A molecular marker combination for identifying Camellia chicken breeds and its application

Abstract

The present invention discloses a molecular marker combination for identifying Camellia chicken varieties and its application, relating to the field of molecular biological detection. The molecular marker combination includes molecular markers whose nucleotide sequences are respectively shown as SEQ ID NO.1-16. The present invention is based on a large-scale population of 25 representative Chinese local chicken breeds including Camellia chicken and 2 introduced breeds, identifies specific SNPs sites, screens out molecular markers for identifying Camellia chicken, combines the existing specific SNP site detection method and the AHP hierarchical analysis method, comprehensively considers the allele frequency and genotype type of the specific site, and improves the detection efficiency and scientific nature of the evaluation while ensuring the accuracy of identification. The present invention uses molecular marker technology to carry out scientific variety-specific identification and evaluation of Camellia chicken, which is simple to operate and the results are reliable, and can provide a scientific basis for the protection and rational utilization of local chicken genetic resources.

Description

Technical Field

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

[0002] Chahua chicken, named for its rooster's crowing that resembles "two camellia flowers," is a local dual-purpose breed. It originated from the red junglefowl, a local breed that was domesticated and bred by locals in tropical and subtropical environments. Its distinctive features include its small size, light weight, and delicate bones.

[0003] Single nucleotide polymorphism (SNP) refers primarily to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. This variation can be caused by a single base transition or transversion, or by a base insertion or deletion. Theoretically, SNPs can be either di-allelic, tri-allelic, or quad-allelic, but in practice, the latter two are extremely rare and almost negligible. Therefore, commonly referred to SNPs are di-allelic, with only two variations: transition and transversion, with a ratio of 2:1. SNPs are numerous, have rich polymorphisms, and possess high genetic stability.

[0004] Restriction-site-associated DNA sequencing (RAD-seq) is a reduced-representation genomic technology developed based on next-generation sequencing. Reduced-representation sequencing (RGS) utilizes restriction enzymes to fragment genomic DNA, selecting specific fragments for high-throughput sequencing. This method generates a large number of genetic polymorphism signatures that fully represent the entire genome of the target species. This method reduces genomic complexity and can obtain genome-wide genetic polymorphism signatures without relying on a reference genome. Its reduced genomic complexity and ease of use, coupled with the freedom from reference genome constraints, allow for rapid identification of high-density single nucleotide polymorphisms (SNPs). RAD sequencing library construction involves enzyme 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. However, currently used molecular markers for Camellia chicken evaluation are limited in functionality, making it difficult to assess genetic diversity in Camellia chickens, and also pose the challenge of inaccurate purebred identification methods. Therefore, screening for single nucleotide polymorphism (SNP) molecular markers in Camellia chickens based on RAD-seq is of great significance. Summary of the Invention

[0005] The purpose of the present invention is to provide a molecular marker combination for identifying the Camellia 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 Camellia 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 Camellia Chicken 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 locus combination for identifying Camellia chicken breeds, including the SNP loci shown in the following table:

[0008] SNP number chromosome SNP location wild type Mutant SNP1 1 84677176 T C SNP2 1 85673580 C G SNP3 1 97714151 G A SNP4 1 97724658 T C SNP5 2 68989386 A T SNP6 2 83716389 C T SNP7 2 83945430 C T SNP8 2 84242111 T G SNP9 2 84267921 G C SNP10 2 106652910 T C SNP11 2 106807600 G A SNP12 5 30644687 T A SNP13 7 30874559 C T SNP14 8 19906710 A G SNP15 10 16184380 T C SNP16 20 2332134 C T .

[0009] The present invention also provides a molecular marker combination for identifying a Camellia chicken breed, wherein the molecular marker combination includes molecular markers whose nucleotide sequences are respectively shown as SEQ ID NO.1-16;

[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 / C mutation;

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

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

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

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

[0022] The base at 11 bp of the molecular marker shown in SEQ ID NO. 13 has a mutation site SNP13, which is a C / T mutation;

[0023] The base at 11 bp of the molecular marker shown in SEQ ID NO. 14 has a mutation site SNP14, which is an A / G mutation;

[0024] The base at 11 bp of the molecular marker shown in SEQ ID NO. 15 has a mutation site SNP15, which is a T / C mutation;

[0025] There is a mutation site SNP16 at the 11 bp position of the molecular marker shown in SEQ ID NO. 16, which is a C / T mutation.

[0026] Furthermore, the genotype of the SNP1 is TT, TC or CC; the genotype of the SNP2 is CC, CG or GG; the genotype of the SNP3 is GG, GA or AA; the genotype of the SNP4 is TT, TC or CC; the genotype of the SNP5 is AA, AT or TT; the genotype of the SNP6 is CC, CT or TT; the genotype of the SNP7 is CC, CT or TT; the genotype of the SNP8 is TT, TG or GG; the genotype of the SNP9 is GG, GC or CC; the genotype of the SNP10 is TT, TC or CC; the genotype of the SNP11 is GG, GA or AA; the genotype of the SNP12 is TT, TA or AA; the genotype of the SNP13 is CC, CT or TT; the genotype of the SNP14 is AA, AG or GG; the genotype of the SNP15 is TT, TC or CC; and the genotype of the SNP16 is CC, CT or TT.

[0027] The present invention also provides a primer pair combination for identifying the Camellia chicken breed, wherein the primer pair combination includes primer pairs 1-16 as shown in the following table:

[0028] .

[0029] The present invention also provides the use of the above-mentioned SNP site combination, molecular marker combination or primer pair combination in identifying Camellia chicken varieties.

[0030] The present invention also provides the use of the primer pair combination in preparing a kit for identifying the variety of Camellia chicken.

[0031] The present invention also provides a Camellia chicken variety identification kit, comprising the above primer pair combination.

[0032] The present invention also provides a method for identifying the variety of Camellia chicken, comprising the following steps:

[0033] (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;

[0034] (2) sequencing the gene fragment combination to identify the genotype of each SNP site in the above SNP site combination;

[0035] (3) Based on the genotype identified in step (2), the total score S of the consanguinity evaluation is calculated using the following formula:

[0036]

[0037] Where Ai is the final weight of the genotype corresponding to SNPi; i is an integer greater than 0 and less than 17;

[0038] The value of Ai is obtained according to the following table:

[0039] ;

[0040] (4) When the total score S of the blood relationship evaluation is greater than 50, the chicken individual to be tested is a Camellia chicken.

[0041] Furthermore, the PCR amplification uses the above primer pair combination.

[0042] 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.

[0043] The present invention discloses the following technical effects:

[0044] This method, based on a large-scale population of 25 representative Chinese indigenous chicken breeds, including the Camellia chicken, and two introduced breeds, identifies specific SNPs and screens molecular markers for identifying Camellia chicken. By combining existing methods for detecting specific SNPs with the Analytic Hierarchy Process (AHP), and comprehensively considering the allele frequencies and genotype types of specific loci, this method improves detection efficiency and the scientific nature of the evaluation while ensuring identification accuracy. This method uses molecular marker technology to scientifically identify and evaluate Camellia chicken breed-specific characteristics. The method is simple to operate and produces reliable results, providing a scientific basis for the conservation and rational utilization of indigenous chicken genetic resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] 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.

[0046] Figure 1 This is a cluster diagram of Camellia chicken and other chicken breeds;

[0047] Figure 2 Flowchart for weight calculation of AHP. DETAILED DESCRIPTION

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0053] Example 1 Screening of SNP Molecular Markers in Camellia Chicken

[0054] 1. Blood sample collection

[0055] Based on the previous genetic evolutionary studies on local chickens (Gallus gallus), 25 local chicken breeds and two introduced breeds (Yuanbao chicken and Dulong chicken) were selected as research objects according to the genetic background of different chicken breeds. The 25 chicken breeds, including Camellia chicken (CH), were derived from the National Local Chicken Breed Gene Bank (Jiangsu), with 10 roosters and 20 hens of each breed; Yuanbao chicken (YB, n = 8) samples were derived from the experimental population of Jiangsu Poultry Science Research Institute; Dulong chicken (DL, n = 10) sequences were derived from the NCBI database (Table 1); 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) to ensure that there would be no interference from hybrid populations, such as Figure 1 Each individual species on the phylogenetic tree shown can form an independent branch.

[0056] 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.

[0057] Table 1 Sample information

[0058]

[0059] 2. DNA sample acquisition

[0060] Genomic DNA from all species was extracted using the conventional phenol-chloroform method. The resulting DNA was subjected to quality control, including preliminary concentration testing using Nanodrop and electrophoresis to assess DNA integrity, including the presence of degradation and contamination by other impurities such as protein and RNA. DNA was accurately quantified using the Qubit 2.0 assay, and samples with a mass of ≥1 μg were selected. Qualified samples were stored at -80°C for library construction and sequencing.

[0061] 3. Simplify genome RAD-seq library construction and sequencing

[0062] 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.

[0063] 4. Data quality control

[0064] 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%.

[0065] 5. Statistical analysis and site screening

[0066] Linkage disequilibrium (LD) analysis was performed using Haploview 4.1 software; mean heterozygosity (Ho), inbreeding coefficient (Fis), and population differentiation index (Fst) were calculated using PopGene software. Cluster analysis was performed using Admixture software, and a phylogenetic tree was constructed using the maximum likelihood (ML) method. 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 using PLINK 1.9 software, with a 100 kb window and a 10 kb step size. Chromosome segments or individuals with poor enzyme digestion were omitted from analysis to ensure accurate allele frequency calculations. Gene functional enrichment analysis was performed using DAVID, and the results were visualized using ggplot2 in the R language.

[0067] 6. Specific site screening and determination

[0068] Statistical analysis and site screening identified 16 specific SNP sites. (See Table 2)

[0069] Table 2 Information of specific SNPs identified

[0070]

[0071]

[0072] Note: The underlined sites in the above table are single nucleotide polypeptide mutation sites; Y is T or C, S is C or G, R is A or G, W is A or T, and K is T or G.

[0073] 7. Calculation of Camellia Chicken-specific SNP locus weights

[0074] Establish a hierarchical model of specific sites (such as Figure 2 As 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:

[0075] 1 means that the two loci have the same importance in terms of allele frequency or dominant allele type;

[0076] 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;

[0077] 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;

[0078] 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;

[0079] 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;

[0080] 2, 4, 6, and 8 represent the intermediate values ​​of the above adjacent judgments.

[0081] Table 3 Construction criterion layer pairwise comparison matrix

[0082] SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 SNP7 SNP8 SNP9 SNP10 SNP11 SNP12 SNP13 SNP14 SNP15 SNP16 SNP1 1.00 0.60 0.60 0.33 1.00 0.33 0.43 0.43 3.00 1.00 1.00 3.00 0.60 3.00 1.00 3.00 SNP2 1.67 1.00 1.00 0.56 1.67 0.56 0.71 0.71 5.00 1.67 1.67 5.00 1.00 5.00 1.67 5.00 SNP3 1.67 1.00 1.00 0.56 1.67 0.56 0.71 0.71 5.00 1.67 1.67 5.00 1.00 5.00 1.67 5.00 SNP4 3.00 1.80 1.80 1.00 3.00 1.00 1.29 1.29 9.00 3.00 3.00 9.00 1.80 9.00 3.00 9.00 SNP5 1.00 0.60 0.60 0.33 1.00 0.33 0.43 0.43 3.00 1.00 1.00 3.00 0.60 3.00 1.00 3.00 SNP6 3.00 1.80 1.80 1.00 3.00 1.00 1.29 1.29 9.00 3.00 3.00 9.00 1.80 9.00 3.00 9.00 SNP7 2.33 1.40 1.40 0.78 2.33 0.78 1.00 1.00 7.00 2.33 2.33 7.00 1.40 7.00 2.33 7.00 SNP8 2.33 1.40 1.40 0.78 2.33 0.78 1.00 1.00 7.00 2.33 2.33 7.00 1.40 7.00 2.33 7.00 SNP9 0.33 0.20 0.20 0.11 0.33 0.11 0.14 0.14 1.00 0.33 0.33 1.00 0.20 1.00 0.33 1.00 SNP10 1.00 0.60 0.60 0.33 1.00 0.33 0.43 0.43 3.00 1.00 1.00 3.00 0.60 3.00 1.00 3.00 SNP11 1.00 0.60 0.60 0.33 1.00 0.33 0.43 0.43 3.00 1.00 1.00 3.00 0.60 3.00 1.00 3.00 SNP12 0.33 0.20 0.20 0.11 0.33 0.11 0.14 0.14 1.00 0.33 0.33 1.00 0.20 1.00 0.33 1.00 SNP13 1.67 1.00 1.00 0.56 1.67 0.56 0.71 0.71 5.00 1.67 1.67 5.00 1.00 5.00 1.67 5.00 SNP14 0.33 0.20 0.20 0.11 0.33 0.11 0.14 0.14 1.00 0.33 0.33 1.00 0.20 1.00 0.33 1.00 SNP15 1.00 0.60 0.60 0.33 1.00 0.33 0.43 0.43 3.00 1.00 1.00 3.00 0.60 3.00 1.00 3.00 SNP16 0.33 0.20 0.20 0.11 0.33 0.11 0.14 0.14 1.00 0.33 0.33 1.00 0.20 1.00 0.33 1.00

[0083] 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:

[0084] First: describe the confidence interval (CI value) obtained by the above calculation [CI = (largest eigenvalue - n) / (n - 1)];

[0085] Second: Combine the judgment matrix order to obtain the average random consistency index (RI value);

[0086] Third: Calculate the CR value and make consistency judgment.

[0087] The calculation formula is as follows, where CI is the confidence interval, λmax is the maximum characteristic root, n is the number of object features, A is the feature matrix, and W is the normalized matrix of the A matrix. The formula is as follows:

[0088]

[0089] 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 in the subsequent consistency test.

[0090] Table 4 AHP hierarchical analysis results

[0091]

[0092]

[0093] 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…16.

[0094] According to the final weight values ​​calculated in Table 4, the scores of the individual Camellia chickens to be tested are evaluated:

[0095]

[0096] Where S is the total score of blood relationship evaluation, Ai is the final weight of SNPi corresponding to the genotype (see Table 4); i = 1, 2, 3...16.

[0097] Example 2 Application of Camellia Chicken Molecular Markers

[0098] 1. Blood collection from individuals of Camellia chicken conservation population

[0099] A medical disposable syringe was used to randomly collect 1.0 mL of whole blood from the wing vein of male and female Camellia chickens and the closely related chicken species, Daweishan miniature chickens and Tibetan chickens (extreme chicken species for comparison; non-closely related chicken species are easier to identify and distinguish). 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 later use.

[0100] 2. DNA extraction and quality testing

[0101] 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.

[0102] 3. SNP marker detection of Camellia chicken breed-specific genes

[0103] Primer Design: The SNPs identified in the Camellia 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 designed using software such as Oligo (see Table 5) were then amplified by PCR.

[0104] PCR amplification and detection:

[0105] The total volume of PCR amplification was 20 μL: 1 μL DNA template, with a concentration of 100 ng / μL; 2 μL 10× PCR Buffer; 1.5 μL dNTP, with a concentration of 10 mmol / L; 1 μL each of upstream and downstream primers, with a concentration of 10 pmol / μL; 0.2 μL Taq enzyme, with a concentration of 5 U / μL; and 13.3 μL ddH2O.

[0106] 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.

[0107] Table 5 PCR amplification primer sequences

[0108]

[0109] 4. Identification of Camellia Chicken Breed

[0110] The genotype identification results of Camellia chicken and its closely related chicken breeds are shown in Table 6.

[0111] Table 6 Genotype identification of Camellia chicken and related chicken breeds

[0112]

[0113]

[0114] 5. Individual identification and evaluation of Camellia chickens and T-test results are shown in Table 7 below.

[0115] Table 7 Individual identification evaluation and T-test of Camellia chicken

[0116]

[0117] Note: ** indicates extremely significant difference, P < 0.01.

[0118] The results in Table 7 show that the molecular markers screened by the present invention can efficiently and accurately evaluate the blood purity of individual Camellia chickens and achieve breed identification. When the total blood purity evaluation score S>50, the chicken individual to be tested can be determined to be Camellia chicken.

[0119] 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 ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A molecular marker combination for identifying Camellia chicken breeds, characterized in that: The molecular marker combination consists of the molecular markers shown in SEQ ID NO.1-16; The base at 11 bp of the molecular marker shown in SEQ ID NO. 1 has a mutation site SNP1, which is a T / C mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 2 has a mutation site SNP2, which is a C / 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 / A mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 4 has a mutation site SNP4, which is a T / C mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 5 has a mutation site SNP5, which is an A / 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 C / T mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 8 has a mutation site SNP8, which is a T / G 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 / C 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 / C mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 11 has a mutation site SNP11, which is a G / A mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 12 has a mutation site SNP12, which is a T / A mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 13 has a mutation site SNP13, which is a C / T mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 14 has a mutation site SNP14, which is an A / G mutation; The base at 11 bp of the molecular marker shown in SEQ ID NO. 15 has a mutation site SNP15, which is a T / C mutation; There is a mutation site SNP16 at the 11 bp position of the molecular marker shown in SEQ ID NO. 16, which is a C / T mutation.

2. The molecular marker combination according to claim 1, characterized in that The genotype of the SNP1 is TT, TC or CC; the genotype of the SNP2 is CC, CG or GG; the genotype of the SNP3 is GG, GA or AA; the genotype of the SNP4 is TT, TC or CC; the genotype of the SNP5 is AA, AT or TT; the genotype of the SNP6 is CC, CT or TT; the genotype of the SNP7 is CC, CT or TT; the genotype of the SNP8 is TT, TG or GG; the genotype of the SNP9 is GG, GC or CC; the genotype of the SNP10 is TT, TC or CC; the genotype of the SNP11 is GG, GA or AA; the genotype of the SNP12 is TT, TA or AA; the genotype of the SNP13 is CC, CT or TT; the genotype of the SNP14 is AA, AG or GG; the genotype of the SNP15 is TT, TC or CC; the genotype of the SNP16 is CC, CT or TT.

3. A primer pair combination for identifying Camellia chicken breeds, characterized in that: The primer pair combination includes primer pairs 1-16 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 Camellia chicken varieties.

5. Use of the primer pair combination as claimed in claim 3 in preparing a kit for identifying the variety of Camellia chicken.

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

7. A method for identifying the variety of Camellia chicken, 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 17; 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 a Camellia chicken; The SNP site combination consists of SNP sites SNP1-16 located in the chicken reference genome GRCg6a as shown in the following table: 。 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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