A molecular marker combination for identifying dahuangshan bantam chicken breed and application thereof

Abstract

The application discloses a molecular marker combination for identifying Daweishan miniature chicken breeds and application thereof, and relates to the field of molecular biology detection. The molecular marker combination comprises molecular markers with nucleotide sequences shown in SEQ ID NO. 1-10. The application identifies specific SNPs sites based on a large-scale population comprising 25 representative Chinese local chicken breeds including Daweishan miniature chicken and two introduced breeds, screens out molecular markers for identifying Daweishan miniature chicken, combines the detection method of the existing specific SNP sites and AHP hierarchical analysis method, and comprehensively considers the allele frequency and genotype type of the specific sites, thereby improving the detection efficiency and the scientificity of evaluation while ensuring the identification accuracy. The application scientifically identifies and evaluates the breed specificity of Daweishan miniature chicken by using the molecular marker technology, is simple in operation and reliable in result, and can provide 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 the Daweishan miniature chicken breed and its application. Background Technology

[0002] The Daweishan Mini Chicken, also known as "Fragrant Chicken," "Golden Chicken," or "Delicate Chicken," is a local breed cultivated for both meat and eggs, as well as for competitive and ornamental purposes. Its origin and main production area is Pingbian Miao Autonomous County, Honghe Hani and Miao Autonomous Prefecture, Yunnan Province, and it is distributed in Pingbian County, Wantang Township, Baihe Township, and other areas. The Daweishan Mini Chicken has a long history and is characterized by its small size, fine bones, low fat content, well-developed breast and leg muscles, agility, fighting spirit, tolerance to roughage, and strong disease resistance.

[0003] Single nucleotide polymorphisms (SNPs) primarily refer to DNA sequence polymorphisms caused by variations in a single nucleotide base (A, T, C, G) in genomic DNA alleles. They possess advantages such as high number density, strong representativeness, genetic stability, and ease of automated analysis. These variations include transformations, transversions, deletions, and insertions. In practice, SNP variations require alleles to have a frequency of at least 1% or more; the latter two are very rare and almost negligible. Therefore, SNP variations are limited to transformations and transversions, with a ratio of 2:1.

[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 (RAD-seq) is a novel sequencing method that utilizes restriction endonucleases to break down genomic DNA, selecting specific fragments for high-throughput sequencing to obtain a large number of genetic polymorphism tag sequences that fully represent the whole genome information of the target species. This method has advantages such as reducing the complexity of the genome, simple experimental procedures, low cost, and no reliance on a reference genome. RAD sequencing library construction involves digesting genomic DNA fragments with restriction enzymes to produce fragments of a certain size, selecting fragments with restriction enzyme sites at one end and random breakpoints at the other end for library construction and sequencing. RAD-seq is simple to operate, has a short experimental cycle, high accuracy, and is not limited by a reference genome, allowing for rapid identification of high-density SNPs. However, the molecular markers currently used for evaluating Daweishan Miniature Chickens have limited functions, making it difficult to assess their genetic diversity and causing inaccuracies in purebred identification. Therefore, screening for single nucleotide polymorphism molecular markers for Daweishan Miniature Chickens based on simplified genome sequencing is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular marker combination for identifying the Daweishan Miniature 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 Daweishan Miniature Chicken individuals, realize breed identification, and help alleviate the problems of lack of molecular markers that can evaluate the genetic diversity of Daweishan Miniature Chicken and the inaccuracy of existing purebred identification methods in the prior art.

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

[0007] This invention provides a combination of SNP loci for identifying the Daweishan miniature chicken breed, including the SNP loci shown in the table below:

[0008] SNP1 1 42284399 A G SNP2 1 99918703 T C SNP3 1 111211903 T C SNP4 1 123493475 T C SNP5 1 161539901 C T SNP6 1 163016285 G A SNP7 2 34143429 G A SNP8 4 50985628 C T SNP9 11 15691201 C G SNP10 14 9284081 C T .

[0009] The present invention also provides a molecular marker combination for identifying the Daweishan miniature chicken breed, the molecular marker combination comprising molecular markers with nucleotide sequences as shown in SEQ ID NO.1-10 respectively;

[0010] The molecular marker shown in SEQ ID NO.1 has a mutation site SNP1 at 11 bp, which is an A / G mutation;

[0011] The molecular marker shown in SEQ ID NO.2 has a mutation site SNP2 at 11 bp, which is a T / C mutation;

[0012] The molecular marker shown in SEQ ID NO.3 has a mutation site SNP3 at 11 bp, which is a T / C mutation;

[0013] The molecular marker shown in SEQ ID NO.4 has a mutation site SNP4 at 11 bp, which is a T / C mutation;

[0014] The molecular marker shown in SEQ ID NO.5 has a mutation site SNP5 at 11 bp, which is a C / T mutation;

[0015] The molecular marker shown in SEQ ID NO.6 has a mutation site SNP6 at 11 bp, which is a G / A mutation;

[0016] The molecular marker shown in SEQ ID NO.7 has a mutation site SNP7 at 11 bp, which is a G / A mutation;

[0017] The molecular marker shown in SEQ ID NO.8 has a mutation site SNP8 at 11 bp, which is a C / T mutation;

[0018] The molecular marker shown in SEQ ID NO.9 has a mutation site SNP9 at 11 bp, which is a C / G mutation;

[0019] The molecular marker shown in SEQ ID NO.10 has a mutation site SNP10 at 11 bp, which is a C / T mutation.

[0020] Further, the genotype of SNP1 is AA, AG, or GG; the genotype of SNP2 is TT, TC, or CC; the genotype of SNP3 is TT, TC, or CC; the genotype of SNP4 is TT, TC, or CC; the genotype of SNP5 is CC, CT, or TT; the genotype of SNP6 is GG, GA, or AA; the genotype of SNP7 is GG, GA, or AA; the genotype of SNP8 is CC, CT, or TT; the genotype of SNP9 is CC, CG, or GG; and the genotype of SNP10 is CC, CT, or TT.

[0021] This invention also provides a primer pair combination for identifying the Daweishan miniature chicken breed, the primer pair combination comprising primer pairs 1-10 as shown in the table below:

[0022]

[0023] The present invention also provides the application of the above-mentioned SNP site combinations, molecular marker combinations or primer pair combinations in the identification of the Daweishan miniature chicken breed.

[0024] This invention also provides the application of the above-mentioned primer pair combination in the preparation of the Daweishan miniature chicken breed identification kit.

[0025] The present invention also provides a kit for identifying the Daweishan miniature chicken breed, comprising the above-mentioned primer pair combination.

[0026] This invention also provides a method for identifying the Daweishan miniature chicken breed, comprising the following steps:

[0027] (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.

[0028] (2) Sequencing the gene fragment combination to identify the genotype of each SNP site in the above SNP site combination;

[0029] (3) Based on the genotypes identified in step (2), calculate the total kinship evaluation score S using the following formula:

[0030]

[0031] Where Ai is the final weight of the genotype corresponding to SNPi; i is an integer greater than 0 and less than 11;

[0032] The value of Ai is obtained from the following table:

[0033]

[0034] (4) When the total score of the bloodline evaluation S>50, the chicken individual to be tested is a Daweishan miniature chicken.

[0035] Furthermore, in step (1), the PCR amplification uses the primer pair combination described above.

[0036] Further, in step (1), the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 58℃ annealing for 40 s, 72℃ extension for 40 s, 35 cycles; and finally 72℃ extension for 10 min.

[0037] The present invention discloses the following technical effects:

[0038] This invention, based on a large-scale population of 25 representative Chinese local chicken breeds, including the Daweishan Miniature Chicken, and 2 introduced breeds, identifies specific SNP loci and screens molecular markers for identifying the Daweishan Miniature Chicken. Combining existing methods for detecting specific SNP loci with AHP hierarchical analysis, and comprehensively considering allele frequencies and genotype types of specific loci, it improves detection efficiency and the scientific rigor of evaluation while ensuring identification accuracy. This invention uses molecular marker technology to scientifically identify and evaluate the breed specificity of the Daweishan Miniature 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. Attached Figure Description

[0039] 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.

[0040] Figure 1 Cluster diagram of Daweishan Miniature Chicken and other chicken breeds;

[0041] Figure 2 A flowchart for weight calculation in the AHP (Analytic Hierarchy Process). Detailed Implementation

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Example 1: Screening of SNP loci in Daweishan miniature chickens

[0048] 1. Blood sample collection

[0049] 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 the Daweishan Miniature Chicken, were obtained from the National Local Chicken Breed Gene Bank (Jiangsu), with 10 roosters and 20 hens for each breed; the Yuanbao chicken (YB, n=8) samples were obtained from the experimental population of the Jiangsu Provincial Institute of Poultry Science; the Dulong chicken (DL, n=10) sequence was obtained from the NCBI database (Table 1); the local chicken breed materials and the 2 introduced breeds mainly came from the purebred conservation population of the National Local Chicken Breed Gene Bank (Jiangsu) to ensure that there was no interference from hybrid populations, such as... Figure 1 As shown, each individual species on the phylogenetic tree can form an independent branch.

[0050] 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.

[0051] Table 1 Sample Information

[0052]

[0053]

[0054] 2. DNA Sample Acquisition

[0055] Genomic DNA was extracted from all varieties using the conventional phenol-chloroform method. The obtained DNA samples were initially tested for concentration using Nanodrop and for integrity using electrophoresis, including the presence of DNA degradation and contamination by proteins, RNA, or other impurities. Qubit 2.0 was used for accurate quantification of the DNA samples, selecting samples with a mass ≥1 μg. Qualified samples were stored at -80℃ for library construction and sequencing.

[0056] 3. Simplified genome RAD-seq library preparation and sequencing

[0057] 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.

[0058] 4. Data quality control

[0059] 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%.

[0060] 5. Statistical analysis and site selection

[0061] 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.

[0062] 6. Specific site screening

[0063] Statistical analysis was used to screen for specific loci, and a total of 10 specific SNPs were identified. (See Table 2)

[0064] Table 2 Information on the specific SNPs identified.

[0065] SNP1 1 42284399 A G AAATAAAGCARCTTAATAAAA SEQ ID NO.1 SNP2 1 99918703 T C GAACTTATCTYGTTAAAATAA SEQ ID NO.2 SNP3 1 111211903 T C GTTCCCTCCTYGGTGCCTCTG SEQ ID NO.3 SNP4 1 123493475 T C GAACCCTCCCYTCTGAAATGT SEQ ID NO.4 SNP5 1 161539901 C T TCAAAGAAAAYGAAGAGGTAA SEQ ID NO.5 SNP6 1 163016285 G A AACTGGAAGGRTATCAACTGC SEQ ID NO.6 SNP7 2 34143429 G A TTTCACCAGTRCTTTAGCAAA SEQ ID NO.7 SNP8 4 50985628 C T GACAGACTACYGTATGATTTA SEQ ID NO.8 SNP9 11 15691201 C G GACTGACTGASTGTTGTTTCC SEQ ID NO.9 SNP10 14 9284081 C T CAGAGGGCAGYGCTGAGCTCT SEQ ID NO.10

[0066] Note: In the table above, underlined sites are single nucleotide polypeptide mutation sites; R represents A or G, Y represents C or T, and S represents C or G.

[0067] 7. Calculation of weights of specific SNP sites in Daweishan miniature chickens

[0068] 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:

[0069] 1 indicates that the two loci are equally important in terms of allele frequency or dominant allele genotype.

[0070] The number 2 indicates that, compared to the previous number 1, the locus is slightly more important.

[0071] 3 indicates that, compared to the previous locus, the locus is more important than the former locus 2 in terms of allele frequency or dominant allele type;

[0072] Similarly, the importance of allele frequencies or dominant alleles increases sequentially for numbers 4, 5, 6, 7, 8, and 9, with higher numerical values ​​indicating greater importance.

[0073] Table 3 Constructs pairwise comparison matrices for the criterion layer.

[0074] SNP1 1.00 0.44 0.57 0.50 1.33 2.00 0.67 1.00 4.00 0.50 SNP2 2.25 1.00 1.29 1.13 3.00 4.50 1.50 2.25 9.00 1.13 SNP3 1.75 0.78 1.00 0.88 2.33 3.50 1.17 1.75 7.00 0.88 SNP4 2.00 0.89 1.14 1.00 2.67 4.00 1.33 2.00 8.00 1.00 SNP5 0.75 0.33 0.43 0.38 1.00 1.50 0.50 0.75 3.00 0.38 SNP6 0.50 0.22 0.29 0.25 0.67 1.00 0.33 0.50 2.00 0.25 SNP7 1.50 0.67 0.86 0.75 2.00 3.00 1.00 1.50 6.00 0.75 SNP8 1.00 0.44 0.57 0.50 1.33 2.00 0.67 1.00 4.00 0.50 SNP9 0.25 0.11 0.14 0.13 0.33 0.50 0.17 0.25 1.00 0.13 SNP10 2.00 0.89 1.14 1.00 2.67 4.00 1.33 2.00 8.00 1.00

[0075] The AHP (Analog-Philosophy of Hierarchical Analysis) method was used to weight and score the selected germplasm-specific SNP loci. When calculating the weights using AHP, a consistency test analysis is required to study and evaluate the consistency of the weight calculation results, specifically calculating the reliability (CR) value of the consistency index combination, as follows:

[0076] First: Describe the confidence interval (CI value) obtained from the above calculation [CI = (maximum eigenvalue - n) / (n-1)];

[0077] Second: Combine the order of the judgment matrix to obtain the average random consistency index (RI value);

[0078] Third: Calculate the CR value and perform a consistency check.

[0079] 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:

[0080]

[0081]

[0082]

[0083] 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. The CI value is used for the consistency test described below.

[0084] Table 4 Results of AHP (Analog-Philosophical Analysis)

[0085]

[0086]

[0087] 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…10.

[0088] Based on the final weight values ​​calculated in Table 4, the scores of the tested Daweishan miniature chicken individuals were evaluated:

[0089]

[0090] Where S is the total score of pedigree evaluation, and Ai is the final weight of the genotype corresponding to SNPi (see Table 4); i = 1, 2, 3...10.

[0091] Example 2: Application of molecular markers for Daweishan microchickens

[0092] 1. Blood collection from individual individuals in the Daweishan miniature chicken breeding population

[0093] Blood samples of 1.0 mL were collected from the wing veins of male and female Daweishan miniature chickens and closely related breeds Piao chicken and Chahua chicken (for comparison of extreme breeds, non-related breeds are easier to identify). Five chickens of each breed were used as an example. The blood samples were placed in enzyme-free tubes containing 2 μL of 0.5 mol / L EDTA-2Na anticoagulant and stored at 4°C for later use.

[0094] 2. DNA extraction and quality testing

[0095] 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.

[0096] 3. Detection of SNP markers for breed-specific genes in Daweishan miniature chickens

[0097] Primer design: SNP loci of the Daweishan miniature 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.

[0098] PCR amplification and detection:

[0099] 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.

[0100] PCR amplification reaction program: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 58℃ 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.

[0101] Table 5. Primer sequences for PCR amplification

[0102]

[0103] 4. The results of the identification of the Daweishan miniature chicken breed are shown in Table 6:

[0104] Table 6. Genotyping of Daweishan Miniature Chicken and Closely Related Chicken Breeds

[0105]

[0106] 5. Individual identification and evaluation of Daweishan miniature chickens and closely related chicken breeds, and the results of the T-test are shown in Table 7.

[0107] Table 7 Individual Identification and T-test of Daweishan Miniature Chickens

[0108]

[0109] Note: ** indicates a highly significant difference, P<0.01.

[0110] Table 7 shows that the molecular markers screened using this invention can efficiently and accurately evaluate the bloodline purity of Daweishan Miniature Chicken individuals and achieve breed identification. When the total bloodline evaluation score S>50, the chicken individual to be tested can be identified as Daweishan Miniature Chicken.

[0111] 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 molecular marker combination for identifying the Daweishan miniature chicken breed, characterized in that, The molecular marker combination includes molecular markers with nucleotide sequences as shown in SEQ ID NO. 1-10, 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 T / C mutation; The molecular marker shown in SEQ ID NO.3 has a mutation site SNP3 at 11 bp, which is a T / C mutation; The molecular marker shown in SEQ ID NO.4 has a mutation site SNP4 at 11 bp, which is a T / C 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 a G / A 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 C / T mutation; The molecular marker shown in SEQ ID NO.9 has a mutation site SNP9 at 11 bp, which is a C / G mutation; The molecular marker shown in SEQ ID NO.10 has a mutation site SNP10 at 11 bp, which is a C / T mutation.

2. The molecular marker combination according to claim 1, characterized in that, The genotypes of SNP1 are AA, AG, or GG; the genotypes of SNP2 are TT, TC, or CC; the genotypes of SNP3 are TT, TC, or CC; the genotypes of SNP4 are TT, TC, or CC; the genotypes of SNP5 are CC, CT, or TT; the genotypes of SNP6 are GG, GA, or AA; the genotypes of SNP7 are GG, GA, or AA; the genotypes of SNP8 are CC, CT, or TT; the genotypes of SNP9 are CC, CG, or GG; and the genotypes of SNP10 are CC, CT, or TT.

3. A primer pair combination for identifying the Daweishan miniature chicken breed, characterized in that, The primer pair combinations include primer pairs 1-10 as shown in the table below: 。 4. A kit for identifying the Daweishan miniature chicken breed, characterized in that, Includes the primer pair combination as described in claim 3.

5. A method for identifying the Daweishan miniature chicken breed, 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 by PCR to obtain a combination of gene fragments containing the mutation sites SNP1-10 as described in claim 1; (2) Sequencing the gene fragment combination to identify the genotype of each SNP locus; (3) Based on the genotypes identified in step (2), calculate the total kinship evaluation score S using the following formula: Where Ai is the final weight of the genotype corresponding to SNPi; i is an integer greater than 0 and less than 11; The value of Ai is obtained from the following table: ； (4) When the total score of the bloodline evaluation S>50, the chicken individual to be tested is a Daweishan miniature chicken.

6. The method according to claim 5, characterized in that, In step (1), the PCR amplification uses the primer pair combination as described in claim 3.

7. The method according to claim 5, characterized in that, In step (1), the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 58℃ annealing for 40 s, 72℃ extension for 40 s, 35 cycles; and finally 72℃ extension for 10 min.

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