Molecular marker combination for identifying tianjin monkey chicken breed and application thereof

Abstract

This invention discloses a molecular marker combination for identifying the Tianjin Monkey Chicken breed and its application, relating to the field of molecular biology detection. The molecular marker combination includes molecular markers with nucleotide sequences as shown in SEQ ID NO. 1-19. Based on a large-scale population of 25 representative local Chinese chicken breeds, including the Tianjin Monkey Chicken, and two introduced breeds, this invention identifies specific SNP loci, screens for molecular markers to identify the Tianjin Monkey Chicken, and combines existing methods for detecting specific SNP loci with AHP hierarchical analysis to comprehensively consider the allele frequency and genotype type of specific loci. This ensures accuracy while improving detection efficiency and the scientific rigor of evaluation. This invention uses molecular marker technology to scientifically identify and evaluate the breed specificity of the Tianjin Monkey Chicken. The operation is simple and the results are reliable, providing a scientific basis for the protection and rational utilization of local chicken genetic resources.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection, and in particular to a molecular marker combination for identifying Tianjin monkey chicken breeds and its application. Background Technology

[0002] Tianjin Monkey Chicken (also known as Tianjin Heyuan Monkey Chicken) is a distinctive dual-purpose local breed in my country. Its origin and central production area are located in the Heyuan region of Tianjin. It is a large breed with excellent egg and meat quality. The breed's distinctive physical characteristic is a bare neck or only a few crest feathers on the head. Compared to conventionally feathered chickens, the bare area between feathered zones is wider, while the feathered areas on the chest, legs, and other parts are significantly smaller, with the most prominent bare-necked area on the neck where feathers are almost completely absent. It represents a valuable genetic resource of bare-necked chickens in my country.

[0003] Single nucleotide polymorphisms (SNPs) refer to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. These include transitions, transversions, deletions, and insertions. The resulting genetic markers are numerous and highly polymorphic. Theoretically, each SNP site can have four different variant forms, but in reality, only transitions and transversions occur, with a ratio of 2:1. As one of the most common forms of polymorphism in the genome, SNPs exhibit high genetic stability.

[0004] Restriction-site-associated DNA sequencing (RAD-seq) is a simplified genome sequencing technology developed based on next-generation sequencing (NGS) technology. Reduced-representation sequencing uses restriction endonucleases to digest the genome, selecting only a specific region for sequencing, thus reducing genomic complexity. RAD sequencing library construction involves digesting genomic DNA fragments with restriction enzymes and randomly cutting them, selecting fragments with restriction sites at one end and random break sites at the other for library construction and sequencing. RAD-seq can reduce genomic complexity without being limited by a reference genome, is simple to operate, and can rapidly identify high-density SNPs. However, current methods for evaluating the function of molecular markers in Tianjin monkeys are limited, making it difficult to assess their genetic diversity, and there are issues such as inaccurate methods for identifying purebreds. Therefore, based on simplified genome sequencing, it is highly significant to screen molecular markers for single nucleotide polymorphisms (SNPs) in Tianjin monkeys and conduct a comprehensive evaluation. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular marker combination for identifying the Tianjin Monkey Chicken breed and its application, in order to solve the problems existing in the prior art. The molecular marker combination provided by this invention can efficiently and accurately evaluate the bloodline purity of individual Tianjin Monkey Chickens and realize breed identification. It helps to alleviate the problems in the prior art due to the lack of molecular markers that can evaluate the genetic diversity of Camellia Chicken and the inaccuracy of existing purebred identification methods.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a combination of SNP sites for identifying the Tianjin monkey chicken breed, including the SNP sites shown in the table below:

[0008]

[0009]

[0010] The present invention also provides a molecular marker combination for identifying the Tianjin monkey chicken breed, the molecular marker combination comprising molecular markers with nucleotide sequences as shown in SEQ ID NO.1-19 respectively;

[0011] The molecular marker shown in SEQ ID NO.1 has a mutation site SNP1 at 11 bp, which is an A / G mutation;

[0012] The molecular marker shown in SEQ ID NO.2 has a mutation site SNP2 at 11 bp, which is a C / T mutation;

[0013] The molecular marker shown in SEQ ID NO.3 has a mutation site SNP3 at 11 bp, which is a C / T mutation;

[0014] The molecular marker shown in SEQ ID NO.4 has a mutation site SNP4 at 11 bp, which is an A / T mutation;

[0015] The molecular marker shown in SEQ ID NO.5 has a mutation site SNP5 at 11 bp, which is a C / T mutation;

[0016] The molecular marker shown in SEQ ID NO.6 has a mutation site SNP6 at 11 bp, which is an A / G mutation;

[0017] The molecular marker shown in SEQ ID NO.7 has a mutation site SNP7 at 11 bp, which is a G / A mutation;

[0018] The molecular marker shown in SEQ ID NO.8 has a mutation site SNP8 at 11 bp, which is a G / A mutation;

[0019] The molecular marker shown in SEQ ID NO.9 has a mutation site SNP9 at 11 bp, which is a G / A mutation;

[0020] The molecular marker shown in SEQ ID NO.10 has a mutation site SNP10 at 11 bp, which is a T / C mutation;

[0021] The molecular marker shown in SEQ ID NO.11 has a mutation site SNP11 at the 11 bp, which is a T / C mutation;

[0022] The molecular marker shown in SEQ ID NO.12 has a mutation site SNP12 at the 11 bp, which is an A / G mutation;

[0023] The molecular marker shown in SEQ ID NO.13 has a mutation site SNP13 at 11 bp, which is a C / T mutation;

[0024] The molecular marker shown in SEQ ID NO.14 has a mutation site SNP14 at the 11 bp, which is an A / C mutation;

[0025] The molecular marker shown in SEQ ID NO.15 has a mutation site SNP15 at 11 bp, which is a T / C mutation;

[0026] The molecular marker shown in SEQ ID NO.16 has a mutation site SNP16 at 11 bp, which is a C / G mutation;

[0027] The molecular marker shown in SEQ ID NO.17 has a mutation site SNP17 at 11 bp, which is a C / A mutation;

[0028] The molecular marker shown in SEQ ID NO.18 has a mutation site SNP18 at 11 bp, which is an A / T mutation;

[0029] The molecular marker shown in SEQ ID NO.19 has a mutation site SNP19 at 11 bp, which is a T / C mutation.

[0030] Further, the genotype of SNP1 is AA, AG, or GG; the genotype of SNP2 is CC, CT, or TT; the genotype of SNP3 is CC, CT, or TT; the genotype of SNP4 is AA, AT, or TT; the genotype of SNP5 is CC, CT, or TT; the genotype of SNP6 is AA, AG, or GG; the genotype of SNP7 is GG, GA, or AA; the genotype of SNP8 is GG, GA, or AA; the genotype of SNP9 is GG, GA, or AA; and the genotype of SNP10 is... The genotypes of SNP11, SNP12, SNP13, SNP14, SNP15, SNP16, SNP17, SNP18, and SNP19 are TT, TC, or CC. The genotypes of SNP11, SNP12, SNP13, SNP14, SNP15, SNP16, SNP17, SNP18, SNP19, SNP18, and SNP19 are TT, TC, or CC.

[0031] This invention also provides a primer pair combination for identifying the Tianjin monkey chicken breed, the primer pair combination comprising primer pairs 1-19 as shown in the table below:

[0032]

[0033] This invention also provides the application of the above-mentioned SNP site combinations, molecular marker combinations, or primer pair combinations in the identification of Tianjin monkey chicken breeds.

[0034] This invention also provides the application of the above-mentioned primer pair combination in the preparation of a kit for identifying Tianjin monkey chicken breeds.

[0035] The present invention also provides a Tianjin monkey chicken breed identification kit, comprising the above-mentioned primer pair combination.

[0036] This invention also provides a method for identifying the Tianjin Monkey Chicken breed, comprising the following steps:

[0037] (1) Obtain the whole genome DNA of the chicken individual to be tested, and use the whole genome DNA as a template to amplify the gene fragment combination containing the above-mentioned SNP site combination by PCR.

[0038] (2) Sequencing the gene fragment combination to identify the genotype of each SNP site in the SNP site combination;

[0039] (3) Based on the genotype identified in step (2), calculate the total bloodline evaluation score S using the following formula:

[0040]

[0041] Where Ai is the final weight of the genotype corresponding to SNPi; i is an integer greater than 0 and less than 20;

[0042] The value of Ai is obtained from the following table:

[0043]

[0044] (4) When the total score of the bloodline evaluation S>50, the chicken individual to be tested is a Tianjin monkey chicken.

[0045] Furthermore, the PCR amplification uses the primer pair combination described above.

[0046] Furthermore, the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 57.2℃ annealing for 40 s, 72℃ extension for 40 s, 35 cycles; and a final extension at 72℃ for 10 min.

[0047] The present invention discloses the following technical effects:

[0048] This invention, based on a large-scale population of 25 representative local Chinese chicken breeds, including the Tianjin Monkey Chicken, and two introduced breeds, identifies specific SNP loci, screens molecular markers for identifying the Tianjin Monkey Chicken, and combines existing methods for detecting specific SNP loci with AHP (Alternating Hierarchical Hierarchy Process) to comprehensively consider allele frequencies and genotype types of specific loci. This approach ensures accuracy while improving detection efficiency and the scientific rigor of the evaluation. This invention utilizes molecular marker technology for the scientific identification and evaluation of the Tianjin Monkey Chicken breed; the operation is simple and the results are reliable, providing a scientific basis for the protection and rational utilization of local chicken genetic resources. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 Cluster diagram of Tianjin monkey chicken and other chicken breeds;

[0051] Figure 2 A flowchart for weight calculation in the AHP (Analytic Hierarchy Process). Detailed Implementation

[0052] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0053] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0054] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0055] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0056] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0057] Example 1: Screening of SNP molecular markers in Tianjin monkeys and chickens

[0058] 1. Blood sample collection

[0059] Based on previous genetic evolution studies of local chicken breeds (Gallus gallus), 25 local chicken breeds and 2 introduced breeds (Yuanbao Chicken and Dulong Chicken) were selected as research subjects according to the genetic background of different chicken breeds. The 25 chicken breeds, including Tianjin Monkey Chicken, were sourced from the National Local Chicken Breed Gene Bank (Jiangsu), with 10 roosters and 20 hens from each breed; the Yuanbao Chicken samples were from the experimental population of the Jiangsu Provincial Poultry Science Research Institute; the Dulong Chicken sequence was obtained from the NCBI database (Table 1); the local chicken breed materials and the 2 introduced breeds were mainly from the purebred conservation population of the National Local Chicken Breed Gene Bank (Jiangsu) to ensure that there would be no interference from hybrid populations, such as... Figure 1Each individual species on the phylogenetic tree shown can form an independent branch.

[0060] 1 mL of blood was collected from the sterile wing vein of the above-mentioned experimental individuals, and sodium citrate anticoagulant was added, mixed well, and stored at -80℃ for later use.

[0061] Table 1 Sample Information

[0062]

[0063] 2. DNA Sample Acquisition

[0064] Genomic DNA was extracted from all varieties using the conventional phenol-chloroform method. The obtained DNA underwent quality control, including preliminary DNA concentration testing with Nanodrop; electrophoresis to check DNA integrity, including the absence of DNA degradation and contamination by proteins, RNA, or other impurities; and accurate quantification of the DNA samples using Qubit 2.0, selecting samples with a mass ≥1 μg. Qualified samples were stored at -80℃ for library construction and sequencing.

[0065] 3. Simplified genome RAD-seq library preparation and sequencing

[0066] DNA was extracted from anticoagulated blood samples. For samples that passed DNA quality control, pair-end libraries with a length range of 300–500 bp were constructed using the ddRAD library construction method. Simplified genome RAD-seq sequencing was performed, and the raw reads (paired-end sequences) obtained from the sequencing were evaluated to obtain the raw reads for each sample. The reads were then aligned to the chicken reference genome (GRCg6a, https: / / www.ncbi.nlm.nih.gov / nuccore / 1375922358?report=fasta) using BWAMEM 0.7.15 software.

[0067] 4. Data quality control

[0068] Quality control was performed on the raw sequencing data using the samtools program, filtering out bases with a 99% accuracy rate and a Q20 ratio ≥ 95%. SNP detection was performed using GATK software, with double-enzyme digestion genome sequencing coverage depth ≥ 60%, SNP single nucleotide polymorphism detection (Callrate) ≥ 70% in the chicken flock, and minimum allele frequency (MAF) ≥ 0.05. Based on these, the SNP detection rate in a single chicken breed was ≥ 90%.

[0069] 5. Statistical analysis and site selection

[0070] Linkage disequilibrium (LD) analysis was performed using Haploview 4.1 software; PopGene software was used to calculate mean heterozygosity (Ho), inbreeding coefficient (Fis), and population differentiation index (Fst); Admixture software was used for population cluster analysis, and a phylogenetic tree was constructed using the maximum likelihood (ML) method, with the Shimodaira-Hasegawa test used to determine the confidence level of each node; selection signal analysis was performed on quality-controlled SNPs using the genetic differentiation coefficient (Fst) method in PLINK 1.9 software, with a sliding window of 100kb and a step size of 10kb. Chromosomal fragments or individuals with poor enzyme digestion were deleted and not analyzed to ensure accurate allele frequency calculations. Gene functional enrichment analysis was performed using DAVID, and the results were visualized using ggplot2 in R.

[0071] 6. Specific site screening and determination

[0072] Nineteen specific SNP loci were identified through statistical analysis and locus screening. (See Table 2)

[0073] Table 2 Information on the specific SNPs identified.

[0074]

[0075] Note: Underlined sites in the table above are single nucleotide polypeptide mutation sites.

[0076] 7. Calculation of weights for specific SNP sites in Tianjin monkeys and chickens.

[0077] Establish a hierarchical model of specific sites (e.g.) Figure 2 As shown in Table 3), a pairwise comparison matrix of the criteria layer was constructed using the 1-9 scaling method. Based on the results in Table 3, considering the weaknesses of low detection efficiency and poor overall evaluation accuracy of existing specific loci, allele frequency and dominant allele genotype were fully taken into account. The initial score was graded using the 1-9 scaling method, and allele frequency and dominant allele genotype were graded respectively:

[0078] 1 indicates that the two loci are equally important in terms of allele frequency or dominant allele genotype.

[0079] 3 indicates that, compared to the two loci, the former is slightly more important than the latter in terms of allele frequency or dominant allele type;

[0080] 5 indicates that, compared to the two loci, the former is significantly more important than the latter in terms of allele frequency or dominant allele type;

[0081] 7 indicates that, compared to the two loci, the former is extremely important than the latter in terms of allele frequency or dominant allele type;

[0082] 9 indicates that, compared to the two loci, the former is significantly more important than the latter in terms of allele frequency or dominant allele type;

[0083] 2, 4, 6, 8 represent the intermediate values ​​of the above adjacent judgments.

[0084] Table 3 Constructs pairwise comparison matrices for the criterion layer.

[0085]

[0086] The selected germplasm-specific SNP loci were weighted and scored using the results of the Analytic Hierarchy Process (AHP). When calculating the weights using AHP, a consistency test analysis is required to evaluate the consistency of the weight calculation results; specifically, the reliability (CR) value of the consistency index combination is calculated, as follows:

[0087] First: Describe the confidence interval (CI value) obtained from the above calculation [CI = (maximum eigenvalue - n) / (n-1)];

[0088] Second: Combine the order of the judgment matrix to obtain the average random consistency index (RI value);

[0089] Third: Calculate the CR value and perform a consistency check.

[0090] The calculation formula is as follows, where CI is the confidence interval, λmax is the maximum eigenvalue, n is the number of object features, A is the feature matrix, and W is the normalization matrix of A. The formula is as follows:

[0091]

[0092] The largest eigenvalue can be calculated by combining the eigenvectors (as shown in Table 4). Then, the CI value is calculated using the largest eigenvalue, which is used in the subsequent consistency test.

[0093] Table 4 Results of AHP (Analog-Philosophical Analysis)

[0094]

[0095]

[0096] Note: Genotype 1 is wild-type homozygous, genotype 2 is heterozygous, genotype 3 is mutant homozygous, and the deletion site weight is 0; i = 1, 2, 3…16.

[0097] Based on the final weight values ​​calculated in Table 3, evaluate the scores of the tested Tianjin monkey-chicken individuals:

[0098]

[0099] Where S is the total score of blood source evaluation, and Ai is the final weight of the genotype corresponding to SNPi (see Table 4); i = 1, 2, 3...16.

[0100] Example 2: Application of molecular markers in Tianjin monkeys and chickens

[0101] 1. Blood collection from individual individuals in the Tianjin monkey-chicken breeding population

[0102] Using a disposable medical syringe, 1.0 mL of whole blood was randomly collected from the wing veins of male and female Tianjin monkey chickens and closely related breeds (for comparison of extreme breeds, non-related breeds are easier to identify and distinguish). Five chickens of each breed were used as an example. After collection, the blood was quickly injected into an enzyme-free tube containing 2 μL of 0.5 mol / L EDTA-2Na anticoagulant. The enzyme-free tube was then stored at 4°C for later use.

[0103] 2. DNA extraction and quality testing

[0104] At room temperature, 0.2 mL of blood from male and female chickens stored in enzyme-free tubes was aspirated, and DNA was extracted from the blood using the conventional animal peripheral blood phenol extraction method. The DNA integrity was analyzed by agarose gel electrophoresis, and the DNA purity was detected by spectrophotometry. Qualified samples were stored at -80℃ for use in SNP detection.

[0105] 3. SNP marker detection of breed-specific genes in Tianjin monkey chickens

[0106] Primer design: SNP loci of the Tianjin monkey chicken breed characteristic genes screened were located on chromosomes in a reference genome to obtain a sequence containing these SNP loci. Using chicken genomic DNA as a template, primers were designed using software such as Oligo (see Table 5) for PCR amplification.

[0107] PCR amplification and detection:

[0108] The total volume of PCR amplification was 20 μL: 1 μL DNA template at a concentration of 100 ng / μL; 2 μL 10× PCR Buffer; 1.5 μL dNTP at a concentration of 10 mmol / L; 1 μL each of forward and reverse primers at a concentration of 10 pmol / μL; 0.2 μL Taq enzyme at a concentration of 5 U / μL; and 13.3 μL ddH2O.

[0109] PCR amplification program: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 57.2℃ annealing for 40 s, 72℃ extension for 40 s, 35 cycles; final extension at 72℃ for 10 min, store at 4℃ for later use; send for testing.

[0110] Table 5. Primer sequences for PCR amplification

[0111]

[0112] 4. Identification of Tianjin Monkey Chicken Breed

[0113] The genotypic identification results of Tianjin Monkey Chicken and closely related chicken breeds are shown in Table 6.

[0114] Table 6. Genotyping of Tianjin Monkey Chicken and Closely Related Chicken Breeds

[0115]

[0116] 5. Individual identification and evaluation of Tianjin monkey chickens and T-test results are shown in Table 7 below.

[0117] Table 7 Individual Identification and T-test of Tianjin Monkey Chickens

[0118]

[0119]

[0120] Note: ** indicates a highly significant difference, P<0.01.

[0121] Table 7 shows that the molecular markers screened by this invention can efficiently and accurately evaluate the bloodline purity of Tianjin Monkey Chicken individuals and achieve breed identification. When the total bloodline evaluation score S>50, the chicken individual to be tested can be identified as a Tianjin Monkey Chicken.

[0122] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A combination of molecular markers for identifying Tianjin monkey breed, characterized in that, The molecular marker combination includes molecular markers with nucleotide sequences as shown in SEQ ID NO.1-19, respectively; The molecular marker shown in SEQ ID NO.1 has a mutation site SNP1 at 11 bp, which is an A / G mutation; The molecular marker shown in SEQ ID NO.2 has a mutation site SNP2 at 11 bp, which is a C / T mutation; The molecular marker shown in SEQ ID NO.3 has a mutation site SNP3 at 11 bp, which is a C / T mutation; The molecular marker shown in SEQ ID NO.4 has a mutation site SNP4 at 11 bp, which is an A / T mutation; The molecular marker shown in SEQ ID NO.5 has a mutation site SNP5 at 11 bp, which is a C / T mutation; The molecular marker shown in SEQ ID NO.6 has a mutation site SNP6 at 11 bp, which is an A / G mutation; The molecular marker shown in SEQ ID NO.7 has a mutation site SNP7 at 11 bp, which is a G / A mutation; The molecular marker shown in SEQ ID NO.8 has a mutation site SNP8 at 11 bp, which is a G / A mutation; The molecular marker shown in SEQ ID NO.9 has a mutation site SNP9 at 11 bp, which is a G / A mutation; The molecular marker shown in SEQ ID NO.10 has a mutation site SNP10 at 11 bp, which is a T / C mutation; The molecular marker shown in SEQ ID NO.11 has a mutation site SNP11 at the 11 bp, which is a T / C mutation; The molecular marker shown in SEQ ID NO.12 has a mutation site SNP12 at the 11 bp, which is an A / G mutation; The molecular marker shown in SEQ ID NO.13 has a mutation site SNP13 at 11 bp, which is a C / T mutation; The molecular marker shown in SEQ ID NO.14 has a mutation site SNP14 at the 11 bp, which is an A / C mutation; The molecular marker shown in SEQ ID NO.15 has a mutation site SNP15 at 11 bp, which is a T / C mutation; The molecular marker shown in SEQ ID NO.16 has a mutation site SNP16 at 11 bp, which is a C / G mutation; The molecular marker shown in SEQ ID NO.17 has a mutation site SNP17 at 11 bp, which is a C / A mutation; The molecular marker shown in SEQ ID NO.18 has a mutation site SNP18 at 11 bp, which is an A / T mutation; The molecular marker shown in SEQ ID NO.19 has a mutation site SNP19 at 11 bp, which is a T / C mutation.

2. The molecular marker combination according to claim 1, characterized in that, The genotypes of SNP1 are AA, AG, or GG; SNP2 is CC, CT, or TT; SNP3 is CC, CT, or TT; SNP4 is AA, AT, or TT; SNP5 is CC, CT, or TT; SNP6 is AA, AG, or GG; SNP7 is GG, GA, or AA; SNP8 is GG, GA, or AA; SNP9 is GG, GA, or AA; and SNP10 is TT. The genotypes of SNP11, SNP12, SNP13, SNP14, SNP15, SNP16, SNP17, SNP18, and SNP19 are TT, TC, or CC. The genotypes of SNP11, SNP12, SNP13, SNP14, SNP15, SNP16, SNP17, SNP18, SNP19, SNP18, and SNP19 are TT, TC, or CC.

3. A primer pair combination for identifying the Tianjin monkey chicken breed, characterized in that, The primer pair combinations include primer pairs 1-19 as shown in the table below: 。 4. The application of a molecular marker combination as described in claim 1 or 2, or a primer pair combination as described in claim 3, in the identification of Tianjin monkey chicken breeds.

5. The application of the primer pair combination as described in claim 3 in the preparation of a kit for identifying Tianjin monkey chicken breeds.

6. A kit for identifying Tianjin monkey-chicken breeds, characterized in that, Includes the primer pair combination as described in claim 3.

7. A method for identifying the breed of Tianjin Monkey Chicken, characterized in that, Includes the following steps: (1) Obtain the whole genome DNA of the chicken individual to be tested, and use the whole genome DNA as a template to amplify the gene fragment combination containing the SNP site combination by PCR. The SNP locus combinations include SNP1-SNP19 located on the reference genome GRCg6a as shown in the table below: ； (2) Sequencing the gene fragment combination to identify the genotype of each SNP site in the SNP site combination; (3) Based on the genotype identified in step (2), calculate the total score for kinship evaluation using the following formula. S : in, Ai The final weights for the genotypes corresponding to SNPi; i An integer greater than 0 and less than 20; The Ai The values ​​are derived from the following table: ； (4) When the total score of the bloodline evaluation S When the value is >50, the tested chicken individual is a Tianjin monkey chicken.

8. The method according to claim 7, characterized in that, The PCR amplification uses the primer pair combination as described in claim 3.

9. The method according to claim 7, characterized in that, The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 57.2℃ annealing for 40 s, 72℃ extension for 40 s, 35 cycles; and a final extension at 72℃ for 10 min.

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