CN117051133B - SNP molecular marker for detecting brucellosis resistance of sheep, detection primer and application thereof - Google Patents

Abstract

The invention relates to the technical field of molecular marker-assisted breeding, and specifically to SNP molecular markers for detecting sheep brucellosis resistance and detection primers and applications thereof. The present invention provides a SNP molecular marker related to sheep brucellosis resistance, which contains a nucleotide sequence in which the polymorphism at position 101 of the sequence shown in SEQ ID NO. 1 is G/A. This SNP molecular marker is significantly related to sheep brucellosis resistance, can more accurately detect the level of sheep resistance to brucellosis, and can be used for the selection and molecular marker-assisted breeding of brucellosis-resistant sheep. , which is conducive to improving breeding efficiency.

Description

SNP molecular markers and their detection primers for detecting sheep brucellosis resistance and application

Technical field

The present invention relates to the technical field of molecular marker-assisted breeding, and in particular to SNP molecular markers for detecting sheep brucellosis resistance and detection primers and applications thereof.

Background technique

Brucellosis (brucellosis), referred to as brucellosis, is a zoonotic systemic infectious disease caused by Brucella. Its clinical symptoms include wave fever, arthritis and reproductive disorders (orchitis and epididymitis in male animals, abortion and female animals). Infertility) etc. Brucella has the characteristics of immune evasion and latent infection. Among them, Brucella melitensis is the most virulent and infective, which not only causes huge economic losses to the sheep breeding industry, but also becomes a major safety hazard to public health. Excavating molecular markers related to brucellosis resistance traits in the host animal sheep and applying them to assist disease-resistant breeding in sheep will not only fundamentally cut off the transmission route of brucellosis, but also reduce the risk of antibiotic abuse. And cause environmental pollution and drug residue hazards. Therefore, it is of great significance to explore the molecular markers related to resistance to brucellosis in sheep.

Contents of the invention

The invention provides SNP molecular markers for detecting sheep brucellosis resistance and detection primers and applications thereof.

By conducting whole-genome association analysis in different breeds of sheep, the present invention discovered SNP molecular markers related to brucellosis resistance traits in the sheep genome. Through verification of a large sample size of sheep populations, it was proved that the SNP molecular markers are related to sheep. There is a significant correlation between brucellosis resistance traits and can be used to detect the resistance of sheep to brucellosis. To facilitate detection, the present invention also developed a primer combination for detecting the SNP molecular marker.

Based on the above findings, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a SNP molecular marker related to sheep brucellosis resistance. The SNP molecular marker contains a polymorphism of G/A at position 101 of the sequence shown in SEQ ID NO.1. nucleotide sequence.

The polymorphic site of the above-mentioned SNP molecular marker is located at position 35698759 on chromosome 3 of the sheep reference genome with version number Oar_v1.0 in November 2017, and the polymorphism is G/A.

The above-mentioned SNP molecular markers are significantly correlated with the resistance of sheep to brucellosis, and can more accurately identify the resistance to brucellosis of different breeds of sheep, and can be used to resist brucellosis in sheep. Molecular marker-assisted breeding and variety improvement to improve molecular breeding efficiency.

Based on the polymorphic site at position 35698759 on chromosome 3 of the sheep reference genome with the version number Oar_v1.0 in November 2017, in order to facilitate detection, the present invention develops the upstream and downstream sequences of this polymorphic site The SNP molecular marker sequence fragment and the primers used to amplify the SNP molecular marker are obtained. Combining the upstream and downstream sequences of the polymorphic site, the present invention obtains the sequence shown in SEQ ID NO.1. Those skilled in the art can understand that sequence fragments of different lengths can be developed as SNP molecular markers based on the above-mentioned SNP sites and their upstream and downstream sequences. Therefore, the sequence shown in SEQ ID NO. 1 does not constitute a reference to the SNP molecular markers of the present invention. Limitation, as long as the sequence fragment containing the polymorphic site at position 35698759 of chromosome 3 of the sheep reference genome with version number Oar_v1.0 in November 2017 is within the protection scope of the SNP molecular marker of the present invention.

In some embodiments of the present invention, the nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1, and the polymorphic site is located at position 101 of the sequence as shown in SEQ ID NO.1. Sex is G/A.

SEQ ID NO.1：

TGTTTTGCTTTCTCTTTCCTTCCATCTTTTCTTGCCCAGGTCACCCAGCTAAGGAATGGCCAAATCAGCTGGGACTCTGCCCTTTCGCTGGAGCTGGGGARAGAAGGTCCCTAGGAGGTAGGGACCAGCCCCTCAGCCTTATCCCTCCCCTTCTCTGCCAGGTCCTGGGGAGTCCGAGAAGATCCAGCAGCTGGAAGAGCA. Among them, R is the polymorphic site of SNP molecular marker, R=G or A.

In some embodiments of the present invention, the above-mentioned SNP molecular markers are amplified by using the sheep genome as a template and using primers with sequences as shown in SEQ ID NO. 2-4.

The genotype of the polymorphic site of the above-mentioned SNP molecular marker is GG or AA, which corresponds to high resistance to brucellosis, and the genotype is GA, which corresponds to low resistance to brucellosis.

Among the above SNP molecular markers, sheep with the "GG" homozygous genotype and "AA" homozygous genotype have stronger brucellosis resistance than the "GA" heterozygous genotype.

In a second aspect, the present invention provides a primer combination for amplifying the above-mentioned SNP molecular markers related to sheep brucellosis resistance.

Based on the genomic location of the polymorphic site of the SNP molecular marker provided above and its upstream and downstream sequences, those skilled in the art can develop various types of primers for amplifying the SNP molecular marker.

The above primers can be any primers that can be used to detect the genotype of SNP molecular markers.

Preferably, the primer combination includes a primer with a sequence shown in SEQ ID NO.2-4, wherein the primer with a sequence shown in SEQ ID NO.2-3 is a forward primer, and a primer with a sequence shown in SEQ ID NO.4 The primer is a reverse universal primer.

SEQ ID NO.2: 5’-CCTACCTCCTAGGGACCTTCTT-3’;

SEQ ID NO.3: 5’-CTACCTCCTAGGGACCTTCTC-3’;

SEQ ID NO. 4: 5'-TCACCCAGCTAAGGAATGCCAAAT-3'.

The primer combinations described above can achieve efficient amplification and genotyping of the above SNP molecular markers.

KASP, competitive allele-specific PCR, is a technology that can perform high-precision biallelic typing of SNPs through specific fluorescent probes. The analysis is stable and accurate, low-cost, high-efficiency, and easy to achieve high-throughput and automation. Therefore, in order to achieve efficient detection, the present invention developed a KASP primer combination for detecting the above-mentioned SNP molecular markers based on KASP technology.

Preferably, the KASP primer combination includes a first forward primer, a second forward primer and a reverse universal primer, wherein the sequence of the first forward primer is a sequentially connected specific fluorescent tag sequence and SEQ ID The sequence shown in NO.2, the sequence of the second forward primer is the sequentially connected specific fluorescent tag sequence and the sequence shown in SEQ ID NO.3; the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.4 Show.

The first forward primer and the second forward primer described above have different fluorescent labels.

In some embodiments of the present invention, the primer combination includes the forward primers shown in SEQ ID NO. 5-6 and the reverse universal primer shown in SEQ ID NO. 4.

In a third aspect, the present invention provides a kit comprising the primer combination described above.

To facilitate detection, the kit may also contain other reagents for PCR amplification, including but not limited to DNA polymerase, PCR reaction buffer, probes, dNTPs, Mg ²⁺ , water, etc.

The above reagents can be packaged individually or mixed and provided as a premix.

The kits described above can be used for any of the following purposes:

1) Detect or assist in detecting the resistance of sheep to brucellosis;

2) Screen or identify sheep with high resistance to brucellosis;

3) Early prediction of sheep’s brucellosis resistance traits;

4) Molecular marker-assisted breeding of sheep for brucellosis resistance;

5) Improvement of sheep breeds resistant to brucellosis.

In the fourth aspect, the present invention provides any one of the following 1)-8) applications of SNP molecular markers or detection primers of said SNP molecular markers:

1) Application in detecting or assisting in detecting the resistance of sheep to brucellosis;

2) Application in the preparation of reagents for detecting or assisting in detecting the resistance of sheep to brucellosis;

3) Application in screening or identifying sheep with high resistance to brucellosis;

4) Application in the preparation of reagents for screening or identifying sheep with high resistance to brucellosis;

5) Application in early prediction of sheep brucellosis resistance traits;

6) Application in the preparation of early prediction reagents for sheep resistance to brucellosis traits;

7) Application in molecular marker-assisted breeding of sheep resistant to brucellosis;

8) Application in the improvement of sheep breeds resistant to brucellosis.

The polymorphic site of the SNP molecular marker is located at position 35698759 on chromosome 3 of the sheep reference genome with version number Oar_v1.0 in November 2017, and the polymorphism is G/A.

In some embodiments of the present invention, in the above application, the SNP molecular marker contains a nucleotide sequence in which the polymorphism at position 101 of the sequence shown in SEQ ID NO. 1 is G/A.

In some embodiments of the present invention, in the above application, the nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1, and the polymorphic site is located at position 101 of the sequence as shown in SEQ ID NO.1 , the polymorphism is G/A.

In some embodiments of the present invention, in the above applications, the detection primers for SNP molecular markers include primers with sequences as shown in SEQ ID NO.2-4, wherein primers with sequences as shown in SEQ ID NO.2-3 is a forward primer, and the primer whose sequence is shown in SEQ ID NO. 4 is a reverse universal primer.

In the above application, the genotype of the polymorphic site of the SNP molecular marker is GG or AA, which corresponds to high resistance to brucellosis, and the genotype is GA, which corresponds to low resistance to brucellosis.

In the above application, sheep whose genotypes of the SNP molecular markers are "GG" and "AA" are selected as parents for breeding for brucellosis resistance traits.

In a fifth aspect, the present invention provides a method for detecting the resistance of sheep to brucellosis. The method includes: detecting the genotype of the SNP molecular marker in the sheep genome that is related to the sheep's resistance to brucellosis traits, so The polymorphic site of the above-mentioned SNP molecular marker is located at position 35698759 on chromosome 3 of the sheep reference genome with version number Oar_v1.0 in November 2017, and the polymorphism is G/A.

The genotype of the polymorphic site of the SNP molecular marker is GG or AA, which corresponds to high resistance to brucellosis, and the genotype is GA, which corresponds to low resistance to brucellosis.

In some embodiments of the present invention, the SNP molecular marker contains a nucleotide sequence in which the polymorphism at position 101 of the sequence shown in SEQ ID NO. 1 is G/A.

In some embodiments of the present invention, the nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1, and the polymorphic site is located at position 101 of the sequence as shown in SEQ ID NO.1. Sex is G/A.

Preferably, the method includes the following steps:

1) Extract the genomic DNA of the sheep to be tested;

2) Use the genomic DNA in step 1) as a template and use the primers with the sequence shown in SEQ ID NO. 2-4 to perform PCR amplification;

3) Analyze the genotype of the SNP molecular marker in the PCR amplification product, and determine the resistance of the sheep to be tested to brucellosis based on the genotype. If the genotype of the polymorphic site of the SNP molecular marker is If the genotype is GG or AA, it is judged to be high resistance to brucellosis. If the genotype is GA, it is judged to be low resistance to brucellosis.

The beneficial effects of the present invention at least include: the present invention provides SNP molecular markers related to sheep brucellosis resistance traits. The SNP molecular markers are significantly related to sheep brucellosis resistance and can more accurately detect sheep's resistance to brucellosis. The level of resistance to brucellosis can be used to predict the resistance to brucellosis in sheep early. It is not restricted by the age, gender, etc. of sheep. Even sheep can be accurately screened just after they are born, and can be used for resistance to brucellosis. The breeding of sheep with fungal disease and the molecular marker-assisted breeding of sheep resistant to brucellosis can significantly promote the breeding process of sheep resistant to brucellosis, effectively improve the breeding efficiency, and contribute to the development and utilization of excellent sheep varieties. Excellent economic characteristics and the protection and rational utilization of variety resources are of great significance.

Description of the drawings

In order to explain the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are of the present invention. For some embodiments of the invention, those of ordinary skill in the art can also obtain other drawings based on these drawings without exerting creative efforts.

Figure 1 shows the genotyping results of polymorphic sites of SNP molecular markers in Example 2 of the present invention.

Figure 2 is the population expansion verification result of SNP molecular markers in the sheep population in Example 2 of the present invention, where ** represents p<0.01.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention. , not all examples. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

The present invention provides a SNP molecular marker related to sheep brucellosis resistance. The polymorphic site of the SNP molecular marker is located at No. 1 of the sheep reference genome with version number Oar_v1.0 in November 2017. There is a G/A base mutation at position 35698759 of chromosome 3, which is significantly related to the resistance to brucellosis in sheep. It is speculated that mutation of this site may affect the clearance of Brucella in sheep. Ability; at this polymorphic site, sheep with the "GG" homozygous genotype and "AA" homozygous genotype have stronger brucellosis resistance than the "GA" heterozygous genotype. The SNP molecular markers provided by the present invention have important guiding significance for genotype differentiation and screening of sheep with brucellosis resistance traits, and can improve the accuracy and efficiency of sheep resistance to brucellosis screening.

The present invention has no special restrictions on the method for detecting the genotype of the SNP molecular marker, and conventional genotype detection methods in the field can be used. In a specific embodiment of the present invention, the KASP method is used to detect the genotype of the SNP molecular marker in the sheep genome to be tested.

The SNP molecular markers related to sheep brucellosis resistance or their detection primers provided by the present invention can be used in combination with other SNP molecular markers related to sheep brucellosis resistance or their detection primers for sheep resistance to brucellosis. Identification of leucellosis traits.

Example 1 Development of SNP molecular markers related to sheep brucellosis resistance

This example uses sheep populations of four breeds: Suffolk, East Frisian, White Suffolk and Texel as samples, and uses genome-wide association analysis (GWAS) to develop the traits related to sheep brucellosis resistance. Relevant SNP molecular markers are as follows:

Suffolk, East Frisian, White Suffolk and Texel breed sheep were raised under the same conditions and naturally infected with brucellosis. Sheep blood samples were collected and competitive enzyme-linked immunosorbent assay (cELSA) was used. Indirect enzyme-linked immunosorbent assay (iELISA) and fluorescence polarization assay (FPA) are used to detect whether sheep are infected with brucellosis. The positive judgment thresholds of each method are: cELISA: 30, iELISA: 15, FPA: 20, which is higher than the above The threshold line is judged as positive. If the test results of the above three methods are all positive, it is judged that the patient is suffering from Brucella, if all are negative, the patient is healthy, and the rest are judged as suspicious. Finally, 25 sheep judged to be suffering from brucellosis were selected as the experimental group, and 25 sheep judged to be healthy were used as the control group. A genome-wide association analysis (GWAS) was performed on the above 50 sheep. Among them, the information of the sick sheep and The blood sample test results are shown in Table 1, and the information of healthy sheep and their blood sample test results are shown in Table 2.

Table 1

Table 2

Whole-genome resequencing was performed on the blood DNA samples of the above 50 sheep. The sequencing platform was BGI T7, the sequencing depth was 20×, and the total sequencing volume was 2.6 T. Sequence alignment and quality control were performed on the sequencing data. The software fastp was used for data filtering, and the software GTX was used for alignment and mutation detection. An aligned VCF file with a size of 71.08 GB was obtained. The software PLINK was used for quality control. The quality control standards were as follows: --mind 0.1 --geno 0.1 --maf 0.05 --hwe 1e-5. The number of valid SNPs after quality control was 24,683,444. Use the software PLINK, code: plink --bfile filename --pca 10 --out filename_pca --chr-set 27 --allow-extra-chr to perform principal component analysis. Select quantitative traits (iELISA values), use GEMMA (Version 0.95) software, and As the basis of the model, Genome assembly Oar_rambouillet_v1.0 is used as the reference genome to perform genome-wide association analysis. Then perform SNP annotation and enrichment analysis. The tool for SNP site annotation is https://asia.ensembl.org/index.html, the reference genome is Genome assembly Oar_rambouillet_v1.0, and the tool for GO enrichment analysis is https: //biit.cs.ut.ee/gprofiler/gost, the reference genome is Homo sapiens (Human), Ovis aries (Sheep), the KEGG enrichment analysis tool is: https://david.ncifcrf.gov, the reference genome is Homosapiens (Human), Ovis aries (Sheep).

In the genome-wide association analysis, the -log ₁₀ (P) value of the Top 1000 SNPs ranged from 4.56 to 7.36, and 100 genes were annotated. Among them, there were 48 SNPs with -log ₁₀ (P) values above 6. , 13 genes were annotated. Among these 48 SNPs, SNP molecular markers that were significantly associated with the disease resistance phenotype of sheep brucellosis were screened. Finally, the present invention obtained a SNP molecular marker related to sheep brucellosis resistance. The polymorphic site of this SNP molecular marker is located at No. 3 of the sheep reference genome with version number Oar_v1.0 in November 2017. Chromosome No. 35698759, the polymorphism is G/A, which has a significant correlation with the resistance to brucellosis in sheep. The polymorphism site is the "GG" homozygous genotype and the "AA" homozygous gene. The cELISA value, iELISA value and FPA value of individual sheep with type 1 were significantly lower than those of the "GA" heterozygous genotype (p<0.01). That is, compared with the "GA" heterozygous genotype, the "GG" homozygous genotype and the "GG" homozygous genotype were significantly lower than those of the "GA" heterozygous genotype. Sheep with the homozygous genotype AA” have greater resistance to brucellosis, and “GG” and “AA” are the dominant genotypes in Brucella-resistant sheep. The above-mentioned SNP molecular marker can correspond to the sequence shown in SEQ ID NO. 1, wherein the polymorphic site is located at position 101 of the sequence shown in SEQ ID NO. 1, and the polymorphism is G/A.

Based on the above SNP molecular markers, the present invention further developed KASP primers for detecting the SNP molecular markers, specifically as follows:

Primer X:

5’-gaaggtgaccaagttcatgctCCTACCTCCTAGGGACCTTCTT-3’ (SEQ ID NO.5, the lowercase letters are the specific fluorescent tag sequence FAM);

Primer Y:

5’-gaaggtcggagtcaacggattCTACCTCCTAGGGACCTTCTC-3’ (SEQ ID NO.6, the lowercase letters are the specific fluorescent tag sequence VIC);

Primer R: 5’-TCACCCAGCTAAGGAATGGCCAAAT-3’ (SEQ ID NO. 4).

The polymorphic sites of the SNP molecular markers obtained above can be used to distinguish the "GA" heterozygous genotype, the "GG" homozygous genotype and the "AA" homozygous gene in the sheep population of this example. type these three genotypes.

Example 2 Application of SNP molecular markers related to sheep brucellosis resistance

The SNP molecular markers related to sheep brucellosis resistance developed in Example 1 were subjected to expanded population verification, as follows:

1. Collect sheep blood samples to be tested and identify serum antibody concentrations

Jugular blood was collected from unvaccinated 127 Suffolk sheep, 129 East Friesian sheep, 130 White Suffolk sheep and 27 Texel sheep from farms naturally infected with Brucella . The indirect enzyme-linked immunosorbent assay (iELISA) method was used to detect the serum Brucella antibody concentration. The iELISA value of an individual can be used as an indicator of its disease resistance. That is, the lower the iELISA value, the stronger the individual's resistance to brucellosis, and vice versa. In the embodiment, the threshold for determining negative and positive iELISA results is set to 15.

2. Extract genomic DNA from the sheep blood sample to be tested

The magnetic bead method was used to extract genomic DNA from the sheep blood samples to be tested.

3. Amplification of SNP molecular marker fragments

Using the genomic DNA extracted in step 2 above as a template, use the KASP primer combination (SEQ ID NO. 5-6 and SEQ ID NO. 4) developed in Example 1 to perform PCR amplification, as follows:

(1) KASP amplification system:

The 1.6 μL reaction system includes: 0.8 μL of 50-100 ng/μL genomic DNA, 0.022 μL of primer mixture (preferred primer mixture ratio: 60 μL of 100 μmol/L forward primers Primer Add 150 μL of primer Primer R, 230 μL of 10 mM Tris HCl), 0.4 μL of 2× Master mix, and 1.6 μL of double-distilled water.

The above reaction system is the preferred reaction system for the Douglas Array Tape platform. Other reasonable reaction systems can also achieve the same detection purpose.

Among them, 2×Master mix includes fluorescent probe A, fluorescent probe B, quenching probe A and quenching probe B, as well as high-fidelity Taq enzyme, dNTP and Mg ²⁺ , etc. The sequence of fluorescent probe A is 5'-GAAGGTGACCAAGTTCATGCT-3', and its 5' end is connected to a fluorescent group FAM; the sequence of fluorescent probe B is 5'-GAAGGTCGGAGTCAACGGATT-3', and its 5' end is connected to a fluorescent group. VIC; the nucleotide sequence of quenching probe A is 5'-AGCATGAACTTGGTCACCTTC-3', and its 3' end is connected to a fluorescent group BHQ; the nucleotide sequence of quenching probe B is 5'-AATCCGTTGACTCCGACCTTC-3 ', with a fluorescent group BHQ connected to its 3' end.

(2) PCR reaction conditions:

DNA fragment amplification: pre-denaturation at 94°C for 15 min, set up one cycle; denaturation at 94°C for 20 s, gradient annealing and extension at 61-55°C for 60 s, set up 10 cycles, with each cycle lowered by 0.6°C.

Fluorescence signal enhancement: denaturation at 94°C for 20 s, annealing and extension at 55°C for 60 s, 26 cycles set.

4. Use the Douglas Array Tape platform to detect the PCR amplification product and obtain the genotype of the polymorphic site of the SNP molecular marker.

The polymorphic site genotypes of the SNP molecular markers in the above 413 sheep were detected. The results are shown in Table 3 and Figure 1. Three genes, "GG", "GA" and "AA", were distinguished in the sheep population. type. Correlation analysis between genotypes and Brucella resistance traits was performed. The results are shown in Figure 2. The iELISA values of individual sheep with "GG" and "AA" genotypes were significantly lower than those of the "GA" genotype (p<0.01). It shows that the brucellosis resistance of individual sheep with "GG" and "AA" genotypes is higher than that of the "GA" genotype, and "GG" and "AA" are the dominant genotypes of sheep resistant to brucellosis. The present invention The provided SNP molecular markers are highly accurate for identification of brucellosis resistance traits.

Table 3 Number of individuals with different genotypes of polymorphic sites of SNP molecular markers in the sheep population

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that it can still be used Modifications are made to the technical solutions described in the foregoing embodiments, or equivalent substitutions are made to some of the technical features; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (3)

1. Use of any one of the following 1) -5) of detection primers for snp molecular markers:

   1) The application in preparing a reagent for detecting or assisting in detecting the resistance of sheep to brucellosis;

   2) Use in the preparation of a reagent for screening or identifying sheep with high resistance to brucellosis;

   3) The application of the method in preparing early prediction reagent for the brucellosis resistance of sheep;

   4) Application in molecular marker assisted breeding of sheep to brucellosis resistance;

   5) The application in the improvement of the brucellosis resistance of sheep;

   the polymorphic site of the SNP molecular marker is positioned at 35698759 of chromosome 3 of sheep reference genome with version number of oar_v1.0 and 2017, 11 months, and the polymorphism is G/A;

   the genotype of the polymorphic locus of the SNP molecular marker is GG or AA, which corresponds to the high resistance of brucellosis, and the genotype is GA, which corresponds to the low resistance of brucellosis.
2. 2. The use according to claim 1, wherein the detection primer of the SNP molecular marker comprises a primer with a sequence shown as SEQ ID NO.2-4, wherein the primer with a sequence shown as SEQ ID NO.2-3 is a forward primer and the primer with a sequence shown as SEQ ID NO.4 is a reverse universal primer.
3. 3. The use according to claim 1, wherein the SNP molecular marker has a nucleotide sequence shown as SEQ ID NO.1, the polymorphic site is located at position 101 of the sequence shown as SEQ ID NO.1, and the polymorphism is G/A.