Method for identifying xiaoshan chicken

Abstract

The present application relates to the technical field of gene detection, and particularly relates to a Xiaoshan chicken identification method.The method provided by the present application uses specific SNP sites to identify the Xiaoshan chicken breed, and reliable and accurate detection results can be obtained.Nine optional specific SNP sites are provided by the present application, which are basically fixed in the Xiaoshan chicken breed and are all single genotypes; and are wild genotypes in other breeds. Xiaoshan chicken breed can be identified by using one or more sites, which has a high Xiaoshan chicken identification accuracy, and the results are stable and reliable. It is beneficial to the conservation and resource utilization and development of Xiaoshan chicken, and provides an important guarantee for carrying out corresponding breeding work and forming a high-quality and high-yield Xiaoshan chicken matching line. In addition, the method provided by the present application is simple in overall operation, high in efficiency, and suitable for popularization and application.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a method for identifying Xiaoshan chicken. Background Technology

[0002] Xiaoshan chicken is one of my country's eight famous chicken breeds. It is relatively large, grows rapidly in its early stages, is disease-resistant and hardy, adaptable, has a high slaughter rate, and boasts a unique meat flavor. It is a typical meat-type breed that combines the advantages of both meat production and flavor. Xiaoshan chickens have a rounded appearance, dense feathers in red and yellow, black tail feathers, and red combs, wattles, and earlobes. Currently, the main breeding area for Xiaoshan chickens is the Xiaoshan district of Hangzhou, but large-scale farming is relatively low, with most chickens being raised by small-scale farmers. This breeding process involves varying degrees of crossbreeding between different breeds, resulting in impure bloodlines for Xiaoshan chickens.

[0003] Pure bloodline is a prerequisite for selective breeding, and ensuring the pure bloodline of Xiaoshan chickens is a prerequisite for carrying out local chicken breeding. Traditional methods of identifying chicken breeds based on body shape and appearance are difficult to evaluate the bloodline and genomic homozygosity of breeds, resulting in significant errors. Identifying specific molecular tags based on high-throughput sequencing technology is beneficial for more accurate and efficient breed identification of large-scale populations, and is simpler and more efficient.

[0004] Based on different breeding directions, a large number of specific SNP markers for modern broiler and laying hens have been identified in existing technologies. However, most of these sites are in a neutral state and have not been fixed in specific breeds, making it difficult to form effective molecular tags. In view of this, the present invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for identifying Xiaoshan chicken, which uses specific SNP sites for identification. These specific SNP sites all have a high accuracy rate in identifying Xiaoshan chicken. Identification based on one or more of these specific SNP sites yields stable and reliable results.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for identifying Xiaoshan chicken, which uses specific SNP sites for identification, wherein the specific SNP sites are selected from one or more of sites 1, 2, 3, 4, 5, 6, 7, 8 and 9;

[0008] Each SNP locus was determined based on the chicken reference genome CRCg7b:

[0009] The locus 1 is rs317723503, located at position 86052625 on chicken chromosome 4.

[0010] The locus 2 is rs312777682, located at position 86052863 on chicken chromosome 4.

[0011] The locus 3 is novel_xs1, located at position 86053043 on chicken chromosome 4.

[0012] The locus 4 is rs313244787, located at position 86067938 on chicken chromosome 4.

[0013] The locus 5 is rs314033806, located at position 86067975 on chicken chromosome 4.

[0014] The locus 6 is rs314273100, located at position 86068042 on chicken chromosome 4.

[0015] The locus 7 is rs314057491, located at position 86068161 on chicken chromosome 4.

[0016] The locus 8 is rs315888209, located at position 86068370 on chicken chromosome 4.

[0017] The locus 9 is rs313352119, located at position 86069234 on chicken chromosome 4.

[0018] The nine specific SNP loci provided by this invention can each be used independently for the identification of Xiaoshan chicken, with an identification rate of over 92.5%. Identifying Xiaoshan chicken breeds based on one or more of these specific SNP loci yields high specificity and accuracy, and the identification results are stable and reliable.

[0019] The nine specific SNP loci provided by this invention are essentially fixed in the genomic DNA of Xiaoshan chickens and exhibit a single genotype in the Xiaoshan chicken breed. Specifically:

[0020] The Xiaoshan chicken has the genotype GG at locus 1;

[0021] The Xiaoshan chicken has the TT genotype at locus 2;

[0022] The Xiaoshan chicken has the AA genotype at locus 3;

[0023] The Xiaoshan chicken has the TT genotype at locus 4;

[0024] The Xiaoshan chicken has the AA genotype at locus 5;

[0025] The Xiaoshan chicken has the AA genotype at locus 6;

[0026] The Xiaoshan chicken has the TT genotype at locus 7;

[0027] The Xiaoshan chicken has the CC genotype at locus 8;

[0028] The Xiaoshan chicken has the TT genotype at locus 9.

[0029] When identifying Xiaoshan chickens, this invention preferably uses the specific genotypes mentioned above directly, which can omit whole-genome sequencing and SNP polymorphism analysis for the Xiaoshan chicken breed, thus simplifying the identification process.

[0030] In this invention, the specific SNP sites preferably include at least one of sites 4, 5, 6, 7, 8, and 9. These preferred SNP sites have higher specificity and accuracy, with an identification rate of 92.5% in Xiaoshan chicken samples; and when other chicken breeds (including Beijing You chicken, Dongxiang black chicken, Jiangshan white-feathered black-bone chicken, Luhua chicken, and Longyou Ma chicken, etc.) are used as test samples, the average identification rate does not exceed 6.0%.

[0031] More preferably, the specific SNP sites include at least one of sites 4, 5, 6, 7, and 8. Sites 4, 5, 6, 7, and 8 have extremely low identification rates (0.0%) in Dongxiang Black Chicken, Jiangshan White-feathered Silkie Chicken, and Longyou Ma Chicken.

[0032] In a preferred embodiment of the present invention, a specific SNP site combination can be used to identify Xiaoshan chicken. For example, the specific SNP site combination is:

[0033] Loci 4, loci 6, loci 7 and loci 8.

[0034] This invention can also simultaneously use nine specific SNP loci to identify Xiaoshan chicken. The combined use of these nine specific SNP loci enables higher accuracy and better specificity in the identification of a wider range of samples and more varieties.

[0035] The location information of each specific SNP locus and the results of genotype polymorphism analysis are shown in Table 1:

[0036] Table 1. Location information and genotype of specific SNP sites in Xiaoshan chickens

[0037]

[0038]

[0039] Furthermore, the primer information used for detecting the specific SNP sites mentioned above is shown in Table 2:

[0040] Table 2 Primers for amplifying specific SNP sites in Xiaoshan chicken

[0041]

[0042] The SNP loci provided in this invention are basically fixed in the Xiaoshan chicken breed, all of which are single genotypes and are reported for the first time. One or more of these loci can be used to identify the Xiaoshan chicken breed, with high accuracy and stable, reliable results. This is beneficial for the preservation, resource utilization, and development of the Xiaoshan chicken breed, providing important support for relevant breeding work and the formation of high-quality, high-yield Xiaoshan chicken breeding lines. It also has significant reference value for identifying the bloodlines of Xiaoshan chickens and for targeted breeding applications.

[0043] The detection method provided by this invention is simple to operate, highly efficient, and produces accurate and reliable results, making it suitable for widespread application.

[0044] Secondly, the present invention provides a Xiaoshan chicken-specific SNP molecular marker (or marker combination), including one or more of the following sites: site 1, site 2, site 3, site 4, site 5, site 6, site 7, site 8 and site 9;

[0045] Preferably, the SNP molecular marker (or marker combination) includes site 4, site 6, site 7 and site 8.

[0046] Thirdly, the present invention provides a specific detection primer for the specific SNP molecular marker of the Xiaoshan chicken;

[0047] Preferably, the specific detection primers for site 1 include XS_1F and XS_1R;

[0048] The specific detection primers for site 2 include XS_2F and XS_2R;

[0049] The specific detection primers for site 3 include XS_3F and XS_3R;

[0050] The specific detection primers for site 4 include XS_4F and XS_4R;

[0051] The specific detection primers for site 5 include XS_5F and XS_5R;

[0052] The specific detection primers for site 6 include XS_6F and XS_6R;

[0053] The specific detection primers for site 7 include XS_7F and XS_7R;

[0054] The specific detection primers for site 8 include XS_8F and XS_8R;

[0055] The specific detection primers for site 9 include XS_9F and XS_9R;

[0056] The nucleotide sequences of XS_1F, XS_1R, XS_2F, XS_2R, XS_3F, XS_3R, XS_4F, XS_4R, XS_5F, XS_5R, XS_6F, XS_6R, XS_7F, XS_7R, XS_8F, XS_8R, XS_9F, and XS_9R are shown in SEQ ID No. 1-18, respectively.

[0057] Fourthly, the present invention provides a detection kit, the detection kit comprising the specific detection primers.

[0058] Preferably, the detection kit further includes genomic DNA extraction reagent, PCR reaction mix and double-distilled water.

[0059] Fifthly, the present invention provides the application of the Xiaoshan chicken-specific SNP molecular marker, the specific detection primer, and the detection kit in chicken breed identification.

[0060] The application scheme provided by this invention has important reference value for identifying the bloodline of Xiaoshan chickens and for targeted breeding applications.

[0061] Beneficial effects:

[0062] The method provided by this invention uses specific SNP loci to identify the Xiaoshan chicken breed, obtaining reliable and accurate detection results. This invention provides nine selectable specific SNP loci, which are basically fixed in the Xiaoshan chicken breed and all have a single genotype. Using one or more of these loci can identify the Xiaoshan chicken breed, which is beneficial for the preservation, resource utilization, and development of the Xiaoshan chicken, providing an important guarantee for the formation of high-quality, high-yield Xiaoshan chicken breeding lines. Furthermore, the method provided by this invention is simple to operate, highly efficient, and provides accurate and reliable identification results, making it suitable for widespread application. Detailed Implementation

[0063] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are all conventional methods; the materials and reagents used are all commercially available.

[0064] Example 1

[0065] In this embodiment, specific SNP sites were amplified and genotype frequency analysis was performed on Silkie Xiaoshan Chicken, Xianju Chicken, Xiaoxiang Chicken, Yandang Ma Chicken, Songyang Golden Pheasant, Wenling Grass Chicken, White-eared Yellow Chicken, and Silkie Black-boned Chicken to identify specific SNP molecular markers for Xiaoshan Chicken. The SNP molecular markers are shown in Table 3.

[0066] Table 3. Annotation information of specific SNP sites in Xiaoshan chickens

[0067]

[0068]

[0069] Example 2

[0070] This embodiment provides a method for identifying Xiaoshan chickens, which uses specific SNP loci for identification. When the genotype corresponding to Xiaoshan chicken is detected at one or more of the following specific SNP loci, the chicken breed can be identified as Xiaoshan chicken.

[0071] The specific SNP site information is as follows:

[0072] rs317723503 is located at 86052625 on chromosome 4 of chicken, downstream of gene ENSGALG00010019007, and the Xiaoshan chicken genotype is GG.

[0073] rs312777682 is located at 86052863 on chromosome 4 of chicken, downstream of gene ENSGALG00010019007, and the Xiaoshan chicken genotype is TT.

[0074] novel_xs1 is located at 86053043 on chromosome 4 of chicken, downstream of gene ENSGALG00010019007, and the Xiaoshan chicken genotype is AA.

[0075] rs313244787 is located at 86067938 on chromosome 4 of chicken, between the gene ENSGALG00010019007 and the gene ENSGALG00010018722. The Xiaoshan chicken genotype is TT.

[0076] rs314033806 is located at 86067975 on chromosome 4 of chicken, between the gene ENSGALG00010019007 and the gene ENSGALG00010018722. The Xiaoshan chicken genotype is AA.

[0077] rs314273100 is located at 86068042 on chromosome 4 of chicken, between the gene ENSGALG00010019007 and the gene ENSGALG00010018722. The Xiaoshan chicken genotype is AA.

[0078] rs314057491 is located at 86068161 on chromosome 4 of chicken, between the gene ENSGALG00010019007 and the gene ENSGALG00010018722. The Xiaoshan chicken genotype is TT.

[0079] rs315888209 is located at 86068370 on chromosome 4 of chicken, between the gene ENSGALG00010019007 and the gene ENSGALG00010018722. The Xiaoshan chicken genotype is CC.

[0080] rs313352119 is located at 86069234 on chromosome 4 of chicken, between the gene ENSGALG00010019007 and the gene ENSGALG00010018722. The Xiaoshan chicken genotype is TT.

[0081] Example 3

[0082] This embodiment provides a primer combination for identifying Xiaoshan chicken, including the following primer pairs for detecting specific SNP molecular markers:

[0083] The primers used to detect the marker rs317723503 are the two DNA sequences shown in XS_1F and XS_1R;

[0084] The primers used to detect the marker rs312777682 are the two DNA sequences shown in XS_2F and XS_2R;

[0085] The primers used to detect the marker novel_xs1 are the two DNA sequences shown in XS_3F and XS_3R;

[0086] The primers used to detect the marker rs313244787 are the two DNA sequences shown in XS_4F and XS_4R;

[0087] The primers used to detect the marker rs314033806 are the two DNA sequences shown in XS_5F and XS_5R;

[0088] The primers used to detect the marker rs314273100 are the two DNA sequences shown in XS_6F and XS_6R;

[0089] The primers used to detect the marker rs314057491 are the two DNA sequences shown in XS_7F and XS_7R;

[0090] The primers used to detect the marker rs315888209 are the two DNA sequences shown in XS_8F and XS_8R;

[0091] The primers used to detect the marker rs313352119 are the two DNA sequences shown in XS_9F and XS_9R.

[0092] The nucleotide sequences of XS_1F, XS_1R, XS_2F, XS_2R, XS_3F, XS_3R, XS_4F, XS_4R, XS_5F, XS_5R, XS_6F, XS_6R, XS_7F, XS_7R, XS_8F, XS_8R, XS_9F, and XS_9R are shown in Table 2 above.

[0093] This embodiment also provides a detection kit for identifying Xiaoshan chicken, the composition of which is shown in Table 4:

[0094] Table 4. Composition of the test kit for identifying Xiaoshan chicken

[0095]

[0096]

[0097] Experimental Example 1

[0098] This experimental example uses the nine specific SNP loci described in Example 1 to identify the chicken flock to be tested. Details are as follows:

[0099] Twenty blood samples from Xiaoshan chickens and 193 blood samples from non-Xiaoshan chickens were collected for identification. The non-Xiaoshan chicken blood samples came from Beijing Oil Chicken (n=20), Dongxiang Black Chicken (n=20), Jiangshan White-feathered Silkie Chicken (n=19), Luhua Chicken (n=20), Longyou Ma Chicken (n=10), White-eared Yellow Chicken (n=10), Huiyang Bearded Chicken (n=8), Sanhuang Chicken (n=6), Wenling Grass Chicken (n=20), Xianghuang Chicken (n=10), Xianju Chicken (n=14), Xiaoxiang Chicken (n=19), Yandang Ma Chicken (n=10), and Wenchang Chicken (n=7). SNP typing was performed using the primers and kits described in Example 3.

[0100] The PCR reaction system is calculated in 25 μl increments:

[0101]

[0102] The PCR reaction conditions were: 95℃ for 10 min, 95℃ for 30 s, 60℃ for 30 s, 72℃ for 50 s, for a total of 30 cycles; 72℃ for 5 min.

[0103] Based on the sequencing results, the genotypes of nine SNP loci were determined. The identification rates of different loci for Xiaoshan chickens are shown in Table 5.

[0104]

[0105] As shown in Table 5, the identification rate of each specific SNP site described in Example 1 for Xiaoshan chicken is above 92.5%. Therefore, the combination of the 9 sites provided by this invention for Xiaoshan chicken breed identification has high specificity and accuracy.

[0106] Experiment Example 2

[0107] This experimental example utilizes the nine specific SNP loci described in Example 1 to form two specific SNP combinations: Combination 1: all nine SNP loci; Combination 2: four SNP loci: rs313244787, rs314273100, rs314057491, and rs315888209. The population to be tested was then identified as follows:

[0108] Twenty blood samples from Xiaoshan chickens and 193 blood samples from non-Xiaoshan chickens were collected for identification. The non-Xiaoshan chicken blood samples came from Beijing Oil Chicken (n=20), Dongxiang Black Chicken (n=20), Jiangshan White-feathered Silkie Chicken (n=19), Luhua Chicken (n=20), Longyou Ma Chicken (n=10), White-eared Yellow Chicken (n=10), Huiyang Bearded Chicken (n=8), Sanhuang Chicken (n=6), Wenling Grass Chicken (n=20), Xianghuang Chicken (n=10), Xianju Chicken (n=14), Xiaoxiang Chicken (n=19), Yandang Ma Chicken (n=10), and Wenchang Chicken (n=7). SNP typing was performed using the primers and kits described in Example 3.

[0109] The PCR reaction system is calculated in 25 μl increments:

[0110]

[0111] The PCR reaction conditions were: 95℃ for 10 min, 95℃ for 30 s, 60℃ for 30 s, 72℃ for 50 s, for a total of 30 cycles; 72℃ for 5 min.

[0112] Based on the sequencing results, the frequencies of two specific combinations were statistically analyzed. The identification rates of different loci for Xiaoshan chicken are shown in Table 6.

[0113]

[0114] The embodiments described above are merely illustrative of several implementations of the present invention, designed to facilitate a detailed understanding of the technical solutions of the present invention. However, they should not be construed as limiting the scope of patent protection. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for identifying Xiaoshan chicken, characterized in that, Identification was performed using specific SNP sites, which consist of sites 1, 2, 3, 4, 5, 6, 7, 8, and 9. The specific SNP sites were determined based on the chicken reference genome CRCg7b. The locus 1 is rs317723503, located at position 86052625 on chicken chromosome 4. The locus 2 is rs312777682, located at position 86052863 on chicken chromosome 4. The locus 3 is novel_xs1, located at position 86053043 on chicken chromosome 4. The locus 4 is rs313244787, located at position 86067938 on chicken chromosome 4. The locus 5 is rs314033806, located at position 86067975 on chicken chromosome 4. The locus 6 is rs314273100, located at position 86068042 on chicken chromosome 4. The locus 7 is rs314057491, located at position 86068161 on chicken chromosome 4. The locus 8 is rs315888209, located at position 86068370 on chicken chromosome 4. The locus 9 is rs313352119, located at position 86069234 on chicken chromosome 4. Among them, the Xiaoshan chicken has the genotype GG at locus 1; The Xiaoshan chicken has the TT genotype at locus 2; The Xiaoshan chicken has the AA genotype at locus 3; The Xiaoshan chicken has the TT genotype at locus 4; The Xiaoshan chicken has the AA genotype at locus 5; The Xiaoshan chicken has the AA genotype at locus 6; The Xiaoshan chicken has the TT genotype at locus 7; The Xiaoshan chicken has the CC genotype at locus 8; The Xiaoshan chicken has the TT genotype at locus 9.

2. The application of the specific detection primers for the SNP site described in claim 1 in the preparation of a Xiaoshan chicken detection kit, characterized in that: The specific detection primers for site 1 are XS_1F and XS_1R; The specific detection primers for site 2 are XS_2F and XS_2R; The specific detection primers for site 3 are XS_3F and XS_3R; The specific detection primers for site 4 are XS_4F and XS_4R; The specific detection primers for site 5 are XS_5F and XS_5R; The specific detection primers for site 6 are XS_6F and XS_6R; The specific detection primers for site 7 are XS_7F and XS_7R; The specific detection primers for site 8 are XS_8F and XS_8R; The specific detection primers for site 9 are XS_9F and XS_9R; The nucleotide sequences of XS_1F, XS_1R, XS_2F, XS_2R, XS_3F, XS_3R, XS_4F, XS_4R, XS_5F, XS_5R, XS_6F, XS_6R, XS_7F, XS_7R, XS_8F, XS_8R, XS_9F, and XS_9R are shown in SEQ ID No. 1-18, respectively.

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