SNP (Single Nucleotide Polymorphism) site, chip and identification method for identifying avian leukosis

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) site related to a chicken anti-avian leukosis character on a chicken chromosome 1. The SNP marker site comprises a 196584812th site Tgt on the chicken chromosome 1 (NC006088.5); a, the 196585463rd site of Agt; c, Cgt at the 196586977 site; a. By preferably selecting the disease-resistant related allele of the SNP, the frequency of the disease-resistant related allele can be increased generation by generation, the resistance character of chicken to avian leukosis is improved, and the genetic improvement progress of chicken is accelerated, so that the economic benefit of chicken breeding is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to chicken ( Gallus gallus ) SNP sites on chromosomes associated with resistance to avian leukosis symptoms and their applications. Background Art

[0002] In China, chicken has become the second most consumed meat after pork, and its demand is increasing every year. Therefore, the scale of chicken farming in China is constantly expanding, and farming technology is also gradually improving. In the context of globalization, the livestock and poultry farming industry is facing unprecedented challenges and opportunities. As one of the world's largest poultry producers, the development and innovation of China's chicken seed industry is not only a key link in the process of national agricultural modernization, but also an important way to improve food safety, increase farmers' income, and ensure market supply. In recent years, with the rapid development of biotechnology and information technology, China's chicken seed industry is undergoing a profound transformation from traditional breeding mode to intelligent and precision breeding. In this process, genetic improvement and disease prevention and control have become the two core issues in the development of the chicken seed industry. On the one hand, genetic improvement can improve the production performance, adaptability and disease resistance of chicken breeds to meet the market demand for high-quality and high-yield chicken breeds; on the other hand, disease prevention and control, especially the prevention and control of highly contagious and pathogenic diseases such as avian leukosis, is directly related to the sustainable development and biosafety of the chicken seed industry.

[0003] Avian leukosis is a chronic infectious tumor disease caused by avian leukosis virus. It is characterized by the production of lymphoid tumors and decreased egg production in adult chickens. There are many clinical manifestations, mainly lymphoid leukosis, followed by erythroblastic leukemia, myeloblastic leukemia, myelocytosis, nephroblastoma, osteolithiasis, hemangioma, sarcoma and mesothelioma. Since Roloff first reported lymphoid leukosis in 1868, it has been considered one of the most important poultry diseases that seriously endangers the poultry industry. It is believed that the disease affects almost all commercial chicken flocks. Although the disease occurs gradually and has a sustained low mortality rate, it is difficult to control the disease because it is mainly transmitted vertically, which seriously affects the weight gain and egg production of the chicken flock. In particular, the resistance of the affected chickens is reduced and they are easily infected with various diseases, which brings great difficulties to the feeding and management of the chicken flock.

[0004] Jingyuan chicken is one of the five regional livestock and poultry genetic resources in Ningxia, China, which are officially included in the "Catalogue of Chinese Livestock and Poultry Genetic Resources". It is widely distributed in the Loess Plateau in southern Ningxia and is an excellent egg-meat dual-purpose chicken breed that is resistant to high cold and drought climates on the Loess Plateau. As an excellent local chicken breed in China, Jingyuan chicken is also deeply troubled by avian leukosis infection. The prevalence of the disease has caused problems such as slow resistance to avian leukosis, decreased production performance, and increased mortality in the chicken flock, which directly led to increased breeding costs and impaired economic benefits. More importantly, the continued existence of avian leukosis has seriously hindered the genetic improvement plan of Jingyuan chicken. Genetic improvement aims to improve the production performance, health status and adaptability of chicken breeds through breeding, while the infection of avian leukosis, especially chronic infection, often leads to the inability to fully demonstrate the genetic potential of the chicken flock, affecting the accuracy and efficiency of breeding work. In addition, the infectiousness of avian leukosis also threatens the biosafety of the chicken breed industry. Once the disease breaks out in the chicken flock, not only will the current chicken flock be damaged, but it may also affect the health and performance of future chicken breeds, causing long-term negative impacts on the entire industry and causing great trouble to the sustainable development of the Jingyuan chicken industry. Therefore, it is of great significance to conduct association analysis on polymorphic sites and avian leukosis resistance traits in the Jingyuan chicken population and screen out sites that affect the avian leukosis resistance traits of Jingyuan chicken, so as to provide a basis for molecular breeding and variety improvement of local chicken breeds, especially Jingyuan chicken. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a SNP that significantly affects the resistance to avian leukosis in chickens, identified through resequencing and GWAS analysis strategies, and used it in molecular marker-assisted selection and genomic selection to select genotypes with favorable avian leukosis resistance traits for seed retention, thereby increasing the gene frequency of disease-resistant related alleles from generation to generation, which can accelerate the process of breeding and improvement of breeder chickens and bring huge economic benefits to local chicken farming.

[0006] The primary purpose is to identify SNP molecular markers associated with resistance to avian leukosis symptoms. The molecular markers are located on chromosome 1 (NC_006088.5) of the chicken genome reference sequence.

[0007] The SNP site of the molecular marker corresponds to the 196584812th T>A mutation on chromosome 1 (NC_006088.5) of the chicken GRCg6a genome, and / or the 196585463rd A>C mutation, and / or the 196586977th C>A mutation.

[0008] The SNP marker is shown in SEQ ID NO: 1, and / or SEQ ID NO: 2, and / or SEQ ID NO: 3, and is specifically shown in the following table

[0009] Another object of the present invention is to provide a chip for detecting the above-mentioned molecular marker sites, wherein the chip comprises primers and probes for amplifying and detecting the above-mentioned sites.

[0010] Another object of the present invention is to provide the use of the above molecular marker in screening chicken individuals with avian leukosis resistance traits. Specifically, the genotype at position 196584812 on chicken chromosome 1 (NC_006088.5), and / or the genotype at position 196585463 and / or the genotype at position 196586977 are detected, and individuals with genotype TT at position 196584812 on chicken chromosome 1 (NC_006088.5), and / or individuals with AA at position 196585463 and / or individuals with CC at position 196586977 are eliminated.

[0011] In a preferred embodiment, individuals having the following genotypes on chicken chromosome 1 (NC_006088.5) are eliminated: the genotype at position 196584812 is TT, the genotype at position 196585463 is AA, and the genotype at position 196586977 is CC.

[0012] In a preferred embodiment, individuals with genotype AA at position 196584812 on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens.

[0013] In a preferred embodiment, individuals with a genotype of CC at position 196585463 on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens.

[0014] In a preferred embodiment, individuals with genotype AA at position 196586977 on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens.

[0015] In a preferred embodiment, individuals containing the following genotypes on chicken chromosome 1 (NC_006088.5) are retained at the same time: the genotype at position 196584812 is AA, the genotype at position 196585463 is CC, and the genotype at position 196586977 is AA.

[0016] Another object of the present invention is to provide a reagent or kit for detecting the above molecular markers for use in identifying or assisting in identifying the resistance of Jingyuan chicken to avian leukosis. Specifically, the reagent or kit is used to detect the genotype at position 196584812 on chicken chromosome 1 (NC_006088.5), and / or the genotype at position 196585463 and / or the genotype at position 196586977, such as individuals with genotype at position 196584812 being AA, genotype at position 196585463 being CC, or genotype at position 196586977 being AA, and individuals with genotype at position 196584812 being TT, genotype at position 196585463 being AA, or genotype at position 196586977 being CC, being disease-resistant individuals, and individuals with genotype at position 196584812 being TT, genotype at position 196585463 being AA, or genotype at position 196586977 being CC, being disease-susceptible individuals.

[0017] Another object of the present invention is to provide a reagent or kit for detecting the above molecular markers for use in chicken genome selection breeding. Detect the genotype at position 196584812 on chicken chromosome 1 (NC_006088.5), and / or the genotype at position 196585463 and / or the genotype at position 196586977, and eliminate individuals with genotype TT at position 196584812 on chicken chromosome 1 (NC_006088.5), and / or individuals with AA at position 196585463 and / or individuals with CC at position 196586977.

[0018] In a preferred embodiment, individuals having the following genotypes on chicken chromosome 1 (NC_006088.5) are eliminated: the genotype at position 196584812 is TT, the genotype at position 196585463 is AA, and the genotype at position 196586977 is CC.

[0019] In a preferred embodiment, individuals with genotype AA at position 196584812 on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens.

[0020] In a preferred embodiment, individuals with a genotype of CC at position 196585463 on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens.

[0021] In a preferred embodiment, individuals with genotype AA at position 196586977 on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens.

[0022] In a preferred embodiment, individuals containing the following genotypes on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens: the genotype at position 196584812 is AA, the genotype at position 196585463 is CC, and the genotype at position 196586977 is AA.

[0023] Another object of the present invention is to provide a method for genetic improvement of chickens, the method comprising: determining the genotype of the molecular marker loci associated with the chicken resistance to avian leukosis in the breeder chicken resource population, and making corresponding selections based on the molecular markers to increase the genotype frequency associated with the resistance to avian leukosis trait at the corresponding loci generation by generation, thereby improving the resistance to avian leukosis trait of the offspring chickens. The chicken population includes the Shizuohara chicken and its synthetic line.

[0024] Optionally, individuals with genotype TT at position 196584812 on chicken chromosome 1 (NC_006088.5) are eliminated.

[0025] Optionally, individuals with AA genotype at position 196585463 on chicken chromosome 1 (NC_006088.5) are eliminated.

[0026] Optionally, individuals with a genotype of CC at position 196586977 on chicken chromosome 1 (NC_006088.5) are eliminated.

[0027] In a preferred embodiment, individuals having the following genotypes on chicken chromosome 1 (NC_006088.5) are eliminated: the genotype at position 196584812 is TT, the genotype at position 196585463 is AA, and the genotype at position 196586977 is CC.

[0028] In a preferred embodiment, individuals with genotype AA at position 196584812 on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens.

[0029] In a preferred embodiment, individuals with a genotype of CC at position 196585463 on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens.

[0030] In a preferred embodiment, individuals with genotype AA at position 196586977 on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens.

[0031] In a preferred embodiment, individuals containing the following genotypes on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens: the genotype at position 196584812 is AA, the genotype at position 196585463 is CC, and the genotype at position 196586977 is AA.

[0032] The specific steps include: (1) Extracting genomic DNA from the chicken to be tested; (2) testing the genotype of the chicken to be tested; (3) Based on the test results, determine the genotype at position 196584812 on chromosome 1 (NC_006088.5) of the chicken reference genome GRCg6a, and / or the genotype at position 196585463 and / or the genotype at position 196586977 of the chicken to be tested.

[0033] (4) Make corresponding selections based on the genotypes detected in step 3) to increase the frequency of genotypes associated with the avian leukosis resistance trait at the corresponding loci generation by generation, thereby improving the avian leukosis resistance trait of subsequent chickens.

[0034] Optionally, individuals with genotype TT at position 196584812 on chicken chromosome 1 (NC_006088.5), and / or individuals with AA at position 196585463, and / or individuals with CC at position 196586977 are eliminated.

[0035] In a preferred embodiment, individuals having the following genotypes on chicken chromosome 1 (NC_006088.5) are eliminated: the genotype at position 196584812 is TT, the genotype at position 196585463 is AA, and the genotype at position 196586977 is CC.

[0036] In a preferred embodiment, individuals with genotype AA at position 196584812 on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens.

[0037] In a preferred embodiment, individuals with a genotype of CC at position 196585463 on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens.

[0038] In a preferred embodiment, individuals with genotype AA at position 196586977 on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens.

[0039] In a preferred embodiment, individuals containing the following genotypes on chicken chromosome 1 (NC_006088.5) are retained as breeder chickens: the genotype at position 196584812 is AA, the genotype at position 196585463 is CC, and the genotype at position 196586977 is AA.

[0040] Compared with the prior art, the present invention has the following advantages and effects: First, new molecular markers affecting the resistance to avian leukosis in chickens were identified.

[0041] Second, it is determined that new molecular markers have a stronger linkage relationship.

[0042] Third, the SNP molecular markers determined by the present invention are applied to the genetic improvement of the avian leukosis resistance trait of breeder chickens, which can improve the disease resistance of offspring to avian leukosis, thereby increasing the market competitiveness of breeding enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The method of the present invention and its beneficial effects are described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0044] Figure 1 Linkage map of the 196584812, 196585463, and 196586977 loci of the chicken reference genome GRCg6a (NC_006088.5) of the Jingyuan chicken. The darker the color and the larger the value (0-100), the tighter the linkage.

[0045] Figure 2 are the genotype distribution frequencies of related SNPs in the susceptible group and the resistant group (* indicates that there is a difference in the genotype distribution frequency between the two groups; **** indicates p≤0.0001; *** indicates p≤0.001; ** indicates p≤0.01; * indicates p≤0.05); Figure A is T>A at position 196584812 on chromosome 1 (NC_006088.5), Figure B is A>C at position 196585463, and Figure C is C>A at position 196586977. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs.

[0048] Example 1 Statistical analysis of the resistance of Jingyuan chicken to avian leukosis The Jingyuan Chicken used in this study were from the Jingyuan Chicken National Breeding Farm (Pengyang, China). First, we conducted preliminary pathological observations on Jingyuan Chickens with avian leukosis symptoms. The symptoms of 126-day-old chickens infected with avian leukosis virus were mainly lymphocytic leukosis (mainly A subtype avian leukosis virus), as follows: Mentality and appetite: Sick chickens were mentally depressed, weak all over, and had decreased or no appetite. Body shape and appearance: progressive weight loss and anemia, pale and wrinkled combs and wattles, and occasionally cyanosis. Excretion and egg production: diarrhea may occur, and egg production stops. Abdominal changes: The abdomen is often significantly enlarged, and an enlarged liver can be felt by pressing with the hand. Bursal changes: The enlarged bursa of Fabricius can be felt through the cloaca with fingers, and small nodular lesions can be seen after the bursa is cut open. Tumor lesions: Multiple tissues and organs have tumor lesions. Soft, gray or gray-yellow tumors of varying sizes can be seen in the liver, bursa of Fabricius, spleen, kidneys, etc. The large ones are as big as eggs and have various shapes. On August 8, 2022, blood samples of Jingyuan chickens sent by the Animal Husbandry Technology Extension Service Center of Pengyang County, Ningxia Hui Autonomous Region were tested for avian leukosis and avian leukosis, with 2,507 samples each. The test results are as follows: 1. Avian leukosis test: The 2,507 blood samples sent were tested for avian leukosis blood virus isolation, and a total of 201 positive individuals were detected. 50 Jingyuan chickens with positive results were selected as the susceptible group. At the same time, 50 Jingyuan chickens with no symptoms and negative test results were selected as the disease-resistant group, with 25 roosters and 25 hens in each group. Blood was collected from the subwing vein of 100 Jingyuan chickens for whole genome resequencing. Anticoagulated blood was collected from the subwing vein of 50 susceptible individuals and 50 resistant individuals in a total of 100 samples of Siberian Gallus gallus domesticus. Genomic DNA was extracted from whole blood using a genomic DNA purification kit (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the DNA quality was measured using a Qubit fluorometer and then electrophoresed on a 1% agarose gel to determine its quality.

[0049] Example 2 Resequencing and SNP analysis of avian leukosis resistance traits 1. Anticoagulated blood was collected from the sub-wing vein of 50 susceptible individuals and 50 resistant individuals in a total of 100 samples of Jingyuan chicken. Genomic DNA was extracted from whole blood using a genomic DNA purification kit (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the DNA quality was measured using a Qubit fluorometer and then electrophoresed on a 1% agarose gel to determine its quality. Qualified DNA samples were tested and whole genome resequencing was performed on the Illumina HiSeq platform.

[0050] 2 Genome-wide association analysis After quality control of the sequencing data, SNP annotation is performed by comparing with the reference genome. Genome-wide association analysis detects common genetic variations (single nucleotide polymorphisms, copy number variations, and structural variations) across the entire genome, and then performs association analysis between the obtained genotypes and the observed trait phenotypes. Individual kinship and population structure are the main factors causing false positive associations during GWAS analysis. Therefore, a mixed linear model (MLM) is used for trait association analysis, with population genetic structure as a fixed effect and individual kinship as a random effect to correct the effects of population structure and individual kinship on the results.

[0051] y = Xα + Zβ + Wμ + e y is the phenotypic trait, X is the indicator matrix of fixed effects, a is the estimated parameter of fixed effects; Z is the indicator matrix of SNPs, β is the effect of SNPs; W is the indicator matrix of random effects, μ is the predicted random individual, e is the random residual, and obeys e ~ (0, δe2).

[0052] EMMAX is a software commonly used in crop GWAS analysis. It can calculate the Kinship matrix and select the first five PCA components as covariates. We use EMMAX to perform association analysis on different traits and screen out potential candidate SNP sites through the significance of the association (P-value).

[0053] In this study, GWAS analysis identified three SNPs on chromosome 1 that were significantly associated with avian leukosis in Shizuohara chicken (Table 1). Linkage disequilibrium analysis showed that the three loci had a strong linkage relationship ( Figure 1 ).

[0054]

[0055] To further determine the relationship between SNP locus genotype and disease resistance and susceptibility characteristics, we used SPSS23.0 to analyze the genotype distribution frequency of related SNPs in the disease resistance group and the susceptible group. The chi-square test was used to test the significance of the frequency distribution difference between the two groups. The results showed that ( Figure 1 ), the following genotypes in the disease-resistant group were significantly different from those in the susceptible group: AA and AT at Chr1_196584812, CC and AC at Chr1_196585463, and AA and AC at Chr1_196586977 were significantly associated with disease resistance, and these genotypes were considered to be beneficial to disease resistance.

[0056] According to the disclosure and teaching of the above description, those skilled in the art to which the present invention belongs may also make appropriate changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present invention should also fall within the scope of protection of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for the convenience of description and do not constitute any limitation to the present invention.

Claims

1. A set of SNP molecular markers for chicken resistance to avian leukosis, characterized in that: The SNP molecular marker group includes at least one of SNP1, SNP2 or SNP3, wherein SNP1 corresponds to the 196584812th T>A mutation on chromosome 1 (NC_006088.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.1; The SNP2 corresponds to the molecular marker of the A>C mutation at position 196585463 on chromosome 1 (NC_006088.5) of the chicken genome GRCg6a, and its nucleotide sequence is shown in SEQ ID NO.2; The SNP1 corresponds to the C>A mutation at position 196586977 on chromosome 1 (NC_006088.5) of the chicken genome GRCg6a, and its nucleotide sequence is shown in SEQ ID NO.

3.

2. A chip for detecting chicken resistance to avian leukosis, the chip comprising primers and probes for amplifying and detecting the molecular markers according to claim 1.

3. A kit for detecting chicken resistance to avian leukosis, the chip comprising primers and probes for amplifying and detecting the molecular markers of claim 1.

4. Use of the SNP molecular marker according to claim 1 and / or the chip according to claim 2 and / or the kit according to claim 3 in improving the avian leukosis resistance trait of chickens.

5. Use of the SNP molecular marker according to claim 1 and / or the chip according to claim 2 and / or the kit according to claim 3 in identifying or assisting in identifying the strength of chicken resistance to avian leukosis.

6. Use of the SNP molecular marker according to claim 1 and / or the chip according to claim 2 and / or the kit according to claim 3 in genomic selection breeding for avian leukosis resistance.

7. A method for identifying or assisting in identifying the strength of chicken resistance to avian leukosis, comprising the following steps: 1) Detect the genotype at position 196584812, and / or the genotype at position 196585463, and / or the genotype at position 196586977 on chicken chromosome 1 (NC_006088.5); 2) Determine the strength of the chicken's resistance to avian leukosis. For example, individuals with a genotype of TT at position 196584812, or a genotype of AA at position 196585463, or a genotype of CC at position 196586977 are susceptible individuals, and individuals with a genotype of AA at position 196584812, or a genotype of CC at position 196585463, or a genotype of AA at position 196586977 are resistant individuals.

8. A method for improving the resistance of chickens to avian leukosis, characterized in that: The method comprises the following steps: 1) Detecting the genotype at position 196584812, and / or the genotype at position 196585463, and / or the genotype at position 196586977 on chicken chromosome 1 (NC_006088.5); 2) Eliminate individuals with genotype TT at position 196584812 on chicken chromosome 1 (NC_006088.5), and / or individuals with AA at position 196585463, and / or individuals with CC at position 196586977 based on the test results, so as to increase the genotype frequency associated with the avian leukosis resistance trait at the corresponding loci generation by generation.

9. The method according to claim 7 or 8, characterized in that: The step 1) comprises the following steps: (1) Extracting genomic DNA from the chicken to be tested; (2) Detecting the genotype of the chicken to be tested; (4) Based on the detection result, determining the genotype of the SNP marker according to claim 1 of the chicken to be tested.

10. The method according to claim 7 or 8, characterized in that: The chicken population includes Shizuohara chicken and its synthetic strains.

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