A SNP molecular marker related to immune traits of landes geese and application thereof

Abstract

The application discloses a SNP (Single Nucleotide Polymorphism) molecular marker related to an immune character of Landes geese and application thereof, and belongs to the technical field of molecular markers. CNTFR The SNP molecular marker is located in a nucleotide sequence shown in SEQ ID No. 1, and polymorphism exists in any one or more of positions 133334, 165805, 166710 and 174733, corresponding to G>A, C>T, G>A and A>G mutations respectively. The SNP molecular marker provided by the application can realize rapid and accurate identification of the level of the heterophil / lymphocyte ratio of the Landes geese, the method is simple in operation, the result is reliable, the method is suitable for molecular marker assisted breeding of the Landes geese, and the efficiency of breeding for selecting the immune performance is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically relating to a SNP molecular marker associated with the immune traits of Landes geese and its application in molecular-assisted breeding. Background Technology

[0002] Landes geese, a major breed for foie gras production in China, are known for their tender foie gras and unique flavor. However, intensive force-feeding can easily trigger stress responses, leading to decreased immune function and increased susceptibility to disease. The heterophile-to-lymphocyte ratio (H / L) is an important physiological indicator for evaluating stress status and overall immune function in poultry. Individuals with lower H / L ratios exhibit better environmental adaptability, production performance, and offspring disease resistance, indicating the significant application value of this indicator in breeding poultry strains with high immune performance. Single nucleotide polymorphisms (SNPs), as third-generation genetic markers, are widely present in the genomes of various species. SNPs located in gene coding or regulatory regions may directly affect protein coding, splicing processes, or expression regulation, leading to phenotypic differences. Therefore, they can serve as important molecular-assisted breeding markers, widely used in genetic resource evaluation and the breeding of important economic traits. However, there are currently no reports of a correlation between SNPs and the H / L value in Landes geese. Therefore, developing SNP molecular markers related to H / L is of great significance for breeding Landes goose strains with high immune performance. Summary of the Invention

[0003] To address the lack of molecular markers for the immune traits of Landes geese in existing technologies, the present invention aims to provide a SNP molecular marker related to the immune traits of Landes geese and its application.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] In a first aspect, the present invention provides SNP molecular loci associated with immune traits in Landes geese, wherein all SNP molecular loci are located on chromosome Z of the Landes goose. CNTFR The full-length gene sequence is significantly correlated with the H / L level of Landes geese, and can be used to efficiently identify Landes geese individuals with different immune performances, and provide a basis for molecular breeding of immune traits.

[0006] Secondly, the present invention provides a SNP molecular marker associated with the immune traits of Landes geese, the nucleotide sequence of which is shown in SEQ ID No. 1. CNTFR The full-length gene sequence, and the SNP molecular marker in CNTFRPolymorphism exists at any one or more of the following sites in the full-length gene sequence: G>A mutation at position 133334, C>T mutation at position 165805, G>A mutation at position 166710, and A>G mutation at position 174733. These molecular markers can be used to assess the H / L level and immune performance of Landes geese and provide a basis for molecular selection of immune traits.

[0007] Thirdly, the present invention provides a primer pair for detecting SNP molecular markers associated with immune traits in Landes geese, wherein the primer pair comprises any one or more of the following, capable of specifically amplifying nucleotide fragments containing the SNP molecular markers:

[0008] Nucleotide sequences such as primers C5-F1 and C5-R1 shown in SEQ ID No. 2 and SEQ ID No. 3;

[0009] Nucleotide sequences such as primers C1-F1 and C1-R1 shown in SEQ ID No. 4 and SEQ ID No. 5;

[0010] Nucleotide sequences such as primers C3-F1 and C3-R1 shown in SEQ ID No. 6 and SEQ ID No. 7;

[0011] The nucleotide sequences are as shown in primers C4-F1 and C4-R1, which are SEQ ID No. 8 and SEQ ID No. 9, respectively.

[0012] Fourthly, this invention provides an application of SNP molecular markers and primer pairs, namely, for identifying high / low levels of H / L in Landes goose serum, and for selecting Landes geese with superior immune performance.

[0013] Fifthly, this invention provides another application of SNP molecular markers and primer pairs, namely, for detecting and identifying the immune performance of Landes geese and screening for immune Landes geese.

[0014] Sixthly, the present invention provides a method for identifying high / low levels of heterophile granulocyte / lymphocyte ratio in Landes goose serum, the method comprising:

[0015] (1) Extract genomic DNA from the Landes goose to be tested;

[0016] (2) Using the extracted genomic DNA as a template, PCR amplification is performed using any one or more primer pairs of the nucleotide sequences shown in SEQ ID No. 2 and SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7, and SEQ ID No. 8 and SEQ ID No. 9 to obtain an amplified fragment containing the SNP molecular site;

[0017] (3) Sequencing the amplified fragment, and determining the H / L level and corresponding immune traits of the Landes goose by sequencing the sequence and peak diagram.

[0018] When the 133334th position of the sequencing sequence is G, the genotype at the locus is GG; when the 165805th position is C, the genotype at the locus is CC; when the 166710th position is A, the genotype at the locus is AA; when the 174733rd position is A, the genotype at the locus is AA; when any one or more of the above genotypes are present at the locus, the Landes goose to be tested is a high H / L individual with a low level of immunity.

[0019] When the 133334th position of the sequencing sequence is A, the genotype at the locus is AA; when the 165805th position is T, the genotype at the locus is TT; when the 166710th position is G, the genotype at the locus is GG; when the 174733rd position is G, the genotype at the locus is GG; when any one or more of the above genotypes are present at the locus, the Landes goose to be tested is a low H / L individual with a high level of immunity.

[0020] The present invention has the following beneficial effects:

[0021] This invention identifies four SNP loci (CNTFR-1, CNTFR-2, CNTFR-3, and CNTFR-4) on chromosome Z of Landes geese that are significantly associated with immune traits through population selection signal screening, and establishes a molecular marker detection system based on these loci. This marker can accurately and efficiently distinguish the H / L levels of individual Landes geese, and is characterized by its ease of operation and stable, reliable results, providing a scientific basis for molecular-assisted selection of immune traits in Landes geese. Using this marker, early and precise screening of individuals with high immune performance can be achieved in breeding. Purification of the breeding population at the target H / L level can be achieved with only one generation of testing and selection, significantly improving breeding efficiency and contributing to the development of new Landes goose strains with superior immune traits. Attached Figure Description

[0022] Figure 1 This is based on F provided in Embodiment 1 of the present invention. ST Figure showing the results of population selection signal analysis related to H / L in Landes geese;

[0023] Figure 2 This is a graph showing the analysis results of the population selection signal based on θπ H / L correlation of Landes geese provided in Embodiment 1 of the present invention;

[0024] Figure 3 This is a graph showing the analysis results of population selection signals based on XP-EHH for Landes geese H / L correlation, provided in Embodiment 1 of the present invention.

[0025] Figure 4 This is a forward sequencing map of the three genotypes at the CNTFR-1 locus provided in Example 2 of this invention;

[0026] Figure 5 This is a forward sequencing map of three genotypes at the CNTFR-2 locus provided in Example 2 of this invention;

[0027] Figure 6 This is a forward sequencing map of the three genotypes at the CNTFR-3 locus provided in Example 2 of this invention;

[0028] Figure 7 This is the forward sequencing diagram of the three genotypes at the CNTFR-4 locus provided in Example 2 of this invention;

[0029] Figure 8 This is a comparison chart of the relative expression levels of serum cytokine mRNA in Landes geese under different H / L levels during 28 days of force-feeding, provided in Example 4 of this invention. Detailed Implementation

[0030] In a first aspect, the present invention provides a SNP molecular locus associated with the immune traits of Landes geese, which is obtained through population selection signal analysis (F... ST The SNPs in this invention were obtained through screening using θπ and XP-EHH. The SNP molecular sites are located at the sites shown in SEQ ID No. 1. CNTFR One or more sites at positions 133334, 165805, 166710, and 174733 of the full-length gene sequence, wherein position 133334 contains a G>A mutation, position 165805 contains a C>T mutation, position 166710 contains a G>A mutation, and position 174733 contains an A>G mutation. For ease of description, the sites at positions 133334, 165805, 166710, and 174733 are designated as CNTFR-1, CNTFR-2, CNTFR-3, and CNTFR-4, respectively.

[0031] Secondly, the present invention provides a SNP molecular marker associated with the immune traits of Landes geese, the nucleotide sequence of which is shown in SEQ ID No. 1. CNTFRFull-length gene sequence. This SNP molecular marker is located in... CNTFR Polymorphism exists at any one or more of the following sites in the full-length gene sequence: G>A mutation at position 133334, C>T mutation at position 165805, G>A mutation at position 166710, and A>G mutation at position 174733. These molecular markers can be used to assess the H / L level and immune performance of Landes geese and provide a basis for molecular selection of immune traits.

[0032] Thirdly, the present invention provides a primer pair for detecting SNP molecular sites, the primer pair comprising any one or more of the following:

[0033] The nucleotide sequences are as shown in SEQ ID No. 2 and SEQ ID No. 3 for primers C5-F1 and C5-R1; wherein primer C5-F1 is 5'-CCCAACTCCCAGTGCTTCTCC-3' and primer C5-R1 is 5'-TCCTCTCCTCCCCTCCCTGAC-3'.

[0034] The nucleotide sequences are as shown in SEQ ID No. 4 and SEQ ID No. 5 for primers C1-F1 and C1-R1; wherein primer C1-F1 is 5'-TTGCCTTTCTCTTCTCGTGGAG-3' and primer C1-R1 is 5'-CCTGAGAGCACTGACGGAGATG-3'.

[0035] The nucleotide sequences are shown in SEQ ID No. 6 and SEQ ID No. 7 for primers C3-F1 and C3-R1; wherein primer C3-F1 is 5'-CTGGCTGGTGGAGAAATGTGT-3' and primer C3-R1 is 5'-GAGGGGAGGAAGGGGAGTT-3'.

[0036] The nucleotide sequences are as shown in SEQ ID No. 8 and SEQ ID No. 9 for primers C4-F1 and C4-R1; wherein primer C4-F1 is 5'-CCCCCTACACCCCTGTGCT-3' and primer C4-R1 is 5'-CCAGTGAGGAGGATGCCAGAG-3'.

[0037] In this invention, the DNA of Landes goose is amplified by PCR using any one or more of the above primer pairs to obtain DNA fragments containing corresponding SNP molecular marker sites. After sequencing, the genotype is determined based on the sequencing sequence and peak diagram.

[0038] Specifically, when the 133334th position of the sequencing sequence is G, the genotype of the CNTFR-1 locus is GG;

[0039] When the 133334th position of the sequencing sequence is A, the genotype of the CNTFR-1 locus is AA;

[0040] When both G and A are present at position 133334 of the sequencing sequence, the genotype at the CNTFR-1 locus is GA;

[0041] When position 165805 of the sequencing sequence is C, the genotype at the CNTFR-2 locus is CC;

[0042] When the 165805th position of the sequencing sequence is T, the genotype of the CNTFR-2 locus is TT;

[0043] When both C and T are present at position 165805 of the sequencing sequence, the genotype of the CNTFR-2 locus is CT.

[0044] When the 166710th position of the sequencing sequence is G, the genotype of the CNTFR-3 locus is GG;

[0045] When position 166710 of the sequencing sequence is A, the genotype at the CNTFR-3 locus is AA;

[0046] When both G and A are present at position 166710 of the sequencing sequence, the genotype at the CNTFR-3 locus is GA;

[0047] When the 174733rd position of the sequencing sequence is G, the genotype of the CNTFR-4 locus is GG;

[0048] When position 174733 of the sequencing sequence is A, the genotype at the CNTFR-4 locus is AA;

[0049] When both G and A are present at position 174733 of the sequencing sequence, the genotype at the CNTFR-4 locus is AG.

[0050] Fourthly, the present invention provides a kit related to immune traits in Landes geese, the kit comprising the primer pairs described in the third aspect. Additionally, the kit includes premixed solutions commonly used in the art. In the embodiments of the present invention, no particular limitation is made to the type of premixed solution, such as, but not limited to, Phusion® Ultra Fidelity PCR Premixed Solution, Platinum Direct PCR Universal Premixed Solution, etc.

[0051] Fifthly, the present invention provides an application of SNP molecular markers, primer pairs, or kits, specifically in the identification of high / low H / L levels in Landes goose serum.

[0052] Sixthly, the present invention provides an application of SNP molecular markers, primer pairs or kits for detecting and identifying the immune performance of Landes geese and screening for immune Landes geese.

[0053] In a seventh aspect, the present invention provides a method for identifying high / low levels of heterophile granulocyte / lymphocyte ratio in Landes goose serum, the method comprising:

[0054] (1) Extract genomic DNA from the Landes goose to be tested;

[0055] (2) Using the genomic DNA as a template, PCR amplification is performed using any one or more primer pairs of the nucleotide sequences shown in SEQ ID No. 2 and SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7, and SEQ ID No. 8 and SEQ ID No. 9 to obtain an amplified fragment containing SNP molecular sites;

[0056] (3) Sequencing the amplified fragment and determining the H / L level of the Landes goose to be tested by the sequencing sequence and peak diagram.

[0057] Specifically, determining the H / L level of the Landes goose being tested using sequencing sequences and peak diagrams involves:

[0058] When position 133334 is G, the genotype at CNTFR-1 is GG; when position 165805 is C, the genotype at CNTFR-2 is CC; when position 166710 is A, the genotype at CNTFR-3 is AA; when position 174733 is A, the genotype at CNTFR-4 is AA. When any one or more of the above genotypes are present, the corresponding Landes goose is a high H / L individual with a low level of immunity.

[0059] When position 133334 is A, the genotype at CNTFR-1 is AA; when position 165805 is T, the genotype at CNTFR-2 is TT; when position 166710 is G, the genotype at CNTFR-3 is GG; when position 174733 is G, the genotype at CNTFR-4 is GG. When any one or more of the above genotypes are present, the corresponding Landes goose is a low H / L individual with a high level of immunity.

[0060] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0061] Example 1 – Acquisition and Determination of SNP Molecular Sites and SNP Molecular Markers

[0062] 90-day-old Landes geese were selected, and 20 individuals were selected from each of the high and low H / L ratios. Blood samples were collected from the wing veins of each goose. Total genomic DNA was extracted using the traditional phenol-chloroform method. The DNA concentration and purity were measured, and the DNA stock solution was aliquoted and diluted to 50 ng / μL and stored at -20°C.

[0063] The quality of the preserved DNA stock solution was tested, and whole-genome resequencing was performed after passing the test. Subsequently, software such as Trinmomtic, BWA-mem, SAMtools, and GATK were used to filter, align, and detect variants in the resequencing data to obtain SNP data for each Landes goose individual. Further population selection signal analysis was performed on high and low H / L populations, including calculating the polymorphism level (θπ) and genetic differentiation coefficient (F) based on a 10 kb window and a 5 kb step size. ST The results of cross-population extended haplotype homozygosity (XP-EHH) are shown in the appendix. Figure 1-3 Subsequently, candidate genes and loci related to H / L in Landes geese were screened using methods such as principal component analysis, functional enrichment analysis, and haplotype analysis.

[0064] Based on the above analysis (screening criteria: weighted F), ST The highest 5% window, the highest and lowest 5% windows of log2 (θπ, high H / L / θπ, low H / L), and the windows containing sites with XP-EHH absolute values ​​≥2 were selected as candidate regions related to the H / L trait (the common windows from the three screenings were taken as candidate regions). A total of 1147 windows and 25257 sites were obtained, and 488 candidate genes were annotated. Principal component analysis and functional enrichment analysis revealed Z chromosome genes. CNTFR This gene, associated with H / L levels in Landes geese, encodes a member of the type 1 cytokine receptor family. The encoded protein is a ligand-specific subunit of the ciliary neurotrophic factor receptor, involved in regulating neuronal cell survival, differentiation, and gene expression.

[0065] Extracting Z chromosome genes CNTFR By comparing the genotype frequencies of all SNP loci in the study and contrasting high and low H / L populations, four SNP loci associated with H / L levels in Landes geese were identified and named CNTFR-1, CNTFR-2, CNTFR-3, and CNTFR-4. (See https: / / www.ncbi.nlm.nih.gov / )

[0066] Based on the reference genome shown in datasets / genome / GCA_040182565.1 / , the location information of the four SNP sites is shown in Table 1, and the forward sequencing is shown in the attached figure. Figure 4-7 .

[0067] Table 1: Location information of CNTFR-1, CNTFR-2, CNTFR-3 and CNTFR-4 loci

[0068]

[0069] Example 2 – Validation of SNP molecular sites and SNP molecular markers

[0070] Thirty Landes geese of different H / L ratios and high were selected at 90 days of age. Blood was collected from the wing vein of each goose, and total genomic DNA was extracted using the traditional phenol-chloroform method. The DNA concentration and purity were tested, and the DNA stock solution was aliquoted and diluted to 50 ng / μL and stored at -20°C.

[0071] Using the bcftools software with the reference genome shown at https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_040182565.1 / as a template, the following data was retrieved: CNTFR The full-length gene sequence was obtained, and the SNP molecular marker shown in SEQ ID No. 1 was acquired. Subsequently, PCR primers were designed near the four candidate SNPs using Oligo 6.0 software, and the primer pairs shown in Table 2 were obtained.

[0072] Using the DNA stock solution as a template, PCR amplification was performed using the primer pairs shown in Table 2 according to the PCR reaction system shown in Table 3 and the PCR reaction conditions shown in Table 4 to obtain the amplification products.

[0073] Table 2: Primer Pair Information

[0074]

[0075] Table 3: PCR reaction system

[0076]

[0077] Table 4: PCR reaction conditions

[0078]

[0079] The amplified products were sent for sequencing, and the genotypes of each individual were obtained using ChromasPro software based on the sequencing results. The genotype frequency distribution of each population was analyzed, and Table 5 was obtained.

[0080] Table 5: Genotype frequency distribution of CNTFR-1, CNTFR-2, CNTFR-3 and CNTFR-4 loci

[0081]

[0082] As shown in Table 5, the four SNP loci exhibited significant differences in genotypic distribution across different H / L populations. At the CNTFR-1 locus, the GG genotype was predominant in individuals with high H / L (73.33%), followed by the GA genotype (26.67%), with no AA genotype detected. In contrast, the AA genotype was predominant in individuals with low H / L (70%), with the GA genotype accounting for 30%, and no GG genotype detected. At the CNTFR-2 locus, the CC genotype was predominant in individuals with high H / L (73.33%), followed by the CT genotype (26.67%), with no TT genotype detected. In contrast, the TT genotype was predominant in individuals with low H / L (73.33%), with the CT genotype accounting for 26.67%, and no CC genotype detected. At the CNTFR-3 locus, the AA genotype was predominant in individuals with high H / L (73.33%), followed by the GA genotype (26.67%). No individuals with the GG genotype were detected. In contrast, the GG genotype was predominant in individuals with low H / L (73.33%), with the GA genotype accounting for 26.67%. No individuals with the AA genotype were detected. At the CNTFR-4 locus, the AA genotype was predominant in individuals with high H / L (73.33%), followed by the AG genotype (26.67%). No individuals with the GG genotype were detected. In contrast, the GG genotype was predominant in individuals with low H / L (70%), with the AG genotype accounting for 30%. No individuals with the AA genotype were detected.

[0083] Based on the above four SNP molecular sites, this embodiment of the invention provides an SNP molecular marker, which is the nucleotide sequence shown in SEQ ID No.1, and has polymorphism at any one or more of the following sites in the nucleotide sequence: G>A mutation at position 133334, C>T mutation at position 165805, G>A mutation at position 166710, and A>G mutation at position 174733.

[0084] Example 3

[0085] This invention provides a kit comprising the primer pairs shown in Table 2, and other commonly used premixes in the art. In this invention, no special limitation is placed on the type of premix; any PCR amplification premix well-known in the art is acceptable, such as Phusion® Ultra Fidelity PCR Premix, Platinum Direct PCR Universal Premix, etc.

[0086] Example 4 - Detection of individual immune traits with high and low H / L ratios

[0087] Ninety 90-day-old Landes geese with high, medium, and low H / L ratios were selected for force-feeding. Before force-feeding, all Landes geese underwent anal swab testing to detect avian influenza and Newcastle disease antigens. All Landes geese tested negative. Blood was collected from the wing vein of each Landes goose, placed on a glass slide, and a blood smear was prepared. 1 mL of Wright-Gymsa A solution was dropped onto the dried blood smear, covering the entire blood film, and fixed for 1 min. Then, 1 mL of PBS solution was added, mixed with dye, and stained for 1 min. The Wright-Gymsa A solution was rinsed off with water, and after natural drying, the smears were examined under a microscope. The numbers of heterophile granulocytes, lymphocytes, and monocytes were recorded, with the total number of these three cell types controlled to be around 100. The H / L ratio was calculated based on the statistical results, as shown in Table 6.

[0088] Table 6: H / L value distribution

[0089]

[0090] Five Landes geese with different H / L levels were randomly selected on day 28 of the force-feeding period. Blood samples were collected from the wing veins of each Landes goose. Total RNA was extracted from the whole blood using the TriZol method. The RNA concentration and purity were determined. The RNA stock solution was aliquoted and diluted to 500 ng / μL and stored at -20℃.

[0091] Using the extracted RNA as a template, reverse transcription was performed according to the reverse transcription reaction system shown in Table 7 and the reverse transcription reaction conditions shown in Table 8. Subsequently, the obtained cDNA was analyzed using a quantitative real-time PCR analyzer with the primer pairs shown in Table 9 and the qPCR reaction system shown in Table 10 and the qPCR reaction conditions shown in Table 11. IgA , IgG , IgM , IL-2, IFN-γ , TNF-β and β-actin Perform qPCR amplification.

[0092] Table 7: Reverse Transcription Reaction System

[0093]

[0094] Table 8: Conditions for Reverse Transcription Reaction

[0095]

[0096] Table 9: Primer Pair Information

[0097]

[0098] Table 10: qPCR reaction system

[0099]

[0100] Table 11: qPCR reaction conditions

[0101]

[0102] The CT values ​​obtained from the machine detection were used to 2 -ΔΔCT The relative expression level was calculated using the method described above, and graphs were created using Graphpad software to obtain the results. Figure 8 .

[0103] In addition, Landes geese with different H / L levels were randomly selected during the 28-day force-feeding period (see Table 6 for details) to detect antibody titers for avian influenza H5 and Newcastle disease. Simultaneously, after slaughter, the carcass weight, bursa of Fabricius, spleen, and thymus were weighed, and the immune organ index was calculated, as shown in Table 12. The antibody titer values ​​are expressed as the logarithm of log2; the immune organ index is equal to organ weight (g) / live body weight (g).

[0104] Table 12: Data on organ weight, immune organ index, and antibody titer at different force-feeding periods

[0105]

[0106] Note: Different lowercase letters in Table 12 indicate significance levels. P <0.05.

[0107] From the appendix Figure 8 It is evident that for individuals with low H / L levels after a force-feeding period of 28 days, their IgG, IgM, IL-2 and IFN-γ Expression was significantly higher than in individuals with high H / L ( P <0.05); As shown in Table 7, the Newcastle disease antibody titer, spleen weight, and spleen index of Landes geese with high H / L levels were significantly lower than those with low H / L levels at 28 days of force-feeding. P <0.05). The above results indicate that individuals with low H / L levels exhibit higher antibody levels, greater development of immune organs, and higher cytokine expression, suggesting that they may have a stronger overall immune response.

[0108] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Use of SNP molecular markers to identify high / low levels of heterophil / lymphocyte ratio in serum of Landes geese, characterized in that, The nucleotide sequence of the SNP molecular marker is located in the full-length sequence of the gene shown in SEQ ID No. 1, and there is a polymorphism at any one or more of positions 133334, 165805, 166710 and 174733, wherein there is a G>A mutation at position 133334, a C>T mutation at position 165805, a G>A mutation at position 166710, and an A>G mutation at position 174733. CNTFR The nucleotide sequence of the SNP molecular marker is located in the full-length sequence of the gene shown in SEQ ID No. 1, and there is a polymorphism at any one or more of positions 133334, 165805, 166710 and 174733, wherein there is a G>A mutation at position 133334, a C>T mutation at position 165805, a G>A mutation at position 166710, and an A>G mutation at position 174733. The method for determining the heterophil / lymphocyte ratio level of the to-be-tested Landes geese comprises the following steps: when the 133334th position in the sequencing sequence is G, the site genotype is GG; when the 165805th position is C, the site genotype is CC; when the 166710th position is A, the site genotype is AA; when the 174733th position is A, the site genotype is AA; when any one or more of the above site genotypes appears, the to-be-tested Landes goose is a high-heterophil / lymphocyte ratio individual; when the 133334th position in the sequencing sequence is A, the site genotype is AA; when the 165805th position is T, the site genotype is TT; when the 166710th position is G, the site genotype is GG; when the 174733th position is G, the site genotype is GG; when any one or more of the above site genotypes appears, the to-be-tested Landes goose is a low-heterophil / lymphocyte ratio individual.

2. Application of the primer pair of the SNP molecular marker in identifying the high / low level of the heterophil / lymphocyte ratio in the serum of the Landes goose, characterized in that, The nucleotide sequence of the SNP molecular marker is located in the full-length sequence of the gene shown in SEQ ID No. 1 CNTFR 133334, 165805, 166710 and 174733, wherein a G>A mutation exists at the 133334th position, a C>T mutation exists at the 165805th position, a G>A mutation exists at the 166710th position, and an A>G mutation exists at the 174733th position. the primer pair comprises one or more of the following primer pairs; the primer C5-F1 with the nucleotide sequence as shown in SEQ ID No. 2: 5'-CCCAACTCCCAGTGCTTCTCC-3' and the primer C5-R1 with the nucleotide sequence as shown in SEQ ID No. 3: 5'-TCCTCTCCTCCCCTCCCTGAC-3'; the primer C1-F1 with the nucleotide sequence as shown in SEQ ID No. 4: 5'-TTGCCTTTCTCTTCTCGTGGAG-3' and the primer C1-R1 with the nucleotide sequence as shown in SEQ ID No. 5: 5'-CCTGAGAGCACTGACGGAGATG-3'; the primer C3-F1 with the nucleotide sequence as shown in SEQ ID No. 6: 5'-CTGGCTGGTGGAGAAATGTGT-3' and the primer C3-R1 with the nucleotide sequence as shown in SEQ ID No. 7: 5'-GAGGGGAGGAAGGGGAGTT-3'; the primer C4-F1 with the nucleotide sequence as shown in SEQ ID No. 8: 5'-CCCCCTACACCCCTGTGCT-3' and the primer C4-R1 with the nucleotide sequence as shown in SEQ ID No. 9: 5'- CCAGTGAGGAGGATGCCAGAG-3'; The method for determining the heterophil / lymphocyte ratio level of the to-be-tested Landes geese comprises the following steps: when the 133334th position in the sequencing sequence is G, the site genotype is GG; when the 165805th position is C, the site genotype is CC; when the 166710th position is A, the site genotype is AA; when the 174733th position is A, the site genotype is AA; when any one or more of the above site genotypes appears, the to-be-tested Landes goose is a high-heterophil / lymphocyte ratio individual; when the 133334th position in the sequencing sequence is A, the site genotype is AA; when the 165805th position is T, the site genotype is TT; when the 166710th position is G, the site genotype is GG; when the 174733th position is G, the site genotype is GG; when any one or more of the above site genotypes appears, the to-be-tested Landes goose is a low-heterophil / lymphocyte ratio individual. When the sequencing sequence is A at position 133334, the site genotype is AA; T at position 165805, the site genotype is TT; G at position 166710, the site genotype is GG; G at position 174733, the site genotype is GG; when any one or more of the above site genotypes occurs, the individual Landes goose to be tested is a low heterophil / lymphocyte ratio individual.

3. The SNP molecular marker of claim 1 or the primer pair of claim 2 is used in the detection and identification of the immune performance of Landes geese, and the screening of high immune performance Landes geese. wherein The level of the heterophil / lymphocyte ratio of the individual Landes goose to be tested is determined by the sequencing sequence and the peak graph, which comprises: When the sequencing sequence is G at position 133334, the site genotype is GG; C at position 165805, the site genotype is CC; A at position 166710, the site genotype is AA; A at position 174733, the site genotype is AA; when any one or more of the above site genotypes occurs, the individual Landes goose to be tested is a high heterophil / lymphocyte ratio individual, and the immune level is low; When the sequencing sequence is A at position 133334, the site genotype is AA; T at position 165805, the site genotype is TT; G at position 166710, the site genotype is GG; G at position 174733, the site genotype is GG; when any one or more of the above site genotypes occurs, the individual Landes goose to be tested is a low heterophil / lymphocyte ratio individual, and the immune level is high.

4. A method of identifying high / low levels of heterophil / lymphocyte ratio in Landes goose serum, characterized in that, The method comprises: (1) extracting the genomic DNA of the individual Landes goose to be tested; (2) using the extracted genomic DNA as a template, performing PCR amplification using any one or more primer pairs in claim 2 to obtain an amplified fragment containing the SNP molecular site; (3) performing gene sequencing on the amplified fragment, and determining the level of the heterophil / lymphocyte ratio of the individual Landes goose to be tested by the sequencing sequence and the peak graph; When the sequencing sequence is G at position 133334, the site genotype is GG; C at position 165805, the site genotype is CC; A at position 166710, the site genotype is AA; A at position 174733, the site genotype is AA; when any one or more of the above site genotypes occurs, the individual Landes goose to be tested is a high heterophil / lymphocyte ratio individual; When the sequencing sequence is A at position 133334, the site genotype is AA; T at position 165805, the site genotype is TT; G at position 166710, the site genotype is GG; G at position 174733, the site genotype is GG; when any one or more of the above site genotypes occurs, the individual Landes goose to be tested is a low heterophil / lymphocyte ratio individual.

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