CN107385090B - Molecular marker for identifying variety of pilose antler, identification method and application - Google Patents
Molecular marker for identifying variety of pilose antler, identification method and application Download PDFInfo
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- CN107385090B CN107385090B CN201710800319.6A CN201710800319A CN107385090B CN 107385090 B CN107385090 B CN 107385090B CN 201710800319 A CN201710800319 A CN 201710800319A CN 107385090 B CN107385090 B CN 107385090B
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
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- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention discloses a molecular marker for identifying a deer antler variety, which comprises 16 SNPs, and the sequences of the SNPs are respectively shown as SEQ ID NO.1-SEQ ID NO. 16. The invention also discloses a method for identifying the variety of the pilose antler and application thereof. The invention discovers the molecular markers shown by SEQ ID NO.1-SEQ ID NO.16 for the first time, can well distinguish cervus elaphus linnaeus, cervus nippon linnaeus and hybrid cervus elaphus linnaeus, has the success rate of identifying the hybrid cervus elaphus linnaeus within 5 generations of more than 95 percent, has high reliability, provides a most economical and effective new molecular identification way for ensuring the legal right of consumers, and can be used for the molecular marker-assisted selective breeding and variety identification of the hybrid cervus elaphus linnaeus.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a molecular marker for identifying a variety of pilose antler, an identification method and application.
Background
The pilose antler is a young horn of the male deer which is not ossified and has pilose antler hair, and is a rare medicinal material. The cornu Cervi Pantotrichum contains phospholipid, glycolipid, gum lipid, hormone, fatty acid, amino acid, protein, and calcium, phosphorus, magnesium, and sodium, wherein the amino acid component accounts for more than half of the total components. According to the record of Chinese pharmacopoeia, the pilose antler varieties which can be used as medicines include hairy antler, hairy antler and spotted deer antler. The cervus elaphus linnaeus is the cervus elaphus linnaeus grown from the cervus elaphus linnaeus, generally large, the weight is generally between 10 and 50 jin, the color of the cervus elaphus linnaeus is black, and the body is large, and the hair of the cervus elaphus linnaeus is long and black; the spotted deer antler is grown from spotted deer, is generally in the shape of single-arm, two-bar and three-fork, has high ornamental value, and has higher economic value than red deer.
The red deer and the sika deer are two deer species, can be crossed positively and negatively, and the offspring can be bred to become the hybrid deer. The appearance of the hybridized antler grown by the hybridized deer has the characteristics of the spotted deer antler, but the hybridized antler has lower market price than the spotted deer antler, so that the hybridized antler is easily used as the spotted deer antler for sale by lawbreakers to deceive consumers.
In the prior art, the quality of the pilose antler is mostly identified from the aspects of color, texture and smell, and the species of the pilose antler is judged, but a scientific identification method is still lacked. Therefore, the invention discloses a method for identifying the crossbred antler of the sika deer and the red deer.
Disclosure of Invention
The molecular marker for identifying the deer antler varieties, the identification method and the application provided by the invention can identify the deer antler varieties of different sika deer, red deer and hybrid deer.
The first purpose of the invention is to provide a molecular marker for identifying a deer antler variety, which comprises 16 SNPs, and the sequences of the SNPs are respectively shown as SEQ ID NO.1-SEQ ID NO. 16.
The second purpose of the invention is to provide a method for identifying the variety of the antler by using the molecular marker, which is characterized by comprising the following steps:
s1, extracting the genome DNA of the material to be identified;
s2, respectively carrying out PCR amplification by using primers corresponding to SEQ ID NO.1-SEQ ID NO.16 to respectively obtain 16 groups of amplification products;
wherein, the primer sequences correspondingly used for obtaining the molecular markers of SEQ ID NO.1-SEQ ID NO.16 are respectively shown in 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 ID NO.47-SEQ ID NO. 48;
(1) the PCR amplification system is 10 μ L including DNA 0.2 μ L, forward primer 0.5 μ L, reverse primer 0.5 μ L, 2 × TapPCR Master Mix 5.0 μ L, ddH2O 3.8μL；
(2) PCR amplification conditions: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45 s; circulating for 35 times; final extension at 72 deg.C for 10min, and cooling at 4 deg.C for 10 min;
carrying out 100V electrophoresis on the 16 groups of amplification products on 2% agarose gel for 35min respectively, observing under a gel imaging system, and recovering 16 groups of amplification products of 101bp respectively;
s3, sequencing identification
Respectively sequencing and identifying 16 groups of amplification product nucleotide sequences, wherein if the genotypes of the corresponding SNP loci are the same as the genotypes of homozygotes of the sika deer, the material to be detected is the sika deer; if the genotype of the detected materials is the same as that of the red deer homozygote, the detected material is the red deer; and if the hybrid deer is heterozygous, the material to be detected is the hybrid deer.
The third purpose of the invention is to provide the application of the molecular marker for identifying the deer antler varieties in hybrid deer breeding.
The third purpose of the invention is to provide the application of the method for identifying the variety of the deer antler in molecular marker-assisted breeding of the hybrid deer.
Compared with the prior art, the molecular marker for identifying the variety of the cartialgenous, the identification method and the application provided by the invention have the following beneficial effects:
in the prior art, the quality of the pilose antler is mostly identified from the aspects of color, texture and smell, and the species of the pilose antler is judged, but a scientific identification method is still lacked. Therefore, the invention discloses a method for identifying the crossbred antler of the sika deer and the red deer. The invention discovers the molecular markers shown in SEQ ID NO.1-SEQ ID NO.16 for the first time, can well distinguish cervus elaphus linnaeus, cervus nippon linnaeus and hybrid cervus elaphus linnaeus, has the success rate of identifying the hybrid cervus elaphus linnaeus within 5 generations of more than 95 percent, has high reliability, and provides a new economic and effective molecular identification way for ensuring the legal right of consumers. Can also be used for molecular marker-assisted selective breeding and variety identification of hybrid deer.
Detailed Description
The present invention is described in detail below with reference to specific examples, but the present invention should not be construed as being limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The molecular marker for identifying the deer antler varieties provided by the invention comprises 16 SNPs, and the sequences of the SNPs are respectively shown as SEQ ID NO.1-SEQ ID NO. 16. Wherein two bases in the [ ] ' represent SNP sites, and if the genotype of a sample to be detected is homozygote which is the same as the base before the '/', the sample to be detected is a spotted deer; if the genotype of the sample to be detected is homozygote with the same basic group as the base after the '/', the sample to be detected is red deer; if the genotype of the sample to be detected is heterozygote of two basic groups in [ ]', the sample to be detected is a filial generation of the sika deer and the red deer, namely a hybridized deer.
Firstly, obtaining a molecular marker:
s1, respectively extracting antler genome of offspring hybrid deer I and hybrid deer II obtained after positive and negative hybridization of the sika deer and the red deer, and extracting antler genome DNA of the sika deer and the red deer; and extracting the genome DNA of other 96 hybrid deer, wherein the female parent and the male parent of the sika deer and the red deer are all commercially available.
S2, simplifying the positioning result of Reads of genome sequencing (dd-RADSseq) on a reference genome, performing Local alignment (Local alignment) and mutation detection by using GATK software, performing mutation detection by using STACKS software, taking intersection mutation sites obtained by the GATK software and the STACKS software, and the like to ensure the accuracy of the detected SNP (single nucleotide polymorphism), detecting species-specific SNP sites on the genomes of sika deer, red deer and hybrid deer by using large samples, detecting the linkage condition of the SNP sites, and finally screening 16 SNPs which are far away from the reference genome and are not easy to be linked as candidate sites for identifying the hybrid deer.
S3, SNP analysis of different deer antler varieties
SNP genotypes of deer antlers for spotted deer, red deer, hybrid deer I and hybrid deer II are shown in Table 1.
TABLE 1 SNP analysis of different deer antler varieties
| SNP sequences | Spotted deer | Cervus elaphus Linnaeus | Hybrid deer 1 | Hybrid deer II |
| 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 |
Secondly, the method for identifying the variety of the pilose antler by utilizing the SNP molecules comprises the following specific steps:
s1, extracting the genome DNA of the material to be identified by using a CTAB method, and storing the genomic DNA in a refrigerator at 4 ℃ for later use.
When used, the DNA concentration was checked with 0.8% agarose and diluted to the working concentration for PCR amplification.
S2, respectively carrying out PCR amplification by using the primers corresponding to SEQ ID NO.1-SEQ ID NO.16 to respectively obtain 16 groups of amplification products. Wherein, the primer sequences correspondingly used for obtaining the molecular markers of SEQ ID NO.1-SEQ ID NO.16 are respectively shown in 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 ID NO.47-SEQ ID NO. 48; specifically, as shown in table 2.
It should be noted that 16 groups of amplification product fragments obtained by amplification using the primers in Table 2 are between 300 and 600bp, the 101bp sequence corresponding to SEQ ID NO.1-SEQ ID NO.16 is the characteristic sequence of deer and is contained in the amplification product fragments, and the amplification products are respectively compared with the corresponding molecular markers SEQ ID NO.1-SEQ ID NO.16 during sequence comparison.
TABLE 2 primer sequences for different molecular markers
(1) The PCR amplification system was 10. mu.L including 0.2. mu.L (1 ng/. mu.L) of DNA, 0.5. mu.L of forward primer, 0.5. mu.L of reverse primer, 5.0. mu.L of 2 × Tap PCR Master Mix (available from Hakka century Co., Ltd.); ddH2O 3.8μL。
The concentration of the primers was 4 pmol/. mu.L.
(2) PCR amplification conditions: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45 s; circulating for 35 times; final extension at 72 deg.C for 10min, and cooling at 4 deg.C for 10 min.
The 16 groups of amplification products were electrophoresed on 5% agarose gel at 100V for 35min and observed under a gel imaging system, and the results were recorded.
S3, judging the variety of the pilose antler according to the electrophoresis result
Respectively sequencing and identifying the nucleotide sequences of the 16 groups of amplification products, wherein if the genotypes of the corresponding SNP sites are the same as the genotypes of the homozygotes of the sika deer shown in the table 1, the material to be detected is the sika deer; if the genotype of the detected material is the same as that of the red deer homozygote shown in the table 1, the detected material is red deer; and if the heterozygotes are heterozygotes shown in the table 1, the material to be detected is the hybrid deer.
The filial generation of the red deer and the sika deer is subjected to progressive hybridization with the sika deer, theoretically (50 percent of recombination rate and 95 percent of SNP typing success rate), and the relationship between the hybridization algebra and the accuracy of the hybridized antler can be identified by utilizing the 16 SNP molecular markers disclosed by the invention and shown in a table 3.
TABLE 3 progressive hybridization with Cervus Nippon Temminck
The results in table 3 show that the success rate of identifying the hybridized antler within 5 generations of hybridization through the sika deer grade is 95%, although the success rate is reduced along with the increase of the hybridization generation number, the experience shows that the hybridized antler is basically consistent with the sika deer antler in the appearance shape, color and nutrient components by hybridizing for more than 5 generations in the mode. Therefore, in the market, the hybrid antler within 5 generations can obtain reliable identification results.
It should be noted that when the following claims refer to numerical ranges, it should be understood that both ends of each numerical range and any value between the two ends can be selected, and since the steps and methods used are the same as those of the above embodiments, the preferred embodiments of the present invention have been described for the purpose of preventing redundancy, but once the basic inventive concept is known, those skilled in the art may make other variations and modifications to these embodiments. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<120> molecular marker for identifying variety of pilose antler, identification method and application
<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 (3)
1. An application of a primer in identifying sika deer and red deer hybridized antler is characterized in that the primer is used for detecting 16 SNPs, and the sequences of the 16 SNPs are respectively shown as SEQ ID NO.1-SEQ ID NO. 16;
the primer sequences used for obtaining the molecular markers of SEQ ID NO.1-SEQ ID NO.16 are shown in 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 ID NO.47-SEQ ID NO.48, respectively.
2. A method for identifying a variety of pilose antler by using the primer of claim 1, which comprises the following steps:
s1, extracting the genome DNA of the material to be identified;
s2, respectively carrying out PCR amplification by using primers corresponding to SEQ ID NO.1-SEQ ID NO.16 to respectively obtain 16 groups of amplification products;
wherein, the primer sequences correspondingly used for obtaining the molecular markers of SEQ ID NO.1-SEQ ID NO.16 are respectively shown in 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 ID NO.47-SEQ ID NO. 48;
(1) the PCR amplification system is 10 μ L, including 0.2 μ L DNA, 0.5 μ L forward primer, 0.5 μ L reverse primer, 2 × Tap PCRMaster Mix 5.0 μ L ddH2O 3.8μL；
(2) PCR amplification conditions: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45 s; circulating for 35 times; final extension at 72 deg.C for 10min, and cooling at 4 deg.C for 10 min;
respectively carrying out 100V electrophoresis on the 16 groups of amplification products on 2% agarose gel for 35min, observing under a gel imaging system, and respectively recovering 16 groups of amplification products;
s3, sequencing identification
Respectively sequencing and identifying 16 groups of amplification product nucleotide sequences, wherein if the genotypes of the corresponding SNP loci are the same as the genotypes of homozygotes of the sika deer, the material to be detected is the sika deer; if the genotype of the detected materials is the same as that of the red deer homozygote, the detected material is the red deer; and if the hybrid deer is heterozygous, the material to be detected is the hybrid deer.
3. Use of the method according to claim 2 for identifying deer antler and red deer antler hybrids.
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| 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 |
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