A molecular marker for identifying Anka Red chicken breed and its application

Abstract

The present invention discloses a molecular marker for identifying Anka Red chicken varieties and its application, and relates to the field of molecular biological detection. The molecular marker combination includes molecular markers whose nucleotide sequences are respectively shown as SEQ ID NO.1-13. The present invention is based on a large-scale population identification of specific SNPs sites of 25 representative Chinese local chicken breeds and 2 introduced breeds including Anka Red chicken, screens out molecular markers for identifying Anka Red chicken, combines the existing specific SNP site detection method with the AHP hierarchical analysis method to comprehensively consider the allele frequency and genotype type of the specific site, while ensuring the accuracy of identification, improving the detection efficiency and the scientific nature of the evaluation. The present invention uses molecular marker technology to carry out scientific breed-specific identification and evaluation of Anka Red chicken, which is simple to operate and has reliable results, and can provide a scientific basis for the protection and rational utilization of chicken genetic resources.

Description

Technical Field

[0001] The present invention relates to the field of molecular biological detection, in particular to a molecular marker for identifying Anka Red chicken breeds and an application thereof. Background Art

[0002] The Anka Red is a fast-growing, yellow-feathered broiler, available in four lines. Originating in Israel, it is the fastest-growing red-feathered broiler in the country. Its plumage is yellowish-red, with some birds having hemp feathers on their necks and backs. Its shanks, toes, skin, and beak are all yellow. Large and rounded, it is one of the fastest-growing colored-feather broilers, characterized by strong adaptability, rapid growth, and high feed conversion rates.

[0003] Single nucleotide polymorphism (SNP) mainly refers to the polymorphism of nucleic acid sequence caused by the change of a single nucleotide at the genomic level. There are many SNPs and the polymorphism is rich. In theory, each SNP site can have four different forms of variation, including transition, transversion, deletion and insertion, but in reality only two occur, namely transition and transversion, with a ratio of 2:1. SNP is the most common form of polymorphism in the genome and has high genetic stability. SNPs are considered to be the main genetic source of phenotypic variability. They have individual differences in a specific population, and the allele frequency of a specific SNP can be different in different populations. SNP markers provide more accurate identification for livestock and poultry breed identification and kinship identification. The increase in marker density provides researchers with a more detailed genetic map.

[0004] Restriction-site-associated DNA sequencing (RAD-seq) is a simplified genomic technology developed based on next-generation sequencing technology. Reduced-representation sequencing (RRGS) is a sequencing strategy that uses restriction enzymes to fragment genomic DNA and then performs high-throughput sequencing of specific fragments to obtain a large number of genetic polymorphism markers, fully representing the entire genome of the target species. RAD sequencing library construction involves enzymatic digestion and random fragmentation of genomic DNA fragments, selecting fragments with a restriction site on one end and a random fragmentation site on the other end for library construction and sequencing. RAD-seq reduces genomic complexity, is easy to operate, and is not limited by a reference genome, allowing for rapid identification of a high density of single nucleotide polymorphisms. However, the molecular markers currently used to evaluate Anka Red chickens are primarily related to economic performance, making them difficult to assess genetic diversity and inaccurate for purebred Anka Red chickens. Therefore, screening for single nucleotide polymorphism (SNP) molecular markers for Anka Red chickens based on reduced genomic sequencing is of great significance. Summary of the Invention

[0005] The purpose of the present invention is to provide a molecular marker for identifying the Anka Red chicken breed and its application, so as to solve the problems existing in the above-mentioned prior art. The molecular marker combination provided by the present invention can efficiently and accurately evaluate the blood purity of Anka Red chicken individuals and realize breed identification, which is conducive to alleviating the problems in the prior art due to the lack of molecular markers that can evaluate the genetic diversity of Anka Red chickens and the inaccuracy of existing purebred identification methods.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a SNP site combination for identifying Anka Red Chicken varieties, including SNP sites SNP1-13 shown in the following table:

[0008] SNP number chromosome SNP location wild type Mutant SNP1 1 22519784 T A SNP2 2 69229744 A G SNP3 2 69264992 G T SNP4 2 69329456 C T SNP5 2 91639328 C T SNP6 2 103467101 C T SNP7 2 106949790 T G SNP8 2 126786478 G A SNP9 3 76559047 G A SNP10 3 77123885 T A SNP11 19 6238586 A G SNP12 23 3687958 A G SNP13 24 2732572 T G .

[0009] The present invention also provides a molecular marker combination for identifying Anka Red Chicken varieties, wherein the molecular marker combination comprises molecular markers whose nucleotide sequences are respectively shown as SEQ ID NO.1-13;

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

[0011] The base at 11 bp of the molecular marker shown in SEQ ID NO. 2 has a mutation site SNP2, which is an A / G mutation;

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

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

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

[0015] The base at 11 bp of the molecular marker shown in SEQ ID NO. 6 has a mutation site SNP6, which is a C / T mutation;

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

[0017] The base at 11 bp of the molecular marker shown in SEQ ID NO. 8 has a mutation site SNP8, which is a G / A mutation;

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

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

[0020] The base at 11 bp of the molecular marker shown in SEQ ID NO. 11 has a mutation site SNP11, which is an A / G mutation;

[0021] The base at 11 bp of the molecular marker shown in SEQ ID NO. 12 has a mutation site SNP12, which is an A / G mutation;

[0022] There is a mutation site SNP13 at the base 11 bp of the molecular marker shown by SEQ ID NO. 13, which is a T / G mutation.

[0023] Furthermore, the genotype of the SNP1 is TT, TA or AA; the genotype of the SNP2 is AA, AG or GG; the genotype of the SNP3 is GG, GT or TT; the genotype of the SNP4 is CC, CT or TT; the genotype of the SNP5 is CC, CT or TT; the genotype of the SNP6 is CC, CT or TT; the genotype of the SNP7 is TT, TG or GG; the genotype of the SNP8 is GG, GA or AA; the genotype of the SNP9 is GG, GA or AA; the genotype of the SNP10 is TT, TA or AA; the genotype of the SNP11 is AA, AG or GG; the genotype of the SNP12 is AA, AG or GG; and the genotype of the SNP13 is TT, TG or GG.

[0024] The present invention also provides a primer pair combination for identifying the Anka Red Chicken variety, wherein the primer pair combination includes primer pairs 1-13 as shown in the following table:

[0025]

[0026] The present invention also provides the use of the above-mentioned SNP site combination, molecular marker combination or primer pair combination in identifying Anka Red Chicken varieties.

[0027] The present invention also provides application of the primer pair combination in preparing an Anka Red Chicken variety identification kit.

[0028] The present invention also provides an Anka Red Chicken variety identification kit, comprising the above primer pair combination.

[0029] The present invention also provides a method for identifying the Anka Red Chicken variety, comprising the following steps:

[0030] (1) obtaining whole genomic DNA of the chicken individual to be tested, and using the whole genomic DNA as a template, performing PCR amplification to obtain a gene fragment combination containing the above-mentioned SNP site combination;

[0031] (2) sequencing the gene fragment combination to identify the genotype of each SNP site in the above-mentioned SNP site combination;

[0032] (3) Based on the genotype identified in step (2), the total score S of the consanguinity evaluation is calculated using the following formula:

[0033]

[0034] Where Ai is the final weight of the genotype corresponding to SNPi; i is an integer greater than 0 and less than 14;

[0035] The value of Ai is obtained according to the following table:

[0036]

[0037]

[0038] (4) When the total score S of the blood relationship evaluation is greater than 50, the chicken individual to be tested is an Anka Red chicken.

[0039] Furthermore, the PCR amplification uses the above primer pair combination.

[0040] Furthermore, the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 40 seconds, annealing at 58°C for 40 seconds, extension at 72°C for 40 seconds, 35 cycles; and finally extension at 72°C for 10 minutes.

[0041] The present invention discloses the following technical effects:

[0042] This study identifies specific SNPs based on a large-scale population analysis of 25 representative Chinese indigenous chicken breeds and two introduced breeds, including the Anka Red chicken. This method screens molecular markers for identifying the Anka Red chicken. By combining existing methods for detecting specific SNPs with the Analytic Hierarchy Process (AHP), the method comprehensively considers the allele frequencies and genotypes of specific loci, ensuring accurate identification while improving detection efficiency and the scientific nature of the evaluation. This method uses molecular marker technology to scientifically identify and evaluate Anka Red chickens for breed-specificity. The method is simple to perform and produces reliable results, providing a scientific basis for the conservation and rational utilization of chicken genetic resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0044] Figure 1 This is a cluster diagram of Anka Red Chicken and other chicken breeds; WX, PJ, DL, ZZ, CH, WC, HX, JH, RW, AK, XS, LY, HOU, WS, YB, LA, LS, SG, BY, BJ, DG, DX, WH, BE, XJ, GS and DJ are the abbreviations of Daweishan Miniature Chicken, Diao Chicken, Dulong Chicken, Tibetan Chicken, Camellia Chicken, Wenchang Chicken, Huiyang Bearded Chicken, Jinhu Black Phoenix Chicken, Recessive White Feather Chicken, Anka Red Chicken, Xiaoshan Chicken, Luyuan Chicken, Tianjin Monkey Chicken, Wenshang Reed Chicken, Yuanbao Chicken, Langya Chicken, Langshan Chicken, Shouguang Chicken, Beijing Oily Chicken, Bian Chicken, Big Bone Chicken, Dongxiang Green Shell Layer, Anyi Gray Chicken, White Ear Chicken, Xianju Chicken, Gushi Chicken and Henan Fighting Chicken respectively;

[0045] Figure 2Flowchart for weight calculation of AHP. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] Example 1 Screening of SNP Molecular Markers in Anka Red Chicken

[0052] 1. Blood sample collection

[0053] Based on the previous genetic evolutionary studies of local chickens (Gallus gallus), 25 local chicken breeds and two introduced breeds were selected as research objects based on the genetic background of different chicken breeds. The 25 local chicken breed materials and the two introduced breeds were mainly derived from the purebred conservation population of the National Local Chicken Breed Gene Bank (Jiangsu), with 10 roosters and 20 hens of each breed; Yuanbao chicken (YB, n = 8) samples were obtained from the experimental population of the Jiangsu Poultry Science Research Institute; Dulong chicken (DL, n = 10) sequences were obtained from the NCBI database (Table 1); each breed was ensured to be free of interference from hybrid populations, such as Figure 1 Each individual species on the phylogenetic tree shown can form an independent branch.

[0054] 1 mL of blood was collected from the wing vein of the above experimental individuals using sterile methods, and sodium citrate anticoagulant was added and mixed, and stored at -80°C for later use.

[0055] Table 1 Sample information

[0056]

[0057]

[0058] 2. DNA sample acquisition

[0059] Genomic DNA from all individuals was extracted using a rapid DNA extraction kit, and DNA quality and concentration were determined using gel electrophoresis and Nanodrop. DNA samples were accurately quantified using the Qubit 2.0, and samples with a mass of ≥1 μg were selected. Qualified samples were stored at −80°C for library construction and sequencing.

[0060] 3. Simplify genome RAD-seq library construction and sequencing

[0061] DNA was extracted from anticoagulated blood samples. Paired-end libraries ranging in length from 300 to 500 bp were constructed using the ddRAD method for samples that passed DNA quality inspection. Simplified genome RAD-seq sequencing was performed. The raw reads (paired-end sequences) obtained by sequencing were evaluated to obtain the raw reads of each sample. The reads were aligned to the chicken reference genome (GRCg6a, https: / / www.ncbi.nlm.nih.gov / nuccore / 1375922358?report=fasta) using BWAMEM 0.7.15 software.

[0062] 4. Data quality control

[0063] The original sequencing data were quality controlled using the samtools program, and the base number ratio (Q20) with an accuracy rate of 99% ≥ 95% was filtered; SNP detection was performed using GATK software, the coverage depth of double-enzyme genome sequencing was ≥ 60%, the SNP single nucleotide polymorphism detection (Callrate) in the chicken population was ≥ 70%, and the minimum allele frequency (MAF) was ≥ 0.05; on this basis, the SNP detection rate in a single chicken breed was ≥ 90%.

[0064] 5. Statistical analysis and site screening

[0065] Haploview 4.1 software was used for linkage disequilibrium (LD) analysis. PopGene software was used to calculate the average heterozygosity (Ho), inbreeding coefficient (Fis), and population differentiation index (Fst). Admixture software was used for population cluster analysis, and the maximum likelihood (ML) method was used to construct a phylogenetic tree. The Shimodaira-Hasegawa test was used to determine the credibility of each node. Quality-controlled SNPs were analyzed for selection signals using the genetic differentiation coefficient (Fst) method with PLINK 1.9 software, using a 100-kb window and a 10-kb step size for sliding detection. Chromosome segments or individuals with poor enzyme digestion were deleted and not analyzed to ensure accurate allele frequency calculations.

[0066] 6. Specific site screening

[0067] Thirteen Anka Red chicken-specific SNPs were identified, distributed on chromosomes 1, 2, 3, 19, 23, and 24 (see Table 2, the sequences corresponding to SNP1-SNP13 are numbered SEQ ID NO.1 to SEQ ID NO.13, respectively).

[0068] Table 2 Information of specific SNPs identified

[0069]

[0070]

[0071] Note: The underlined sites in the above table are single nucleotide polypeptide mutation sites; W is A or T, R is A or G, K is T or G, and Y is T or C.

[0072] 7. Calculation of Anka Red Chicken-specific SNP locus weights

[0073] Establish a hierarchical model of specific sites (such as Figure 2As shown in Figure 3, a 1-9 scale method was used to construct a pairwise comparison matrix at the criterion level (Table 3). Based on the results in Table 3, in order to address the weaknesses of low detection efficiency of existing specific sites and poor comprehensive evaluation accuracy, allele frequencies and dominant allele types were fully considered. The initial scores were graded using a 1-9 scale method, with allele frequencies and dominant allele types graded separately:

[0074] 1 means that the two loci have the same importance in terms of allele frequency or dominant allele type;

[0075] 3 means that compared with the two loci, the former is slightly more important than the latter in terms of allele frequency or dominant allele type;

[0076] 5 means that the former is significantly more important than the latter in terms of allele frequency or dominant allele type compared with the two loci;

[0077] 7 represents that the former is more important than the latter in terms of allele frequency or dominant allele type compared with the two loci;

[0078] 9 represents that the former is more important than the latter in terms of allele frequency or dominant allele type when comparing the two loci;

[0079] 2, 4, 6, and 8 represent the intermediate values ​​of the above adjacent judgments.

[0080] Table 3 Construction criterion layer pairwise comparison matrix

[0081] SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 SNP7 SNP8 SNP9 SNP10 SNP11 SNP12 SNP13 SNP1 1.00 0.56 0.56 0.56 1.00 5.00 0.71 1.00 0.56 5.00 5.00 1.00 1.67 SNP2 1.80 1.00 1.00 1.00 1.80 9.00 1.29 1.80 1.00 9.00 9.00 1.80 3.00 SNP3 1.80 1.00 1.00 1.00 1.80 9.00 1.29 1.80 1.00 9.00 9.00 1.80 3.00 SNP4 1.80 1.00 1.00 1.00 1.80 9.00 1.29 1.80 1.00 9.00 9.00 1.80 3.00 SNP5 1.00 0.56 0.56 0.56 1.00 5.00 0.71 1.00 0.56 5.00 5.00 1.00 1.67 SNP6 0.20 0.11 0.11 0.11 0.20 1.00 0.14 0.20 0.11 1.00 1.00 0.20 0.33 SNP7 1.40 0.78 0.78 0.78 1.40 7.00 1.00 1.40 0.78 7.00 7.00 1.40 2.33 SNP8 1.00 0.56 0.56 0.56 1.00 5.00 0.71 1.00 0.56 5.00 5.00 1.00 1.67 SNP9 1.80 1.00 1.00 1.00 1.80 9.00 1.29 1.80 1.00 9.00 9.00 1.80 3.00 SNP10 0.20 0.11 0.11 0.11 0.20 1.00 0.14 0.20 0.11 1.00 1.00 0.20 0.33 SNP11 0.20 0.11 0.11 0.11 0.20 1.00 0.14 0.20 0.11 1.00 1.00 0.20 0.33 SNP12 1.00 0.56 0.56 0.56 1.00 5.00 0.71 1.00 0.56 5.00 5.00 1.00 1.67 SNP13 0.60 0.33 0.33 0.33 0.60 3.00 0.43 0.60 0.33 3.00 3.00 0.60 1.00

[0082] The AHP hierarchical analysis method was used to perform weighted scoring on the selected germplasm-specific SNP sites. When using the AHP hierarchical analysis method for weight calculation, a consistency test analysis is required to study and evaluate the consistency test results of the weight calculation results, that is, to calculate the consistency index combination reliability (CR value), as follows:

[0083] First: describe the confidence interval (CI value) obtained by the above calculation [CI = (largest eigenvalue - n) / (n - 1)];

[0084] Second: Combine the judgment matrix order to obtain the average random consistency index (RI value);

[0085] Third: Calculate the CR value and make consistency judgment.

[0086] The calculation formula is as follows, where CI is the confidence interval, λ max is the maximum characteristic root, n is the number of object features, A is the characteristic matrix, W is the normalized matrix of A matrix, and the formula is as follows:

[0087]

[0088]

[0089]

[0090] The maximum eigenvalue can be calculated by combining the eigenvectors (as shown in Table 4), and then the CI value is calculated using the maximum eigenvalue. The CI value is used for the following consistency test.

[0091] Table 4 AHP hierarchical analysis results

[0092]

[0093]

[0094] Note: Genotype 1 is the wild-type homozygote, genotype 2 is the heterozygote, genotype 3 is the mutant homozygote, and the deletion site weight is 0; i = 1, 2, 3…13.

[0095] According to the final weight values ​​calculated in Table 4, the scores of the Anka Red Chicken individuals to be tested were evaluated.

[0096]

[0097] Where S is the total score of blood source evaluation, Ai is the final weight of SNPi corresponding to the genotype (see Table 4); i = 1, 2, 3...13.

[0098] Example 2 Application of Anka Red Chicken Molecular Markers

[0099] 1. Blood collection from individuals of Anka Red Chicken, Recessive White Feather Chicken and Camellia Chicken conservation populations

[0100] A medical disposable syringe was used to randomly collect 1.0 mL of whole blood from the wing vein of male and female Anka Red chickens and their closely related chicken species (recessive white-feathered chickens and Camellia chickens). Five chickens of each species were used as an example. After collection, the whole blood was quickly injected into an enzyme-free tube containing 2 μL of 0.5 mol / LEDTA-2Na anticoagulant, and then the enzyme-free tube was stored at 4°C for future use.

[0101] 2. DNA extraction and quality testing

[0102] At room temperature, 0.2 mL of blood from individual male and female chickens was aspirated and stored in enzyme-free tubes for future use. DNA was extracted from the individual blood using the conventional animal peripheral blood benzene-phenol extraction method. DNA integrity was analyzed by agarose gel electrophoresis, and DNA purity was tested by spectrophotometry. Qualified samples were stored at -80°C for use in SNP detection.

[0103] 3. SNP marker detection of Anka Red chicken breed-specific genes

[0104] Primer Design: The identified SNPs in the Anka Red chicken's characteristic genes were mapped to chromosomes within the reference genome to obtain a sequence containing these SNPs. Using chicken genomic DNA as a template, primers were designed using software such as Oligo (see Table 5) for PCR amplification.

[0105] PCR amplification and detection:

[0106] 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 upstream and downstream 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.

[0107] PCR amplification program: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 40 s, annealing at 58°C for 40 s, extension at 72°C for 40 s, 35 cycles; final extension at 72°C for 10 min, storage at 4°C until use; and submission for testing.

[0108] Table 5 SNP sites and PCR amplification primer sequences

[0109]

[0110] 4. Identification of Anka Red Chicken Breed

[0111] The genotype identification results of Anka Red chicken and its closely related chicken breeds are shown in Table 6.

[0112] Table 6 Genotype identification of Anka Red chicken and related chicken breeds

[0113]

[0114]

[0115] 5. Individual identification and evaluation of Anka Red Chicken and T-test. The results are shown in Table 7 below.

[0116] Table 7 Individual identification evaluation and T-test of Anka Red Chicken

[0117]

[0118]

[0119] Note: ** indicates extremely significant difference, P < 0.01.

[0120] The results in Table 7 show that the molecular markers screened by the present invention can efficiently and accurately evaluate the blood purity of Anka Red chicken individuals and achieve breed identification. When the total blood purity evaluation score S>50, the chicken individual to be tested can be determined to be Anka Red chicken.

[0121] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A molecular marker combination for identifying the Anka Red chicken variety, characterized in that: The molecular marker combination consists of the molecular markers shown in SEQ ID NO.1-13; The base at 11 bp of the molecular marker shown in SEQ ID NO. 1 has a mutation site SNP1, which is a T / A mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 2 has a mutation site SNP2, which is an A / G mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 3 has a mutation site SNP3, which is a G / T mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 4 has a mutation site SNP4, which is a C / T mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 5 has a mutation site SNP5, which is a C / T mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 6 has a mutation site SNP6, which is a C / T mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 7 has a mutation site SNP7, which is a T / G mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 8 has a mutation site SNP8, which is a G / A mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 9 has a mutation site SNP9, which is a G / A mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 10 has a mutation site SNP10, which is a T / A mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 11 has a mutation site SNP11, which is an A / G mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 12 has a mutation site SNP12, which is an A / G mutation; There is a mutation site SNP13 at the base 11 bp of the molecular marker shown by SEQ ID NO. 13, which is a T / G mutation.

2. The molecular marker combination according to claim 1, characterized in that The genotype of the SNP1 is TT, TA or AA; the genotype of the SNP2 is AA, AG or GG; the genotype of the SNP3 is GG, GT or TT; the genotype of the SNP4 is CC, CT or TT; the genotype of the SNP5 is CC, CT or TT; the genotype of the SNP6 is CC, CT or TT; the genotype of the SNP7 is TT, TG or GG; the genotype of the SNP8 is GG, GA or AA; the genotype of the SNP9 is GG, GA or AA; the genotype of the SNP10 is TT, TA or AA; the genotype of the SNP11 is AA, AG or GG; the genotype of the SNP12 is AA, AG or GG; the genotype of the SNP13 is TT, TG or GG.

3. A primer pair combination for identifying the Anka Red Chicken variety, characterized in that: The primer pair combination includes primer pairs 1-13 as shown in the following table: 。 4. Use of the molecular marker combination according to claim 1 or 2 or the primer pair combination according to claim 3 in identifying the Anka Red chicken variety.

5. Use of the primer pair combination as claimed in claim 3 in preparing an Anka Red Chicken variety identification kit.

6. A kit for identifying the variety of Anka Red Chicken, characterized in that: The method comprises the primer pair combination according to claim 3.

7. A method for identifying the Anka Red Chicken variety, characterized in that: The following steps are involved: (1) Obtaining the whole genome DNA of the chicken individual to be tested, and using the whole genome DNA as a template, performing PCR amplification to obtain a gene fragment combination containing the SNP site combination; The PCR amplification uses the primer pair combination as claimed in claim 3; (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), the total score S of the consanguinity evaluation is calculated 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 14; The value of Ai is obtained according to the following table: ； (4) When the total score S of the blood relationship evaluation is greater than 50, the chicken to be tested is an Anka Red chicken; The SNP site combination is located in the chicken reference genome GRCg6a SNP site SNP1-13 shown in the following table composition: 。 8. The method according to claim 7, characterized in that The PCR amplification reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 40 s, annealing at 58°C for 40 s, and extension at 72°C for 40 s, for 35 cycles; and finally extension at 72°C for 10 min.

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