CN112126692A - Molecular marker for identifying sika Lujijikuai subspecies, identification method and application - Google Patents
Molecular marker for identifying sika Lujijikuai subspecies, identification method and application Download PDFInfo
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Abstract
The invention provides a molecular marker for identifying sika Lujijuan subspecies, an identification method and application. Aiming at the fact that a molecular marker and an identification method which can be used for identifying the semispecies of the Halidays sika deer do not exist in the prior art, the molecular marker of the Halidays sika deer is deeply researched to obtain an SNP molecular marker which can be used for identifying the semispecies of the Halidays sika deer, and the molecular marker comprises 11 SNP marker sites. The 11 SNP markers are located on 6 gene fragments, and have the characteristics of strong specificity and good stability. According to the gene fragment, 6 pairs of SNP marker primers which can be used for identifying the Jiujima subspecies of the spotted deer are designed, and an accurate and reliable detection method is established. The invention makes up the defect that no molecular marker detection technology for identifying the subfamily of the sika deer exists in the field at present, realizes the accurate identification of the subfamily of the sika deer, and has important theory and application value on the aspects of protection and management of the sika deer resources.
Description
Technical Field
The invention relates to a molecular marker for identifying sika jiujima subspecies, an identification method and application, belonging to the technical field of molecular biology.
Background
Sika deer (Cervus nippon) belongs to Mammalia (Mammalia), Artiodactyla (Artiodactyla), Cervidae (Cervidae) and Cervus (Cervus), is a landmark animal in the east Asian monsoon region, and is distributed from Wusuliang to Vietnam. As the ecological environment is damaged and the habitat is continuously reduced, the number of wild sika deer in China is very rare, the wild sika deer is the I-type protective animal in China at present and is listed in red directory of endangered species of the world nature protection alliance (IUCN). At present, the subspecies division of the sika deer is greatly disputed and disordered according to geographical distribution and morphological characteristics.
It is now well recognized that Japanese sika is divided into 6 subspecies. The Hokkaido subspecies (Cervus nipponeyosensis) is distributed in the Hokkaido island. The state subspecies (Cervus nippon centralis) are distributed in the state and para-horse islands. Subspecies of nomenclature (Cervus nippon nippon) are distributed in the Jiuzhou and the Quadrature islands. The Ornithodium subspecies (Cervus nipponeyakushimae) is distributed in the Ornithodium and the surrounding islets, and is small in size. Malus mauritiana subspecies (Cervus nippon mageshimae) is distributed in Malus mauritiana. Lucuma subspecies (Cervus nippon keramae) were distributed in the lucuma island and its surrounding islets. Japanese sika deer is further divided into a southern pedigree and a northern pedigree, the genetic difference between the two pedigrees is larger than that between subspecies, the northern pedigree comprises northern Hakkaido subspecies and northern population of the subspecies of the state, the southern pedigree comprises southern population of the subspecies of the state, named subspecies, Kyohima subspecies, Makino subspecies and Youkibo subspecies, and no obvious morphological characteristic difference exists between the subspecies, so that the molecular difference between the subspecies is urgently needed as an effective tool for identifying the subspecies.
At present, the subspecies division of sika deer is mainly based on morphological difference and geographical distribution, but scholars also propose that the morphological difference of artillery hoofs cannot be always used as the basis for classification, because morphological characteristics are different due to different environments. Therefore, the problem of genetic resource classification confusion exists among the current sika deer subspecies. The molecular identification method is a hotspot of the current research, the SNP marker has the advantages of high accuracy, rich variation, simple operation and the like, and the molecular marker is not influenced by environmental factors and can directly reflect the difference of animal gene levels, so the molecular marker is widely applied to animal individual identification and seed source identification, and the mitochondrial DNA marker is effective to solve the problems of evolution models among different species and historical problems of colonial places. At present, no molecular marker and method for identifying the subspecies of sika deer exist.
Disclosure of Invention
Aiming at the defects of the prior art, the invention deeply researches the molecular marker of the sika deer to obtain the SNP molecular marker and the detection primer which can be used for identifying the semispecies of the sika deer wujiu island. The invention also provides a method for identifying the sika deer wujima subspecies, so that the accurate identification of the sika deer subspecies is realized, and the method has important theoretical and application values in the aspects of protection and management of sika deer resources.
In view of the above, the present invention provides, in a first aspect, a molecular marker for identifying gecko's island subspecies of sika deer, the molecular marker comprising 11 SNP marker sites: s-1, S-2, S-3, S-4, S-5, S-6, S-7, S-8, S-9, S-10 and S-11; the site information is shown in table 1:
TABLE 1 SNP marker loci of the present invention for identifying Halidaria sanguinea subspecies of Cervus nippon
| Marking | Sika mtDNA genome | Site of the body | Base |
| S-1 | 495bp fragment of ND1 gene | 301bp in length | A/G |
| S-2 | COX3 gene 500bp fragment | 140bp of | G/A |
| S-3 | COX3 gene 500bp fragment | 335bp in length | T/C |
| S-4 | 670bp fragment of ND4 gene | 148bp | T/C |
| S-5 | 670bp fragment of ND4 gene | 362bp in length | T/C |
| S-6 | 670bp fragment of ND4 gene | 559bp | T/C |
| S-7 | ND5 gene 500bp fragment | 83bp in length | G/A |
| S-8 | ND5 gene 500bp fragment | 244bp of | C/T |
| S-9 | 483bp fragment of ND6 gene | 93bp of | G/A |
| S-10 | 483bp fragment of ND6 gene | 302bp of | C/T |
| S-11 | 946bp fragment of Cytb gene | 523bp of | C/T |
The 11 SNP loci S-1-S-11 provided by the invention are A, G, T, T, T, T, G, C, G, C, C in the Halenia solium subspecies respectively; g, A, C, C, C, C, A, T, A, T, T in the non-spotted deer jiuhuadao subspecies respectively. The non-sika Lujijuan subspecies includes sika northeast subspecies, Sichuan subspecies, south China subspecies, Taiwan subspecies, Hokkaido subspecies, Benzhou subspecies, named subspecies, etc.
The invention also provides a primer for identifying the semiaquilegia of the sika deer, wherein the primer is one or more of the following primer pairs: a primer pair 1 with the sequence shown in SEQ ID NO.1 and SEQ ID NO. 2; a primer pair 2 with sequences shown as SEQ ID NO.3 and SEQ ID NO. 4; a primer pair 3 with the sequence shown in SEQ ID NO.5 and SEQ ID NO. 6; a primer pair 4 with the sequence shown as SEQ ID NO.7 and SEQ ID NO. 8; a primer pair 5 with the sequence shown in SEQ ID NO.9 and SEQ ID NO. 10; a primer pair 6 with the sequence shown in SEQ ID NO.11 and SEQ ID NO. 12.
The primer pair 1 is used for amplifying 495bp fragments of the ND1 gene; the primer pair 2 is used for amplifying 500bp segments of COX3 genes; the primer pair 3 is used for amplifying a 670bp fragment of the ND4 gene; the primer pair 4 is used for amplifying a 500bp fragment of the ND5 gene; the primer pair 5 is used for amplifying 483bp segments of ND6 genes; the primer pair 6 is used for amplifying 946bp segments of the Cytb gene.
The invention also provides application of the molecular marker for identifying the sika Lujikuai subspecies or the primer for identifying the sika Lujikuai subspecies in identifying the sika Lujikuai subspecies; and the application in the preparation of a kit or a detection method, wherein the kit or the detection method is used for identifying the semiaquilegia of sika deer.
The invention also provides a method for identifying the sika jujima subspecies, which comprises the following steps: extracting DNA of a sample to be detected, and carrying out PCR amplification by using any one or more pairs of the primer pairs 1-6; sequencing the PCR product, and identifying whether the sample to be detected is the sika lujijuan subspecies or not according to the SNP marker locus base.
If the sequences of the amplification sequencing results of the primer pairs 1-6 are respectively shown as SEQ ID NO.13 (the base at the position of 301bp is A), SEQ ID NO.15 (the base at the position of 140bp is G, the base at the position of 335bp is T), SEQ ID NO.17 (the base at the position of 148bp is T, the base at the position of 362bp is T, the base at the position of 559bp is T), SEQ ID NO.19 (the base at the position of 83bp is G, the base at the position of 244bp is C), SEQ ID NO.21 (the base at the position of 93bp is G, the base at the position of 302bp is C) and SEQ ID NO.23 (the base at the position of 523bp is C), the sample to be detected can be judged to be the semiaquilegia. If the sequencing results are respectively shown in SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22 and SEQ ID NO.24, namely the bases at the corresponding sites are respectively G, A, C, C, C, C, A, T, A, T, T, the sample to be detected can be judged to be non-sika lujiujima subspecies.
The molecular marker and the identification method for identifying the semivariety of the sika Lujijuan have the following advantages:
(1) the invention obtains 11 SNP loci for identifying specificity of the Halenia solium subspecies of the spotted deer, and the 11 SNP loci are positioned on 6 gene segments and have the characteristics of strong specificity and good stability. Provides a stable and reliable molecular detection method for identifying the subspecies of the sika deer resources and provides a new idea and method for identifying the sika deer germplasm resources. Has important theoretical and application values in the aspects of DNA fingerprint drawing, wild subspecies protection management and the like of the sika deer.
(2) The invention designs 6 pairs of SNP marker primers which can be used for identifying the Jiujima subspecies of the sika deer, and establishes an accurate and reliable detection method. Establishes a method for identifying the specific SNP of the sika deer wujikuai subspecies, and makes up the defect that no molecular marker detection technology for identifying the sika deer subspecies exists in the field at present.
(3) The identification method has extremely high accuracy. 90 sika samples from china and japan were tested by the method of the present invention. The results show that: 15 of the 90 samples were languajima subspecies, and the remaining samples were not languajima subspecies. The detection accuracy rate is 100%.
(4) Low cost and high practicability. Because the SNP locus is stable and reliable, one or more pairs of primers can be adopted, and when the marker identified by 1 SNP molecule is the same as the base of the specific SNP locus of the Haliday cunninghamia sublateritifer, the Haliday cunninghamia sublateritifer can be judged. Thus greatly saving time and reducing cost. Moreover, the morphological characteristics of the spotted deer to be detected do not need to be mastered, and the DNA sample of the spotted deer can be collected to realize identification.
(5) Compared with the existing problem of inaccurate classification of genetic resources, the method has the greatest advantage of accurate identification. Namely, the method realizes the accurate identification of the longisland subspecies of the sika deer, and has important theoretical and application values in the aspects of protection and management of sika deer resources.
Drawings
FIG. 1 shows the difference SNP sites of mtDNA sequences of sika jiujima subspecies and other subspecies. In the figure:
cervus nippon Hortulorum, northeast subspecies; cervus nippon sichuanicus, subspecies Sichuan; cervus nippon kopschi south China subspecies; cervus nippon taiouanus subspecies Taiwan; cervus nippon yesoensis subspecies Hokkaido; cervus nippon centralis, subspecies, Benzhou; cervus nippon nippon, named subspecies; cervus nippon yakushimae Ornithodium subspecies
FIG. 2 is a graph showing the PCR results of example 2. In the figure, M: DNAker 1000; 1: a primer pair 1; 2: a primer pair 2; 3: a primer pair 3; 4: a primer pair 4; 5: a primer pair; 6: and (3) a primer pair 6.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the present invention is not limited thereto. The methods of the present invention are those commonly used in the art unless otherwise specified, and the reagents of the present invention may be commercially available without further specification.
Example 1 acquisition of specific SNP marker for identifying Haliotis sika subspecies
1. Specific SNP site screening of sika deer hokkaido subspecies
According to mtDNA sequences of different subspecies samples such as northeast subspecies, Sichuan subspecies, south China subspecies, Taiwan subspecies, Hokkaido subspecies, and the like of the spotted deer, Mega6.0 comparison analysis is utilized, sites which are shared by the same subspecies and are specific to other subspecies are mainly screened, and 11 different SNP sites on 6 genes are obtained by screening, as shown in figure 1. The Mega6.0 is used to intercept ND1 gene segment, COX3 gene segment, ND4 gene segment, ND5 gene segment, ND6 gene segment and Cytb gene segment, the sequence length is about 500bp, which are used to design identifying primer.
2. Primer design
According to the intercepted target gene fragment, a discrimination primer is designed by combining Primer5.0 and Oligo7.0. Obtaining:
1F：5'-CTTAGCCGTGGCATTCCTCA-3'(SEQ ID NO.1)；
1R：5'-CGGTGAGATCAAATGGGGCT-3'(SEQ ID NO.2)。
2F：5'-TCCTGCTAACACTCGGCCTA-3'(SEQ ID NO.3)；
2R：5'-AGTGTCAGTATCAGGCAGCG-3'(SEQ ID NO.4)。
3F：5'-CACATTTTACCCTGGCAAGCA-3'(SEQ ID NO.5)；
3R：5'-ATTGCTGCAAGGACCATGGA-3'(SEQ ID NO.6)。
4F：5'-CGCCCCCGTGTATAATAACAC-3'(SEQ ID NO.7)；
4R：5'-GTATTTGCGTCTGCTCGTCC-3'(SEQ ID NO.8)。
5F：5'-ACAAAGACCACCCAGTCACA-3'(SEQ ID NO.9)；
5R：5'-TGGGTGGGTTTTTCGGATGT-3'(SEQ ID NO.10)。
primer pair for amplifying Cytb gene fragment 6:
6F：5'-CCACAACCAACTCCACCACT-3'(SEQ ID NO.11)；
6R：5'-GGGCTATGGCTTCTTCCTTGA-3'(SEQ ID NO.12)。
the primers were designed and synthesized by Biotechnology engineering (Shanghai) GmbH.
Example 2 establishment of a method for identifying Cervus Nippon Temminck subspecies and other subspecies
According to the primer pair shown in SEQ ID NO.1-SEQ ID NO.12, a PCR experiment is carried out to establish a method for identifying the sika jikuai island subspecies and other subspecies, and the PCR amplification conditions are shown in Table 2.
TABLE 2 PCR amplification conditions
The PCR reaction system is shown in Table 3.
TABLE 3 PCR System
The PCR amplification process comprises the following steps: pre-denaturation at 94 ℃ for 5 min; 30 cycles of denaturation at 94 ℃ for 30sec, annealing at 59 ℃ for 30sec, and elongation at 72 ℃ for 30 sec; further extension at 72 deg.C for 5min, and storage at 4 deg.C.
The PCR result is shown in figure 2, the electrophoresis bands of the PCR results of the primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4, the primer pair 5 and the primer pair 6 are single and bright, and the experimental result shows that the method for effectively identifying the hokkaido subspecies and other subspecies of the sika deer is successfully established.
Example 3 specific application of the method of the present invention to the identification of the Liangjima subspecies of Cervus Nippon Temminck and other subspecies
Selecting 90 sika deer samples collected from China and Japan, respectively extracting genome DNA, randomly mixing the samples by an experimenter A, numbering again, carrying out PCR amplification on the samples by an experimenter B by adopting the primer designed by the invention, and sending the amplified products to a biological engineering (Shanghai) corporation for sequencing. And checking a sequencing peak map through Bioedit 7.0, wherein peaks and troughs of the sequencing result map are separated, are not overlapped and are not misread, and the sequencing result is credible. Mega6.0 is used for comparing sequences, and the base of the specific SNP locus of the semiaquilegia of the invention is used as a judgment basis.
According to the mtDNA specificity SNP molecular marker determined by the invention. The results of the identification are shown in Table 4. The results are indicated as yes/no for the langoustine island subspecies/other subspecies, respectively. The results show that among 90 sika deer samples, the S-1, S-2, S-3, S-4, S-5, S-6, S-7, S-8, S-9, S-10 and S-11 sites of 75 samples Y1-Y75 are G, A, C, C, C, C, A, T, A, T, T respectively; the S-1, S-2, S-3, S-4, S-5, S-6, S-7, S-8, S-9, S-10 and S-11 sites of the 15 samples Y76-Y90 are A, G, T, T, T, T, G, C, G, C, C respectively, and through checking with an experimenter A, the 75 samples Y1-Y75 are determined to be non-languian subspecies with the accuracy rate of 100 percent, and the 15 samples Y76-Y90 are determined to be languian subspecies with the accuracy rate of 100 percent.
TABLE 4 Cervus Nippon Temminck sample test results
In summary, it is demonstrated that when the 11 SNP sites proposed by the present invention are used for identifying the sika lujiu jiu island subspecies and other subspecies, the accuracy is high and the stability is good, and considering the cost problem, one or more pairs of primers can be adopted, because when the marker identified by 1 SNP molecule is the same as the base of the specific SNP site of the sika lujiu jiu island subspecies, the sika lujiu jiu island subspecies can be identified. Preferably, only any one of primer pairs 2, 3, 4, 5 is used, and most preferably, only primer pair 3 is used.
Sequence listing
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tctatcaaca ccactattaa ttttaaccat atgacttctt cccttaatat taatagctag 300
ccaacaccac ctatcaaaag aaaatcttac ccgaaaaaaa ctatatatta ccatattaat 360
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tctatttgaa gcaacactag tcccaacact cattattatt acccgatggg gaaaccaaac 480
agaacgccta aacgctggcc tctacttcct gttttataca ctagtaggtt ctctcccact 540
actagtcgca ctagtttatc tccaaaacat tactggatct ctaaactttc tagtactcca 600
atactgagta caacccctat ccaactcctg atcaaacgtt ttcatgtgac tagcatgcat 660
aatagccttt 670
<210> 18
<211> 670
<212> DNA
<213> Spotted deer (Cervus nippon)
<400> 18
tactagttaa agtatcaaat acatatggca ctgactatgt tcaaaatctt aacctacttc 60
aatgctaaaa tatattatcc ctacaataat actcatacct ctgacctgac tatcaaaagg 120
caatataatt tgaattaatt ctacaaccca tagcctgtta attagcctca caagccttct 180
cctcataaat caattcagtg acaacagcct caacttctcg ttaatattct tttccgactc 240
cctatcaaca ccactattaa ttttaaccat atgacttctt cccttaatat taatagctag 300
ccaacaccac ctatcaaagg aaaatcttac ccgaaaaaaa ctatatatta ccatattaat 360
tctacttcaa ctattcttaa tcatgacttt tactgctata gaactaatct ttttctatat 420
tctatttgaa gcaacactag tcccaacact cattattatt acccgatggg gaaaccaaac 480
agaacgccta aacgctggcc tctacttcct gttttataca ctagtaggtt ctctcccact 540
actagtcgca ctagtctacc tccaaaacat tactgggtct ctaaactttc tagtactcca 600
atactgagta caacccctat ccaactcctg atcaaacgtt ttcatgtgac tagcatgcat 660
aatagctttt 670
<210> 19
<211> 500
<212> DNA
<213> Spotted deer (Cervus nippon)
<400> 19
ggtgcaactc caaataaaag taataaacct attctcctcc ttcactctag ttaccctatt 60
actactaact atccccatca tagccacaag ttctgacaac tataaaactt ccaactaccc 120
actctacgta aaaacaacca tctcatatgc tttcatcacc agtataattc ccacaataat 180
atttattcat actggccaag aaataattat ctcaaactga cactgactaa ctatccaaac 240
tatcaaacta tcacttagct tcaagataga ttatttctca ataatatttg taccagtagc 300
attattcgtt acatgatcca ttatagaatt ctcaatgtga tatatacact cagaccccaa 360
cattaatcaa ttcttcaaat atcttctcct atttctcatt actatactta tcctcgtcac 420
agcaaataat ctatttcaat tattcatcgg atgagagggt gtaggaatca tatcattttt 480
acttattgga tgatgatatg 500
<210> 20
<211> 500
<212> DNA
<213> Spotted deer (Cervus nippon)
<400> 20
ggtgcaactc caaataaaag taataaacct attctcctcc ttcacgctag ttaccctatt 60
actactaact atccccatca taactacaag ctctgacaac tataaaactt ccaactaccc 120
gctctacgta aaaacaacta tctcatatgc tttcatcacc agtataattc ccacaataat 180
attcatttat actggccaag aaataattat ctcaaactga cactgactaa ctatccaaac 240
tattaaacta tcgcttagct tcaaaataga ttatttctca ataatatttg taccagtagc 300
actattcgtt acatggtcca ttatagaatt ctcaatgtga tatatacact cagaccccaa 360
cattaatcaa ttcttcaaat atcttctcct atttctcatt actatactta tcctcgtcac 420
agcaaataat ctatttcaat tattcatcgg atgagaaggt gtaggaatca tatcattttt 480
acttatcgga tgatgatatg 500
<210> 21
<211> 483
<212> DNA
<213> Spotted deer (Cervus nippon)
<400> 21
aatctcctgt gtcataaata acccaatctc ctaacccatt aaactgaaac acaatctcca 60
cctcctcatc ctttaacaca taataaacta tcgtaacttc catcaacaga ccagtaacaa 120
atgcccctaa cacagtctta ttagacaccc aaatctcagg gtattgctcc gtagctattg 180
ccgttgtata accaaaaacc accatcattc ctcctaaata aattaagaat accattaaac 240
ccagaaagga cccaccaaaa tttaacacaa taccacaacc aactccacca ctcacaatta 300
accccaaccc cccataaata ggcgaaggtt tcgaagaaaa tcctacaaaa ccaagcacaa 360
aaataatact taagataaat acaatgtatg ttatcattat tctcacatgg aatctaacca 420
tgactaatga tatgaaaaac catcgttgtc attcaactac aagaacacta atgatcaaca 480
tcc 483
<210> 22
<211> 483
<212> DNA
<213> Spotted deer (Cervus nippon)
<400> 22
aatcccctgt atcataaata acccaatctc ctaatccatt aaactgaaac acaatctcca 60
cctcctcatc ctttaacaca taataaacta tcataacttc catcaacaga ccagtgacaa 120
atgcccctaa cacagtctta ttagacaccc aaatctcagg gtactgctcc gtagctattg 180
ccgttgtata accaaaaacc accatcattc ctcccaaata aattaagaat accattaaac 240
ccagaaagga cccaccaaaa tttaacacaa taccacaacc aactccacca ctcacaatta 300
atcccaaccc cccataaata ggcgaaggtt tcgaagaaaa tcctacaaaa ccaagcacaa 360
aaataatact taagataaat acaatgtatg ttatcattat tctcacatgg aatctaacca 420
tgactaatga tatgaaaaat catcgttgtc attcaactac aagaacacta atgaccaaca 480
tcc 483
<210> 23
<211> 946
<212> DNA
<213> Spotted deer (Cervus nippon)
<400> 23
tcctacaaaa ccaagcacaa aaataatact taagataaat acaatgtatg ttatcattat 60
tctcacatgg aatctaacca tgactaatga tatgaaaaac catcgttgtc attcaactac 120
aagaacacta atgatcaaca tccgaaaaac ccacccatta ataaaaattg taaacaacgc 180
attcattgac ctccccgccc catcaaatat ttcatcctga tgaaatttcg gctccttact 240
aggaatttgc ctaatcctac aaatcctcac aggcctattc ctagcaatac actatacatc 300
cgacacaata acagcattct cctctgtcac ccatatctgt cgagatgtca attatggttg 360
aattattcga tacatacacg caaacggggc atcaatattt ttcatctgcc tattcataca 420
cgtaggacga ggtctgtact acggatcata tacttttcta gagacatgaa acatcggagt 480
aattctccta tttacagtca tagccacagc attcgtagga tacgtcctac catgaggaca 540
aatatcattc tgaggagcaa cagtcattac caaccttctc tcagcaattc catacattgg 600
cacaaaccta gtcgaatgga tctgaggagg cttttcagta gataaagcaa ccctaacccg 660
atttttcgcc ttccacttta ttcttccatt tatcatcaca gcactcgcta tagtacactt 720
actcttcctt cacgagacag gatccaacaa cccaacagga atcccatcgg acgcagacaa 780
aatccccttc catccttact ataccattaa agatatccta ggcatcttac ttctagtact 840
cttcctgata tcactagtat tattcgcacc agacctgctt ggagatccag acaactacac 900
cccagcaaat ccgctcaaca caccccctca catcaaacct gaattt 946
<210> 24
<211> 946
<212> DNA
<213> Spotted deer (Cervus nippon)
<400> 24
tcctacaaaa ccaagcacaa aaataatact taagataaat acaatgtatg ttatcattat 60
tctcacatgg aatctaacca tgactaatga tatgaaaaat catcgttgtc attcaactac 120
aagaacacta atgaccaata tccgaaaaac ccacccatta ataaaaattg taaacaacgc 180
attcattgac ctccccgccc catcaaatat ttcatcctga tgaaatttcg gctccctact 240
aggaatttgt ctaatcctac aaatccttac aggcctattc ctagcaatac actatacatc 300
tgacacaata acagcatttt cctctgtcac ccatatctgt cgagatgtca actatggttg 360
aattatccga tacatacacg caaacggggc atcaatattt ttcatctgcc tattcataca 420
tgtaggacga ggcctgtact acggatcata tacttttcta gagacatgaa acatcggagt 480
aattctccta tttacagtta tagccacagc attcgtagga tatgtcctac catgaggaca 540
aatatcattc tgaggagcaa cagtcattac caacctcctc tcagcaattc catatattgg 600
cacaaaccta gtcgaatgga tctgaggggg cttctcagta gataaagcaa ccctaacccg 660
atttttcgct ttccacttta ttcttccatt tatcatcgca gcacttgcta tagtacactt 720
actcttcctt cacgagacag gatccaacaa cccaacagga atcccatcgg acgcagacaa 780
aatccccttc catccttact acaccattaa agatatctta ggcatcttac ttctagtact 840
cttcctaata ttactagtat tattcgcacc agacctgctt ggagatccag acaactatac 900
cccagcaaat ccactcaaca caccccctca catcaaacct gaattt 946
Claims (10)
1. The molecular marker for identifying the semivariety of the Halidaria sika is characterized by comprising 11 SNP sites:
。
2. The molecular marker for identifying the semivariety of the Haliday sika deer as claimed in claim 1, wherein the 11 SNP sites S-1 to S-11 are A, G, T, T, T, T, G, C, G, C, C in the Haliday sika deer subspecies; g, A, C, C, C, C, A, T, A, T, T in the non-spotted deer jiuhuadao subspecies respectively.
3. The molecular marker for identifying the jiu dao subspecies of sika deer as claimed in claim 2, wherein the non-jiu dao subspecies of sika deer includes northeast subspecies of sika deer, sichuan subspecies, south china subspecies, taiwan subspecies, north sea subspecies, honzhou subspecies, and nominated subspecies.
4. The primer for identifying the semispecies of the sika judai is characterized in that the primer is one or more of the following primer pairs:
a primer pair 1 with the sequence shown in SEQ ID NO.1 and SEQ ID NO. 2; a primer pair 2 with sequences shown as SEQ ID NO.3 and SEQ ID NO. 4; a primer pair 3 with the sequence shown in SEQ ID NO.5 and SEQ ID NO. 6; a primer pair 4 with the sequence shown as SEQ ID NO.7 and SEQ ID NO. 8; a primer pair 5 with the sequence shown in SEQ ID NO.9 and SEQ ID NO. 10; a primer pair 6 with the sequence shown in SEQ ID NO.11 and SEQ ID NO. 12.
5. The primer for identifying the semispecies nigella praecox as claimed in claim 4, wherein the primer pair 1 is used for amplifying 495bp segment of ND1 gene; the primer pair 2 is used for amplifying 500bp segments of COX3 genes; the primer pair 3 is used for amplifying a 670bp fragment of the ND4 gene; the primer pair 4 is used for amplifying a 500bp fragment of the ND5 gene; the primer pair 5 is used for amplifying 483bp segments of ND6 genes; the primer pair 6 is used for amplifying 946bp segments of the Cytb gene.
6. Use of the molecular marker for identifying the semivariety of aegilops islanding of sika as claimed in claim 1 or the primer for identifying the semivariety of aegilops islanding of sika as claimed in claim 4 for identifying the semivariety of aegilops islanding of sika.
7. The use of the molecular marker for identifying the semivariety of aegilops tauschii of sika as claimed in claim 1 or the primer for identifying the semivariety of aegilops tauschii as claimed in claim 4 in the preparation of a kit or in a detection method, wherein the kit or the detection method is used for identifying the semivariety of aegilops tauschii.
8. A method for identifying sika Lujijuan subspecies is characterized by comprising the following steps: extracting the DNA of a sample to be detected,
performing PCR amplification using the primers of claim 4; sequencing the PCR product, and identifying whether the sample to be detected is the sika lujijuan subspecies or not according to the SNP marker locus base.
9. The method for identifying the semispecies Fujima sika as claimed in claim 8, wherein the primer is any one or more of the primer pairs 1-6.
10. A kit for identifying Halidaria cuneata subspecies, which is characterized by comprising one or more pairs of primers in the primers of claim 4.
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| CN113308556A (en) * | 2021-07-07 | 2021-08-27 | 大连海关技术中心 | Quantitative PCR (polymerase chain reaction) kit for rapidly detecting sika deer-derived deer products and preparation method thereof |
| CN114045349A (en) * | 2021-12-02 | 2022-02-15 | 锡林郭勒职业学院 | Primer, probe and kit for synchronously detecting sources of sika deer and red deer |
| CN114672575A (en) * | 2022-04-19 | 2022-06-28 | 中国检验检疫科学研究院 | Kit for rapidly detecting hybrid antler based on RAA amplification |
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| CN114045349A (en) * | 2021-12-02 | 2022-02-15 | 锡林郭勒职业学院 | Primer, probe and kit for synchronously detecting sources of sika deer and red deer |
| CN114045349B (en) * | 2021-12-02 | 2023-08-01 | 锡林郭勒职业学院 | Primer, probe and kit for synchronously detecting source of sika deer and red deer |
| CN114672575A (en) * | 2022-04-19 | 2022-06-28 | 中国检验检疫科学研究院 | Kit for rapidly detecting hybrid antler based on RAA amplification |
| CN114672575B (en) * | 2022-04-19 | 2024-04-12 | 中国检验检疫科学研究院 | Kit for rapidly detecting hybridized pilose antler based on RAA amplification |
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