13 Listed as male); Columns 14-19 are Landaise Goose (columns 14-16 are female and 17-19 are male). From Figure 23, CGSex-F/CGSex_R (SEQ ID N0: 22) /SEQ ID NO: 23) The pair of primers, indicated by the arrow, shows that the genomic DNA of female goose such as Chinese goose, Landaise goose and white roman goose can increase a single specific fragment of about 91 lbp, so it can be applied to different varieties. Sex identification of the geese. Implementation of your J: 菜 duck / 7 / no power) i singularity specific 1 8.1 ducks of the sex-specific sequence according to the embodiment of 1. 1 to 1. 4 The method has the RAPD-PCR electrophoresis gel map shown in the twenty-fourth figure, and is found on the DNA fragment distribution map of the female duck with a gender-specific sequence. The sex-specific sequence was purified and inserted into the pCRI Ι-Τ0Ρ0 vector according to the procedure of 1.4 to form a plastid DNA (Fig. 25). After the plastid DNA was transformed, the plastid DNA was extracted and the sex-specific sequence of the duck was sequenced as SEQ ID NO: 24. According to SEQ ID NO: 24, the oligonucleotide primer pair designed to increase the sex-specific sequence of the duck is TsaiSex-F (SEQ ID NO: 25): 5, -ACACAGTCCA ATAAACTCGA TTGTC-3' TsaiSex-R (SEQ ID NO:26): 5,-TCGTGTCAAA ATAGAGAGCT TGAAG3' Take the above primers to increase the genomic DNA (5Ong/ // 1) of the duck with the following conditions: the denaturation test conditions are as follows: denaturation at 94 °C for 5 minutes, then each increase 22 1288780 The reaction was carried out for 30 temperature cycles (denaturation at 94 ° C for 1 minute, bonding at 66 ° C for 1 minute, and extension at 72 ° C for 2 minutes), and then extension at 72 ° C for 7 minutes to completely double the DNA. For electrophoresis analysis, the results are shown in Figure 26. The first column of the twenty-sixth figure is a lOObp molecular marker, the second column is a female duck, and the eighth column is a male duck. The figure shows that the female duck can increase the sex-specific sequence of about lkb. Therefore, the oligonucleotide primer of the present invention can specifically increase the sex-specific sequence in the genomic DNA of female ducks by TsaiSex-F/TsaiSex-R (SEQ ID NO: 25/SEQ ID NO: 26). Accurately provide gender identification methods. The method described in the first embodiment of the present invention, the method of the first embodiment of the present invention, the method of the first embodiment of the present invention The RAPD-PCR electrophoresis gel map shown in Fig. 27 is found on the DNA fragment distribution map of the female ostrich, and has a gender-specific sequence, as indicated by the arrow. The sex-specific sequence was purified and inserted into the pCRI Ι-Τ0Ρ0 vector according to the procedure of 1.4 to form a plastid DNA (Fig. 28). After the plastid DNA was transformed, the plastid DNA was extracted and the sex-specific sequence of ostrich was sequenced as SEQ ID NO: 27. According to SEQ ID NO: 27, the oligonucleotide primer pair designed to amplify the ostrich sex-specific sequence is OST-F (SEQ ID NO: 28): 5, -CTTGAGGCCAAACAAATAG-3' OST-R (SEQ ID NO: 29): GGTACAAAAACTCACAAGG-3 23 1288780 Take the speed primer to carry out the test on the genome of the remaining birds (9) ng/chuan. The conditions of the amplification test are as follows: 9 generations of denaturation for 5 minutes, and then each amplification reaction is carried out for 25 temperature cycles (9 generations of denaturation). In the minute, the 6th generation entered the pair of minutes and the extension was 2 minutes. For electrophoresis analysis, the results are shown in the second: Figure 9. In the first column of the twenty-ninth figure, the i〇〇bP molecular marker n6 is listed as male 4, and the 7th-n is female. The arrow indicates that although the bird can increase - about 642 bp of gender-specific sequence. Therefore, the ST-F/〇ST-R (SEQ ID N〇: 28/SEQ ID NO-29) of the present invention can specifically increase the gender-specific column in the genome face of female dry birds, and provide the gender quickly and accurately. Identification method. * The above-described method for identifying avian and mammalian sex sequences and its sex-specific sequences of the present invention have the above-described effects. ^There are various modifications and variations that can be made by the present invention. It is apparent that the scope and spirit of the invention are not deviated. Although the present invention is specific to specific preferences, it must be understood that the present invention should not be unnecessarily limited to the specific preferences. In fact, the stated mode aspect of the invention is true to those skilled in the art. [Simple description of the diagram] (1) Figure 2 of the diagram is a picture of the buffalo genomic DNA in the electrophoresis gel. The second picture shows the genetic map of Taiwan buffalo-specific sequence annihilation to vector. 24 2488780 The second picture shows the electrophoresis gel of Taiwan buffalo genomic DNA amplified by SEQ ID NO: 4/SEQ ID Ν 0··5. The fourth figure is the genomic DNA of each of the different breeds of cattle with SEq ID N〇: 4/SEQ ID Ν〇: 5. The fifth picture shows the electrophoresis gel of the yellow cattle genome ΜΑ and the amplitude test. Gene map to the vector. Ν0··8 increase of yellow cattle genome DM Ν0··8 increase of different varieties

The sixth figure is the chimeric specific sequence chimerism. The seventh figure is the electrophoresis gel map of SEQ ID NO.. 7/SEQ ID. The eighth figure is DMA with SEQ ID NO: 7/SEQ bovine genome. The ninth image shows the electrophoresis gel map of the Pankesi pig genome. The tenth figure is the chimeric sequence of the hexagram sequence. The eleventh figure is SEQ ID NO: 1 〇 / SEQ ID N 〇: u m map & Pig and other breeds of cattle are genetically engineered. The eleventh figure of the Θ 克 克 夏 子 对 对 对 对 对 对 对 对 对 对 对 对 对 对 对 对 对 对 对 对The thirteenth is the chimeric sequence of the heterozygous sequence. The fourteenth figure is SEQ ID NO: 13/SEQ ID NO: 14 and " Due to group DNA. · The base of the pigeons The increase test The fifteenth image is the electrophoresis map of the Muscle duck genome. The sixteenth figure is an electrophoresis gel map of the Musca duck genome. (10) A by the introduction of the machine to the worm (4) cut to increase the duck test 25 1288780 The seventeenth figure is the gene mapping of the specific sequence of the Muscovy duck to the vector. The eighteenth figure is a genetic map of the muscovy duck specific sequence chimeric to the vector. The nineteenth figure is the genomic DNA of Muscovy ducks increased by SEQ ID NO: 17/SEQ ID NO: 18.曰 The tenth figure is the genomic DNA of Muscovy ducks increased by SEq ID N〇:19/SEQ ID Ν〇··2〇.曰 The first tenth-picture system is an electrophoresis gel map of the increase test of the Chinese crane gene and the group ship. Putian: The twenty-two map is a genetic map of the medium-specific sequence chimeric to the vector. The twenty-third figure is the genomic DNA of the same species of crane SEQ ID NO: 22/SEq ID N〇: 23. Group DNA is subjected to an increase test by the machine

The twenty-fourth figure is an electrophoresis gel image of the duck duck gene test. The twenty-fifth image shows that the duck-specific sequence is chimeric to the vector. The twenty-sixth image is the genomic DNA of _I"〇: 25/SEq ID N〇::.

The seventeenth figure is an electrophoresis gel image of the genome of the scorpion scorpion and the sputum. τ 仃 仃 ‘ 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 『 a toweling field 焉匕 J 70 pieces of representative symbols No 26 1288780 References:

1 · SHIAU, JW & HUANG, MC (1997) The probe of repetitive sequence for DNA fingerprinting in Holstein cattle. Journal of the Agriculture Association of China, 177: 1-10. 2 e Sambrook, J.5 EF Fritsch & T. Maniatis. 1989. In vitro amplification of DNA by the polymerase chain reaction, chapter 14. In: Molecular cloning: A Labortory Manual, ed. by Cold Spring Harbor Labortory Press. Cold Spring Harbor New York. 3. HEDGES, SB· , MOBERG, KD· & MAXSON, LR (1990) Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote

Relationships. Molecular Biology and Evolution, 7:607-633. 4·Lin Zhisheng, 1991. The casein gene was selected from a pool of cattle and goat genomes. Master's thesis of Zhongxing University.

27 1288780 Sequence Listing <110> Executive Yuan Agriculture Committee <120> Screening Uzbekistan or Mammals <130><160> 29 <170> Patentln version 3.1 <210> 1 <211> 20 &lt ;212> DNA <213> 18SrRNA <400> 1 agctctttct cgattccgtg 20 0 12 3 1A 11 ία lx <2<2<2<2

2 20 DNA 18SrRNA <400〉 2 gggtagacac aagctgagcc 20 0 12 3 1111 <2<2<2<2 3 321 DNA Bubalus bubal is <400> 3 cacactccag ttccacaagt gtgtttcagt aatagtatgt gtgttgggtc tctaacacag cttcttcatg gcagtgacat caccaatgtt atagttgtgt tcagtagatt cacaccatgt gattcaagtg tcacctgtga Cacataagat atcatcatcc caaacatacc tttcttcatt catgattcct agtcctcgag aatattttaa cagtagctta ttctgcaggt cttaatttat acaagccaac ctgtggaaca tcaccatccc aaacacactc ttagttggct tctcaccata 60 120 180 240 300 Page 1 1288780 gcatgcagtc tctggagtgt g 321

<210> 4 <211> 25 <212> DNA <213> Bubalus bubal is <400> 4 cactccagtc taacacagtc agtag 25 >>>> 0 12 3 1X 11 1i 1A 2 2^2 << V < 5 25

DNA

Bubalus bubal is <400> 5 ctccagagac tgcatgctat ggtga 25 <210> 6 <211> 1529 <212> DNA <213> Bos indicus <400> 6 ggtggccaag cacgtgaaga atcctcctgc aatgtgtgag acctgcacac aatgtgtttg 60 agccttgggt tgagaagatc ctctggagaa gggaaaggct atctattcca gtattctggc 120 ctgaagaatt acaaattcca tacactgtat gatccatggg attgcaaaaa gtcagacatg 180 actaagcaac tttcacttac ccagagagat acaaaataag cattgatgaa agaaagcaaa 240 acacatatag atggagaaac ataccatgtt cttagattgg attaattgat atagttaaaa 300 tgagtatact acccaaaaca atcaatagat tcaatgcaat acctacaata tttttcataa 360 aactagaaca aattatttta tgatttgcat ggaaacacat aagactttga agagacaaag 420 caattttgag aaagaagaat gaaactagaa gagtcaaact ttttgataag actacaccac 480 aaagatatca agagagtatg aaacttgcac aaacatagaa atacagacca atggaacaaa 540 atataaagtc caaagataaa ttcatgcacc cattatcttt gacaatggag gcaaaaatat 600 Page 2 660 1288780 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1529 gcaaccaaaa aatatatttt cttcaaaaag tgttactggg aaaactggac agctatgtgt agaaactaga acagtatcta acatcaagtt caaaataaac tcaaaatgga tttacagatg taaatgtaag accagaaaat ataaaaccct tagagtaaaa cataggtata accactctaa aataagacac agcaagatcc tctatgaccc attgcctaga gtaaaggaaa taaaaacaaa attaaaaaaa aaaagcaacc taattaagct ctatagcttg tgtacaatga ggaaactata attaaggtga atatgtagcc ttcagtttta tgtgattatg gtttcagtgt atctgtcctc tgatgccctc ttgcaacacc taccatcttt tttggatttc tcttaccttg gtcatggggt atctcttcac ggctgctcca gcaaagcgca gccattgctc cttaccttgg atgaggggta tctcctcacg gcacctatcc tgaaattgaa catggaatag ctcctctagg ccctcctgag cccatgcagc caccgctcct tggatctggg gttactcatc tcagctgctg ccccaacttt gggtgtgggg tagctgctct tggctgctct tgtgctgtcg cagccttgca ctgtccgcca cagcccctga cttctgacac ggtttagctc ctccctgctg tgcccctgac ctttgacacg ggtaagcttc tctcggctac agcccctgac ctcggacgtg gggtagctcc tcccagccat cgcccctgac ctcagacgtg gggtagctcc tatgggctgc tccagcgctg ttgtagcctt gctctctcag ctgctggccc tgacctcgga catggggtag ctcctctggg ctgctcctgc gctgttgcag ccttggaatc ttggccacc) > >; > & gt 0 12 3 lx 1x lx 11 <2≪2<2<2 7 24

DNA

Bos indicus <400〉 7 tcctcctgca atgtgtgaga cctg 24 0>1>2> 11 lx 11 2 2 2

Page 3 1288780 <213> Bos indicus <400> 8 caagattcca aggctgcaac agcg 24 <210> 9 <211> 1098 <212> DNA <213> Sus scrofa <400> 9 ttgctcacgg tagataacaa gagagactcc tcaaattcca caactgtgta ccccacaaac 60 aagccagcag tggaactagt caccccactt ccatccagca ccccagtaga caggacgtaa 120 tcttatcaac ctgtgaaata cccaccagac gtatcccagc ttcattcttg ggacatgtgt 180 tactgtggat agctagtgtg tgctttcatc tattgacgtg tacgttcaga agcagggaag 240 ggtccttgcc cccattcaag tatttcctca gggttgatat tgagggcctc gtccttggtg 300 gaaggcagag gggcctattg tcatggtttg agctctggaa tgagatttat catcccggtc 360 attcttggga gacgttattc ctctgttgga aaagactgga agggataagc tcactggtcc 420 ctctcagatg tatcataatc caaaagcaag cctgagccgt ggctatttcc tcagcagcct 480 gatgagaatg gtgggcatca gaagaatgct ggaaagaata cttgtcatcc aattgtccaa 540 tacatccaat ggatcctcct ttcatttagt atcaacatac ctccagatca tgagctcagg 600 ctgcccgagg taccacaaat gatgggtgac ggtgagagct tcccaccttg ggcccatcac 660 ttgcctctct tctcacagag agtcagtggt gtcacaatgc ttggtgggcg tcatgccatt 720 gccatggcca ccattgggcc tgaacttctc agatggaagg caactccacc tcctctgtgc 780 ccacctgctc tctacagaag gatggctctt cccctgaacg tgtgtcaggc tcaacacggt 840 ggttccagaa ctgcttccca ggaatacaca gtctacaatc ccatttggaa agaaaatatg 900 ggattgaaaa cgacccactg gctaaagatt gggcaagaga gcggaggagt agctggacac 960 gctgaacact tcataggtac ttttgtcggt agggtcccat atgggacgca gagagcaaca 1020 Page 4 1288780 gacttgaaac aacgttcaca tgagtgctct cccaagcaat ggcttctctg cagcattccc 1080 tacctggtcc gtgagcaa 1098 <210> 10 <211> 25 <212> DNA <213> Sus scrofa <400> 10 ttgctcacgg tagataacaa gagag 25 <210> 11 <211> 25 <212> DNA <213> Sus Scrofa <400> 11 ttgctcacgg accaggtagg gaatg 25 <210> 12 <211> 732

≪ 212 > DNA < 213 > Columba livia < 400 > 12 acttcgccac aatttcagtg gttcctgtac tgtattttac ctgtatcttt gtgaaatttt 60 tagcagtgta ggtcccgtat attgcctgta tacccgccac atattttgcg gtgcaggtaa 120 cgtattttac ctgtatgttt tcacattttt agcagtgtgt ttaccgtata tttcctgtat 180 attcgacaaa attttagcca tgcagctacc gtgttttacc tgtatgttct tcacatttta 240 agccatgcag ctttttcacc tgtatattca ccagtttttt acctgtatat tttaccagta 300 tattcaccag ttttttattg gttcagctac cgtattttac ctgtatattt gccgcatttt 360 tagtagtgtg tttcctgtat tttgctagta tcgtctcccc atttttatca gtggaagtag 420 catattttag ctgtatattc atcacagttt tagtggtgca gttaccatag tttacctgta 480 tattccccac ttctttatcg gtgcagctac ggtattttac ccgtatgttc accacatttt 540 page 5 1288780 tagtagtgcg tgtacagtat tttacctgta tagtcaccac gtttgatagt ggtgcaactg 600 ccgtatatta cctgtatatt catcacagtt ttagcggtgc aggtaccata ttcttcctat 660 acattcacca tatatttagc ggtgcagctc Ctgtatatta cctgtatatt tttacatttt 720 tagtggcgaa gt 732 )>>>> 0 12 3 1X 1A 1± 1A <2<2<2<2 13 25 DNA C Olumba livia <400> 13 acttcgccac aatttcagtg gttcc 25 <210> 14 <211> 25 <212> DNA <213> Columba livia <400> 14 acttcgccac taaaaatgta aaaat 25 b >>> 0 12 3 lx 11 11 11 < 2 < 2 < 2 < 2 15 685 DNA Cairina moschata < 400> 15 tcccggtctc tgttgttccc tgacagggct gaaagaagag gacaaaaacc atgaaaccaa accacataaa caaaaaaacc ctacccatat gctctgaaat gctctgaaca ttagtacatt agtatcatgg tatgcttctt atctgtaaag atgtgaaaaa ttgtttttct gatgcatttt gctgtaaacc ctgaacacaa tcaccgatgt catgttctgc aaaccagaat tcattccatt tacatcagtg caacctcatg Gtgtaaattc aggaaaggtc acccaaatta ggcctatgat ttattcttta gcataggagg cctgattctc agactcagat gtgccttgat tttccccaaa 60 120 180 240 300

Page 6 360 1288780 gtaaactgga ggtcaaatat tgcaccaata aaaacgactg gagattttta agataatggc 420 aatgggaagt cagaccgcta actgtttaac cacgctaact gtttaaacac cacaactgtt 480 taacttaaag acactaaacc gatgcatgtc atcacaaatt tcatgtcttc cttcctccag 540 ccatccttgt tttcccttta gtagttagtg taagctggca attagtaata ctttgcctgt 600 ttatagaacc ttccatctga ggatctagaa acactttgta aacgctaatg aactaagctt 660 cttagcaccc cctgtgagac cggga 685 > > >> 0 12 3 1X ix 1X 1X 2 2 2 2 << V < 16 1501

DNA

Cairina moschata < 400 > 16 tgagaagcgg tagaacggag agagtcattt ctttggtggt tcgggtttga gccctgggaa 60 ttcggtaaga agggggaggg cgaatttatt taggcctcaa taccttttta acccccccat 120 attgacggag acacaggagt gattccttgt tttgtatggg cttactaaga aagtgcgtga 180 gaaattcggt cctgttgaaa tgtaaatttt gtggaaaagg tggtaatgtt tgtctagaag 240 aggaaggctg agtgcttcag ctgttttcct gaagttccgt ggatttatga ttggaacggg 300 gctcattaat aatctgaaat agtaaagttt gtacgtggga ggaagcgttt aatccagaga 360 attgggacat tagggaagaa gattaggaaa agatagtaaa aaaaaaaaaa aaaaaaaaaa 420 aaacaacccg cagtaaaaag gtttgcgtgg aaattgaagc aaaggacagt aactagtaag 480 gaatagtaat aaacaaataa aagggaatgg gaaatcaagg aaatgggtaa tatccaaaac 540 aaagacgttt taaagaaagc ctccttgggt gcatattggc acattcggag gatattgtta 600 gaactggggg cacggaaagg aagaggactt tcactaagta tggtggccac ttgcattcaa tctttctaaa aggtacaaaa 780 660 tacataagct aaatggccac tttatggatc aatcgactat aacactgttt taagtgaggt 720 ctcaattcaa tctgaatttt tgtaatattt ataaaacaaa gctataaaag gtataaaagg Caagtaaaca gcgctgggtg tgcggggagt ctat gctcca 840 page 7 1288780 ccaacacgca cacacgaaca tcaaacattc taaatacatg tatacagaac attgctttac 900 ataataaccc gagtatgcct cagtaaagga gcccgcagct 1200 atacatacgt acaatcagga taggtaacaa acaatccttt 960 aagttccata acttaacagt ctccattttc cattcctttt cttgcttcag gccttatgta 1020 tcccgttctt cccagattga agaaaaacac gtccgtcttc ttgtggagac caagaggcct 1080 gggtcaaaag gcacgtccag gtcagcaggg ggcgtaacag caaaagcctt cgatggctgt 1140 agcagcagag aggccgatgg cgatggcgta gcagtcggat ccctcactgt ctggcaagcc acacgtttgt gattgtggct ccacatggct ctttaaggcc 1260 tcagagattg ctcgccaggt gccaagcaat cccactgcga ccttgtcatt tttagttgca 1320 gagtcccaca ccttgacctc agtttggtcc catacttcag gctcgtaaac tgtccaatcg 1380 ttaataagga gaataagccg taaggttaca ggacggtcct tgttcctaag gacgtatgat 1440 tccccataat gcgcaactgc ttaccagcga gaggcagttg tgtgcacagg tccgcttctc 1500 a 1501

<210> 17 <211> 21 <212> DNA <213> Cairina moschata <400> 17 tgttgttccc tgacagggct g 21

<210> 18 <211> 19 <212> DNA <213> Cairina moschata <400> 18 acagggggtg ctaagaagc 19 <210> 19 <211> 22 1288780

<212> DNA <213> Cairina moschata <400> 19 aagcggtaga acggagagag tc 22

<210> 20 <211> 20 <212> DNA <213> Cairina moschata <400> 20 aactgcctct cgctggtaag 20 <210> 21 <211> 939 <212> DNA <213> Anser anser < 400 > 21 ggggaagaca gacctaacag acagggattc actgaagtac atgcccatga attgcatcct 60 cccaattatc cagtctaaca attcactagc tattattcca tgctgagtta tcagggagta 120 gctgctttga acttactgcc atcttactcc tttcagattt actctctaca ctggaagtat 180 tcagttttct gacatcagta cccttccctg tattgcccgc tatgaataag cctgtttcaa 240 acacttgtac aaaatcaagt caacggtttt gttttaccac tatcatgatg gagcctccac 300 tgcaaaacaa cagaaatagc ttttgctttg ataaagtagt ctgacaaaca ctattccatt 360 aaaccgagaa gtccagcgct ttctgaaaaa cagtatttcc ttcaagcata ccttctttat 420 aaaactgaaa ttgggatgta aagggggtgg ggggagtgga gagaaggggc cacacatttc 480 ataacttgaa acatacttta gtttcaagaa taacaagata gttaagttca cttcatagaa 540 tcacagaata tcccaagttg gaaggcaccc acaagaatca agtacaaccc ctggacaaag 600 gaccatccaa aaaatctgac catgtgtctg agcactgttc aaatgcttcc tgaactgtaa 660 caggctcact gctgtgacca gttccccggg gagcctgttc cagtgcctga ccactctctc 720 page 9 1288780 agtgaagaac cttctcctga tatctagccc gaacctcccg tcacaacttc aaaccatttc 780 ctcaagtcct attgctggtc accagagaca agatcagcgc ctgaccctct gctccccctc 840 atgaggaagc tgtagaccgt gatgaggtca tccctcagtt tcctcttctc caggctgaac 900 aatacaagtg acttcagccg ctcctcatgt gtcttcccc 939 < 210> 22 < 211 > 21 < 212 > DNA <213> Anser anser <400> 22 gggaagacag acctaacaga c 21 <210> 23 <211> 21 <212> DNA <213> Anser anser <400> 23 agtcacttgt attgttcagc c 21

≪ 210 > 24 < 211> 1137 < 212 > DNA < 213 > Anas platyrhynchos < 400> 24 ggaacccaca cagtccaata aactcgattg tctctttcct ccccctggct ggaggcaggg 60 cccctctaat cctggtcata atattcatca ctgtggctga gggattgccc gctggaaatt 120 ggagcaacaa gttattcaga gaagcccctt tcccgattgg tttttcttgc aacttacata 180 cccgtgcctc tagggtcgag gtagatttcc catcccattt tctcatgtcc tcttcatggt 240 cacgtaggta aaaccacagg gtggcatggg gtgagtaccc actatattgt cttccttgaa 300 cagaaggttg cttacccctg atagctgaga cactgctttg tacaggtggg gaggaaaata 360 aactctcttt aagttggtgg acctcttcag aaagtttctc caaaatattc tcttttagtt 420 page 10 1288780 ggtggaactc ttgagaaagt cttccatagc cgagacacac gcctgtaggg aagataagag 480 atgttcttca aaatgccaga gttgtttagc catgttataa atagctcagt cccaaaatag 540 gattataatc aaagtttaca atttattaaa agaatagagg taagcaaaca gcactgggtg 600 Cgccgggagt ctccgctcca cctagtcgca cacccgttac atcaagatgc tgatttttta 660 tgctcctaga ctaatacaga ttcattacta tttctaaaaa aaaaaggagt tattataatt 720 agtatccgga atccaaaccc tcctactgga gcatacgtat cagtggtc cc tctgggggtc 780 tctgggggtc tttcatgctg aaggctcata gtcttcctct cagttttttt tcttgtcctt 840 cttctttgta ctccttggct gcatgtcaaa agcttacaca gcgtcccctg caagctttgt 900 cttttagctg tccttcagct tttcccttct ccttaatctc ctggccagat atcaggggct 960 tgcatagtgt cccattcaga agtcataaca gttagcagtt gttagcagtt gttctgaaac 1020 ccccagatat cagagacttc aaggaaagaa catacaaata cattttcctt caagctctct 1080 attttgacac gaattgttaa aacattcctc acagccactt catccactgt gggttcc 1137 0 12 3 1X IX Lx 1X 2 2 2 2 <<<< 25 25

DNA

Anas platyrhynchos <400> 25 acacagtcca ataaactcga ttgtc 25 7777 0 12 3 ix I _-Η·*1lx 2 2 2 2 << V < 26 25

DNA

Anas platyrhynchos <400> 26 tcgtgtcaaa atagagagct tgaag <210> 27 <211> 760 Page ll 25 1288780

≪ 212 > DNA < 213 > Struthio camelus < 400 > 27 cagccgttcc ttgaggccaa acaaatagca tatgaagaaa caatgggttt ctagcttgct ccctctggaa aaacagcaga aatattttga aataatatac ataatagcct acatctagcc actgtacata ggcaccatgt caccttttct aaaatgcagc gtcccaatgc tacagcacat tagtttatct ttcaaatctc tcatttttgt tgcacttctc tgaatctttt ctatttctgg gcaaccagat ggaacatagt tttcaaagca agggcatatt cataatattt tgtcagtttt ttttctttat ccatttccta tggtggtttt ttaacctctg Taactcactg agaaggagtt tatgactagt ctttttctga gtggttgcat tcaatttaga tcagggattt tttttccttt cagtacgtgt tgccttgtga gttgactaaa cacatacgtc caatctagga aacaagcaga gttagacaga tgccttaagc aaatgttttg ggaacggctg

<210> 28 <211> 19 <212> DNA <213> Struthio camelus <400> 28 cttgaggcca aacaaatag

<210> 29 <211> 19 <212> DNA <213> Struthio camelus <400> 29 Page 12 cactttctgt cacaaaagag 60 attgctgagt aaacgaatca 120 tacagccttc ctgtttacac 180 cactttgggt ctggcatgta 240 ttctttttct gttacttcag 300 tattttgtat tggaggtgag 360 atcagtttat ataatagcat 420 Atacaaccta acattattgt 480 ttcattatgc aatccaaaat 540 accacttgat atttagtagc 600 gtttttgtac ctactattgt 660 aaacttagtg tctacttcaa 720 760

19 1288780 19 ggtacaaaaa ctcacaagg第13页

Claims (1)

1288780 m Jji ^ r( , I; Pickup, Patent Range: 1 · An oligonucleotide primer pair of the ostrich sex-specific sequence of the ostrich includes the primer pair of SEQ ID NO: 28 / SEQ ID NO: 29. 2. The primer pair according to claim 1 of the patent application, further comprising an internal control group primer pair is SEQ ID NO: 1 / SEQ ID Ν〇 _ 2 2. 3 - a method for detecting the sex of an ostrich, The method includes: providing a sample; increasing the amplitude of the nucleic acid contained in the sample and the primer pair of SEQ ID N:28/SEQ...Ν〇Μ; and detecting the presence or absence of the amplification product to determine the sex of the scorpion The method wherein the internal control group primer pair in the sample is increased by 4. The nucleic acid contained in the third aspect of the patent application is a gender-specific primer pair and the amplitude is 0. : 1 / SEQ ID NO: 2 6. The sample according to any one of claims 3 to 5 is whole blood, blood cells, wing urine, semen or feathers. 7) as specified in item 6 of the patent application scope The sex sequence is SEQ IDN 〇 27. The method wherein the obtained sex is 8 · one is identified as its bird sex-specific SEQ ID NO. 27 sequence. /, contains early departure, pick up, schema: as the next page