CN107385090A - A kind of molecular labeling, authentication method and application for identifying pilose antler kind - Google Patents
A kind of molecular labeling, authentication method and application for identifying pilose antler kind Download PDFInfo
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- CN107385090A CN107385090A CN201710800319.6A CN201710800319A CN107385090A CN 107385090 A CN107385090 A CN 107385090A CN 201710800319 A CN201710800319 A CN 201710800319A CN 107385090 A CN107385090 A CN 107385090A
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Abstract
The invention discloses a kind of molecular labeling for identifying pilose antler kind, including 16 SNP, its sequence is respectively as shown in SEQ ID NO.1 SEQ ID NO.16.The invention also discloses a kind of methods and applications for identifying pilose antler kind.Present invention firstly discovers that the molecular labeling shown in SEQ ID NO.1 SEQ ID NO.16, it can be very good to distinguish cervus elaphus linnaeus, sika deer velvet antler and hybridization pilose antler, hybridization young pilose antler within 5 generations identifies that success rate is more than 95%, reliability is high, to ensure that consumer's lawful right provides a kind of most economical effective Molecular Identification new way, it may also be used for hybridization deer molecular marker assisted selection breeding and cultivar identification.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to a kind of molecular labeling for identifying pilose antler kind, identification
Method and application.
Background technology
Pilose antler is that stag is unossified and young horn with fine hair, is rare medicinal herbs.In pilose antler containing phosphatide, glycolipid, glue fat, swash
The composition such as element, aliphatic acid, amino acid, protein and calcium, phosphorus, magnesium, sodium, wherein aminoacid ingredient account for more than half of total composition.
According to《Chinese Pharmacopoeia》Record, the pilose antler kind that can be used as medicine has pilose antler to have cervus elaphus linnaeus and sika deer velvet antler.Cervus elaphus linnaeus is by red deer
The pilose antler grown, typically all bigger than normal, weight is typically between 10-50 jin, and the color of cervus elaphus linnaeus is partially black, and only body is larger, fine hair
It is long and black；Sika deer velvet antler is the pilose antler that sika deer grows, and shape is generally single branch, and two thick sticks, trident, ornamental value is high, Er Qiemei
The economic value of pilose antler of sika is also higher than red deer.
Red deer and sika deer are two deer kinds, positive and negative can hybridize and offspring can breed, turn into hybridization deer.Hybridize deer
The hybridization young pilose antler outward appearance grown has sika deer velvet antler feature, but its market price is less than sika deer velvet antler, so easily by illegal point
Son is useed sika deer velvet antler as and sold, and cheats consumer.
In the prior art, the quality of pilose antler quality is identified in terms of color and luster, quality, smell are fixed mostly, judges pilose antler species,
But still lack a kind of authentication method of science.Therefore, it is imperative to invent a kind of method for identifying sika deer and red deer hybridization young pilose antler.
The content of the invention
A kind of molecular labeling, authentication method and application for identifying pilose antler kind provided by the invention, can be identified different
Sika deer, red deer and the pilose antler kind for hybridizing deer.
First purpose of the present invention is to provide a kind of molecular labeling for identifying pilose antler kind, including 16 SNP, its sequence
Respectively as shown in SEQ ID NO.1-SEQ ID NO.16.
Second object of the present invention is to provide a kind of method using above-mentioned molecular markers for identification pilose antler kind, its feature
It is, comprises the following steps：
S1, extract material genomic DNA to be identified；
S2, it is utilized respectively each self-corresponding primers of SEQ ID NO.1-SEQ ID NO.16 and enters performing PCR amplification, respectively obtain
16 groups of amplified productions；
Wherein, the primer sequence used corresponding to the molecular labeling for obtaining SEQ ID NO.1-SEQ ID NO.16 is respectively such as
SEQ ID NO.17-SEQ ID NO.18、SEQ ID NO.19-SEQ ID NO.20、SEQ ID NO.21-SEQ ID
NO.22、SEQ ID NO.23-SEQ ID NO.24、SEQ ID NO.25-SEQ ID NO.26、SEQ ID NO.27-SEQ ID
NO.28、SEQ ID NO.29-SEQ ID NO.30、SEQ ID NO.31-SEQ ID NO.32、SEQ ID NO.33-SEQ ID
NO.34、SEQ ID NO.35-SEQ ID NO.36、SEQ ID NO.37-SEQ ID NO.38、SEQ ID NO.39-SEQ ID
NO.40、SEQ ID NO.41-SEQ ID NO.42、SEQ ID NO.43-SEQ ID NO.44、SEQ ID NO.45-SEQ ID
Shown in NO.46, SEQ ID NO.47-SEQ ID NO.48；
(1) PCR amplification system is 10 μ L：Including μ L of DNA 0.2, μ L of forward primer 0.5, the μ L of reverse primer 0.5；2×Tap
PCR Master Mix 5.0μL；ddH2O 3.8μL；
(2) PCR amplification conditions：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 60 DEG C of annealing 45s, 72 DEG C of extension 45s；Follow
Ring 35 times；72 DEG C extend 10min, 4 DEG C of cooling 10min eventually；
16 groups of amplified productions 100V electrophoresis 35min on 2% Ago-Gel, and being seen under gel imaging system respectively
Examine, 16 groups of 101bp amplified production is separately recovered；
S3, sequencing identification
16 groups of amplified production nucleotide sequences of sequencing identification respectively, if the genotype of corresponding SNP site is and sika deer
Homozygote genotype is identical, then detected materials are sika deer；If identical with red deer homozygote genotype, detected materials are
Red deer；If being heterozygote, detected materials are hybridization deer.
Third object of the present invention is to provide a kind of molecular labeling of above-mentioned identification pilose antler kind in deer seed selection is hybridized
Application.
It is auxiliary in hybridization deer molecular labeling that third object of the present invention is to provide a kind of method of above-mentioned identification pilose antler kind
The application helped in seed selection.
Compared with prior art, a kind of molecular labeling, authentication method and application for identifying pilose antler kind provided by the invention,
Have the advantages that：
In the prior art, the quality of pilose antler quality is identified in terms of color and luster, quality, smell are fixed mostly, judges pilose antler species,
But still lack a kind of authentication method of science.Therefore, it is imperative to invent a kind of method for identifying sika deer and red deer hybridization young pilose antler.
Present invention firstly discovers that the molecular labeling shown in SEQ ID NO.1-SEQ ID NO.16, can be very good to distinguish cervus elaphus linnaeus, plum
Pilose antler of sika and hybridization pilose antler, the hybridization young pilose antler within 5 generations identify that success rate is more than 95%, and reliability is high, to ensure that consumer closes
Method right provides a kind of most economical effective Molecular Identification new way.It can also be used to hybridize deer molecular marker assisted selection breeding
And cultivar identification.
Embodiment
With reference to specific embodiment, the present invention is described in detail, but should not be construed as the limitation of the present invention.It is following
Experimental method in embodiment, it is conventional method unless otherwise specified, material used, reagent etc. in following embodiments, such as
Without specified otherwise, commercially obtain.
A kind of molecular labeling for identifying pilose antler kind provided by the invention, including 16 SNP, its sequence is respectively such as SEQ ID
Shown in NO.1-SEQ ID NO.16.Two bases wherein in " [] " represent SNP site, if the genotype of testing sample be with
Base identical homozygote before "/", then the testing sample is sika deer；If the genotype of testing sample is and base phase after "/"
Same homozygote, then the testing sample is red deer；If the genotype of testing sample is the heterozygote of two bases in " [] ",
The testing sample is the filial generation of sika deer and red deer, that is, hybridizes deer.
First, the acquisition of molecular labeling：
S1, the advanced generation cross deer one obtained after sika deer, the positive and negative hybridization of red deer and the pilose antler base for hybridizing deer two are extracted respectively
Because of group, extraction sika deer, the pilose antler genomic DNA of red deer；The another genomic DNA for taking other 96 hybridization deer, above-mentioned plum
Hua Lu, red deer are maternal and male parent is commercially available.
S2, by simplifying positioning results of the Reads of gene order-checking (dd-RADseq) in reference gene group, utilize
GATK softwares carry out local comparison (Local Realignment) again and variation detection, while are become using STACKS softwares
Different detection, takes GATK softwares and the step such as common factor variant sites that two methods of STACKS software obtain, to ensure that detection obtains
SNP (single nucleotide polymorphism, SNP) accuracy, plum is detected by large sample
The special SNP site of species on Hua Lu, red deer and hybridization deer genome, while the chain situation of SNP site is detected, it is final to amount to
Filter out the distance difference in reference gene group and be far not susceptible to 16 chain SNP candidate bits fine and soft as identification hybridization
Point.
S3, the snp analysis of different pilose antler kinds
It is as shown in table 1 to the SNP genotype of the pilose antler of sika deer, red deer, hybridization deer one and hybridization deer two.
The snp analysis of the different pilose antler kinds of table 1
| SNP sequences | Sika deer | Red deer | Hybridize deer one | Hybridize deer two |
| SEQ ID NO.1 | T | G | T/G | T/G |
| SEQ ID NO.2 | G | A | G/A | G/A |
| SEQ ID NO.3 | T | C | T/C | T/C |
| SEQ ID NO.4 | T | C | T/C | T/C |
| SEQ ID NO.5 | A | G | A/G | A/G |
| SEQ ID NO.6 | T | G | T/G | T/G |
| SEQ ID NO.7 | T | C | T/C | T/C |
| SEQ ID NO.8 | T | G | T/G | T/G |
| SEQ ID NO.9 | A | G | A/G | A/G |
| SEQ ID NO.10 | A | G | A/G | A/G |
| SEQ ID NO.11 | A | C | A/C | A/C |
| SEQ ID NO.12 | G | A | G/A | G/A |
| SEQ ID NO.13 | G | A | G/A | G/A |
| SEQ ID NO.14 | C | A | C/A | C/A |
| SEQ ID NO.15 | T | C | T/C | T/C |
| SEQ ID NO.16 | T | C | T/C | T/C |
2nd, comprised the following steps that using the method for above-mentioned SNP Molecular Identifications pilose antler kind：
S1, using CTAB methods material genomic DNA to be identified is extracted, 4 DEG C of refrigerators save backup.
In use, the concentration of the agar sugar detection DNA with 0.8%, is diluted to working concentration and is expanded for PCR.
S2, be utilized respectively each self-corresponding primers of SEQ ID NO.1-SEQ ID NO.16 enter performing PCR amplification, respectively obtain
16 groups of amplified productions.Wherein, the primer sequence used corresponding to the molecular labeling for obtaining SEQ ID NO.1-SEQ ID NO.16
Respectively such as SEQ ID NO.17-SEQ ID NO.18, SEQ ID NO.19-SEQ ID NO.20, SEQ ID NO.21-SEQ ID
NO.22、SEQ ID NO.23-SEQ ID NO.24、SEQ ID NO.25-SEQ ID NO.26、SEQ ID NO.27-SEQ ID
NO.28、SEQ ID NO.29-SEQ ID NO.30、SEQ ID NO.31-SEQ ID NO.32、SEQ ID NO.33-SEQ ID
NO.34、SEQ ID NO.35-SEQ ID NO.36、SEQ ID NO.37-SEQ ID NO.38、SEQ ID NO.39-SEQ ID
NO.40、SEQ ID NO.41-SEQ ID NO.42、SEQ ID NO.43-SEQ ID NO.44、SEQ ID NO.45-SEQ ID
Shown in NO.46, SEQ ID NO.47-SEQ ID NO.48；It is specific as shown in table 2.
It should be noted that using the primer in table 2 expand obtained 16 groups of amplified production fragments 300-600bp it
Between, 101bp sequence is the characteristic sequence of deer corresponding to SEQ ID NO.1-SEQ ID NO.16, included in amplified production fragment
Within, by amplified production respectively with corresponding when sequence alignment, SEQ ID NO.1-SEQ ID NO.16 molecular labeling phases
Contrast.
The primer sequence of the different molecular of table 2 mark
(1) PCR amplification system is 10 μ L：Including the μ L of DNA 0.2 (1ng/ μ L), μ L of forward primer 0.5, the μ of reverse primer 0.5
L；2 × Tap PCR Master Mix (band dyestuff, health are bought for ShiJi Co., Ltd) 5.0 μ L；ddH2O 3.8μL。
The concentration of wherein above-mentioned primer is 4pmol/ μ L.
(2) PCR amplification conditions：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 60 DEG C of annealing 45s, 72 DEG C of extension 45s；Follow
Ring 35 times；72 DEG C extend 10min, 4 DEG C of cooling 10min eventually.
16 groups of amplified productions 100V electrophoresis 35min on 5% Ago-Gel, and being seen under gel imaging system respectively
Examine, record result.
S3, pilose antler kind is judged according to electrophoresis result
Respectively the above-mentioned 16 groups of amplified production nucleotide sequences of sequencing identification, if the genotype of corresponding SNP site with table 1
Shown sika deer homozygote genotype is identical, then detected materials are sika deer；If with red deer homozygote genotype shown in table 1
Identical, then detected materials are red deer；If being heterozygote shown in table 1, detected materials are hybridization deer.
The filial generation of red deer and sika deer and sika deer are subjected to grading up, and in theory (50% recombination fraction, SNP points
Type success rate 95%), it can identify the fine and soft relation for hybridizing algebraically and accuracy of hybridization using 16 SNP markers of the present invention
It is shown in Table 3.
Table 3 carries out grading up with sika deer
The result of table 3 shows, identifies that success rate is 95% by the hybridization young pilose antler within the generation of sika deer grading up 5, although into
Power reduces with algebraically increase is hybridized, but experience have shown that it is more than generation to hybridize 5 for this mode, hybridization young pilose antler and spotted deer antler are outside
It is basically identical in table shape, color and nutritional ingredient.Therefore, commercially, pilose antler is hybridized within 5 generations and can obtain reliable mirror
Determine result.
It should be noted that when being related to number range in claims of the present invention, it is thus understood that each number range
Any one numerical value can be selected between two end points and two end points, due to step method and above-described embodiment phase of use
Together, in order to prevent from repeating, description of the invention preferred embodiment, but those skilled in the art once know substantially
Creative concept, then other change and modification can be made to these embodiments.So appended claims are intended to be construed to wrap
Include preferred embodiment and fall into having altered and changing for the scope of the invention.
Obviously, those skilled in the art can carry out the essence of various changes and modification without departing from the present invention to the present invention
God and scope.So, if these modifications and variations of the present invention belong to the scope of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to comprising including these changes and modification.
Sequence table
<120>A kind of molecular labeling, authentication method and application for identifying pilose antler kind
<160> 64
<170> SIPOSequenceListing 1.0
<210> 1
<211> 101
<212> DNA
<213>Deer
<400> 1
tcattgataa tgactccatt ttggcgggag ggccatcgag catccatctg [t/g]gcacagtgg 60
tgctcctgtg ggtacaggct ggctgccctg cagctgggtc c 101
<210> 2
<211> 101
<212> DNA
<213>Deer
<400> 2
taccaaaaca atgacaaggt tttctgcagg ctcttcccat cacatagtct [g/a]aggctcctc 60
cgtcgggagt ctcagctgta aagcgacccc tactgttcct a 101
<210> 3
<211> 101
<212> DNA
<213>Deer
<400> 3
ggaactgaga gcacctcctg cccatgccca caaacccaca ctcactcttt [t/c]tcctgtaga 60
tttgttccag ctgctcagat gatgacgttt gcctcctctg g 101
<210> 4
<211> 101
<212> DNA
<213>Deer
<400> 4
ctctactatt aacaaacagt tcaagaagaa atgaaggaag ctgaatcccc [t/c]tggggacat 60
actcagtttt gccccagtgg gatcttagca gcatctgaga g 101
<210> 5
<211> 101
<212> DNA
<213>Deer
<400> 5
agcctagagg gagaggcaga gaggccctgc aggccagtga gttgtgacat [a/g]ggagtctga 60
ggaacctgct ccaggcagcc gggagcacag caggggccgc a 101
<210> 6
<211> 101
<212> DNA
<213>Deer
<400> 6
aagctgcccg tctccgtggg gcatgaagaa gcagtgtttt ctgcagcatg [t/g]tagtttgct 60
ggagaacatc aaagtaggtg acgtcagggt gcctcctcca g 101
<210> 7
<211> 101
<212> DNA
<213>Deer
<400> 7
tatctgtttg gtctgcattg ggagaacaca tccattcaga tgctgcagca [t/c]aatcattct 60
tgtgaccata aaattatcag cgaagccaag ccctacatct t 101
<210> 8
<211> 101
<212> DNA
<213>Deer
<400> 8
gtgagcaaag tagtagatgt gggtattggc atcaaactta atgacatcac [t/g]aaagcagtt 60
tacatcagaa cactgctctt cttcagctgc taatgctgcc a 101
<210> 9
<211> 101
<212> DNA
<213>Deer
<400> 9
ctgtgggctc atgacccctg ctctggtggc cctgccaggg ggcctcaggg [a/g]ctctgaaat 60
ctagaaggga gcagcccccg aacacacctg gctccaaggt c 101
<210> 10
<211> 101
<212> DNA
<213>Deer
<400> 10
acagctggtg aggagctgga cttcctcccc tgcccaggct ggctctgtga [a/g]agtgctggg 60
agctagaaca gagtggcagg gtctgtgaag gatgctcaag t 101
<210> 11
<211> 101
<212> DNA
<213>Deer
<400> 11
ccagggccac ccctgcagct aagcaagggg gccagcttgc aatgcaagaa [a/c]ttgagggta 60
taaatcaaag tgtcagcatc catgcctggg cttctttctc t 101
<210> 12
<211> 101
<212> DNA
<213>Deer
<400> 12
gcttctcaca aaactgtatg aatttctggt attaggtcat ccataaatac [g/a]cataaatca 60
gggtacagat ttccacatct cagattcact gtcttcttgg a 101
<210> 13
<211> 101
<212> DNA
<213>Deer
<400> 13
ggccgaagcc aagaaagcag acttggggcc ccttggctca acacctttaa [g/a]gctggttac 60
gagtccagga tgtttttctc cagggcctgg ggagcccctg a 101
<210> 14
<211> 101
<212> DNA
<213>Deer
<400> 14
cttctatatt ttttaaagca acaccttcca tattgcactg cagttattgt [c/a]cttacacat 60
cattcttaca cacaaaaaag gatcatgcag ttatctatac t 101
<210> 15
<211> 101
<212> DNA
<213>Deer
<400> 15
gtccccctgc ggggcactct gaccctgggc aactcacagt gtctagtcat [t/c]accttcccc 60
ctggcctcat tagagtaggc acagatgggg acctcagaaa a 101
<210> 16
<211> 101
<212> DNA
<213>Deer
<400> 16
gaagggggag aaaagtctgt taagttcccc tccctactca gcagtgtgaa [t/c]gctagtccc 60
ctctggctct gggaggaaca gcgtcagcag ttgtggtctg c 101
<210> 17
<211> 20
<212> DNA
<213>Artificial sequence
<400> 17
agaagttccc tcctcgttga 20
<210> 18
<211> 18
<212> DNA
<213>Artificial sequence
<400> 18
gagtaagatg ttacctgg 18
<210> 19
<211> 21
<212> DNA
<213>Artificial sequence
<400> 19
ggagggtggg ctttttgagc c 21
<210> 20
<211> 19
<212> DNA
<213>Artificial sequence
<400> 20
ctctgatacc ctctggtca 19
<210> 21
<211> 22
<212> DNA
<213>Artificial sequence
<400> 21
tccactgcag gaacactgcc tg 22
<210> 22
<211> 20
<212> DNA
<213>Artificial sequence
<400> 22
accatccagg tgcctcataa 20
<210> 23
<211> 22
<212> DNA
<213>Artificial sequence
<400> 23
gagaattagg aaacataaag ga 22
<210> 24
<211> 19
<212> DNA
<213>Artificial sequence
<400> 24
tggaatagtt gagacctct 19
<210> 25
<211> 23
<212> DNA
<213>Artificial sequence
<400> 25
aaggtggctg aaggtctgct ccg 23
<210> 26
<211> 16
<212> DNA
<213>Artificial sequence
<400> 26
gactcctctc cgtttg 16
<210> 27
<211> 21
<212> DNA
<213>Artificial sequence
<400> 27
taactgactg ggggctatgg g 21
<210> 28
<211> 17
<212> DNA
<213>Artificial sequence
<400> 28
gatgcaaata tggtccg 17
<210> 29
<211> 19
<212> DNA
<213>Artificial sequence
<400> 29
taccaaaaca atgacaagg 19
<210> 30
<211> 18
<212> DNA
<213>Artificial sequence
<400> 30
aggctcctcc gtcgggag 18
<210> 31
<211> 22
<212> DNA
<213>Artificial sequence
<400> 31
gtcagcgcct tcttattaca gc 22
<210> 32
<211> 20
<212> DNA
<213>Artificial sequence
<400> 32
cgtccaggcc ccggcatgag 20
<210> 33
<211> 21
<212> DNA
<213>Artificial sequence
<400> 33
tgtagttaat gacagactta g 21
<210> 34
<211> 19
<212> DNA
<213>Artificial sequence
<400> 34
atagccaata catgcgtgg 19
<210> 35
<211> 20
<212> DNA
<213>Artificial sequence
<400> 35
aggattactt tcttgaaaca 20
<210> 36
<211> 21
<212> DNA
<213>Artificial sequence
<400> 36
aggataatga ttatttacaa g 21
<210> 37
<211> 21
<212> DNA
<213>Artificial sequence
<400> 37
ttaatcacaa aagcatgtag c 21
<210> 38
<211> 20
<212> DNA
<213>Artificial sequence
<400> 38
tcatcctgcc cctcccctgc 20
<210> 39
<211> 20
<212> DNA
<213>Artificial sequence
<400> 39
gaacaaatac tgcagaaatg 20
<210> 40
<211> 19
<212> DNA
<213>Artificial sequence
<400> 40
tctagctttt aggacaccc 19
<210> 41
<211> 22
<212> DNA
<213>Artificial sequence
<400> 41
gttctctttc tccaccagac tc 22
<210> 42
<211> 21
<212> DNA
<213>Artificial sequence
<400> 42
tatgtccaca cattctcgac c 21
<210> 43
<211> 20
<212> DNA
<213>Artificial sequence
<400> 43
agtcacctct caaaatgata 20
<210> 44
<211> 19
<212> DNA
<213>Artificial sequence
<400> 44
gccgatactc aatcccttc 19
<210> 45
<211> 21
<212> DNA
<213>Artificial sequence
<400> 45
tgaataacct gctatcctgt g 21
<210> 46
<211> 20
<212> DNA
<213>Artificial sequence
<400> 46
tggcagggaa ctagaggtag 20
<210> 47
<211> 22
<212> DNA
<213>Artificial sequence
<400> 47
ctagaggttt taggggtggg ag 22
<210> 48
<211> 21
<212> DNA
<213>Artificial sequence
<400> 48
gagagtgaga agccagtggc g 21
Claims (4)
1. a kind of molecular labeling for identifying pilose antler kind, it is characterised in that including 16 SNP, its sequence is respectively such as SEQ ID
Shown in NO.1-SEQ ID NO.16.
2. a kind of method of molecular markers for identification pilose antler kind using described in claim 1, it is characterised in that including following step
Suddenly：
S1, extract material genomic DNA to be identified；
S2, it is utilized respectively each self-corresponding primers of SEQ ID NO.1-SEQ ID NO.16 and enters performing PCR amplification, respectively obtain 16 groups
Amplified production；
Wherein, the primer sequence used corresponding to the molecular labeling for obtaining SEQ ID NO.1-SEQ ID NO.16 is respectively such as SEQ
ID NO.17-SEQ ID NO.18、SEQ ID NO.19-SEQ ID NO.20、SEQ ID NO.21-SEQ ID NO.22、SEQ
ID NO.23-SEQ ID NO.24、SEQ ID NO.25-SEQ ID NO.26、SEQ ID NO.27-SEQ ID NO.28、SEQ
ID NO.29-SEQ ID NO.30、SEQ ID NO.31-SEQ ID NO.32、SEQ ID NO.33-SEQ ID NO.34、SEQ
ID NO.35-SEQ ID NO.36、SEQ ID NO.37-SEQ ID NO.38、SEQ ID NO.39-SEQ ID NO.40、SEQ
ID NO.41-SEQ ID NO.42、SEQ ID NO.43-SEQ ID NO.44、SEQ ID NO.45-SEQ ID NO.46、SEQ
Shown in ID NO.47-SEQ ID NO.48；
(1) PCR amplification system is 10 μ L：Including μ L of DNA 0.2, μ L of forward primer 0.5, the μ L of reverse primer 0.5；2×Tap PCR
Master Mix 5.0μL；ddH2O 3.8μL；
(2) PCR amplification conditions：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 60 DEG C of annealing 45s, 72 DEG C of extension 45s；Circulation 35
It is secondary；72 DEG C extend 10min, 4 DEG C of cooling 10min eventually；
16 groups of amplified productions 100V electrophoresis 35min on 2% Ago-Gel, and being observed under gel imaging system respectively, point
16 groups of amplified productions are not reclaimed；
S3, sequencing identification
16 groups of amplified production nucleotide sequences of sequencing identification respectively, if the genotype of corresponding SNP site is homozygous with sika deer
Sub- genotype is identical, then detected materials are sika deer；If identical with red deer homozygote genotype, detected materials are horse
Deer；If being heterozygote, detected materials are hybridization deer.
3. molecular labeling the answering in deer molecular marking supplementary breeding is hybridized of identification pilose antler kind according to claim 1
With.
4. application of the method for identification pilose antler kind according to claim 2 in deer molecular marking supplementary breeding is hybridized.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110331216A (en) * | 2019-08-06 | 2019-10-15 | 吉林农业大学 | The specificity amplification primer of sika deer microsatellite locus M027 a kind of and its application |
| CN111378767A (en) * | 2020-05-16 | 2020-07-07 | 中国农业科学院特产研究所 | SNP (Single nucleotide polymorphism) marker for identifying red deer and Chinese red deer, detection primer pair, kit and application |
| CN111793700A (en) * | 2020-08-21 | 2020-10-20 | 长春科技学院 | SNP (Single nucleotide polymorphism) marker for yield traits of pilose antler, detection primer pair, kit and application |
| CN113832236A (en) * | 2021-09-07 | 2021-12-24 | 江汉大学 | Primer group and kit for identifying sika deer, red deer and hybrid deer and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586448A (en) * | 2012-03-06 | 2012-07-18 | 北华大学 | Deer antler capillary electrophoresis DNA (deoxyribonucleic acid) fingerprint spectrum and identification method |
| CN104830870A (en) * | 2015-05-21 | 2015-08-12 | 塔里木大学 | c-fos gene of cervus elaphusyarkandensis antler and application of c-fos gene |
| CN105176988A (en) * | 2015-10-14 | 2015-12-23 | 中国中医科学院中药研究所 | Method for identifying traditional Chinese medical material corn cervi pantotrichum based on PCR-RFLP |
-
2017
- 2017-09-07 CN CN201710800319.6A patent/CN107385090B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586448A (en) * | 2012-03-06 | 2012-07-18 | 北华大学 | Deer antler capillary electrophoresis DNA (deoxyribonucleic acid) fingerprint spectrum and identification method |
| CN104830870A (en) * | 2015-05-21 | 2015-08-12 | 塔里木大学 | c-fos gene of cervus elaphusyarkandensis antler and application of c-fos gene |
| CN105176988A (en) * | 2015-10-14 | 2015-12-23 | 中国中医科学院中药研究所 | Method for identifying traditional Chinese medical material corn cervi pantotrichum based on PCR-RFLP |
Non-Patent Citations (2)
| Title |
|---|
| HENGXING BA等: "Genome-Wide SNP Discovery and Analysis of Genetic Diversity in Farmed Sika Deer (Cervus", 《G3》 * |
| 杜智恒等: "梅花鹿生长激素基因单核苷酸多态与产茸量性状的相关性", 《遗传》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110331216A (en) * | 2019-08-06 | 2019-10-15 | 吉林农业大学 | The specificity amplification primer of sika deer microsatellite locus M027 a kind of and its application |
| CN110331216B (en) * | 2019-08-06 | 2023-03-28 | 吉林农业大学 | Specificity amplification primer of sika deer microsatellite locus M027 and application thereof |
| CN111378767A (en) * | 2020-05-16 | 2020-07-07 | 中国农业科学院特产研究所 | SNP (Single nucleotide polymorphism) marker for identifying red deer and Chinese red deer, detection primer pair, kit and application |
| CN111378767B (en) * | 2020-05-16 | 2023-09-12 | 中国农业科学院特产研究所 | SNP (Single nucleotide polymorphism) marker for identifying Chinese red deer and red deer, detection primer pair, kit and application |
| CN111793700A (en) * | 2020-08-21 | 2020-10-20 | 长春科技学院 | SNP (Single nucleotide polymorphism) marker for yield traits of pilose antler, detection primer pair, kit and application |
| CN111793700B (en) * | 2020-08-21 | 2023-06-30 | 长春科技学院 | SNP (Single nucleotide polymorphism) marker, detection primer pair, kit and application of deer antler yield trait |
| CN113832236A (en) * | 2021-09-07 | 2021-12-24 | 江汉大学 | Primer group and kit for identifying sika deer, red deer and hybrid deer and application |
| CN113832236B (en) * | 2021-09-07 | 2023-06-06 | 江汉大学 | Primer group, kit and application for identifying sika deer, red deer and hybridized deer |
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