Application of a primer for detecting SNP molecular markers associated with chicken skin color in identifying skin color and chicken genetic breeding.

Abstract

The present invention belongs to the technical field of poultry genetic engineering, and specifically relates to an application of a detection primer of a SNP molecular marker related to chicken skin color in identifying skin color and chicken genetic breeding. The site of the SNP molecular marker is the 1571th site of the CRP gene, which is C or T; the CRP gene sequence is shown in SEQ ID NO: 1. Based on the SNP molecular marker and the primer pair for detecting the marker, the genotype of the trait of the skin color of the target chicken group can be determined by detecting the C / T mutation site therein, and the skin color of an individual can also be determined by the genotype, so as to provide a basis for purposefully selecting the skin color trait of the chicken group and improve the consistency of the skin color of the offspring chicken group.

Description

[0001] This application is a subsequent patent application that takes the patent application with application number 202311058315.7 and application date 2023.08.22 as priority. Technical Field

[0002] The invention belongs to the technical field of poultry gene engineering, and in particular relates to an application of a detection primer of a SNP molecular marker associated with chicken skin color in identifying skin color and chicken genetic breeding. Background Art

[0003] Skin color is an important economic trait of chickens. The skin color of chickens is mainly divided into three colors: white, yellow and black. Black-bone chickens are valuable poultry breed resources in my country, and black skin is one of the important characteristics of black-bone chickens. Black-bone chickens are rich in beneficial ingredients such as protein, amino acids, minerals, black chicken peptides and trace elements. They have rich nutritional value and medicinal value and are deeply favored by consumers. The depth of skin color of black-bone chickens is related to the degree of melanin deposition in the skin tissue, and the level of melanin directly affects its medicinal value; compared with light skin, black-bone chickens with dark skin have better nutritional value and medicinal value.

[0004] In actual breeding and production applications, natural pigments can be used to meet people's requirements for chicken skin color, such as adding melanin to feed black-bone chickens to increase the skin depth of black-bone chickens. However, adding colorants has no nutritional value for broiler production, and there are safety risks. The use of colorants will also increase breeding costs. Therefore, in the long run, breeding and selection can be used to improve the consistency of the skin color of offspring chickens, reduce breeding costs and avoid the risks brought by colorants. Therefore, establishing a method for quickly identifying chicken skin color-related genotypes is important for high-quality chicken breeding.

[0005] Single nucleotide polymorphism (SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. As a third-generation molecular marker, SNP has the characteristics of large number, wide distribution, strong representativeness and good genetic stability. At present, it has been widely used in the genetic diversity analysis, gene mapping, molecular marker-assisted breeding and functional genetics research of animals and plants. Therefore, studying molecular markers related to chicken skin color and applying them in molecular breeding can achieve early selection of chicken skin color, especially black-bone chickens, which can save production costs and accelerate genetic progress.

[0006] CRP (C-reactive protein) is a common inflammatory marker that is involved in regulating the production of inflammatory factors, such as interleukin-6 and tumor necrosis factor (TNFα). Inflammatory factors also play a key role in the synthesis of melanin. CRP may be a key gene that plays an important role in melanin synthesis and immune regulation. Studies have found that CRP and OCA2 are significantly differentially expressed in chicken tibia tissue at three age groups (1 day, 10 days, and 20 days) through differential expression gene analysis and functional analysis. At the same time, the downstream gene of CRP in the PPI network analysis is the OCA2 gene, indicating that CRP may be involved in the regulation of melanin production through OCA2. There are few reports on the effect of the CRP gene on chicken skin color, and there are no reports on SNP molecular markers related to chicken skin color traits in the prior art. Summary of the invention

[0007] In view of the above problems, the present invention aims to provide a detection primer of SNP molecular markers related to chicken skin color for use in identifying skin color and chicken genetic breeding. The present invention can accurately identify the genotype of the skin color trait of a target chicken flock, and can also discriminate the skin color of an individual by genotype.

[0008] The present invention provides the following technical solutions:

[0009] On the one hand, the present invention provides a detection primer for a SNP molecular marker associated with chicken skin color for use in identifying skin color and chicken genetic breeding, wherein the SNP site is site 1571 of a CRP gene, which is C or T; the CRP gene sequence is as shown in SEQ ID NO:1; the detection primer comprises a detection primer pair P1 and a detection primer pair P2, wherein the detection primer pair P1 comprises an upstream primer as shown in SEQ ID NO:2 and a downstream primer as shown in SEQ ID NO:3; the detection primer pair P2 comprises an upstream primer as shown in SEQ ID NO:4 and a downstream primer as shown in SEQ ID NO:5; the detection primer pair P1 is used for determining the SNP site, and the detection primer pair P2 is used for determining the genotype of the SNP site.

[0010] On the other hand, the present invention provides a detection kit for SNP molecular markers related to chicken skin color for use in identifying skin color and chicken genetic breeding, wherein the SNP site is site 1571 of the CRP gene, which is C or T; the CRP gene sequence is as shown in SEQ ID NO:1; the detection kit comprises a detection primer pair P1 and a detection primer pair P2; the detection primer pair P1 comprises an upstream primer as shown in SEQ ID NO:2 and a downstream primer as shown in SEQ ID NO:3; the detection primer pair P2 comprises an upstream primer as shown in SEQ ID NO:4 and a downstream primer as shown in SEQ ID NO:5; the detection primer pair P1 is used to determine the SNP site, and the detection primer pair P2 is an enzyme cutting primer, which is used to determine the genotype of the SNP site.

[0011] Furthermore, the above application includes the following steps: (1) using the detection primer pair P1 to perform mixed pool amplification of the chicken genomic DNA sample to be tested, and obtaining the PCR amplification product corresponding to the P1 detection primer pair; (2) sequencing the PCR amplification product corresponding to the obtained P1 detection primer pair, and obtaining the SNP molecular marker site through analysis; (3) using the detection primer pair P2 to amplify the genomic DNA of the chicken to be tested, and obtaining the PCR amplification product corresponding to the P2 detection primer pair; (4) using the endonuclease BlpI to digest the PCR product corresponding to the obtained P2 detection primer pair, and obtaining the genotype of the SNP molecular marker; (5) counting the genotypes of the individuals to be tested, and performing correlation analysis between different genotypes and chicken skin color traits, so as to obtain the favorable genotype of the SNP molecular marker.

[0012] The beneficial effects of the present invention include at least: based on the SNP molecular markers related to chicken skin color provided by the present invention and the primer pairs for detecting the markers, the genotype of the skin color trait of the target chicken flock can be determined by detecting the C / T mutation sites therein, and the skin color of an individual can also be distinguished by the genotype, providing a basis for purposefully selecting the skin color trait of the chicken flock and improving the consistency of the skin color of the offspring chicken flock. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the SNP sequencing peak diagram of the C1104T site (CT) in the CDS region of the chicken CRP gene;

[0014] Figure 2 This is the SNP sequencing peak diagram of the C1104T site (CC) in the CDS region of the chicken CRP gene;

[0015] Figure 3 This is the SNP sequencing peak diagram of the C1104T site (TT) in the CDS region of the chicken CRP gene;

[0016] Figure 4This is the electrophoresis diagram of PCR-RFLP of chicken CRP gene with BlpI endonuclease. DETAILED DESCRIPTION

[0017] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0018] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0019] The embodiment of the present invention provides a detection primer for a SNP molecular marker related to chicken skin color for use in identifying skin color and chicken genetic breeding, wherein the SNP site is site 1571 of the CRP gene, which is C or T; the CRP gene sequence is as shown in SEQ ID NO:1; the detection primers include a detection primer pair P1 and a detection primer pair P2, the detection primer pair P1 includes an upstream primer shown in SEQ ID NO:2 and a downstream primer shown in SEQ ID NO:3; the detection primer pair P2 includes an upstream primer shown in SEQ ID NO:4 and a downstream primer shown in SEQ ID NO:5; the detection primer pair P1 is used to determine the SNP site, and the detection primer pair P2 is used to determine the genotype of the SNP site.

[0020] Specifically, the sequence shown in SEQ ID NO: 1 is the GenBank accession number: KC963999.1 sequence; the SNP site is also the 1104th position in the CDS region of the chicken CRP gene.

[0021] Another embodiment of the present invention provides a detection kit for SNP molecular markers related to chicken skin color for use in identifying skin color and chicken genetic breeding, wherein the SNP site is site 1571 of the CRP gene, which is C or T; the CRP gene sequence is as shown in SEQ ID NO:1; the detection kit comprises a detection primer pair P1 and a detection primer pair P2; the detection primer pair P1 comprises an upstream primer as shown in SEQ ID NO:2 and a downstream primer as shown in SEQ ID NO:3; the detection primer pair P2 comprises an upstream primer as shown in SEQ ID NO:4 and a downstream primer as shown in SEQ ID NO:5; the detection primer pair P1 is used to determine the SNP site, and the detection primer pair P2 is an enzyme cutting primer, which is used to determine the genotype of the SNP site.

[0022] Specifically, the above detection kit includes, in addition to the above primers P1 and P2, Taq DNA polymerase, 10× PCR buffer (containing Mg 2+ )、dNTPs、ultrapure water (ddH 2 O), DNA Marker I, DNA Marker 2000, BlpⅠ endonuclease, 10× buffer, agarose. In addition, as mentioned above, the sequence shown in SEQ ID NO: 1 is the GenBank accession number: KC963999.1 sequence; the SNP site is also the 1104th position in the CDS region of the chicken CRP gene.

[0023] In some specific examples, the above application includes the following steps: (1) using the detection primer pair P1 to amplify the mixed pool of the chicken genomic DNA sample to be tested, and obtaining the PCR amplification product corresponding to the P1 detection primer pair; (2) sequencing the PCR amplification product corresponding to the obtained P1 detection primer pair, and obtaining the SNP molecular marker site through analysis; (3) using the detection primer pair P2 to amplify the genomic DNA of the chicken to be tested, and obtaining the PCR amplification product corresponding to the P2 detection primer pair; (4) using the endonuclease BlpI to digest the PCR product corresponding to the obtained P2 detection primer pair, and obtaining the genotype of the SNP molecular marker; (5) counting the genotypes of the individual chickens to be tested, and performing correlation analysis between different genotypes and chicken skin color traits, so as to obtain the favorable genotype of the SNP molecular marker. In the above step (1), the P1 forward primer sequence used for PCR amplification is shown in SEQ ID NO:2, and the reverse primer sequence is shown in SEQ ID NO:3; in step (2), the SNP molecular marker site is located at the 1104th site of the CDS region of the CRP gene, and the nucleotide of the site is C or T; in step (3), the P2 forward primer sequence used is shown in SEQ ID NO:4, and the reverse primer sequence is shown in SEQ ID NO:5; in step (4), the genotype is CC, CT or TT; in step (5), the trait is the chicken skin color trait.

[0024] Specifically, the SNP molecular marker site in the present invention is located at the 1571st site of the CRP gene (the CRP gene sequence is shown in SEQ ID NO: 1), and the nucleotide at the site is C or T, resulting in a single nucleotide polymorphism at the site. PCR amplification is performed using primers P1 and P2 to obtain a sequence containing a SNP molecular marker associated with the chicken skin color trait (as shown in SEQ ID NO: 6 and SEQ ID NO: 7), and the SNP site is located at the 1318th position of the sequence shown in SEQ ID NO: 6 and at the 26th position of the sequence shown in SEQ ID NO: 7.

[0025] Another embodiment of the present invention provides a method for detecting the SNP molecular marker associated with the chicken skin color trait, the method comprising the following steps:

[0026] (1) Use primer pair P1 to amplify the chicken genomic DNA sample pool to obtain a PCR amplification product.

[0027] (2) After sequencing the obtained PCR amplification product, the SNP molecular marker site is obtained through analysis.

[0028] (3) Furthermore, the upstream and downstream primer sequences used for sequencing the PCR product are shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0029] (4) Amplify the genomic DNA of the chicken to be tested using primer pair P2 to obtain a PCR amplification product.

[0030] (5) The obtained PCR product is digested with endonuclease BlpI to obtain the genotype of the SNP molecular marker.

[0031] (6) Furthermore, the upstream and downstream primer sequences used for restriction digestion of the PCR product are shown in SEQ ID NO: 4 and SEQ ID NO: 5.

[0032] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0033] (1) Experimental materials and genomic DNA extraction

[0034] The chicken population to be tested was from the high-quality broiler S2 line paternal line bred by the Jiangsu Poultry Science Research Institute and the Silky-bone chicken maternal line for skin color test cross, and a resource family F2 generation population of 224 chickens was established, which was raised and managed by a dedicated person and immunized according to the procedure. Blood from the wing veins of all test individuals was collected, added to 70% ethanol, and genomic DNA was extracted by phenol / chloroform method and stored at -20℃.

[0035] (2) Primer P1 design and synthesis

[0036] According to the DNA sequence of the chicken CRP gene published on GenBank (GenBank accession number: KC963999.1, sequence see SEQ ID NO: 1), primer pair P1 was designed using Primer5.0 software. The primer pair P1 amplified fragments included the CDS region of the CRP gene. The primer P1 sequence, target fragment length, amplification region and annealing temperature are shown in Table 1. The primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0037] Table 1 Primer sequences, target fragment length, amplification region and annealing temperature for PCR amplification and restriction digestion of CRP gene

[0038]

[0039] Note: The amplified region in the table refers to the location in GenBank accession number: KC963999.1

[0040] The PCR amplification system of primer P1 is 25 μL, including 10× PCR buffer (containing Mg 2+) 2.5 μL, 1.0 μL each of 10 μmol / L upstream and downstream primers (see Table 1), 2.0 μL of 2 mmol / L dNTPs, 0.5 μL of 5.0 U / μL Taq DNA polymerase, 1.0 μL of 50 ng / μL DNA template, add ultrapure water to 25 μL; PCR amplification conditions: 94°C pre-denaturation for 5 min; then denaturation at 94°C for 30 s, renaturation for 15 s (annealing temperature is 55°C as shown in Table 1), extension at 72°C for 30 s, 35 cycles; finally extension at 72°C for 10 min, and storage at 4°C; the amplified product was detected by 1.5% agarose gel electrophoresis, and the PCR product was 1600 bp.

[0041] (4) PCR product sequencing and SNPs site determination

[0042] The PCR amplification product was obtained by amplification using primer P1 according to the above PCR amplification system and amplification method. The PCR amplification product was sequenced by Shanghai Yingjun Biotechnology Co., Ltd. using ABI3730 sequencer. The sequencing results were analyzed using DNAstar and Primer5.0 software to determine the SNPs sites and restriction sites. The results are as follows Figure 1 , Figure 2 and Figure 3 As shown, the results showed that there was a C→T mutation at position 1104 in the coding region (CDS region).

[0043] (5) Design and synthesis of restriction endonuclease primer P2

[0044] According to the SNP site determined by sequencing with primer P1 (there is a C→T mutation at position 1104 in the CDS region), Prime5 software analysis showed that there was no inherent restriction site at the mutation site, so a BlpI site was created by designing primer P2. The restriction site of BlpI is "5'GC ∧ TNAGC3'", when the C1104T site in the CDS region is a C base, the endonuclease BlpI can cut the DNA amplified fragment, and when the C1086T site in the CDS region is a T base, the endonuclease BlpI cannot cut the DNA amplified fragment. The genotype of the fragment at this site can be identified by the above principle. The primer P2 sequence, target fragment length, amplification region and annealing temperature are shown in Table 1. The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0045] (6) PCR amplification with primer P2

[0046] The PCR amplification system for primer P2 was 25 μL, including 10× PCR buffer (containing Mg 2+) 2.5μL, 1.0μL each of 10μmol / L upstream and downstream primers (see Table 1), 2.0μL of 2mmol / L dNTPs, 0.5μL of 5.0U / μL Taq DNA polymerase, 1.0μL of 50ng / μL DNA template, add ultrapure water to 25μL; PCR amplification conditions: 94℃ pre-denaturation for 5min; then denaturation at 94℃ for 30s, renaturation for 15s (annealing temperature is 68℃ as shown in Table 1), extension at 72℃ for 30s, 35 cycles; finally extension at 72℃ for 10min, and storage at 4℃; the amplified product was detected by 1.5% agarose gel electrophoresis, and the PCR product was 113bp.

[0047] (7) Enzyme digestion, genotyping and chi-square test

[0048] The enzyme digestion reaction system was 20 μL: 7 μL of PCR amplification product, 0.5 μL of restriction enzyme, 2.0 μL of 10× buffer, and ultrapure water was added to 20 μL. The BlpⅠ endonuclease was used for overnight digestion at 37°C, and the result was detected by 3% agarose gel electrophoresis. TM 2200 and 1220 Doumentation and Analysis Systems (Alpha Innotech Corporation, San Leandro, CA, USA) were used for photography, analysis, and statistical genotypes.

[0049] The PCR amplification product of BlpⅠ digestion at site 1104 of CDS region is 113 bp (as shown in SEQ ID NO:7). Figure 4 ) and sequencing results showed that there were three genotypes: TT (113bp), CT (113bp / 91bp / 22bp) and TT (91bp / 22bp). Among them, the 22bp fragment was too small and ran out of the gel during electrophoresis, so it was not shown in the enzyme digestion map.

[0050] The designed BlpI was used to detect the individual genotypes of the F2 generation population, and the distribution of skin genes was statistically analyzed. The results of genotype frequency, gene frequency and chi-square independence test are shown in Table 2.

[0051] Table 2 Genotype frequency, gene frequency and chi-square test of C1104T locus in CDS region of chicken CRP gene

[0052]

[0053] From Table 2 above, we can know that the C allele is a high-quality gene in the white skin group, while the T allele is a high-quality gene in the black skin group. The χ2 independence test between population genotypes reached a significant level, and statistical analysis showed that the C1104T site in the CDS region of the CRP gene had a certain correlation with skin color.

[0054] (8) Determination of skin color index and analysis of its association with genotype

[0055] The L, a, and b values ​​of the chest skin color of 224 F1 generation chickens were measured using a Minolta CR400 colorimeter (Konica Minolta, Japan). The results are shown in Table 3 (in the table, L is the lightness value (0-100), 0 represents black, and 100 represents white; a is the red-green light value, a positive value indicates reddish, and a negative value indicates greenish; b is the yellow-blue light value, a positive value indicates yellowish, and a negative value indicates bluish).

[0056] Table 3 Correlation analysis between C1104T in the CDS region of chicken CRP gene and skin color index

[0057] genotype Number of individuals L value a value b-value CC 73 <![CDATA[75.13 b ±8.19]]> <![CDATA[1.16 a ±0.53]]> <![CDATA[-1.11 a ±0.05]]> CT 65 <![CDATA[68.92 b ±7.25]]> <![CDATA[1.04 a ±0.47]]> <![CDATA[-0.87 a ±0.08]]> TT 86 <![CDATA[54.78 a ±9.11]]> <![CDATA[1.45 a ±0.59]]> <![CDATA[-1.54 a ±0.14]]>

[0058] It can be seen from Table 3 above that the correlation analysis between the three genotypes and the skin color L, a, and b values ​​shows that the TT genotype is significantly correlated with the L value, and the L value of the TT genotype is the smallest, indicating that TT is associated with dark skin color.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. An application of a detection primer for a SNP molecular marker associated with chicken skin color in identifying skin color and chicken genetic breeding, characterized in that: The SNP site is the 1571th site of the CRP gene, which is C or T; the CRP gene sequence is shown in SEQ ID NO:1; the detection primers include a detection primer pair P1 and a detection primer pair P2, and the detection primer pair P1 includes the upstream primer shown in SEQ ID NO:2 and the downstream primer shown in SEQ ID NO:3; the detection primer pair P2 includes the upstream primer shown in SEQ ID NO:4 and the downstream primer shown in SEQ ID NO:5; the detection primer pair P1 is used to determine the SNP site, and the detection primer pair P2 is an enzyme cutting primer, which is used to determine the genotype of the SNP site, and the enzyme is the endonuclease BlpI enzyme; skin color identification is to distinguish gray, black or white; chicken genetic breeding is skin color breeding, and the skin color is gray, black or white; the C allele is a high-quality gene in the white skin color population, and the T allele is a high-quality gene in the black skin color population.

2. A detection kit for SNP molecular markers related to chicken skin color for use in identifying skin color and chicken genetic breeding, characterized in that: The SNP site is the 1571th site of the CRP gene, which is C or T; the CRP gene sequence is shown in SEQ ID NO: 1; the detection kit includes a detection primer pair P1 and a detection primer pair P2; the detection primer pair P1 includes an upstream primer shown in SEQ ID NO: 2 and a downstream primer shown in SEQ ID NO: 3; the detection primer pair P2 includes an upstream primer shown in SEQ ID NO: 4 and a downstream primer shown in SEQ ID NO: 5; the detection primer pair P1 is used to determine the SNP site, and the detection primer pair P2 is an enzyme cutting primer, which is used to determine the genotype of the SNP site, and the enzyme is the endonuclease BlpI enzyme; skin color identification is to identify gray, black or white; chicken genetic breeding is skin color breeding, and the skin color is gray, black or white; the C allele is a high-quality gene in the white skin color population, and the T allele is a high-quality gene in the black skin color population.

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