CN110484635A - A kind of double Casuarius casuarius gender method for identifying molecules and primers of hanging down - Google Patents
A kind of double Casuarius casuarius gender method for identifying molecules and primers of hanging down Download PDFInfo
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- CN110484635A CN110484635A CN201910843635.0A CN201910843635A CN110484635A CN 110484635 A CN110484635 A CN 110484635A CN 201910843635 A CN201910843635 A CN 201910843635A CN 110484635 A CN110484635 A CN 110484635A
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Abstract
The invention discloses a kind of double vertical Casuarius casuarius gender method for identifying molecules and primers.A kind of double Casuarius casuarius gender Molecular Identification primers that hang down, CAW2 shown in CAW1 the and SEQ ID NO.2 shown in SEQ ID NO.1 are formed.Double vertical applications of the Casuarius casuarius gender Molecular Identification primer in double vertical Casuarius casuarius sex identifications of the present invention.A kind of double vertical Casuarius casuarius gender Molecular Identification kits, include primer of the present invention.I.e. blood sample can be used can also be used feather sample to the present invention, can obtain accurate qualification result.This method easily and effectively can carry out sex identification to double vertical Casuarius casuarius, and repeatability preferably, is laid a good foundation for the species in molecular level sex identification.
Description
Technical field
The invention belongs to field of biological detection, are related to a kind of double Casuarius casuarius gender Molecular Identification primer and kits of hanging down.
Background technique
Double vertical Casuarius casuarius (Casuarius casuarius) are classified as endangered species by IUCN, estimate its adult within 2011
Sum is at 2500 or so, and wherein wild individual is less than 1000.Double vertical Casuarius casuarius its be distributed mainly on New Guinea south and Australia
In the low land forest in big Leah the north, but with the increasing for situations such as increase of mankind's activity range, road and rail are built, hunted
Add, the quantity of double vertical Casuarius casuarius sharply declines.The representative of endemic species as rainforest flagship species peace chest mesh birds, double vertical cranes
Ostrich is selected as the preferential species for managing and breeding by the numerous well-known zoos in the whole world.Nanjing Red Hill forest zoo now possesses 23 pairs
It hangs down in Casuarius casuarius, adult has 6, has matched 2 to (2 male, 2 mothers), 17 individuals of Shang You do not complete sex identification and pairing, are
Largest double vertical Casuarius casuarius populations in national zoo.
Double vertical Casuarius casuarius belong to monomorphism bird, their gender is to be difficult to judge difference from appearance and other behaviors, therefore
Sexing birds protect Rare Birds, breeding has biggish meaning.Since double vertical Casuarius casuarius have certain aggressiveness, again
Belong to flat chest mesh, although male has the insertion organ an of penis, is difficult to observe.
At present, conventional identification birds property method for distinguishing turns over anus identification, steroids identification, karyotyping method, PCR identification
Deng.Inventor attempts to turn over anus 3 months the organ that double vertical Casuarius casuarius greatly have a kraftlos similar penis, the property of cannot be distinguished from
Not.Z and W karyotype difference is found not when the karyotyping Casuarius casuarius sex chromosome such as Chizuko Nishida-Umehara
Greatly, it is difficult to distinguish.With the development of molecular engineering, the application of polymerase chain reaction (PCR) amplification is so that prominent chest mesh gender
Identification makes a breakthrough, wherein more certified sequence has CHD1 gene, ATP5A1 gene, EE0.6 sequence, the technology
Extensive utilization is to sex identifications such as flamingo, Mei Zhou Hong Ibis, crane classes.Due to the gene of flat chest mesh sex chromosome and sex identification
Sequence difference is less, so commonly prominent chest mesh sex identification primer is not used to flat chest purpose sex identification.Chean-Ping
W etc. establishes the RAPD method of female ostrich sex identification.Pay the molecule labelling method that crystalline substance etc. establishes emu sex identification.
Huynen etc. is marked using RAPD, is carried out using random primer K1 and K7 to existing ratite class (including ostrich and emu)
The identification of gender, female individuals obtain the band of about 350bp or so, although these methods can distinguish the property of ratite class
Not, but the label repeatability is bad.Therefore, being badly in need of one kind easily and effectively can carry out sex identifications to double vertical Casuarius casuarius, repeat
Property preferable method and reagent, provide technical basis for the double vertical captive breedings of Casuarius casuarius species in industry, ensure stable breeding population
Scientific breeding, to realize preferably in situ conservation.
Summary of the invention
The purpose of the present invention is being directed to the above-mentioned deficiency of the prior art, a kind of double vertical Casuarius casuarius gender Molecular Identification sides are provided
Method.
It is a further object of the present invention to provide a kind of double vertical Casuarius casuarius gender Molecular Identification primers.
It is yet another object of the invention to provide a kind of double vertical Casuarius casuarius gender Molecular Identification kits.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of double vertical Casuarius casuarius gender Molecular Identification primers, shown in CAW1 the and SEQ ID NO.2 as shown in SEQ ID NO.1
CAW2 composition.
Double vertical applications of the Casuarius casuarius gender Molecular Identification primer in double vertical Casuarius casuarius sex identifications of the present invention.
A kind of double vertical Casuarius casuarius gender Molecular Identification kits, include primer of the present invention.
The kit is preferably also comprising the PCR conventional reagent in addition to primer.
A kind of double vertical Casuarius casuarius gender method for identifying molecules, include the following steps:
(1) double vertical Casuarius casuarius genomic DNAs are extracted；
(2) genomic DNA extracted using step (1) carries out PCR amplification using primer of the present invention as template；
(3) pcr amplification product electrophoresis on 2% Ago-Gel indicates this individual gender without band in electrophoresis result
For male, biobelt, which is presented, in 1000-750bp, 500-750bp indicates that this individual gender is female.
PCR reaction system is preferred are as follows: 12.5 0.5 0.5 μ L of μ L, CAW2 of μ L, CAW1 of Mix, 6.5 μ L, DNA mould of distilled water
5 μ L of plate.
PCR reaction condition is preferred are as follows: 94 DEG C of 1min；94 DEG C of 15s, 55 DEG C of 20s, 72 DEG C of 20s, 10 circulations；94℃
15s, 50 DEG C of 20s, 72 DEG C of 20s, 10 circulations；94 DEG C of 15s, 47 DEG C of 20s, 72 DEG C of 20s, 20 circulations；72℃ 2min.
The utility model has the advantages that
Herein for the kW1 sequence of double vertical Casuarius casuarius, devises specific primer (caw1/caw2) and carry out sex identification.
The present invention verifies the validity of the primer using the individual of 2 known genders, with the specificity being related to
Primer (caw1/caw2) is expanded, and agarose gel electrophoresis detection female is 2 bands, and 1 is 750bp or so band, male
Property is without band, and negative control (DNA profiling is replaced with distilled water) is without band.Therefore, this method can easily and effectively to Casuarius casuarius into
Row sex identification (such as Fig. 1).I.e. blood sample can be used can also be used feather sample to the present invention, can obtain accurately identifying knot
Fruit.This method easily and effectively can carry out sex identification to double vertical Casuarius casuarius, and repeatability is the species in molecular level gender preferably
Identification is laid a good foundation.
Detailed description of the invention
Fig. 1 validation verification
Male 2,5. 2,6. blank pair of male sample of sample Isosorbide-5-Nitrae female sample of the female sample 1,3. of 1.2000Maker 2.
According to Fig. 2 repetitive test
1.2000Maker 2-4. male sample 1,5-7. female sample 1,8-10. male sample 2,11-13. female sample
The bis- Casuarius casuarius blood sample sex identifications of hanging down of 2 Fig. 3
1.2000Maker 2-18. is followed successively by 1-17 childhood double vertical Casuarius casuarius, and the double vertical Casuarius casuarius of 19. males, 20. females are double to hang down
The bis- Casuarius casuarius feather sex identifications of hanging down of Casuarius casuarius Fig. 4
1.2000Maker 2-5.4 double vertical Casuarius casuarius feather samples, the double Casuarius casuarius of hanging down of 6. females, the double vertical Casuarius casuarius of 7. males
Specific embodiment
1. materials and methods
1.1 material
17 childhood unknown gender, 4 known genders of the sample under the Red Hill forest zoo cage conditions of Nanjing
Double vertical Casuarius casuarius blood samples and 4 unknown genders double vertical Casuarius casuarius hairs；Wherein, 4 parts of blood samples are from two pairs by the bodily form
State and behavior carried out sex identification, and the double vertical Casuarius casuarius of adult of gender are confirmed by breeding.Sampling mode is anticoagulant
5 μ l of whole blood, 5, the abdomen plumage with hair follicle.
1.2 main agents and instrument
Poba gene group extracts kit, minigene group extracts kit, Taq Plus MasterMix, D2000
DNA Marker, GeneGreen nucleic acid dye are purchased from TIANGEN Biotech (Beijing) Co., Ltd..Primer (caw1/caw2) is equal
There is the synthesis of Jin Sirui biology Co., Ltd.
Grads PCR instrument is purchased from Hangzhou Long Yang instrument company.Gel imaging system is purchased from Shanghai Ou Xiang instrument company.Horizontal electricity
Instrument of swimming is purchased from the east Beijing Jun Yi Co., Ltd.High speed frigorimeter centrifuge is purchased from Shanghai Hu Yueming scientific instrument Co., Ltd.
1 pair, table vertical Casuarius casuarius sex identification primer sequence
1.3 experimental method
1.3.1 extracting genome DNA
The genomic DNA in double vertical Casuarius casuarius blood is extracted by poba gene group extracts kit specification.Base after extraction
It is spare because group DNA sample is saved in -20 DEG C.
1.3.2 efficiency assay
Double vertical Casuarius casuarius genomes of known gender and blank control are expanded using primer (caw1/caw2).Anti-
Answer system are as follows: Mix 12.5 μ L, CAW1 0.5 μ L, CAW2 0.5 μ L, 6.5 μ L of distilled water, 5 μ L of DNA profiling.Reaction condition are as follows:
94℃ 1.5min；94 DEG C of 20s, 55 DEG C of 20s, 72 DEG C of 20s, 10 circulations；94 DEG C of 20s, 50 DEG C of 20s, 72 DEG C of 20s, 10
Circulation；94 DEG C of 20s, 47 DEG C of 20s, 72 DEG C of 20s, 20 circulations；72℃ 2min.
10 μ l pcr amplification products are taken, electrophoresis 30min, is clapped by gel imager on 2% Ago-Gel
According to record result.In electrophoresis result, no band indicates that this individual gender is male, and about 500-750bp is indicated stripe size respectively
This individual gender is female.
1.3.3 objective gene sequence measurement and analysis
It send Jin Sirui Bioisystech Co., Ltd to clone PCR product, and is connected to carrier T and is sequenced.
1.3.4 repetitive test
PCR amplification is carried out to the sample of 4 known genders (2 female 2 is male), three times, agarose is solidifying for each sample repeat amplification protcol
Gel electrophoresis detection.
1.3.5 double vertical Casuarius casuarius sex identifications
PCR amplification, agar are carried out using the double vertical Casuarius casuarius blood samples of 17 unknown genders of the primer pair and 4 parts of feather samples
Sugared detected through gel electrophoresis.
2. result
2.1 primer validation verifications
The validity of the primer is verified using the individual specimen (2 female 2 is male) of 4 known genders, uses primer
(caw1/caw2) it is expanded, agarose gel electrophoresis detection female is 1 band, and 1 is 750bp or so band, male nothing
Band, negative control (DNA profiling is replaced with distilled water) is without band.Therefore, this method can easily and effectively to Casuarius casuarius progressive
It Jian Ding (such as Fig. 1).
2.2 repetitive test
Repetitive test carried out to the sample of 4 known genders (2 female 2 is male), each sample repeat amplification protcol three times, agarose
Detected through gel electrophoresis institute is consistent (such as Fig. 2) at result
2.3 pairs of vertical Casuarius casuarius gender blood sample qualification results
Identified that identification of blood result such as Fig. 3,17 unknown using the individual of the double vertical unknown genders of Casuarius casuarius of the primer pair
There are 9 in double vertical Casuarius casuarius of gender for female, 8 are male.
2.4 nucleotide sequence analysis
Jin Sirui Bioisystech Co., Ltd is sent to carry out cloning and sequencing sequencing the PCR product of purpose band.Sequencing result
Display sequence size is 715bp (shown in SEQ ID NO.3).
2.5 pairs of vertical Casuarius casuarius gender feather qualification results
The genomic DNA that double vertical Casuarius casuarius feathers are extracted by minigene group extracts kit specification, utilizes caw1/caw2
Primer pair carries out sex identification to four in double vertical Casuarius casuarius of 17 unknown genders (No. 12-15 double vertical Casuarius casuarius in 2.3).Identification
As a result see Fig. 4, it is consistent with blood sample qualification result.
Sequence table
<110>Nanjing Red Hill forest zoo administrative office
<120>a kind of double vertical Casuarius casuarius gender method for identifying molecules and primer
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gacttggtgg atccgcaaca gttt 24
<210> 2
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tccgcaacag ttggaattct gagaa 25
<210> 3
<211> 715
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gacttggtgg atccgcaaca gttttcagct tcctttgtgg tgggaaggga aacaaaggta 60
gtcaagggga caggttctct agtgcacatc aacttcagtg ctggtagctt tgcacctctt 120
gagccagatg gtttaaactg cttcaatttg tccatggaca acagtcactg aagtatagac 180
gtagcctaag tcttctgggt ggcctgccaa gtggtgggct ctcagctctg gggtgcttgt 240
tttcaggtct tcccttgcct gtggtcttaa cacagtgcat atgaacagat actttaatgg 300
agaataaccc tgcaagaaaa tagcatgagg ccaccccttc cttctttgta ttgctagggg 360
acaattataa ctctgtgagt aacaggtata ttgaaagccc ttgcactgcc agatgctctc 420
gtcactgaaa ctgtaccacc aagacactct tggagctggt cccctgccag tctaggtgaa 480
agaaataggg ttcattggaa tcagagcttc ttactcatat aatccacaaa aagaaataat 540
cctggaaaat gtatgcttcc aatatacaat ttctaaatct catgtggtcc gagacctgca 600
tatgaaccta gttcgcatga attctaagct gttggcagat ccagctggat tcatttcctt 660
ttctaaactg ttgcggatcc accaagtcaa tctctagagg atccccgggt accga 715
Claims (7)
1. a kind of double vertical Casuarius casuarius gender Molecular Identification primers, it is characterised in that CAW1 the and SEQ ID as shown in SEQ ID NO.1
The composition of CAW2 shown in NO.2.
2. double vertical applications of the Casuarius casuarius gender Molecular Identification primer in double vertical Casuarius casuarius sex identifications described in claim 1.
3. a kind of double vertical Casuarius casuarius gender Molecular Identification kits, it is characterised in that include primer described in claim 1.
4. kit according to claim 3, it is characterised in that also comprising the PCR conventional reagent in addition to primer.
5. a kind of double vertical Casuarius casuarius gender method for identifying molecules, it is characterised in that include the following steps:
(1) double vertical Casuarius casuarius genomic DNAs are extracted；
(2) genomic DNA extracted using step (1) carries out PCR amplification using primer described in claim 1 as template；
(3) pcr amplification product electrophoresis on 2% Ago-Gel indicates that this individual gender is hero without band in electrophoresis result
Property, purpose band, which is presented, in 500-750bp indicates that this individual gender is female.
6. double vertical Casuarius casuarius gender method for identifying molecules according to claim 5, it is characterised in that PCR reaction system are as follows: Mix
12.5 μ L, CAW1 0.5 μ L, CAW2 0.5 μ L, 6.5 μ L of distilled water, 5 μ L of DNA profiling.
7. double vertical Casuarius casuarius gender method for identifying molecules according to claim 5, it is characterised in that PCR reaction condition are as follows: 94
℃1.5min；94 DEG C of 20s, 55 DEG C of 20s, 72 DEG C of 20s, 10 circulations；94 DEG C of 20s, 50 DEG C of 20s, 72 DEG C of 20s, 10 circulations；94
DEG C 20s, 47 DEG C of 20s, 72 DEG C of 20s, 20 circulations；72℃2min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111621574A (en) * | 2020-05-29 | 2020-09-04 | 广州动物园 | Primer for identifying sex of ostrich animals, sex identification method and kit |
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| US5377618A (en) * | 1992-07-31 | 1995-01-03 | Reproductive Sciences, Inc. | Method for preferential production of ratites of a desired sex |
| CN102356158A (en) * | 2008-12-17 | 2012-02-15 | 联邦科学技术研究组织 | Methods of modulating the sex of avians |
| CN106701910A (en) * | 2015-11-18 | 2017-05-24 | 深圳华大农业与循环经济科技有限公司 | Primer pair, kit and method for identifying gender of dove |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111621574A (en) * | 2020-05-29 | 2020-09-04 | 广州动物园 | Primer for identifying sex of ostrich animals, sex identification method and kit |
| CN111621574B (en) * | 2020-05-29 | 2022-11-11 | 广州动物园 | Primer for identifying sex of ostrich animals, sex identification method and kit |
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