SNP (Single Nucleotide Polymorphism) site and primer combination for identifying Ningdu yellow chicken as well as application and identification method of Ningdu yellow chicken

Abstract

The invention relates to the technical field of biology, in particular to an SNP (Single Nucleotide Polymorphism) site for identifying Ningdu yellow chicken, a primer combination as well as application and an identification method of the Ningdu yellow chicken. The invention provides eight specific SNP loci related to the identification of the Ningdu yellow chicken variety, and by detecting the combined genotype of any one or more of the eight SNP loci and combining the gene frequency corresponding to the genotype of each SNP locus, the probability that an individual to be detected is the Ningdu yellow chicken can be calculated through the Bayesian theorem, so that the Ningdu yellow chicken variety is identified. Meanwhile, as long as the genotype of one SNP site 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; the probability of accurately identifying the Ningdu yellow chicken can reach 99.17% or above by optionally selecting five SNP site combinations. The method is easy and convenient to operate and high in accuracy, and the flooding degree of the fake Ningdu yellow chickens in the market can be effectively attacked.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a SNP site, a primer combination and an application and an identification method thereof for identifying Ningdu yellow chicken. Background Art

[0002] Ningdu Yellow Chicken, also known as Ningdu Three Yellow Chicken, is native to Ningdu County, Jiangxi Province. It is a high-quality yellow chicken breed for both meat and eggs. It is small in size, with short and thin shanks, and has the characteristics of "three yellows (yellow beak, yellow shanks, and yellow feathers)" and "five reds (red comb, red whiskers, red face, red eye circles, and red ear leaves)". It is mainly domesticated, raised in pens, and raised in the mountains. Its muscles contain a variety of sweet substances, the muscle fibers are thin in diameter, the meat is tender and delicious, and it is rich in protein, amino acids, and unsaturated fatty acids. Moreover, the indicators such as protein, total amino acids, and muscle fiber density of Ningdu Yellow Chicken are better than those of many domestic broiler breeds.

[0003] With the continuous improvement of consumer standards, most of the livestock and poultry on the market are currently sold in carcasses, and there are many cultivated varieties and a few local varieties on the market that have similar appearance traits to Ningdu Yellow Chicken, which makes it more difficult to distinguish Ningdu Yellow Chicken from other yellow chicken varieties, greatly affecting the sales of Ningdu Yellow Chicken in the market. It not only has a certain impact on the genetic quality of Ningdu Yellow Chicken, but also seriously affects the Ningdu Yellow Chicken brand, making it difficult for consumers to buy real Ningdu Yellow Chicken, and farmers also suffer huge losses. At present, the identification of Ningdu Yellow Chicken in the market mainly relies on appearance traits, and the identification error rate is greatly increased, especially when identifying the hybrid offspring of Ningdu Yellow Chicken and other local chicken varieties, 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 genome level. It is a dimorphic marker with the characteristics of rich sites, wide distribution, high genetic stability, representativeness, and convenient and fast detection. SNP markers use the SNP sites with large variety differences in individual genomic DNA to identify varieties through genotypes of different site combinations, and 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] At present, it is impossible to correctly distinguish Ningdu Yellow Chicken from other local chicken breeds. Therefore, it has become more urgent to develop a product and method based on SNP that can accurately and effectively identify the Ningdu Yellow Chicken breed. Summary of the invention

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

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

[0008] The present invention provides a SNP site for identifying Ningdu yellow chicken, wherein the SNP site includes one or more of the SNP sites 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 sites include 3 to 8 of the SNP sites shown in the aforementioned table.

[0012] Preferably, when the SNP site is ND1P, the genotype of the site includes TT, CC or TC;

[0013] When the SNP site is ND2P, the genotype of the site includes CC, AA or CA;

[0014] When the SNP site is ND3P, the genotype of the site includes CC, AA or CA;

[0015] When the SNP site is ND4P, the genotype of the site includes GG, AA or GA;

[0016] When the SNP site is ND5P, the genotype of the site includes CC, TT or CT;

[0017] When the SNP site is ND6P, the genotype of the site includes AA, GG or AG;

[0018] When the SNP site is ND7P, the genotype of the site includes CC, TT or CT;

[0019] When the SNP site is ND8P, the genotype of the site includes GG, CC or GC.

[0020] The present invention provides a primer combination for identifying Ningdu yellow chicken, wherein the primer combination comprises one or more pairs of primer pairs as described in the following table:

[0021]

[0023] The present invention provides application of the primer pair in preparing a kit for identifying the Ningdu yellow chicken variety.

[0024] The invention provides a kit for identifying the variety of Ningdu yellow chicken, and the kit comprises the above primer combination.

[0025] The present invention provides the use of the above-mentioned SNP site, the above-mentioned primer combination or the above-mentioned kit in identifying Ningdu yellow chicken.

[0026] The present invention provides a method for identifying Ningdu yellow chicken, comprising the following steps:

[0027] Using the genomic DNA of the chicken individual to be tested as a template, PCR amplification is performed using the above primer combination to obtain amplified products;

[0028] Sequencing the amplified product to determine the genotype of the SNP site;

[0029] If one of the genotypes corresponding to the selected SNP sites does not match 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 site combination match the genotype corresponding to the Ningdu Yellow Chicken, the probability that the chicken to be tested belongs to the Ningdu Yellow Chicken is determined according to the Bayesian theorem.

[0030] Preferably, when the SNP site is any one of the above-mentioned SNP sites, if the probability that the chicken individual to be tested belongs to Ningdu Yellow Chicken is greater than 71.03%, the chicken individual to be tested is determined to be Ningdu Yellow Chicken;

[0031] When the SNP loci are any two of the above SNP loci, if the probability that the chicken individual to be tested belongs to Ningdu Yellow Chicken is greater than 85.90%, the chicken individual to be tested is determined to be Ningdu Yellow Chicken;

[0032] When the SNP loci are any three of the above SNP loci, if the probability that the chicken individual to be tested belongs to Ningdu Yellow Chicken is greater than 93.97%, the chicken individual to be tested is determined to be Ningdu Yellow Chicken;

[0033] When the SNP loci are any four of the SNP loci, if the probability that the chicken individual to be tested belongs to the Ningdu Yellow Chicken is greater than 97.65%, the chicken individual to be tested is determined to be the Ningdu Yellow Chicken;

[0034] When the SNP loci are any five of the SNP loci, if the probability that the chicken individual to be tested belongs to the Ningdu Yellow Chicken is greater than 99.17%, the chicken individual to be tested is determined to be the Ningdu Yellow Chicken;

[0035] When the SNP loci are any six of the SNP loci, if the probability that the chicken individual to be tested belongs to the Ningdu Yellow Chicken is greater than 99.74%, the chicken individual to be tested is determined to be the Ningdu Yellow Chicken;

[0036] When the SNP loci are any seven of the SNP loci, if the probability that the chicken individual to be tested belongs to the Ningdu Yellow Chicken is greater than 99.92%, the chicken individual to be tested is determined to be the Ningdu Yellow Chicken;

[0037] When the SNP loci are eight of the SNP loci, if the probability that the chicken individual to be tested belongs to the Ningdu Yellow Chicken is greater than 99.97%, the chicken individual to be tested is determined to be the Ningdu Yellow Chicken.

[0038] Preferably, the calculation formula of the Bayesian theorem is:

[0039]

[0040] Where 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 site in the SNP site combination, and i is an integer from 1 to 8;

[0041] The values ​​of P and Pi are obtained according to the following table:

[0042]

[0043]

[0045] Preferably, the reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 5 min, followed by denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles, and post-extension at 72°C for 10 min.

[0046] The present invention discloses the following technical effects:

[0047] The present invention provides 8 specific SNP sites related to the identification of Ningdu Yellow Chicken varieties. By detecting the combined genotype of any one or several of the 8 SNP sites, combined with the gene frequency corresponding to the genotype of each SNP site, the probability that the individual to be tested is a Ningdu Yellow Chicken can be calculated by Bayes' theorem, thereby identifying the Ningdu Yellow Chicken variety. At the same time, the probability of excluding Ningdu Yellow Chicken by the genotype of any one of the 8 SNP sites reaches 100%. Therefore, as long as there is a SNP site genotype that 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 combination of 5 SNP sites, the probability of accurately identifying Ningdu Yellow Chicken can reach more than 99.17%. This method is easy to operate and has high accuracy, and can effectively combat the prevalence of counterfeit Ningdu Yellow Chickens in the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0049] Figure 1 This is a PCA result diagram of Ningdu Yellow Chicken and Non-Ningdu Yellow Chicken based on 8 SNP sites in an embodiment of the present invention; wherein NND represents Non-Ningdu Yellow Chicken, and ND represents Ningdu Yellow Chicken;

[0050] Figure 2 This is the NJtree result diagram of Ningdu Yellow Chicken and non-Ningdu Yellow Chicken based on 8 SNP sites in the embodiment of the present invention;

[0051] Figure 3 It is a histogram of the genotype frequency of the ND1P locus in the embodiment of the present invention;

[0052] Figure 4 It is a histogram of the genotype frequency of the ND2P locus in the embodiment of the present invention;

[0053] Figure 5 It is a histogram of the genotype frequency of the ND3P locus in the embodiment of the present invention;

[0054] Figure 6 It is a histogram of the genotype frequency of the ND4P locus in the embodiment of the present invention;

[0055] Figure 7 It is a histogram of the genotype frequency of the ND5P locus in the embodiment of the present invention;

[0056] Figure 8 ND6P is a genotype frequency bar graph of the ND6P locus in the embodiment of the present invention;

[0057] Fig. 9 It is a histogram of the genotype frequency of the ND7P locus in the embodiment of the present invention;

[0058] Fig.10 It is a histogram of the genotype frequency of the ND8P locus in the embodiment of the present invention. DETAILED DESCRIPTION

[0059] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0060] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0061] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content of this specification shall prevail.

[0062] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0063] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0064] Example 1

[0065] Firstly, yellow-skinned chickens and local chicken breeds with similar appearance and wide circulation in the market, such as Ningdu yellow chicken, Chongren Ma 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 oily chicken, Wenchang chicken, and Jinghai yellow chicken, were identified and divided into two groups: Ningdu yellow chicken and other chickens. The allele frequency of each SNP site was calculated using the resequencing data of the above chicken breeds, and the two groups of results were compared to screen out SNP sites with a frequency of 0 or 1 in Ningdu yellow chicken and which were extremely different from those of other chickens. Finally, 8 SNP sites with extremely different differences between Ningdu yellow chicken and other chickens were screened out, namely ND1P site-ND8P site, as shown in Table 1.

[0066] Table 1 8 SNP loci affecting the phenotype of Ningdu yellow chicken

[0067] SNP site name chromosome Location 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

[0068] Note: The chromosomes in Table 1 refer to chicken chromosomes. All loci are based on the chicken reference genome (GRCg6a). CHR is the abbreviation of chromosome, and CHR 1-CHR 33 refers to autosomes 1-33.

[0069] The principal component analysis (PCA) method was used to analyze the 8 SNP loci of Ningdu yellow chicken and other chickens. The analysis results are as follows Figure 1 As shown; then the population genetic structure analysis was carried out and the evolutionary tree was constructed using the neighbor-joining method, as shown Figure 2 As shown in the figure, NND means non-Ningdu yellow chicken, and ND means Ningdu yellow chicken. Figure 1 and Figure 2 It can be seen that these 8 SNP sites 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.

[0070] According to the SNP site information in Table 1, the primer sequence corresponding to each SNP site was designed, that is, the primer combination for identifying the Ningdu yellow chicken variety. The specific information of the primers is shown in Table 2.

[0071] Table 2 Primer information of 8 SNP sites

[0072]

[0073] Embodiment 2 the establishment of Ningdu yellow chicken identification method

[0074] Individuals of representative domestic yellow-skinned chicken breeds such as Ningdu yellow chicken, Chongren hemp chicken, Guangfeng white-eared yellow chicken, Huiyang bearded chicken, Guangxi three-yellow chicken (breed name), Hetian chicken, Huaixiang chicken, Huanglang chicken, Wuhua yellow chicken, Zhengyang yellow chicken, Xianju chicken, Pengxian yellow chicken, Xiangdong chicken, Beijing oily chicken, Wenchang chicken, and Jinghai yellow chicken were selected, blood was collected from the wing vein, and genomic DNA was extracted by the phenol-chloroform method. According to the 8 SNP sites obtained by screening in Table 1, the corresponding primer pairs in Table 2 were selected for PCR amplification.

[0075] The PCR amplification system (20 μL) was as follows: 2 μL of DNA template, 1.0 μL of each forward and reverse primer (10 ng / μL), 10 μL of 2×PCR reagent in the Tiangen PCR kit, and the rest was made up with ultrapure water.

[0076] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, followed by denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles, and finally post-extension at 72°C for 10 min, followed by storage at 4°C. The PCR instrument was the Bio-Rad T100 gradient PCR instrument from the United States.

[0077] The amplified products were sent to Beijing Qingke Biotechnology Co., Ltd. (Changsha Branch) for sequence polymorphism detection and genotype determination.

[0078] The different allele frequencies of each locus of yellow-skinned chicken varieties such as Ningdu Yellow Chicken, Chongren Ma Chicken, Guangfeng White-eared Yellow Chicken, Huiyang Bearded Chicken, Guangxi Three Yellow Chicken (variety name), Hetian Chicken, Huaixiang Chicken, Huanglang Chicken, Wuhua Yellow Chicken, Zhengyang Yellow Chicken, Xianju Chicken, Pengxian Yellow Chicken, Xiangdong Chicken, Beijing Oil Chicken, Wenchang Chicken, and Jinghai Yellow Chicken were calculated. And according to the genotype, it is judged whether the chicken to be tested belongs to the Ningdu Yellow Chicken variety. If one of the genotypes corresponding to the selected SNP locus combination does not conform to the genotype corresponding to the Ningdu Yellow Chicken, it is judged that the chicken to be tested does not belong to the Ningdu Yellow Chicken; if the genotypes corresponding to the selected SNP locus combination all conform to the genotype corresponding to the Ningdu Yellow Chicken, the probability of the chicken to be tested belonging to the Ningdu Yellow Chicken is determined according to the Bayesian theorem, and the gene frequencies of the genotypes of the 8 SNP loci in Table 1 in the Ningdu Yellow Chicken and other varieties are determined as shown in Tables 3 and Figure 3-Figure 10 shown.

[0079] Table 3 Genotype and gene frequency of 8 SNP loci in Ningdu yellow chicken and other breeds

[0080]

[0081]

[0082] Note: The genotype frequency of other breeds refers to the detection of the genotype frequency of this group by taking Chongren Ma chicken, Guangfeng white-eared yellow chicken, Huiyang bearded chicken, Guangxi three-yellow chicken (breed name), Hetian chicken, Huaixiang chicken, Huanglang chicken, Wuhua yellow chicken, Zhengyang yellow chicken, Xianju chicken, Pengxian yellow chicken, Xiangdong chicken, Beijing oily chicken, Wenchang chicken and Jinghai yellow chicken as a group.

[0083] According to Bayes' theorem, the probability that an individual belongs to Ningdu Yellow Chicken can be determined. The calculation formula is as follows:

[0084]

[0085] Among them, 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 site in the SNP site combination, and i is an integer from 1 to 8.

[0086] For example, if three SNP sites, ND1P, ND2P, and ND3P, are selected and the combined genotypes are CC, AA, and AA, then the probability that the individual belongs to the Ningdu yellow chicken is P = 1 / (1 + 0.3 × 0.3056 × 0.3444) = 0.9693.

[0087] The probability of excluding Ningdu Yellow Chicken by any SNP locus genotype is 100%.

[0088] According to Bayes' theorem, the probability of any one SNP site genotype being determined as Ningdu Yellow Chicken is as low as 71.03% and as high as 77.92%; the probability of any two SNP sites combined genotype being determined as Ningdu Yellow Chicken reaches more than 85.90%; the probability of any three SNP sites combined genotype being determined as Ningdu Yellow Chicken reaches more than 93.97%; the probability of any four SNP sites combined genotype being determined as Ningdu Yellow Chicken reaches more than 97.65%; the probability of any five SNP sites combined genotype being determined as Ningdu Yellow Chicken reaches more than 99.17%; the probability of any six SNP sites combined genotype being determined as Ningdu Yellow Chicken reaches more than 99.74%. The probability of determining Ningdu Yellow Chicken by the combined genotype of any 7 SNP sites reaches more than 99.92%; the probability of determining Ningdu Yellow Chicken by the combined genotype of any 8 SNP sites reaches more than 99.97%; when the probability of determining Ningdu Yellow Chicken by the combined genotype of any 6 or more SNP sites is greater than 99.7%, the probability of excluding Ningdu Yellow Chicken by the genotype of any SNP site reaches 100%. Therefore, as long as the genotype of one SNP site does not meet the corresponding genotype of Ningdu Yellow Chicken, the individual can be completely excluded from Ningdu Yellow Chicken; the probability of accurately identifying Ningdu Yellow Chicken by any combination of 5 SNP sites can reach more than 99.17%.

[0089] Example 3

[0090] In this embodiment, 15 Ningdu yellow chickens, 15 Chongren hemp chickens, 15 Guangfeng white-eared yellow chickens, 15 Guangxi three-yellow chickens (variety name) and 15 Huiyang bearded chickens were randomly purchased from the seed conservation farm, and blood was collected from the wing vein to identify the chicken species according to the following steps:

[0091] (1) Extraction of total DNA

[0092] First, genomic DNA was extracted using the phenol-chloroform method. The specific steps are as follows:

[0093] ① Take 20 μL of anticoagulated whole blood and place it in a sterilized 1.5 mL centrifuge tube, number it, add 600 μL of DNA extraction solution, and shake it in a constant temperature shaker at 65°C and 180 rpm for 30 minutes;

[0094] ② Centrifuge at 12000r / min for 5min at room temperature and transfer the supernatant to a new 1.5mL centrifuge tube;

[0095] ③ Add 1 / 10 volume (about 60 μL) of 3 mol / L NaAc (pH 5.2), then add 300 μL chloroform-isoamyl alcohol (the volume ratio of chloroform to isoamyl alcohol is 24:1), mix for 1 min, let stand at 4°C for 10 min, and centrifuge at 12000 r / min for 10 min;

[0096] ④ Transfer the supernatant to a new 1.5 mL centrifuge tube, add an equal volume (about 500 μL) of -20°C anhydrous ethanol, gently invert and mix, and wait for the precipitate to precipitate;

[0097] ⑤ Use a yellow gun tip to pick out the DNA and transfer it to a new 0.5mL centrifuge tube, add 400μL 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 2h), add 200μL TE solution to dissolve it, use a UV spectrophotometer to determine the DNA concentration and purity, and store at -20℃.

[0098] (2) PCR amplification

[0099] After DNA was extracted, PCR amplification was performed using primers for the five SNP sites in Table 4.

[0100] Table 4 Primers used for variety identification in this example

[0101]

[0102] The PCR system (20 μL) was as follows: 2 μL of DNA template, 1.0 μL of each forward and reverse primer (10 ng / μL), 10 μL of 2×PCR reagent in the Tiangen PCR kit, and the rest was made up with ultrapure water.

[0103] The PCR program was as follows: first, pre-denaturation at 94°C for 5 min, then denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles, and finally post-extension at 72°C for 10 min, and storage at 4°C. The PCR instrument used was the American Bio-Rad T100 gradient PCR instrument.

[0104] (3) Sequencing and analysis

[0105] 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 5-site joint genotypes of the 15 Ningdu Yellow Chickens were CC, AA, AA, GA and CC, and the probability that the 15 individuals were determined to be Ningdu Yellow Chickens was 99.67%. Therefore, all 15 individuals were identified as Ningdu Yellow Chickens, and the identification accuracy was 100%. The remaining individuals did not fully meet the genotypes of CC, AA, AA, GA and CC, and were therefore determined to be non-Ningdu Yellow Chickens.

[0106] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design 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 all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for identifying SNP sites of Ningdu yellow chicken, characterized in that: The SNP sites include one or more of the SNP sites shown in the following table:

2. The SNP site according to claim 1, characterized in that: When the SNP site is ND1P, the genotype of the site includes TT, CC or TC; When the SNP site is ND2P, the genotype of the site includes CC, AA or CA; When the SNP site is ND3P, the genotype of the site includes CC, AA or CA; When the SNP site is ND4P, the genotype of the site includes GG, AA or GA; When the SNP site is ND5P, the genotype of the site includes CC, TT or CT; When the SNP site is ND6P, the genotype of the site includes AA, GG or AG; When the SNP site is ND7P, the genotype of the site includes CC, TT or CT; When the SNP site is ND8P, the genotype of the site includes GG, CC or GC.

3. A primer combination for identifying Ningdu yellow chicken, characterized in that: The primer combination includes one or more pairs of primer pairs as described in the following table:

4. Use of the primer combination described in claim 3 in preparing a kit for identifying the Ningdu yellow chicken variety.

5. A kit for identifying the variety of Ningdu yellow chicken, characterized in that: The kit comprises the primer combination according to claim 3.

6. Use of the SNP site described in claim 1 or 2, the primer combination described in claim 3, or the kit described in claim 5 in identifying Ningdu yellow chicken.

7. A method for identifying Ningdu yellow chicken, characterized in that: The following steps are involved: Using the genomic DNA of the chicken individual to be tested as a template, PCR amplification is performed using the primer combination described in claim 3 to obtain amplified products; Sequencing the amplified product to determine the genotype of the SNP site in claim 1; If one of the genotypes corresponding to the selected SNP loci does not match 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 the genotypes corresponding to the selected SNP site combination all match the genotypes corresponding to the Ningdu Yellow Chicken, the probability that the tested chicken belongs to the Ningdu Yellow Chicken is determined according to the Bayesian theorem.

8. The method according to claim 7, characterized in that When the SNP locus is any one of the SNP loci described in claim 1, if the probability that the chicken individual to be tested belongs to the Ningdu Yellow Chicken is greater than 71.03%, the chicken individual to be tested is determined to be the Ningdu Yellow Chicken; When the SNP loci are any two of the SNP loci described in claim 1, if the probability that the chicken individual to be tested belongs to Ningdu Yellow Chicken is greater than 85.90%, the chicken individual to be tested is determined to be Ningdu Yellow Chicken; When the SNP loci are any three of the SNP loci described in claim 1, if the probability that the chicken individual to be tested belongs to Ningdu Yellow Chicken is greater than 93.97%, the chicken individual to be tested is determined to be Ningdu Yellow Chicken; When the SNP loci are any four of the SNP loci described in claim 1, if the probability that the chicken individual to be tested belongs to Ningdu Yellow Chicken is greater than 97.65%, the chicken individual to be tested is determined to be Ningdu Yellow Chicken; When the SNP loci are any five of the SNP loci described in claim 1, if the probability that the chicken individual to be tested belongs to Ningdu Yellow Chicken is greater than 99.17%, the chicken individual to be tested is determined to be Ningdu Yellow Chicken; When the SNP loci are any six of the SNP loci described in claim 1, if the probability that the chicken individual to be tested belongs to the Ningdu Yellow Chicken is greater than 99.74%, the chicken individual to be tested is determined to be the Ningdu Yellow Chicken; When the SNP loci are any seven of the SNP loci described in claim 1, if the probability that the chicken individual to be tested belongs to Ningdu Yellow Chicken is greater than 99.92%, the chicken individual to be tested is determined to be Ningdu Yellow Chicken; When the SNP loci are eight of the SNP loci described in claim 1, if the probability that the chicken individual to be tested belongs to the Ningdu Yellow Chicken is greater than 99.97%, the chicken individual to be tested is determined to be the Ningdu Yellow Chicken.

9. The method according to claim 7, characterized in that: The calculation formula of the Bayesian theorem is: Where 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 site in the SNP site combination, and i is an integer from 1 to 8; The values ​​of P and Pi are obtained according to the following table:

10. The method according to claim 7, characterized in that The reaction procedure of the PCR amplification was: pre-denaturation at 94°C for 5 min, followed by denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and 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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