An indel molecular marker related to immune traits of landes geese and application thereof

Abstract

The present application relates to an InDel molecular marker related to the immune traits of Landes geese and an application thereof, and belongs to the technical field of molecular markers. CNTFR The InDel molecular marker in the present application is located in the full-length sequence of SEQ ID No. 1, and a single-base T insertion exists at the 165833th position. The InDel molecular marker of the present application can be used to quickly and accurately distinguish the high / low ratio of heterophil granulocyte / lymphocyte of Landes geese, has the advantages of simple operation and reliable results, and provides an effective molecular basis for the directional selection of the immune-related traits of Landes geese.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker-assisted selection and genetic breeding technology, specifically relating to an InDel molecular marker related to immune traits in Landes geese and its application. Background Technology

[0002] Landes geese are the main breed for foie gras production, known for their tender texture and unique flavor. Foie gras production is primarily achieved through force-feeding; however, intensive force-feeding induces significant stress responses, persistently impacting the physiological functions and immune status of Landes geese. Prolonged force-feeding stress can lead to a decline in immune function, increasing susceptibility to various pathogens such as reovirus, potentially triggering disease outbreaks and causing severe economic losses. Therefore, studying the mechanisms by which force-feeding stress affects the immune performance of Landes geese and screening breeding indicators related to stress-induced immune traits is crucial for improving the stress resistance of Landes geese and cultivating new strains of Landes geese with strong immune capabilities and tolerance to force-feeding.

[0003] Heterophiles are an important component of innate immunity in avian birds, possessing the functions of phagocytosis and pathogen killing, and playing a crucial role in host immune defense. Lymphocytes, on the other hand, constitute the core of the adaptive immune system and participate in specific immune responses. The heterophile / lymphocyte ratio (H / L) directly reflects the dynamic balance of immune cells in poultry and is an important physiological indicator for evaluating stress status and overall immune function. Compared with some immediate and invasive detection methods, the H / L ratio is relatively stable, and the detection process has less impact on the organism, making it more suitable as an evaluation indicator in stress-related breeding practices. Studies show that individuals with lower H / L ratios generally perform better in terms of environmental adaptability, production performance, and disease resistance in their offspring, indicating that this indicator has significant application value in breeding poultry strains with high immune performance.

[0004] InDels (Insertion / Deletion) are a class of DNA polymorphic markers formed by the insertion or deletion of one or more nucleotides in the genome. They belong to the third generation of genetic markers and are widely present in the genomes of various species. Studies have shown that InDels located in gene coding or regulatory regions may lead to phenotypic differences by affecting protein coding, splicing processes, or expression regulation. Therefore, they can serve as important functional molecular markers for the evaluation of germplasm genetic resources and molecular-assisted breeding of economic traits. However, there are currently no reports on the association between InDel markers and the H / L ratio in Landes geese. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide an InDel molecular marker related to the immune traits of Landes geese and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an InDel 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 contains a single T-base insertion at position 165833. The polymorphism of this InDel molecular marker is significantly correlated with the heterophile granulocyte / lymphocyte ratio in Landes geese, and can be used to rapidly and accurately identify Landes geese individuals with different immune capabilities, providing a basis for molecular selection of immune traits.

[0008] Secondly, the present invention provides a primer pair for detecting InDel molecular markers associated with immune traits in Landes geese. The primer pair comprises primer F with nucleotide sequences as shown in SEQ ID No. 2 and primer R with SEQ ID No. 3, wherein primer F is 5'-TTGCCTTTCTCTTCTCGTGGAG-3'; and primer R is 5'-CCTGAGAGCACTGACGGAGATG-3'. This primer pair can specifically amplify molecules containing InDel molecular markers. CNTFR Gene fragments.

[0009] Thirdly, the InDel molecular marker provided by this invention is used to identify the immune traits of Landes geese and to screen Landes geese individuals with good immune performance.

[0010] Fourthly, the InDel molecular markers or primer pairs provided by this invention are used to identify high / low heterophile granulocyte / lymphocyte ratios in Landes goose serum.

[0011] Fifthly, the present invention provides a method for identifying a high / low heterophile granulocyte / lymphocyte ratio in Landes goose serum, the method comprising the following steps:

[0012] (1) Extract genomic DNA from the Landes goose to be tested;

[0013] (2) Using the extracted genomic DNA as a template, PCR amplification was performed using primer pairs with nucleotide sequences as shown in SEQ ID No. 2 and SEQ ID No. 3 to obtain the target fragment containing the InDel molecular marker;

[0014] (3) Sequencing the target fragment, and determining the H / L level and corresponding immune traits of the Landes goose by sequencing the sequence and peak diagram.

[0015] When position 165833 of the sequence is A, the genotype of the InDel molecular marker is A / A, indicating that the tested Landes goose is a low H / L Landes goose individual, representing a high immune trait; when position 165833 of the sequence is AT, the genotype of the InDel molecular marker is AT / AT, indicating that the tested Landes goose is a high H / L Landes goose individual, representing a low immune trait.

[0016] The present invention has the following beneficial effects:

[0017] This invention utilizes population selection signal analysis to screen for InDel molecular markers located on chromosome Z of Landes geese that are significantly associated with immune traits, and establishes a molecular marker detection system based on this locus. These InDel molecular markers can accurately and efficiently distinguish the H / L levels of individual Landes geese, and are characterized by simple operation and stable, reliable results, providing a scientific basis for molecular-assisted selection of immune traits in Landes geese. Using these InDel molecular markers, early and precise screening of individuals with high immune performance (H / L ≤ 0.2) can be achieved in breeding. Only one generation of testing and selection is needed to purify the breeding population at the target H / L level, significantly improving breeding efficiency and contributing to the development of new Landes goose strains with superior immune traits. Attached Figure Description

[0018] Figure 1 This is based on F provided in Embodiment 1 of the present invention. ST The analysis results of the population selection signal related to H / L in Landes geese are shown in the figure.

[0019] 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;

[0020] Figure 3 This is a graph showing the analysis results of the population selection signal based on XP-EHH of Landes geese H / L correlation provided in Embodiment 1 of the present invention;

[0021] Figure 4 This is the forward sequencing diagram of the three genotypes of the InDel molecular marker provided in Example 2 of the present invention;

[0022] Figure 5 This is a comparison chart of the relative expression levels of serum cytokine mRNA in Landes geese under different H / L levels during 12 days of force-feeding, provided in Example 4 of this invention. Detailed Implementation

[0023] In a first aspect, the present invention provides an InDel molecular marker associated with immune traits in Landes geese, which is used in population selection signal analysis (F... STThe InDel molecular site in this application is obtained through screening (θπ and XP-EHH). The site is located at the location shown in SEQ ID No. 1. CNTFR The InDel molecular site is located at position 165833 of the full-length gene sequence, where a single T base is inserted. For ease of description, this application names the InDel molecular site the CNTFR-2 site.

[0024] Secondly, the present invention provides an InDel 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 contains a single T insertion at position 165833.

[0025] Thirdly, the present invention provides a primer pair for detecting InDel molecular markers, comprising primer F with nucleotide sequences as shown in SEQ ID No. 2 and primer R with SEQ ID No. 3. Primer F is 5'-TTGCCTTTCTCTTCTCGTGGAG-3'; primer R is 5'-CCTGAGAGCACTGACGGAGATG-3'.

[0026] The Landes goose genomic DNA was amplified using the primer pairs described above, and a DNA fragment containing the InDel molecular marker was obtained. After sequencing, the genotype was determined based on the sequence characteristics at position 165833.

[0027] When the sequencing result shows A at position 165833, the genotype of this InDel locus is determined to be A / A;

[0028] When the sequencing result shows AT at position 165833, the genotype of this InDel locus is determined to be AT / AT.

[0029] When the sequencing result shows that both A and AT are present at position 165833, the genotype of this InDel locus is determined to be A / AT.

[0030] Fourthly, the present invention provides a kit related to immune traits in Landes geese, the kit comprising the primer pairs described in the second aspect. The kit may also contain PCR amplification premixes conventional in the art, such as, but not limited to, Phusion® Ultra Fidelity PCR Premixes, Platinum Direct PCR Universal Premixes, etc., and the specific type is not limited.

[0031] Fifthly, the present invention provides the application of InDel molecular markers, primer pairs, or kits in identifying the H / L level in Landes goose serum.

[0032] Sixthly, the present invention provides a method for identifying a high / low heterophile granulocyte / lymphocyte ratio in Landes goose serum, the method comprising:

[0033] (1) Extract genomic DNA from the Landes goose to be tested;

[0034] (2) Using the extracted genomic DNA as a template, PCR amplification was performed using primer pairs with nucleotide sequences as shown in SEQ ID No. 2 and SEQ ID No. 3 to obtain the target fragment containing the InDel molecular marker;

[0035] (3) Perform gene sequencing on the target fragment and determine the H / L level of the Landes goose to be tested by the sequencing sequence and peak diagram.

[0036] Specifically, determining the H / L level of the Landes goose being tested using sequencing sequences and peak diagrams involves:

[0037] When the 165833rd position of the sequencing sequence is A, the genotype at the InDel locus is A / A, indicating that the Landes goose being tested is a low H / L Landes goose individual; when the 165833rd position of the sequencing sequence is AT, the genotype at the InDel locus is AT / AT, indicating a higher immune trait; therefore, the Landes goose being tested is a high H / L Landes goose individual with a lower immune trait.

[0038] In a seventh aspect, the present invention provides another application of InDel molecular markers, primer pairs, or kits for screening Landes geese individuals with different immune properties.

[0039] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0040] Example 1 – Obtaining and Determining InDel Molecular Sites and InDel Molecular Markers

[0041] 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, and 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.

[0042] The quality of the preserved DNA stock solution was tested, and whole-genome resequencing was performed after it passed the quality test. Software such as Trinmomtic, BWA-mem, SAMtools, and GATK was used to filter, align, and detect variants in the resequencing data to obtain InDel information for each individual. Further 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. STThe results of extended haplotype homozygosity across populations (XP-EHH) are shown in the appendix. Figure 1-3 The screening criterion is: weighted F-value. ST The highest 5% window, the highest and lowest 5% windows of log2 (θπ, high H / L / θπ, low H / L), and the window containing the site with an absolute XP-EHH value ≥2 were selected as candidate regions associated with the H / L trait. Subsequently, principal component analysis, functional enrichment analysis, and haplotype analysis were used to screen candidate genes and loci associated with H / L in Landes geese.

[0043] The above analysis yielded 1178 windows and 25673 loci, resulting in the annotation of 659 candidate genes. Principal component analysis and functional enrichment analysis revealed that the CNTFR gene, located on the Z chromosome, is associated with H / L levels in Landes geese. This gene encodes a member of the type 1 cytokine receptor family and is a ligand-specific subunit of the ciliary neurotrophic factor receptor, involved in regulating neuronal cell survival, differentiation, and gene expression.

[0044] All InDel loci within the CNTFR gene were extracted, and the genotype frequency differences between high and low H / L populations were compared. One InDel locus significantly associated with H / L levels was identified and named CNTFR-5. Based on the Taihu goose T2T reference genome shown at https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_040182565.1 / , the location information of the CNTFR-5 locus is shown in Table 1, and its forward sequencing is attached. Figure 4 .

[0045] Table 1: CNTFR-5 locus location information

[0046]

[0047] Example 2 – Validation of InDel molecular sites and InDel molecular markers

[0048] Thirty Landes geese of different H / L ratios and high were selected at 90 days of age. 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 tested. The DNA stock solution was aliquoted and diluted to 50 ng / μL and stored at -20°C.

[0049] 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: CNTFRThe full-length sequence was obtained, and the InDel molecular marker shown in SEQ ID No. 1 was acquired. Subsequently, PCR primers were designed near CNTFR-5 using Oligo 6.0 software, and the primer pairs shown in Table 2 were obtained.

[0050] Using the DNA stock solution as a template, PCR amplification was performed using the primers 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.

[0051] Table 2: Primer Pair Information

[0052]

[0053] Table 3: PCR reaction system

[0054]

[0055] Table 4: PCR reaction conditions

[0056]

[0057] 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.

[0058] Table 5: Genotype frequency distribution of CNTFR-5 loci

[0059]

[0060] As shown in Table 5, the CNTFR-5 locus exhibited significant differences in genotypic distribution across different H / L populations. In individuals with high H / L, the AT / AT genotype was predominant (73.33%), followed by the A / AT genotype (26.67%), with no A / A individuals detected. Conversely, in individuals with low H / L, the A / A genotype was predominant (70%), with the A / AT genotype accounting for 30%, and no AT / AT individuals detected. This indicates a significant correlation between the CNTFR-5 locus and H / L levels in Landes geese, with the AT / AT genotype serving as a potential indicator genotype for high H / L, while the A / A genotype corresponds to low H / L.

[0061] Based on the CNTFR-5 site mentioned above, this embodiment of the invention provides an InDel molecular marker, which is the nucleotide sequence shown in SEQ ID No. 1, and has an A / AT polymorphism at position 165833 of the nucleotide sequence.

[0062] Example 3

[0063] 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.

[0064] Example 4 - Detection of individual immune performance with high and low H / L ratios

[0065] 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, and 4 μL was placed on a glass slide to prepare a blood smear. 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.

[0066] Table 6: H / L value distribution

[0067]

[0068] Five Landes geese with different H / L levels were randomly selected during the 12-day force-feeding period. Blood was collected from the wing vein of each Landes goose, and total RNA was extracted from the whole blood using the Trizol method. The RNA concentration and purity were determined, and the RNA stock solution was aliquoted and diluted to 500 ng / μL and stored at -20℃.

[0069] 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 analyzer with the primer pairs shown in Table 9, the qPCR reaction system shown in Table 10, and the qPCR reaction conditions shown in Table 11. IgA , IgM , IFN-γ , TNF-β , IL-2 and β-actin Perform qPCR amplification.

[0070] Table 7: Reverse Transcription Reaction System

[0071]

[0072] Table 8: Conditions for Reverse Transcription Reaction

[0073]

[0074] Table 9: Primer Pair Information

[0075]

[0076] Table 10: qPCR reaction system

[0077]

[0078] Table 11: qPCR reaction conditions

[0079]

[0080] The CT values ​​obtained from the machine detection were used to 2 -ΔΔCT The relative expression levels were calculated using the method described above, and graphs were created using GraphPad software to obtain the results. Figure 5 .

[0081] During the 12-day force-feeding period, Landes geese with different H / L levels (see Table 6 for details) were randomly selected to test 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).

[0082] Table 12: Data on organ weight, immune organ index, and antibody titer at different force-feeding periods

[0083]

[0084] Note: Different lowercase letters in Table 12 indicate significance levels. P <0.05.

[0085] As attached Figure 5 As shown, after 12 days of force-feeding, the serum of individuals with high H / L levels... IgA , IL-2, IFN-γ and TNF-β Expression was significantly lower than in individuals with low H / L ( P <0.05); meanwhile, as shown in Table 12, the Newcastle disease antibody titer and avian influenza H5 antibody titer, as well as indicators such as infectious bursal disease and infectious bursal disease index, were significantly lower in individuals with high H / L than in individuals with low H / L. 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.

[0086] 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. Application of InDel molecular marker in identification of immune traits of Landes geese and screening of Landes geese with high immune traits, characterized in that, The InDel molecular marker for identifying high / low of the heterophil granulocyte / lymphocyte ratio in Landes geese serum is located at the position of 165833 of the full-length sequence of SEQ ID No. 1 CNTFR a single-base T insertion exists at the position of 165833 of the full-length sequence of SEQ ID No. 1 The level of heterophil / lymphocyte ratio of the to-be-tested Landes geese is determined by sequencing the sequence and the peak graph, including: when the base at the 165833th position of the sequencing sequence is A, the genotype of the InDel site is A / A, and the to-be-tested Landes goose is a Landes goose individual with low heterophil / lymphocyte ratio; when the base at the 165833th position of the sequencing sequence is AT, the genotype of the InDel site is AT / AT, and the to-be-tested Landes goose is a Landes goose individual with high heterophil / lymphocyte ratio.

2. The primer pair of the InDel molecular marker is applied in identifying immune traits of Langshan geese and screening Langshan geese with high immune traits, characterized in that, The InDel molecular marker is located at the position of 165833 of the full-length sequence of SEQ ID No. 1 CNTFR a single base T insertion exists at the position of 165833 of the full-length sequence of the gene the nucleotide sequence of the primer pair is shown in the primer F of SEQ ID No. 2 and the primer R of SEQ ID No. 3; wherein the primer F is 5'-TTGCCTTTCTCTTCTCGTGGAG-3'; and the primer R is 5'-CCTGAGAGCACTGACGGAGATG-3'; The level of heterophil / lymphocyte ratio of the to-be-tested Landes geese is determined by sequencing the sequence and the peak graph, including: when the base at the 165833th position of the sequencing sequence is A, the genotype of the InDel site is A / A, and the to-be-tested Landes goose is a Landes goose individual with low heterophil / lymphocyte ratio; when the base at the 165833th position of the sequencing sequence is AT, the genotype of the InDel site is AT / AT, and the to-be-tested Landes goose is a Landes goose individual with high heterophil / lymphocyte ratio.

3. A method for identifying high / low heterophil / lymphocyte ratio in Landes goose serum, characterized in that, The method comprises: (1) extracting the genomic DNA of the to-be-tested Landes goose; (2) using the extracted genomic DNA as a template, performing PCR amplification by using a primer pair with the nucleotide sequences shown in SEQ ID No. 2 and SEQ ID No. 3, to obtain a target fragment containing an InDel site; (3) performing gene sequencing on the target fragment, and determining the level of heterophil / lymphocyte ratio of the to-be-tested Landes goose by sequencing the sequence and the peak graph; wherein the level of heterophil / lymphocyte ratio of the to-be-tested Landes goose is determined by sequencing the sequence and the peak graph, including: when the base at the 165833th position of the sequencing sequence is A, the genotype of the InDel site is A / A, and the to-be-tested Landes goose is a Landes goose individual with low heterophil / lymphocyte ratio; when the base at the 165833th position of the sequencing sequence is AT, the genotype of the InDel site is AT / AT, and the to-be-tested Landes goose is a Landes goose individual with high heterophil / lymphocyte ratio.

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