An SNP locus, primer combination, their applications and identification method for identifying Ningdu Yellow Chickens

Abstract

The present invention relates to the field of biotechnology, and particularly to an SNP locus, a primer combination, and their applications and identification methods for identifying Ningdu Yellow Chickens. The present invention provides 8 specific SNP loci related to the identification of the Ningdu Yellow Chicken breed. By detecting the combined genotypes of any one or several of these 8 SNP loci and combining the gene frequencies corresponding to the genotypes of each SNP locus, the probability that the individual to be tested is a Ningdu Yellow Chicken can be calculated through Bayes' theorem, so as to identify the Ningdu Yellow Chicken breed. At the same time, as long as the genotype of one SNP locus does not conform to the corresponding genotype of the Ningdu Yellow Chicken, the individual can be completely excluded from belonging to the Ningdu Yellow Chicken; by randomly selecting a combination of 5 SNP loci, the probability of accurately identifying the Ningdu Yellow Chicken can reach more than 99.17%. This method is simple to operate and highly accurate, and can effectively combat the prevalence of counterfeit Ningdu Yellow Chickens in the market.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an SNP locus, primer combination, application and identification method for identifying Ningdu yellow chickens. Background Art

[0002] Ningdu yellow chickens, also known as Ningdu three-yellow chickens, are native to Ningdu County, Jiangxi Province. They are high-quality yellow chicken breeds for both meat and eggs. They are small in size, with thin and short shanks, and have characteristics such as "three yellows (yellow beak, yellow shank, yellow feathers)" and "five reds (red comb, red beard, red face, red eye circle, red ear lobe)". They are mainly raised at home, in cages or in mountain forests. Their muscles contain a variety of sweet substances, the muscle fiber diameter is thin, the meat is tender and delicious, rich in protein, amino acids and unsaturated fatty acids. Moreover, the indexes such as the total amount of protein, amino acids and muscle fiber density of Ningdu yellow chickens are superior to those of many domestic broiler breeds.

[0003] With the continuous improvement of consumers' living standards, most livestock and poultry on the current market are sold in carcass form. And there are many cultivated breeds and a few local breeds on the market with similar appearance traits to Ningdu yellow chickens, which makes it more difficult to distinguish Ningdu yellow chickens from other yellow chicken breeds. This has greatly affected the sales of Ningdu yellow chickens in the market, not only causing a certain impact on the germplasm of Ningdu yellow chickens, but also seriously affecting the brand of Ningdu yellow chickens, making it difficult for consumers to buy real Ningdu yellow chickens. At the same time, farmers also suffer huge losses. At present, the identification of Ningdu yellow chickens on the market mainly relies on appearance traits, and the error rate of identification has increased greatly. Especially when identifying the hybrid offspring of Ningdu yellow chickens and other local chicken breeds, it becomes more difficult to make correct classification and identification.

[0004] Single nucleotide polymorphism (SNP) mainly refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level. It is a two-state marker, with characteristics such as rich loci, wide distribution, high genetic stability, representativeness, and convenient and rapid detection. SNP markers use the SNP loci with large differences in the unique individual genomic DNA, and identify the breed through the genotypes of different locus combinations. The identification results are more objective and accurate. At the same time, SNP can be genotyped through technical means such as DNA molecular hybridization and primer extension.

[0005] Since at the present stage, Ningdu yellow chickens cannot be correctly distinguished from other local chicken breeds, therefore, it becomes more urgent to develop a product and method based on SNP that can accurately and effectively identify the breed of Ningdu yellow chickens. Summary of the Invention

[0006] The object of the present invention is to provide an SNP locus, a primer combination, and their application and identification method for identifying Ningdu yellow chickens, so as to solve the problems existing in the above-mentioned prior art. The SNP locus provided by the present invention can accurately distinguish Ningdu yellow chickens from non-Ningdu yellow chickens.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an SNP locus for identifying Ningdu yellow chickens, and the SNP locus includes one or several of the SNP loci shown in the following table:

[0009]

[0010] ND1P CHR 3 23289355 T C ND2P CHR 6 34343857 C A ND3P CHR 8 4084029 C A ND4P CHR 22 3906249 G A ND5P CHR 15 1678798 C T ND6P CHR 12 15103217 A G ND7P CHR 14 7371379 C T ND8P CHR 21 2591010 G C 。

[0011] Further preferably, the SNP locus includes 3 to 8 of the SNP loci shown in the aforementioned table.

[0012] Preferably, when the SNP locus is ND1P, the genotypes of this locus include TT, CC or TC;

[0013] When the SNP locus is ND2P, the genotypes of this locus include CC, AA or CA;

[0014] When the SNP locus is ND3P, the genotypes of this locus include CC, AA or CA;

[0015] When the SNP locus is ND4P, the genotypes of this locus include GG, AA or GA;

[0016] When the SNP locus is ND5P, the genotypes of this locus include CC, TT or CT;

[0017] When the SNP locus is ND6P, the genotypes of this locus include AA, GG or AG;

[0018] When the SNP locus is ND7P, the genotypes of this locus include CC, TT or CT;

[0019] When the SNP locus is ND8P, the genotypes of this locus include GG, CC or GC.

[0020] The present invention provides a primer combination for identifying Ningdu yellow chickens, and the primer combination includes one or several pairs of primer pairs shown in the following table:

[0021]

[0022] The present invention provides the application of the above primer pair in the preparation of a kit for identifying the Ningdu yellow chicken breed.

[0023] The present invention provides a kit for identifying the Ningdu yellow chicken breed, and the kit comprises the above primer combination.

[0024] The present invention provides the application of the above SNP locus, the above primer combination or the above kit in identifying the Ningdu yellow chicken.

[0025] The present invention provides a method for identifying the Ningdu yellow chicken, comprising the following steps:

[0026] Using the genomic DNA of the chicken individual to be tested as a template, performing PCR amplification respectively by using the above primer combination to obtain amplification products;

[0027] Sequencing the amplification products to determine the genotypes of the above SNP loci;

[0028] If one of the genotypes corresponding to the selected SNP loci does not conform to the genotype corresponding to the Ningdu yellow chicken, it is determined that the chicken individual to be tested does not belong to the Ningdu yellow chicken; if all the genotypes corresponding to the selected SNP locus combination conform to the genotype corresponding to the Ningdu yellow chicken, the probability that the chicken individual to be tested belongs to the Ningdu yellow chicken is determined according to Bayes' theorem.

[0029] Preferably, when the SNP locus is any one of the above SNP loci, if the probability that the chicken individual to be tested belongs to the Ningdu yellow chicken is greater than 71.03%, then the chicken individual to be tested is determined to be a Ningdu yellow chicken;

[0030] When the SNP locus is any two of the above SNP loci, if the probability that the chicken individual to be tested belongs to the Ningdu yellow chicken is greater than 85.90%, then the chicken individual to be tested is determined to be a Ningdu yellow chicken;

[0031] When the SNP locus is any three of the above SNP loci, if the probability that the chicken individual to be tested belongs to the Ningdu yellow chicken is greater than 93.97%, then the chicken individual to be tested is determined to be a Ningdu yellow chicken;

[0032] When the SNP locus is any four of the above SNP loci, if the probability that the chicken individual to be tested belongs to the Ningdu yellow chicken is greater than 97.65%, then the chicken individual to be tested is determined to be a Ningdu yellow chicken;

[0033] When the SNP locus is any five of the above SNP loci, if the probability that the chicken individual to be tested belongs to the Ningdu yellow chicken is greater than 99.17%, then the chicken individual to be tested is determined to be a Ningdu yellow chicken;

[0034] When any six of the SNP loci are involved, if the probability that the chicken to be tested belongs to Ningdu Yellow Chicken is greater than 99.74%, then the chicken to be tested is determined to be Ningdu Yellow Chicken;

[0035] When any seven of the SNP loci are involved, if the probability that the chicken to be tested belongs to Ningdu Yellow Chicken is greater than 99.92%, then the chicken to be tested is determined to be Ningdu Yellow Chicken;

[0036] When all eight of the SNP loci are involved, if the probability that the chicken to be tested belongs to Ningdu Yellow Chicken is greater than 99.97%, then the chicken to be tested is determined to be Ningdu Yellow Chicken.

[0037] Preferably, the calculation formula of Bayes' theorem is:

[0038]

[0039] Wherein, P represents the probability that the chicken to be tested belongs to Ningdu Yellow Chicken, Pi represents the frequency of the corresponding genotypes of other breeds in the i-th SNP locus in the SNP locus combination, and i is an integer from 1 to 8;

[0040] The values of P and Pi are obtained according to the following table:

[0041]

[0042]

[0043] Preferably, the reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 5 min, then denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles, and final extension at 72°C for 10 min.

[0044] The present invention discloses the following technical effects:

[0045] The present invention provides 8 specific SNP loci related to the identification of Ningdu Yellow Chicken breed. By detecting the combined genotypes of any one or several of these 8 SNP loci and combining the gene frequencies corresponding to the genotypes of each SNP locus, the probability that the individual to be tested is Ningdu Yellow Chicken can be calculated by Bayes' theorem to identify the Ningdu Yellow Chicken breed. At the same time, the probability of excluding Ningdu Yellow Chicken by the genotype of any one of the 8 SNP loci reaches 100%. Therefore, as long as the genotype of one SNP locus does not conform to the corresponding genotype of Ningdu Yellow Chicken, the individual can be completely excluded from belonging to Ningdu Yellow Chicken; by selecting any 5 SNP locus combinations, the probability of accurately identifying Ningdu Yellow Chicken can reach more than 99.17%. This method is simple to operate and highly accurate, and can effectively combat the prevalence of counterfeit Ningdu Yellow Chicken in the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0047] Figure 1 It is the PCA result diagram of Ningdu yellow chickens and non-Ningdu yellow chickens based on 8 SNP loci in the embodiments of the present invention; among them, NND represents non-Ningdu yellow chickens, and ND represents Ningdu yellow chickens.

[0048] Figure 2 It is the NJtree result diagram of Ningdu yellow chickens and non-Ningdu yellow chickens based on 8 SNP loci in the embodiments of the present invention.

[0049] Figure 3 It is the bar chart of the genotype frequencies of the ND1P locus in the embodiments of the present invention.

[0050] Figure 4 It is the bar chart of the genotype frequencies of the ND2P locus in the embodiments of the present invention.

[0051] Figure 5 It is the bar chart of the genotype frequencies of the ND3P locus in the embodiments of the present invention.

[0052] Figure 6 It is the bar chart of the genotype frequencies of the ND4P locus in the embodiments of the present invention.

[0053] Figure 7 It is the bar chart of the genotype frequencies of the ND5P locus in the embodiments of the present invention.

[0054] Figure 8 It is the bar chart of the genotype frequencies of the ND6P locus in the embodiments of the present invention.

[0055] Figure 9 It is the bar chart of the genotype frequencies of the ND7P locus in the embodiments of the present invention.

[0056] Figure 10 It is the bar chart of the genotype frequencies of the ND8P locus in the embodiments of the present invention. Detailed implementation manners

[0057] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0058] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0059] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, 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 related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0060] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0061] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0062] Example 1

[0063] First, determine yellow-skinned chickens such as Ningdu Yellow Chicken, Chongren Partridge Chicken, Guangfeng White-Eared Yellow Chicken, Huiyang Bearded Chicken, Guangxi Three-Yellow Chicken (breed name), Hetian Chicken, Huaixiang Chicken, Huanglang Chicken, Wuhua Yellow Chicken (Wuhua Three-Yellow Chicken), Zhengyang Yellow Chicken (Zhengyang Three-Yellow Chicken), Xianju Chicken, Pengxian Yellow Chicken, Xiangdong Chicken, Beijing Fatty Chicken, Wenchang Chicken, Jinghai Yellow Chicken, etc., and local chicken breeds with similar appearances and wide circulation in the market. Divide them into two groups: Ningdu Yellow Chicken and other chickens; calculate the allele frequencies of each SNP locus using the resequencing data of the above chicken breeds, and then compare the two groups of results to screen out SNP loci with a frequency of 0 or 1 in Ningdu Yellow Chicken and a large difference from other chickens. Finally, 8 SNP loci with a large difference between Ningdu Yellow Chicken and other chickens are obtained, a total of 8, namely ND1P locus - ND8P locus, as shown in Table 1 specifically.

[0064] Table 1. 8 SNP Loci Affecting the Phenotype of Ningdu Yellow Chicken

[0065] SNP locus name Chromosome Locus Reference genome Mutation site ND1P CHR 3 23289355 T C ND2P CHR 6 34343857 C A ND3P CHR 8 4084029 C A ND4P CHR 22 3906249 G A ND5P CHR 15 1678798 C T ND6P CHR 12 15103217 A G ND7P CHR 14 7371379 C T ND8P CHR 21 2591010 G C

[0066] Note: The chromosomes in Table 1 refer to the chromosomes of chickens. All loci are referenced to the chicken reference genome (GRCg6a). CHR is the abbreviation of chromosome, and CHR 1 - CHR 33 are autosomes 1 - 33.

[0067] The situation of 8 SNP loci of Ningdu Yellow Chickens and other chickens was analyzed by the principal component analysis (PCA) method, and the analysis results are as Figure 1 shown; then population genetic structure analysis was carried out, and a phylogenetic tree was constructed by the neighbor-joining method, as Figure 2 shown; among them, NND represents non-Ningdu Yellow Chickens, and ND represents Ningdu Yellow Chickens. From Figure 1 and Figure 2 it can be seen that these 8 SNP loci can divide Ningdu Yellow Chickens and non-Ningdu Yellow Chickens into two clusters, and can clearly distinguish Ningdu Yellow Chickens and non-Ningdu Yellow Chickens.

[0068] According to the SNP locus information in Table 1, the corresponding primer sequences for each SNP locus were designed, that is, the primer combination for identifying the Ningdu Yellow Chicken breed. The specific information of the primers is shown in Table 2.

[0069] Table 2 Primer Information for 8 SNP Loci

[0070]

[0071] Example 2 Establishment of the Ningdu Yellow Chicken Identification Method

[0072] Individuals of representative domestic breeds of yellow-skinned chickens such as Ningdu Yellow Chickens, Chongren Partridge Chickens, Guangfeng White-Eared Yellow Chickens, Huiyang Bearded Chickens, Guangxi Sanhuang Chickens (breed name), Hetian Chickens, Huaixiang Chickens, Huanglang Chickens, Wuhua Yellow Chickens, Zhengyang Yellow Chickens, Xianju Chickens, Pengxian Yellow Chickens, Xiangdong Chickens, Beijing You Chickens, Wenchang Chickens, Jinghai Yellow Chickens, etc. were selected. Blood was collected from the wing vein, and genomic DNA was extracted by the phenol-chloroform method. According to the 8 SNP loci screened in Table 1, the corresponding primer pairs in Table 2 were selected for PCR amplification respectively.

[0073] The PCR amplification system (20 μL) was: 2 μL of DNA template, 1.0 μL each of forward and reverse primers (10 ng / μL), 10 μL of 2×PCR reagent in the Tiangen PCR kit, and the rest was made up with ultrapure water.

[0074] The PCR amplification program was: pre-denaturation at 94 °C for 5 min, then denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, for a total of 35 cycles, and finally post-extension at 72 °C for 10 min, and stored at 4 °C; the PCR instrument was the American Bio-Rad T100 gradient PCR instrument.

[0075] The amplified products were sent to Beijing Tsingke Biotechnology Co., Ltd. (Changsha Branch) for polymorphism detection and genotype determination of the sequences.

[0076] Calculate the different allele frequencies at each locus of yellow - skinned chicken breeds such as Ningdu Yellow Chicken, Chongren Partridge Chicken, Guangfeng White - eared Yellow Chicken, Huiyang Bearded Chicken, Guangxi Sanhuang Chicken (breed name), Hetian Chicken, Huaixiang Chicken, Huanglang Chicken, Wuhua Yellow Chicken, Zhengyang Yellow Chicken, Xianju Chicken, Pengxian Yellow Chicken, Xiangdong Chicken, Beijing Youji, Wenchang Chicken, Jinghai Yellow Chicken, etc. And determine whether the chicken to be tested belongs to the Ningdu Yellow Chicken breed according to the genotype. If one of the genotypes corresponding to the selected SNP locus combination does not match the genotype of the Ningdu Yellow Chicken, it is determined that the chicken to be tested does not belong to the Ningdu Yellow Chicken; if all the genotypes corresponding to the selected SNP locus combination match the genotype of the Ningdu Yellow Chicken, then determine the probability that the chicken to be tested belongs to the Ningdu Yellow Chicken according to Bayes' theorem. Determine the gene frequencies of the genotypes of 8 SNP loci in Ningdu Yellow Chicken and other breeds as shown in Table 3 and Figures 3 - 10 shown below.

[0077] Table 3 Genotypes and gene frequencies of 8 SNP loci in Ningdu Yellow Chicken and other breeds

[0078]

[0079]

[0080] Note: The genotype frequency of other breeds refers to detecting the genotype frequency of this group with Chongren Partridge Chicken, Guangfeng White - eared Yellow Chicken, Huiyang Bearded Chicken, Guangxi Sanhuang Chicken (breed name), Hetian Chicken, Huaixiang Chicken, Huanglang Chicken, Wuhua Yellow Chicken, Zhengyang Yellow Chicken, Xianju Chicken, Pengxian Yellow Chicken, Xiangdong Chicken, Beijing Youji, Wenchang Chicken, Jinghai Yellow Chicken as a group.

[0081] According to Bayes' theorem, the probability that an individual belongs to the Ningdu Yellow Chicken can be determined, and the calculation formula is as follows:

[0082]

[0083] Among them, P represents the probability that the chicken to be tested belongs to the Ningdu Yellow Chicken, Pi represents the frequency of the corresponding genotype of other breeds in the i - th SNP locus in the SNP locus combination, and i is an integer from 1 to 8.

[0084] For example, if three SNP loci ND1P, ND2P, and ND3P are selected, and the combined genotype is CC, AA, AA, then the probability that this individual belongs to the Ningdu Yellow Chicken is P = 1 / (1 + 0.3×0.3056×0.3444)=0.9693.

[0085] The probability of excluding the Ningdu Yellow Chicken for the genotype of any one SNP locus reaches 100%.

[0086] According to Bayes' theorem, the probability of determining the genotype of any one SNP locus as Ningdu yellow chicken is at least 71.03% and at most 77.92%; the probability of determining the combined genotype of any two SNP loci as Ningdu yellow chicken reaches more than 85.90%; the probability of determining the combined genotype of any three SNP loci as Ningdu yellow chicken reaches more than 93.97%; the probability of determining the combined genotype of any four SNP loci as Ningdu yellow chicken reaches more than 97.65%; the probability of determining the combined genotype of any five SNP loci as Ningdu yellow chicken reaches more than 99.17%; the probability of determining the combined genotype of any six SNP loci as Ningdu yellow chicken reaches more than 99.74%. The probability of determining the combined genotype of any seven SNP loci as Ningdu yellow chicken reaches more than 99.92%; the probability of determining the combined genotype of any eight SNP loci as Ningdu yellow chicken reaches more than 99.97%; when the probability of determining the combined genotype of more than six SNP loci as Ningdu yellow chicken is greater than 99.7%, the probability of excluding Ningdu yellow chicken for any one SNP locus genotype reaches 100%. Therefore, as long as the genotype of one SNP locus does not conform to the corresponding genotype of Ningdu yellow chicken, the individual can be completely excluded from being Ningdu yellow chicken; by selecting any five SNP locus combinations, the probability of accurately identifying Ningdu yellow chicken can reach more than 99.17%.

[0087] Example 3

[0088] In this example, 15 Ningdu yellow chickens, 15 Chongren partridge chickens, 15 Guangfeng white-eared yellow chickens, 15 Guangxi Sanhuang chickens (breed name), and 15 Huiyang bearded chickens were randomly selected from the conservation farm, and blood was collected from the wing vein. The chicken breeds were identified according to the following steps:

[0089] (1) Extract total DNA

[0090] First, the genomic DNA was extracted by the phenol-chloroform method. The specific operations are as follows:

[0091] ① Take 20 μL of anticoagulated whole blood and place it in a sterilized 1.5 mL centrifuge tube, and number it. Add 600 μL of DNA extraction solution, and shake well in a constant temperature shaker at 65 °C and 180 rpm for 30 min;

[0092] ② Under room temperature conditions, centrifuge at 12000 r / min for 5 min, and transfer the supernatant to a new 1.5 mL centrifuge tube;

[0093] ③ Add 1 / 10 volume (about 60 μL) of 3 mol / L NaAc (pH value is 5.2), then add 300 μL of chloroform-isoamyl alcohol (the volume ratio of chloroform to isoamyl alcohol is 24:1), mix well for 1 min, let it stand at 4 °C for 10 min, and centrifuge at 12000 r / min for 10 min;

[0094] ④ Transfer the supernatant to a new 1.5 mL centrifuge tube, add an equal volume (about 500 μL) of absolute ethanol at -20 °C, gently invert and mix well, and wait for the precipitate to form.

[0095] ⑤ Use a yellow pipette tip to pick out the DNA and transfer it to a new 0.5 mL centrifuge tube. Add 400 μL of 75% ethanol (v / v) to wash the precipitate two or three times, and then aspirate the excess 75% (v / v) ethanol in the centrifuge tube. After air drying (about 2 h), add 200 μL of TE solution to dissolve it, and measure the DNA concentration and purity using a UV spectrophotometer, and store it at -20 °C.

[0096] (2) PCR amplification

[0097] After extracting the DNA, perform PCR amplification according to the primers of the 5 SNP loci in Table 4.

[0098] Table 4 Primers for variety identification in this example

[0099]

[0100] The PCR system (20 μL) is as follows: 2 μL of DNA template, 1.0 μL each of forward and reverse primers (10 ng / μL), 10 μL of 2×PCR reagent in the Tiangen PCR kit, and the rest is made up with ultrapure water.

[0101] The PCR program is as follows: First, pre-denature at 94 °C for 5 min, then denature at 94 °C for 30 s, anneal at 55 °C for 30 s, extend at 72 °C for 30 s, for a total of 35 cycles, and finally extend at 72 °C for 10 min and store at 4 °C. The PCR instrument used is the Bio-Rad T100 thermal cycler from the United States.

[0102] (3) Sequencing and analysis

[0103] The amplified products were sent to Beijing Qingke Biotechnology Co., Ltd. (Changsha Branch) for polymorphism detection and genotype determination of the sequences. According to the sequencing results, the combined genotypes of the 5 loci of the 15 Ningdu yellow chickens were all CC, AA, AA, GA, and CC. The probability of identifying these 15 individuals as Ningdu yellow chickens was 99.67%. Therefore, all 15 individuals were identified as Ningdu yellow chickens, and the identification accuracy rate was 100%. The remaining individuals did not fully conform to the genotypes of CC, AA, AA, GA, and CC. Therefore, they were determined to be non-Ningdu yellow chickens.

[0104] The above embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a primer combination for identifying Ningdu yellow chickens in the preparation of a kit for identifying the breed of Ningdu yellow chickens, characterized in that, The primer combination includes 5 pairs of primer pairs in the following table:

2. A method for identifying the Ningdu Yellow Chicken breed, characterized in that, Comprising the following steps: Using the genomic DNA of the individual chicken to be tested as a template, and respectively performing PCR amplification by using the primer combination to obtain amplification products; the primer combination includes 5 pairs of primer pairs in the following table: Sequencing the amplification products to determine the genotypes of the SNP sites; If one of the genotypes corresponding to the SNP sites does not conform to the genotype corresponding to Ningdu Yellow Chicken, it is determined that the individual chicken to be tested does not belong to Ningdu Yellow Chicken; if all the genotypes corresponding to the SNP site combination conform to the genotype corresponding to Ningdu Yellow Chicken, the probability that the chicken to be tested belongs to Ningdu Yellow Chicken is determined according to Bayes' theorem; The calculation formula of Bayes' theorem is: Wherein, P represents the probability that the chicken to be tested belongs to Ningdu Yellow Chicken, and Pi represents the frequency of the corresponding genotypes of other breeds in the i-th SNP site in the SNP site combination; The position information of the SNP sites, the distribution of the genotypes and gene frequencies of the SNP sites in Ningdu Yellow Chicken and other breeds are shown in the following table: All sites are referenced to the chicken reference genome GRCg6a.

3. The method according to claim 2, wherein The reaction program of the PCR amplification is: pre-denaturation at 94°C for 5 min, then denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles, and post-extension at 72°C for 10 min.

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