CN112126692B

CN112126692B - Molecular marker for identifying sika deer long island subspecies, identification method and application

Info

Publication number: CN112126692B
Application number: CN202011206082.7A
Authority: CN
Inventors: 邢秀梅; 董依萌; 王天骄; 李洋; 刘华淼
Original assignee: Institute Special Animal and Plant Sciences CAAS
Current assignee: Institute Special Animal and Plant Sciences CAAS
Priority date: 2020-11-02
Filing date: 2020-11-02
Publication date: 2023-06-23
Anticipated expiration: 2040-11-02
Also published as: CN112126692A

Abstract

The invention provides a molecular marker for identifying sika deer long island subspecies, an identification method and application. Aiming at the fact that no molecular marker and identification method for identifying the sika deer long island subspecies exist in the prior art, the invention carries out intensive research on the sika deer molecular marker to obtain the SNP molecular marker for identifying the sika deer long island subspecies, and the molecular marker comprises 11 SNP marker loci. The 11 SNP markers are positioned on 6 gene segments and have the characteristics of strong specificity and good stability. According to the gene segment, 6 pairs of SNP marker primers for identifying the subspecies of the sika-deer room Jiudao are designed, and an accurate and reliable detection method is established. The invention makes up the defect that the molecular marker detection technology for identifying the sika subspecies does not exist in the field at present, realizes the accurate identification of the sika subspecies, and has important theoretical and application values in the aspects of protection and management of sika resources.

Description

Molecular marker for identifying sika deer long island subspecies, identification method and application

Technical Field

The invention relates to a molecular marker for identifying sika deer house Jiuzhao subspecies, an identification method and application thereof, and belongs to the technical field of molecular biology.

Background

Sika deer (Cervus nippon) belongs to the class Mammalia (Mammalia), artiodactyla (Artiodactyla), deer (Cervidae), deer genus (Cervus), which are characteristic animals in the eastern quarter, and are distributed from the ukaribetween to vietnam. Because of the damage of ecological environment and the continuous reduction of habitat, the number of wild sika deer in China is very rare, and the wild sika deer is a class I protective animal in China at present and is also listed in the red directory of endangered species in the world natural protection alliance (IUCN). At present, subspecies of sika deer are divided mainly according to geographic distribution and morphological characteristics, so that great disputes and classification confusion exist.

The presently accepted statement is that Japanese spotted deer is divided into 6 subspecies. North sea subspecies (Cervus nipponyesoensis) are distributed throughout the North sea island. Subspecies (Cervus nippon centralis) are distributed among the islands of the state and the pair Ma Dao. Subspecies (Cervus nippon nippon) are distributed over the islands of September and four countries. The roof island subspecies (Cervus nipponyakushimae) are distributed on the roof island and the surrounding islands, and have smaller body types. The horseshoe subspecies (Cervus nippon mageshimae) are distributed in the horseshoe islands. The ball subspecies (Cervus nippon keramae) are distributed on the ball islands and the surrounding islands. Japanese spotted deer is further divided into southern and northern lineages, the genetic difference between the two lineages being greater than that between subspecies, northern lineages including North sea subspecies and the northern population of the current subspecies, and southern lineages including the southern population of the current subspecies, the famous subspecies, the Hmonda subspecies, the Massa subspecies, and the globus-ball subspecies, without obvious morphological feature differences between subspecies, so that molecular differences between subspecies are highly desirable as an effective tool for subspecies identification.

At present, subspecies of sika deer are divided mainly according to morphological differences and geographical distribution, but students also propose that the morphological differences of artiodactyls cannot be always used as the basis of classification, because morphological characteristics can be different according to different environments. Therefore, the problem of disordered classification of genetic resources exists among the current sika subspecies. The molecular identification method is a hotspot of current research, the SNP marker has the advantages of high accuracy, abundant variation, simple operation and the like, and the molecular marker is not influenced by environmental factors, can directly reflect the difference of animal gene levels, so that the molecular marker is widely applied to animal individual identification and species source identification, and the mitochondrial DNA marker is effective in solving the problems of evolutionary models and colonial histories among different species. At present, no molecular marker and no identification method for identifying sika subspecies exist.

Disclosure of Invention

Aiming at the defects of the prior art, the invention carries out intensive research on the sika deer molecular marker to obtain the SNP molecular marker and the detection primer which can be used for identifying sika deer Jiuzhaodao subspecies. The invention also provides a method for identifying the sika deer long island subspecies, which realizes the accurate identification of the sika deer subspecies and has important theoretical and application values in the aspects of protection and management of sika deer resources.

In view of the above object, the present invention provides, in a first aspect, a molecular marker for identifying subspecies of sika sikushima, 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, S-11; the site information is shown in table 1:

TABLE 1 SNP marker loci for identifying sika deer Dioscorea subspecies

Marking	Sika mtDNA genome	Site(s)	Base group
				S-1	495bp fragment of ND1 gene	301bp	A/G
S-2	500bp fragment of COX3 gene	140bp	G/A
				S-3	500bp fragment of COX3 gene	335bp	T/C
S-4	ND4 gene 670bp fragment	148bp	T/C
				S-5	ND4 gene 670bp fragment	362 th bp	T/C
S-6	ND4 gene 670bp fragment	559bp	T/C
				S-7	ND5 gene 500bp fragment	83bp	G/A
S-8	ND5 gene 500bp fragment	244bp	C/T
				S-9	ND6 gene 483bp fragment	93bp	G/A
S-10	ND6 gene 483bp fragment	302bp	C/T
				S-11	946bp fragment of Cytb gene	523bp	C/T

The 11 SNP loci S-1 to S-11 provided by the invention are A, G, T, T, T, T, G, C, G, C, C in the sika long island subspecies respectively; g, A, C, C, C, C, A, T, A, T, T in the non-sika house island subspecies respectively. The non-sika long island subspecies comprise sika northeast subspecies, sichuan subspecies, south China subspecies, taiwan subspecies, hokkaido subspecies, benzhou subspecies, named subspecies and the like.

In another aspect, the invention provides primers for identifying the subspecies of sika deer long island, wherein the primers are one or more of the following primer pairs: primer pair 1 with sequences shown as SEQ ID NO.1 and SEQ ID NO. 2; primer pair 2 with sequences shown as SEQ ID NO.3 and SEQ ID NO. 4; primer pair 3 with sequences shown as SEQ ID NO.5 and SEQ ID NO. 6; primer pair 4 with sequences shown as SEQ ID NO.7 and SEQ ID NO. 8; primer pair 5 with sequences shown as SEQ ID NO.9 and SEQ ID NO. 10; and the sequence is shown as a primer pair 6 shown as 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 a 500bp fragment of the COX3 gene; the primer pair 3 is used for amplifying 670bp fragments 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 a 483bp fragment of the ND6 gene; the primer pair 6 is used for amplifying 946bp fragments of the Cytb gene.

The invention also provides application of the molecular marker for identifying the sika long island subspecies or the primer for identifying the sika long island subspecies in identifying the sika long island subspecies; and application of the kit or the detection method in preparation of the kit or the detection method is used for identifying sika deer room Jiudao subspecies.

The invention also provides a method for identifying the sika deer room Jiuzhao 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 of the primer pairs 1-6; and sequencing the PCR product, and identifying whether the sample to be detected is the sika long island subspecies according to the SNP marker locus base.

If the sequence of the primer pair 1-6 amplified sequencing result is respectively shown as SEQ ID NO.13 (the base at 301bp is A), SEQ ID NO.15 (the base at 140bp is G, the base at 335bp is T), SEQ ID NO.17 (the base at 148bp is T, the base at 362bp is T, the base at 559bp is T), SEQ ID NO.19 (the base at 83bp is G, the base at 244bp is C), SEQ ID NO.21 (the base at 93bp is G, the base at 302bp is C) and SEQ ID NO.23 (the base at 523bp is C), the sample to be detected can be judged to be the sika deer room Jiudao subspecies. If the sequencing results are respectively shown as 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, i.e. the bases at the corresponding sites are respectively G, A, C, C, C, C, A, T, A, T, T, the sample to be tested can be judged to be the non-sika deer long island subspecies.

The molecular marker for identifying the sika house Jiudao subspecies and the identification method thereof have the following advantages:

(1) The invention obtains 11 SNP loci for identifying the subspecies of the sika long island, 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 the subspecies identification of sika deer resources and provides a new thought and method for the germplasm identification of sika deer. Has important theoretical and application values in the aspects of DNA fingerprint drawing of sika deer, protection management of wild subspecies and the like.

(2) The invention designs 6 pairs of SNP marker primers for identifying the subspecies of the sika house Jiudao, and establishes an accurate and reliable detection method. Establishes a method for identifying sika deer subspecies of Jiudao, and makes up the defect that the molecular marker detection technology for identifying sika deer subspecies is not available in the field at present.

(3) The identification method has extremely high accuracy. 90 sika deer samples from china and japan were tested using the method of the present invention. The results show that: 15 of the 90 samples were subspecies of the Jiujiu island, and the remaining samples were subspecies of the Jiujiu island. The detection accuracy is 100%.

(4) Low cost and practicability. Because the SNP locus is stable and reliable, one or more pairs of primers can be adopted, and when the identification mark of 1 SNP molecule is identical with the base of the sika long island subspecies specific SNP locus, the sika long island subspecies can be judged. Thus, the time is greatly saved and the cost is reduced. And the morphological characteristics of the sika deer to be detected are not required to be mastered, and the identification can be realized by collecting the DNA sample.

(5) Compared with the existing problem of inaccurate genetic resource classification, the method has the greatest advantage of accurate identification. Namely, the invention realizes the accurate identification of the sika house Jiuzhaoya subspecies and has important theoretical and application values in the aspects of protection and management of sika resources.

Drawings

FIG. 1 shows the sequence-differential SNP loci of the mtDNA of the sika deer long island subspecies and other subspecies. In the figure:

1.Cervus nippon hortulorum subspecies northeast; 2.Cervus nippon sichuanicus Sichuan subspecies; 3.Cervus nippon kopschi subspecies North China; 4.Cervus nippon taiouanus Taiwan subspecies; 5.Cervus nippon yesoensis North sea duct subspecies; 6.Cervus nippon centralis subspecies of Benzhou; 7.Cervus nippon nippon subspecies are named; 8.Cervus nippon yakushimae subspecies of Jiujiu island

FIG. 2 is a graph showing the PCR results of example 2. In the figure, M: DNAMaker1000;1: primer pair 1;2: primer pair 2;3: primer pair 3;4: primer pair 4;5: a primer pair; 6: primer pair 6.

Detailed Description

The invention is further described in connection with the following embodiments, but the invention is not limited thereto. The methods of the present invention, if not specified, are methods commonly used in the art, and the reagents involved, if not specified, are commercially available.

Example 1 acquisition of specific SNP markers for identifying the subspecies of the Dioscorea nipponica

1. Sika deer Hokkaido subspecies specific SNP locus screening

According to mtDNA sequences of different subspecies such as sika northeast subspecies, sichuan subspecies, south China subspecies, taiwan subspecies, north sea subspecies, benzhou subspecies, named subspecies and Jiujiu subspecies, screening is carried out mainly aiming at sites shared by the same subspecies and specific to other subspecies by utilizing Mega6.0 comparison analysis, and 11 differential SNP sites positioned on 6 genes are obtained through screening, as shown in figure 1. The ND1 gene fragment, the COX3 gene fragment, the ND4 gene fragment, the ND5 gene fragment, the ND6 gene fragment and the Cytb gene fragment are intercepted by Mega6.0, and the sequences are about 500bp in length and are used for designing identification primers.

2. Primer design

According to the intercepted target gene fragment, the identification primer is designed by combining Primer5.0 and oligo 7.0. The method comprises the following steps:

primer set 1 for amplifying ND1 gene fragment:

1F：5'-CTTAGCCGTGGCATTCCTCA-3'(SEQ ID NO.1)；

1R：5'-CGGTGAGATCAAATGGGGCT-3'(SEQ ID NO.2)。

primer pair 2 for amplifying COX3 gene fragment:

2F：5'-TCCTGCTAACACTCGGCCTA-3'(SEQ ID NO.3)；

2R：5'-AGTGTCAGTATCAGGCAGCG-3'(SEQ ID NO.4)。

primer set 3 for amplifying ND4 gene fragment:

3F：5'-CACATTTTACCCTGGCAAGCA-3'(SEQ ID NO.5)；

3R：5'-ATTGCTGCAAGGACCATGGA-3'(SEQ ID NO.6)。

primer set 4 for amplifying ND5 gene fragment:

4F：5'-CGCCCCCGTGTATAATAACAC-3'(SEQ ID NO.7)；

4R：5'-GTATTTGCGTCTGCTCGTCC-3'(SEQ ID NO.8)。

primer pair 5 for amplifying ND6 gene fragment:

5F：5'-ACAAAGACCACCCAGTCACA-3'(SEQ ID NO.9)；

5R：5'-TGGGTGGGTTTTTCGGATGT-3'(SEQ ID NO.10)。

primer pair 6 for amplifying the Cytb gene fragment:

6F：5'-CCACAACCAACTCCACCACT-3'(SEQ ID NO.11)；

6R：5'-GGGCTATGGCTTCTTCCTTGA-3'(SEQ ID NO.12)。

the primers were designed and synthesized by the division of biological engineering (Shanghai).

Example 2 establishment of methods for identifying the subspecies of the sika Cervus Nippon Temminck and other subspecies

PCR experiments were performed according to the primer pairs shown in SEQ ID No.1-SEQ ID No.12 to establish a method for identifying the subspecies and other subspecies of sika deer, 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 5min; denaturation at 94℃for 30sec, annealing at 59℃for 30sec, elongation at 72℃for 30sec, 30 cycles in total; further extending at 72℃for 5min and preserving at 4 ℃.

The PCR results are shown in figure 2, and 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 show that the electrophoresis strips are single and bright, and the experimental results show that the invention successfully establishes a method for effectively identifying the sika deer Hokkaido subspecies and other subspecies.

Example 3 specific application of the method of the invention for identifying the subspecies of Cervus Nippon Temminck and other subspecies

90 sika deer samples taken from China and Japan are selected, genomic DNA is extracted respectively, the samples are randomly mixed and renumbered by an experimenter A, PCR amplification is carried out on the samples by using the primers designed by the invention, and amplified products are sent to a biological engineering (Shanghai) stock company for sequencing. And checking a sequencing peak diagram through Bioedit 7.0, wherein the peaks and the troughs of the sequencing result diagram are separated, have no overlapping and have no misreading, so that the sequencing result is credible. The Mega6.0 comparison sequence is used, and the base of the sika deer long island subspecies specific SNP locus is used as a judgment basis.

The mtDNA specific SNP molecular markers determined according to the present invention. The identification results are shown in Table 4. The result is yes/no, which indicates the roof 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 the 75 samples of 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 15 samples of Y76-Y90 are A, G, T, T, T, T, G, C, G, C, C respectively, 75 samples of Y1-Y75 are confirmed to be non-HgJiuzu island subspecies by checking with an experimenter A, the accuracy is 100%, 15 samples of Y76-Y90 are HgJiuzu island subspecies, and the accuracy is 100%.

Table 4 sika deer sample test results

In summary, it is illustrated that the 11 SNP sites proposed by the present invention have high accuracy and good stability when used for identifying sika long island subspecies and other subspecies, and one or more pairs of primers may be used in consideration of cost, because sika long island subspecies can be determined when the marker identified by 1 SNP molecule is identical to the base of the sika long island subspecies-specific SNP site. Preferably, only any one of

primer pair

2, 3, 4, 5 is used, most preferably, only primer pair 3 is used.

Sequence listing

<110> institute of specialty of national academy of agricultural sciences

<120> molecular marker for identifying sika deer room Jiudao subspecies, identification method and application

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<213> sika deer (Cervus nippon)

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ccttataaat caattcagtg ataacagcct caacttctcg ctagtattct tttccgactc 240

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<213> sika deer (Cervus nippon)

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actattcgtt acatggtcca ttatagaatt ctcaatgtga tatatacact cagaccccaa 360

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<212> DNA

<213> sika 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> sika 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> sika 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> sika 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

1. The application of the sika deer molecular marker in identifying sika deer long island subspecies is characterized in that the molecular marker comprises 11 SNP loci:

；

If the base at the 301 th bp of the ND1 gene 495bp fragment SEQ ID NO.13 is A, COX3 gene 500bp fragment SEQ ID NO.15 is G at the 140 th bp, the base at the 335 th bp is T, ND4 gene 670bp fragment SEQ ID NO.17 is T at the 148 th bp, the base at the 362 th bp is T, the base at the 559 th bp is T, ND gene 500bp fragment SEQ ID NO.19 is G at the 83 th bp, the base at the 244 th bp is C, ND gene 483bp fragment SEQ ID NO.21 is G at the 93 th bp, the base at the 302 th bp is C, cytb gene 946bp fragment SEQ ID NO.23 is C, the detected sample can be judged to be the sika room long island subspecies.

2. The use of a sika molecular marker according to claim 1 for identifying sika long island subspecies, wherein the non-sika long island subspecies comprise northeast subspecies, sichuan subspecies, south China subspecies, taiwan subspecies, hokkaido subspecies, benzhou subspecies, and famous subspecies of sika.

3. The use of the sika deer molecular marker according to claim 1 for identifying sika deer subspecies of long islands, characterized in that the primer pair is used for identifying sika deer subspecies of long islands and amplifying gene fragments of the sika deer molecular marker locus in claim 1; the primer pair is as follows: primer pair 1 with sequences shown as SEQ ID NO.1 and SEQ ID NO. 2; primer pair 2 with sequences shown as SEQ ID NO.3 and SEQ ID NO. 4; primer pair 3 with sequences shown as SEQ ID NO.5 and SEQ ID NO. 6; primer pair 4 with sequences shown as SEQ ID NO.7 and SEQ ID NO. 8; primer pair 5 with sequences shown as SEQ ID NO.9 and SEQ ID NO. 10; primer pair 6 with sequences shown as SEQ ID NO.11 and SEQ ID NO. 12;

4. Use of the sika molecular marker of claim 1 or the primer pair of claim 3 in the preparation of a kit or in a detection method, characterized in that the use of the kit or the detection method is to identify sika long island subspecies.

5. A method for identifying sika deer house Jiuzhao subspecies is characterized by comprising the following steps: extracting DNA of a sample to be detected,

performing PCR amplification using the primer set of claim 3; sequencing the PCR product, and identifying whether the sample to be tested is sika deer long island subspecies according to the SNP marker locus base set forth in claim 1;

the primer pair is a primer pair 1 with sequences shown as SEQ ID NO.1 and SEQ ID NO. 2; primer pair 2 with sequences shown as SEQ ID NO.3 and SEQ ID NO. 4; primer pair 3 with sequences shown as SEQ ID NO.5 and SEQ ID NO. 6; primer pair 4 with sequences shown as SEQ ID NO.7 and SEQ ID NO. 8; primer pair 5 with sequences shown as SEQ ID NO.9 and SEQ ID NO. 10; and the sequence is shown as a primer pair 6 shown as SEQ ID NO.11 and SEQ ID NO. 12.

6. An application of a kit in identifying sika deer long island subspecies, which is characterized by comprising: primer pair 1 with sequences shown as SEQ ID NO.1 and SEQ ID NO. 2; primer pair 2 with sequences shown as SEQ ID NO.3 and SEQ ID NO. 4; primer pair 3 with sequences shown as SEQ ID NO.5 and SEQ ID NO. 6; primer pair 4 with sequences shown as SEQ ID NO.7 and SEQ ID NO. 8; primer pair 5 with sequences shown as SEQ ID NO.9 and SEQ ID NO. 10; primer pair 6 with sequences shown as SEQ ID NO.11 and SEQ ID NO. 12; performing PCR amplification by using the primer pair; sequencing the PCR product, and identifying whether the sample to be tested is sika deer long island subspecies according to the SNP marker locus base set forth in claim 1.