CN109988851B - Specific primers and detection methods for Tibetan mastiff molecular markers - Google Patents

Abstract

The invention relates to the technical field of single nucleotide variation and microsatellite marked specific primers and detection methods of domestic dog variety Tibetan mastiff. The method comprises the following steps of 1) extracting genomic DNA of Tibetan mastiff and genomic DNA of other domestic dogs; 2) respectively carrying out PCR amplification by using the genomic DNA extracted in the step (1) as a template and SNP molecular marker primers or primer groups; 3) sequencing the PCR amplification product by using a sequencer; 4) and detecting the variation condition of the single nucleotide polymorphic sites. The specific primer for the Tibetan mastiff molecular marker provided by the invention is handed over to a detailed detection method, and has important application values in the analysis of genetic characteristics, pedigree geographic patterns and population structures of Tibetan mastiff dogs and the scientific protection and utilization of germplasm resources.

Description

Specific primers and detection methods for Tibetan mastiff molecular markers

technical field

The invention belongs to the technical field of molecular markers, and in particular relates to the technical field of specific primers and detection methods for single nucleotide variation and microsatellite markers of domestic dog breed Tibetan mastiffs.

Background technique

The Tibetan Mastiff (Tibetan Mastiff) belongs to the domestic dog subspecies (Canis lupusfamiliaris) of the Canis genus Canis in the carnivorous order Canis. It is a unique domestic dog breed on the Qinghai-Tibet Plateau. Dan. Historically, Tibetan mastiffs were mainly raised by local herdsmen to protect livestock from predators in the wild. In recent years, due to its unique phenotype and ability to adapt to the plateau, it has been highly sought after by the market. Market behaviors such as excessive trade, wrong introduction, random breeding, and artificial speculation have made Tibetan mastiff germplasm resources face a huge threat. In order to scientifically protect Tibetan mastiff germplasm resources and realize sustainable development and utilization of resources, it is of great significance to carry out the specific genetic variation markers and population genetic characteristics analysis and molecular marker breeding of this species. At present, there are no reports on the development of genetic molecular markers for Tibetan mastiffs.

SUMMARY OF THE INVENTION

In order to solve the above problems and defects, the present invention provides a specific primer for molecular markers of Tibetan mastiffs and provides a detailed detection method. Both scientific protection and utilization have important application value.

The present invention is realized by the following technical solutions.

A SNP molecular marker primer, the molecular marker primer of the present invention optionally comprises the following primer pairs;

TM-P1: F:TGCAAGGAGGAGGAAGAAGG,

R:CTCCCAGTCTCTTGCCTCTG;

TM-P2: F:TGCAAGGAGGAGGAAGAAGG,

R:CTCCCAGTCTCTTGCCTCTG;

TM-E: F:CATTCAGTCAGCATTTCTGC

R:GGGCTGCAGGTTGCGAGGGT;

TM-M: F:TTCTTACATGGCAGCTGGTG,

R:CTTTTCCCTGTGAGCTCTGG;

TM-M1: F:GCTGCAATCCACAATGAAGA,

R:ATGCAGTCAGAACCCCAAGT;

TM-H1: F:GAGGCTTCAAATGCCTTGAG,

R:CATGCCTTAGGGGCAGTAAA;

TM-H2: F:GAGGCTTCAAATGCCTTGAG,

R:CATGCCTTAGGGGCAGTAAA;

TM-C: F:AGTCCCCAGAAGGTTCCATG,

R:GCAACCGGTGGAAAGTTTCT;

TM-S: F:GGCCTCCATGCTTAGCTCTA,

R: AGCACCTTACAGACTCCACC; wherein F is the upstream primer, and R is the downstream primer.

A SNP molecular marker primer set, the primer set of the present invention comprises any of the following two sets of primer pairs and combinations thereof; TM-P1, TM-P2, TM-E, TM-M, TM-M1, TM-H1, TM-H2, TM-C, TM-S; the specific sequences are as described above.

A SSR molecular marker primer, the molecular marker primer of the present invention optionally comprises the following primer pairs;

TM-CAS5: F:TCCCTCCTCAGCAGAGAGTC,

R:AACCTCAGGGCTATTCATTTCA;

TM-CAS6: F:TCCCTCTCCCTGTGTCTCTG,

R:CAATCCTTGAGCATGAAACG;

TM-CAS7: F:ACTGTCCTGGTGCCACCTAC,

R: CAACATCCTCTCCCCCTGAAA;

TM-CAS8: F:CCACAAAAGACCCACCCATA,

R:CTTCATGGAGCCTGCTTTTC;

TM-CAS9: F:GCTTTTGCTGTGTCCCAAAG,

R:AACTGTGGCCATCATTAGCA;

TM-CAS10: F:TGGCAACATGCTGAAAGTGT,

R:AGGTGGGCTCTGTGACCATA;

TM-CAS11: F:CTCCCTCTGCCTGTCTTCTG,

R:CTTGGGTGCAGAGCTAGTCC;

TM-CAS12: F:TACATCAGCCCCTGCTTTTCT,

R: TTGGCTTTAGTTCAGATGGAAG; wherein F is the upstream primer, and R is the downstream primer.

An SSR molecular marker primer set, the primer set of the present invention comprises any of the following two sets of primer pairs and combinations thereof; TM-CAS5, TM-CAS6, TM-CAS7, TM-CAS8, TM-CAS9, TM-CAS10, TM-CAS11, TM-CAS12; the specific sequences are as described above.

A primer set combining SNP and SSR molecular marker, the primer set of the present invention comprises at least two sets of primer pairs, namely: primer pair A and primer pair B; described primer pair A is a primer pair belonging to the SNP molecular marker primer set ; Described primer pair B is a primer pair belonging to the SSR molecular marker primer set; The primer pair in the SNP molecular marker primer set is, TM-P1, TM-P2, TM-E, TM-M, TM-M1, TM-H1, TM-H2, TM-C, TM-S; the specific sequences are as described above. The primer pairs belonging to the SSR molecular marker primer set are TM-P1, TM-P2, TM-E, TM-M, TM-M1, TM-H1, TM-H2, TM-C, TM-S; the specific sequence as described above.

Use of the above primer or primer set for identifying or distinguishing Tibetan mastiffs from other domestic dogs.

The present invention provides a detection method for Tibetan mastiff mononucleotide labeling, comprising the following steps:

(1), extract Tibetan mastiff genomic DNA;

(2), using the genomic DNA extracted in step (1) as a template, using the specific primers of each single nucleotide polymorphism site in the specific primers marked by the Tibetan mastiff single nucleotides to carry out PCR amplification respectively increase;

(3), using ABI3730 sequencer to sequence the PCR amplification product;

(4) Detection of single nucleotide polymorphism sites.

By constructing a Tibetan mastiff population genome database, the present invention screens the single nucleotide polymorphism sites with Tibetan mastiff-specific variation, designs specific primers for the single nucleotide polymorphism sites, and analyzes the single nucleotide polymorphism sites in these single nucleotide polymorphism sites. The specificity in Tibetan Mastiff was tested, and 9 SNPs with specific variation in Tibetan Mastiff were developed. The numbers of the 9 SNPs are: TM-P1, TM-P2, TM-E, TM-M, TM-M1, TM-H1, TM-H2, TM-C, TM-S ; Its nucleotide sequences are shown in Table 1, respectively.

100 domestic dog samples (including 50 Tibetan mastiffs and 50 other dog breeds) were detected by using the specific primers labeled with Tibetan mastiff single nucleotides in the present invention, and the results showed that 9 single nucleotide polymorphism sites It can be effectively amplified, and the amplification efficiency reaches 100%. The detection by ABI3730 sequencer shows that the 9 single nucleotide polymorphism sites all show Tibetan mastiff-specific variation, indicating that the 9 pairs of single nucleotide markers in the present invention The specific primers can be used for the identification of the genetic characteristics of Tibetan mastiffs.

The present invention discloses a set of specific primers and detection methods capable of efficiently amplifying the single nucleotide marker of Tibetan mastiff population, and the single nucleotide polymorphism site of the present invention has Tibetan mastiff population-specific variation. Therefore, the present invention provides The specific primers and detection methods for Tibetan mastiff single nucleotide markers can be applied in the fields of identification of the genetic characteristics of Tibetan mastiffs.

The present invention provides a kind of detection method of Tibetan mastiff microsatellite marker, it is characterized in that, comprises the following steps:

(1), extract Tibetan mastiff genomic DNA;

(2), take the genomic DNA extracted in step (1) as a template, utilize the specific primers of each microsatellite site in the specific primers of the described Tibetan mastiff microsatellites to carry out PCR amplification respectively;

(3), using ABI3730 sequencer to detect the polymorphism of PCR amplification products.

By constructing a Tibetan mastiff population genome database, screening DNA sequences containing microsatellite sequences, designing specific primers for microsatellite loci, and performing polymorphism detection on these microsatellite loci, the present invention develops 8 polymorphism-rich The Tibetan Mastiff microsatellite locus. The numbers of the 8 microsatellite sites are: TM-CAS5, TM-CAS6, TM-CAS7, TM-CAS8, TM-CAS9, TM-CAS10, TM-CAS11, TM-CAS12; their nucleotide sequences are as follows shown in Table 2.

100 domestic dog samples (including 50 Tibetan mastiffs and 50 other dog breeds) were detected by using the specific primers marked by the Tibetan mastiff microsatellite in the present invention. The results show that 9 microsatellite sites can be effectively amplified, and The increase rate reached 100%, and the detection by ABI3730 sequencer showed that the 9 microsatellite loci showed abundant polymorphisms, with Tibetan mastiff-specific variation characteristics, and all of them were in line with Hardy-Weinberg equilibrium. It is indicated that the 9 pairs of specific primers for microsatellite markers in the present invention can be used for the identification of the genetic characteristics of Tibetan mastiffs, the analysis of the genetic structure of the population, the identification of kinship and the like.

The invention discloses a set of specific primers and a detection method capable of efficiently amplifying the microsatellite markers of Tibetan mastiff population, and the microsatellite loci of the invention are rich in polymorphism and have Tibetan mastiff population-specific variation. Therefore, the Tibetan mastiff provided by the invention The specific primers and detection methods of microsatellite markers can be applied in the fields of identification of genetic characteristics of Tibetan mastiffs, analysis of population genetic structure, and identification of kinship, laying a foundation for the next step of molecular genetics research.

The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments.

Description of drawings

Fig. 1a is an agarose gel electrophoresis image of a single nucleotide PCR product amplified by primer pair TM-E according to the present invention.

Fig. 1b is an agarose gel electrophoresis image of a single nucleotide PCR product amplified by using the primer pair TM-M according to the present invention.

Fig. 1c is an agarose gel electrophoresis image of the single nucleotide PCR product amplified by the primer pair TM-M1 according to the present invention.

Fig. 2 is the schematic diagram that adopts the method of the present invention to distinguish Tibetan mastiff and other domestic dogs.

Fig. 3 is the schematic diagram that adopts the method of the present invention to distinguish the sub-population of Tibetan Mastiff.

Detailed ways

The following examples are further illustrations of the present invention, rather than limitations of the present invention. The specific experimental conditions and methods are not indicated in the following examples, and the technical means used are usually conventional means well known to those skilled in the art.

Example:

1. Extraction of Tibetan Mastiff Genomic DNA

1. Reagent preparation:

Digestion buffer: Weigh Tris1.2114g, EDTA37.224g, SDS 5g, and make up to 1000mL with sterilized ultrapure water (final concentration is Tris10mmol/L, EDTA0.1mol/L, SDS0.5%), adjust to pH value To 8.0, 4 degrees to save the spare.

2. DNA extraction:

Extract 200 μL of blood samples from 100 domestic dogs, add 400 μL of digestion buffer, 40 μL of proteinase K (10 mg/mL), mix well and digest at 55°C for 5 hours; after digestion is clear, add 600 μL of water-saturated phenol, gently invert and mix Homogenize for 30 minutes, centrifuge at 6000rpm/min for 10 minutes, extract the supernatant; then add 300μL of water-saturated phenol, 300μL of chloroform/isoamyl alcohol (24:1), gently invert and mix for 30 minutes, centrifuge at 6000rpm/min for 10 minutes, Extract the supernatant; then add 600 μL of chloroform/isoamyl alcohol (24:1), invert and mix for 30 minutes, centrifuge at 6000 rpm/min for 10 minutes, and extract the supernatant; Let stand overnight at -20°C, centrifuge at 13,000rpm/min for 10 minutes, discard the supernatant; add pre-cooled 70% ethanol, carefully wash the precipitate by pipetting, centrifuge at 13,000rpm/min for 10 minutes, discard the supernatant; Dissolve the precipitate in pure water and store at -20°C for later use.

2. Mining of Tibetan Mastiff-specific Mononucleotide and Microsatellite Molecular Markers

The domestic dog population genome database was constructed by large-scale genome sequencing, and by comparing the genomic differences between the Tibetan mastiff population and other domestic dog populations, the variation of the Tibetan mastiff-specific single nucleotide polymorphisms and the microsatellite loci in the selected regions of the Tibetan mastiff genome were identified.

3. Design of specific primers

Primer 3 software was used to design single nucleotide and microsatellite primers, and the primer annealing temperature was controlled above 50℃. The length of single nucleotide PCR product was controlled between 350 and 800 bp, and the length of microsatellite PCR product was controlled between 150 and 500 bp.

8 SNPs TM-P1, TM-P2, TM-E, TM-M, TM-M1, TM-H1, TM-C, TM-S and 1 indel variant site TM The primer sequences, mutation types, and annealing temperatures of -H2 are shown in Table 1.

Table 1. Specific primer sequences, mutation types and annealing temperatures for 8 single nucleotide polymorphism sites and 1 indel variant site

In the table, F represents the upstream primer, and R represents the downstream primer.

The primer sequences, core repeats, and annealing temperatures of the eight microsatellite sites CAST5, CAST6, CAST7, CAST8, CAST9, CAST10, CAST11, and CAST12 are shown in Table 2.

Table 2. 8 microsatellite site-specific primer sequences, core repeats and annealing temperatures

In the table, F represents the upstream primer, and R represents the downstream primer.

4. Amplification of single nucleotide polymorphism primers and microsatellite primers in the domestic dog population

PCR amplification was performed on 100 domestic dog samples by using the single nucleotide polymorphism site primer and the microsatellite primer of the present invention. The PCR reaction system is shown in Table 3:

Table 3. PCR reaction system

The PCR reaction program is shown in Table 4.

Table 4. PCR Reaction Program

5. Detection of PCR products by agarose gel electrophoresis

1. Reagent preparation:

10×TBE: 54.495g Tris, 27.81g boric acid, 14.615g EDTA, adjust pH to 8.0～8.2, dilute to 500ml (final concentration 0.9mol/LTris, 0.9mol/L boric acid, 0.1mol/LEDTA), autoclave, room temperature save for backup;

0.5×TBE: Measure 50ml of 10×TBE solution, add ultrapure water to make up to 1000ml, and store at room temperature for later use. 2. Making glue:

Weigh 0.2 g of agarose powder, add 0.5 × TBE to the volume to 100 ml (final concentration 2%), dissolve at high temperature, and the gel solidifies.

3. Electrophoresis

1. Add 10 μL of single nucleotide PCR product, add 5 μL of 6× loading buffer, mix well, load the sample, and run electrophoresis. Voltage 120V/cm, constant voltage electrophoresis for 40 minutes.

2. Add 1 μL of microsatellite PCR product, add 0.5 μL of 6× loading buffer, mix well, load the sample, and run electrophoresis. Voltage 120V/cm, constant voltage electrophoresis for 40 minutes.

The agarose gel electrophoresis images of some PCR products are shown in Figures 1a, 1b, and 1c. Due to space constraints, only the agarose gel electrophoresis images of some PCR products are provided. DNA Macker is DL2000 (2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom)

6. Sequencing of single nucleotide PCR products

1. PCR product recovery:

Under the irradiation of UV lamp, cut the target band, put it into a clean EP tube, and recover and purify it according to the steps provided by the general "DNA product purification and recovery kit".

2. Use the recovered product as a DNA template to perform sequencing reactions with forward and downstream primers respectively. The reaction system is shown in Table 5.

Table 5. Sequencing reaction system

The sequencing reaction program is shown in Table 6.

Table 6. Sequencing Reaction Procedure

3. Purify the sequencing reaction product:

Pipette 30 μL of pre-chilled 75% isopropanol, add the sequencing reaction product, mix well and place at -20°C for 30 minutes.

Centrifuge at 3800rpm/min for 30 minutes at 4°C.

Discard the supernatant and dry the sequencing reaction product.

Add 75 μL of ddH2O and place at 4°C for 2 hours to dissolve.

4. Sequencing:

Sequencing was performed by ABI 3730 sequencer.

Seven, allele analysis

8. Use seqman software to analyze the allele types of SNP loci

9. Calculation of genetic parameters of single nucleotide polymorphisms

Use plink software to calculate allele frequency, observed heterozygosity and expected heterozygosity; check whether the locus conforms to Hardy-Weinberg equilibrium; evaluate the degree of differentiation of the locus in Tibetan mastiff population and other domestic dog populations, and calculate F _ST value. The present invention uses the above method to obtain 9 SNP markers, the genetic parameters of each SNP are shown in Table 7, and all the loci are expressed between Tibetan Mastiff and other domestic dog groups They are highly differentiated, and they are all in line with the Hardy-Weinberg balance.

Table 7. Genetic parameters of single nucleotide polymorphisms in domestic dog populations

Nine, microsatellite PCR product length determination

1. Denaturation of PCR products:

Pipette 900 μL of Hidye into an EP tube, add 7.4 μL of LIZ500 molecular internal standard, mix well and dispense into a 96-well plate, and add 9 μL of mixed solution to each well.

Pipette 1 μL PCR product into the aliquoted Hidye mixture

Denaturation at 95°C for 10 minutes

Immediately put it into -20 ℃ to cool for 5 minutes

4°C for 2 hours

2. PCR product length determination

Length determination by ABI3730 sequencer

10. Calculation of genetic parameters of microsatellite loci

Use genescan software to read the length of each microsatellite PCR product; use geneAlEx software to calculate the number of alleles, population differentiation coefficient Fst, Hardy-Weinberg equilibrium; use Cervus3.0 software to calculate the multiplicity of each microsatellite locus. state information content. The present invention uses the above method to obtain 8 microsatellite markers, the genetic parameters of each microsatellite site are shown in Table 8, all sites are highly polymorphic sites (polymorphism information content>0.5), and all meet Hardy-Weinberg equilibrium (P>0.05).

Table 8. Genetic parameters of microsatellite loci in the domestic dog population

11. Clustering of domestic dog individuals by integrating SNP and microsatellite loci information

The genetic information of single nucleotide polymorphisms and microsatellite loci of each domestic dog individual was analyzed by STRUCTURE software, and all domestic dog individuals were clustered. The present invention obtains 9 single nucleotide polymorphism site markers and 8 microsatellite markers by the above method, integrates the genetic information of 17 sites and conducts MCMC analysis through STRUCTURE software, as shown in Figure 2 to distinguish Tibetan mastiffs from other domestic dogs , as shown in Figure 3 to distinguish the subgroups of Tibetan mastiffs.

Figure 2. Cluster analysis of 17 loci, assuming 2 populations.

Figure 3. Cluster analysis of 17 loci, assuming 3 populations.

The above descriptions are only specific embodiments of the present invention, and common knowledge such as well-known specific parameters in the solution are not described too much here. It should be pointed out that the above-mentioned embodiments do not limit the present invention in any way. For those skilled in the art, all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention. The scope of protection claimed in this application should be based on the content of the claims, and the descriptions of the specific implementation manners in the description can be used to interpret the content of the claims.

<110> Yunnan Zhongke Tibetan Mastiff Germplasm Resources Technology Development Co., Ltd.

<120> Specific primers and detection methods for Tibetan mastiff molecular markers

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TGCAAGGAGGAGGAAGAAGG

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CTCCCAGTCTCTTGCCTCTG

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TGCAAGGAGGAGGAAGAAGG

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CTCCCAGTCTCTTGCCTCTG

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CATTCAGTCAGCATTTCTGC

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GGGCTGCAGGTTGCGAGGGT

<210> 7

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TTCTTACATGGCAGCTGGTG

<210>8

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<213> Artificial sequences

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CTTTTCCCTGTGAGCTCTGG

<210> 9

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<212> DNA

<213> Artificial sequences

<400> 1

GCTGCAATCCACAATGAAGA

<210> 10

<211> 20

<212> DNA

<213> Artificial sequences

<400> 1

ATGCAGTCAGAACCCCAAGT

<210> 11

<211> 20

<212> DNA

<213> Artificial sequences

<400> 1

GAGGCTTCAAATGCCTTGAG

<210> 12

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<212> DNA

<213> Artificial sequences

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CATGCCTTAGGGGCAGTAAA

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GAGGCTTCAAATGCCTTGAG

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CATGCCTTAGGGGCAGTAAA

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<212> DNA

<213> Artificial sequences

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AGTCCCCAGAAGGTTCCATG

<210> 16

<211> 20

<212> DNA

<213> Artificial sequences

<400> 1

GCAACCGGTGGAAAGTTTCT

<210> 17

<211> 20

<212> DNA

<213> Artificial sequences

<400> 1

GGCCTCCATGCTTAGCTCTA

<210> 18

<211> 20

<212> DNA

<213> Artificial sequences

<400> 1

AGCACCTTACAGACTCCACC

<210> 19

<211> 20

<212> DNA

<213> Artificial sequences

<400> 1

TCCCTCCTCAGCAGAGAGTC

<210> 20

<211> 22

<212> DNA

<213> Artificial sequences

<400> 1

AACCTCAGGGCTATTCATTTCA

<210> 21

<211> 20

<212> DNA

<213> Artificial sequences

<400> 1

TCCCTCTCCCTGTGTCTCTG

<210> 22

<211> 20

<212> DNA

<213> Artificial sequences

<400> 1

CAATCCTTGAGCATGAAACG

<210> 23

<211> 20

<212> DNA

<213> Artificial sequences

<400> 1

ACTGTCCTGGTGCCACCTAC

<210> 24

<211> 20

<212> DNA

<213> Artificial sequences

<400> 1

CAACATCCTCTCCCCCTGAAA

<210> 25

<211> 20

<212> DNA

<213> Artificial sequences

<400> 1

CCACAAAAGACCCCACCCATA

<210> 26

<211> 20

<212> DNA

<213> Artificial sequences

<400> 1

CTTCATGGAGCCTGCTTTTC

<210> 27

<211> 20

<212> DNA

<213> Artificial sequences

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GCTTTTGCTGTGTCCCAAAG

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<211> 20

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AACTGTGGCCATCATTAGCA

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TGGCAACATGCTGAAAGTGT

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AGGTGGGCTCTGTGACCATA

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<211> 20

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CTCCCTCTGCCTGTCTTCTG

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<211> 20

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CTTGGGTGCAGAGCTAGTCC

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TACATCAGCCCCTGCTTTTCT

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TTGGCTTTAGTTCAGATGGAAG

Claims (8)

1. A molecular marker primer set, comprising the following SNP primer pairs;

TM-P1 ：F: TGCAAGGAGGAGGAAGAAGG，

R: CTCCCAGTCTCTTGCCTCTG；

TM-P2：F: TGCAAGGAGGAGGAAGAAGG，

R: CTCCCAGTCTCTTGCCTCTG；

TM-E：F: CATTCAGTCAGCATTTCTGC，

R: GGGCTGCAGGTTGCGAGGGT；

TM-M：F: TTCTTACATGGCAGCTGGTG，

R: CTTTTCCCTGTGAGCTCTGG；

TM-M1：F: GCTGCAATCCACAATGAAGA，

R: ATGCAGTCAGAACCCCAAGT；

TM-H1：F: GAGGCTTCAAATGCCTTGAG，

R: CATGCCTTAGGGGCAGTAAA；

TM-H2：F: GAGGCTTCAAATGCCTTGAG，

R: CATGCCTTAGGGGCAGTAAA；

TM-C：F: AGTCCCCAGAAGGTTCCATG，

R: GCAACCGGTGGAAAGTTTCT；

TM-S：F: GGCCTCCATGCTTAGCTCTA，

AGCACCTTACAGACTCCACC is the ratio of R to R; wherein F is an upstream primer and R is a downstream primer.

2. The molecular marker primer set according to claim 1, wherein the molecular marker primer set comprises the following SSR primer pairs;

TM-CAS5：F: TCCCTCCTCAGCAGAGAGTC，

R: AACCTCAGGGCTATTCATTTCA；

TM-CAS6：F: TCCCTCTCCCTGTGTCTCTG，

R: CAATCCTTGAGCATGAAACG；

TM-CAS7：F: ACTGTCCTGGTGCCACCTAC，

R: CAACATCCTCTCCCCTGAAA；

TM-CAS8：F: CCACAAAAGACCCACCCATA，

R: CTTCATGGAGCCTGCTTTTC；

TM-CAS9：F: GCTTTTGCTGTGTCCCAAAG，

R: AACTGTGGCCATCATTAGCA；

TM-CAS10：F: TGGCAACATGCTGAAAGTGT，

R: AGGTGGGCTCTGTGACCATA；

TM-CAS11：F: CTCCCTCTGCCTGTCTTCTG，

R: CTTGGGTGCAGAGCTAGTCC；

TM-CAS12：F: TACATCAGCCCCTGCTTTCT，

TTGGCTTTAGTTCAGATGGAAG is the ratio of R to R; wherein F is an upstream primer and R is a downstream primer.

3. The method for identifying and distinguishing Tibetan mastiff dogs from other domestic dogs by using the molecular marker primer set according to claim 1, characterized by comprising the steps of, 1) extracting genomic DNA of Tibetan mastiff and genomic DNA of other domestic dogs; 2) respectively carrying out PCR amplification by using the SNP molecular marker primer groups by using the genomic DNA extracted in the step (1) as a template; 3) sequencing the PCR amplification product by using a sequencer; 4) and detecting the variation condition of the single nucleotide polymorphic sites.

4. The method for identifying and distinguishing Tibetan mastiff dogs from other domestic dogs by using the molecular marker primer set according to claim 2, characterized by comprising the steps of, 1) extracting genomic DNA of Tibetan mastiff and genomic DNA of other domestic dogs; 2) respectively carrying out PCR amplification by using the SNP molecular marker primer groups by using the genomic DNA extracted in the step (1) as a template; 3) sequencing the PCR amplification product by using a sequencer; 4) and detecting the variation condition of the single nucleotide polymorphic sites.

5. The method of claim 4, comprising the steps of, 1) extracting genomic DNA of Tibetan mastiff and genomic DNA of other domestic dog; 2) respectively carrying out PCR amplification by using the SSR molecular marker primer group by taking the genomic DNA extracted in the step (1) as a template; 3) and carrying out polymorphism detection on the PCR amplification product by using a sequencer.

6. The method according to claim 3, 4 or 5, characterized in that it comprises the following, DNA extraction: respectively extracting 200 muL of 100 blood samples of the domestic dogs, adding 400 muL of digestion buffer solution and 40 muL of protease K10mg/mL, and digesting for 5 hours at 55 ℃ after uniformly mixing; after digestion and clearing, adding 600 mu L of water saturated phenol, slightly reversing and uniformly mixing for 30 minutes, centrifuging at 6000rpm/min for 10 minutes, and extracting supernatant; then adding 300 muL of water saturated phenol and 300 muL of chloroform/isoamyl alcohol 24:1, slightly reversing and uniformly mixing for 30 minutes, centrifuging at 6000rpm/min for 10 minutes, and extracting supernatant; adding 24:1 parts of 600 mu L chloroform/isoamyl alcohol, slightly reversing and uniformly mixing for 30 minutes, centrifuging at 6000rpm/min for 10 minutes, and extracting supernatant; then adding 800 muL of precooled isopropanol, standing overnight at-20 ℃, centrifuging for 10 minutes at 13000rpm/min, and discarding the supernatant; adding pre-cooled 70% ethanol, carefully blowing, washing and precipitating, centrifuging at 13000rpm/min for 10 minutes, and discarding the supernatant; drying the ethanol, adding 50 mu L of ultrapure water to dissolve the precipitate, and storing at-20 ℃ for later use.

7. The method of claim 3, 4 or 5, wherein the method comprises the following steps:

components Adding amount of Concentration of 10×Buffer 1 µL 1× 2.5 mmol/L dNTPs 1 µL 250 µmol/L Forward Primer 0.2 µL 0.2 µmol/L Reverse Primer 0.2 µL 0.2 µmol/L Taq Enzyme 0.1 µL 0.05 U/µL DNA 1 µL 5 ng/µL ddH₂O 6.5 µL Total up to 10 µL

。

8. Use of the molecular marker primer set according to claim 1 or 2, characterized in that the use is for identifying or differentiating Tibetan mastiff dogs from other domestic dogs.

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