Pullorum disease resistance related SNP (Single Nucleotide Polymorphism) site on chromosome 2 and application

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) site related to chicken pullorum disease resistance on a chicken chromosome 2. The site of an SNP marker is the 129338261th site Agt on the chicken chromosome 2 (NC006089.5); g, Tgt at the 129345608th site; and C. By preferably selecting the disease-resistant related alleles of the SNP, the frequency of the disease-resistant related alleles can be increased generation by generation, the resistance character of chickens to pullorum disease is improved, and the genetic improvement progress of the chickens is accelerated, so that the economic benefit of chicken breeding is effectively improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a SNP site on a chicken chromosome related to resistance to pullorum and an application thereof. 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 pullorum, is directly related to the sustainable development and biosafety of the chicken seed industry.

[0003] Salmonellosis was discovered in the late 19th century. It is an acute, septicemic infectious disease caused by Salmonella pullorum. Many poultry (such as chickens, ducks, turkeys, pheasants, quails, etc.) can be infected, but it is mainly prevalent in chickens and turkeys, especially chickens, which are most susceptible. Due to its high morbidity and mortality rate, it is still one of the important diseases affecting the poultry industry.

[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 climate in the Loess Plateau. As an excellent local chicken breed in China, Jingyuan chicken is also deeply troubled by pullorum infection. According to the disease monitoring data in the past two years, the positive rate of non-immune antibodies to pullorum is as high as 23%. This data not only reflects the urgency of disease prevention and control, but also highlights the necessity of taking effective measures to curb the spread of the epidemic. The prevalence of the disease has caused problems such as slow resistance to pullorum, decreased production performance, and increased mortality in the chicken flock, which directly led to increased breeding costs and damaged economic benefits. More importantly, the continued existence of pullorum 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 pullorum infection, 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.

[0005] In addition, the infectiousness of pullorum disease also threatens the biosafety of the chicken breed industry. Once the disease breaks out in a flock, not only will the current 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 loci and pullorum resistance traits in the Jingyuan chicken population and screen out loci that affect the pullorum 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

[0006] In view of the problems existing in the prior art, the present invention provides a SNP that significantly affects the pullorum resistance of chickens identified through resequencing and GWAS analysis strategies, which is used in molecular marker-assisted selection and genomic selection to select genotypes that are favorable for the pullorum resistance trait 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.

[0007] The primary purpose is to determine the SNP molecular marker that affects the resistance to pullorum. The molecular marker is located on chromosome 2 (NC_006089.5) of the chicken genome reference sequence.

[0008] The SNP site of the molecular marker corresponds to the A>G mutation at position 129338261 and / or the T>C mutation at position 129345608 on chromosome 2 (NC_006089.5) of the chicken GRCg6a genome.

[0009] 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

[0010]

[0011]

[0012] Another object of the present invention is to provide the use of the above molecular marker in screening chicken individuals with resistance to pullorum. Specifically, the genotype at position 129338261 and / or the genotype at position 129345608 on chicken chromosome 2 (NC_006089.5) is detected, and the individuals with the genotype at position 129338261 on chicken chromosome 2 (NC_006089.5) being GG are eliminated.

[0013] Optionally, individuals with the CC genotype at position 129345608 on chicken chromosome 2 (NC_006089.5) are eliminated.

[0014] In a preferred embodiment, individuals with genotype AG at position 129338261 on chicken chromosome 2 (NC_006089.5) are retained as breeder chickens.

[0015] In a preferred embodiment, individuals with genotype CT at position 129345608 on chicken chromosome 2 (NC_006089.5) are retained as breeder chickens.

[0016] In a preferred embodiment, individuals with genotype AG at position 129338261 and CT at position 129345608 on chicken chromosome 2 (NC_006089.5) are retained as breeder chickens.

[0017] 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 pullorum. Specifically, the reagent or kit is used to detect the genotype at position 129338261 and / or the genotype at position 129345608 on chicken chromosome 2 (NC_006089.5), such as individuals with genotype GG at position 129338261 or genotype CC at position 129345608 are susceptible individuals, and individuals with genotype AG at position 129338261 or genotype CT at position 129345608 are resistant individuals.

[0018] Another object of the present invention is to provide a reagent or kit for detecting the above molecular markers for use in chicken genomic selective breeding. Specifically, the reagent or kit is used to detect the genotype at position 129338261 on chicken chromosome 2 (NC_006089.5) and / or the genotype at position 129345608, and eliminate individuals with the genotype at position 129338261 on chicken chromosome 2 (NC_006089.5) being GG.

[0019] Optionally, individuals with the CC genotype at position 129345608 on chicken chromosome 2 (NC_006089.5) are eliminated.

[0020] In a preferred embodiment, individuals with genotype AG at position 129338261 on chicken chromosome 2 (NC_006089.5) are retained as breeder chickens.

[0021] In a preferred embodiment, individuals with genotype CT at position 129345608 on chicken chromosome 2 (NC_006089.5) are retained as breeder chickens.

[0022] In a preferred embodiment, individuals with genotype AG at position 129338261 and CT at position 129345608 on chicken chromosome 2 (NC_006089.5) are retained as breeder chickens.

[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 affecting the excellent pullorum resistance of chickens in the breeder chicken resource population, and making corresponding selections based on the molecular markers to increase the genotype frequency related to the pullorum resistance trait at the corresponding loci generation by generation, thereby improving the pullorum resistance trait of the offspring chickens. The chicken population includes the Jingyuan chicken and its synthetic line.

[0024] Optionally, individuals with genotype GG at position 129338261 on chicken chromosome 2 (NC_006089.5) are eliminated.

[0025] Optionally, individuals with the CC genotype at position 129345608 on chicken chromosome 2 (NC_006089.5) are eliminated.

[0026] In a preferred embodiment, individuals with genotype AG at position 129338261 on chicken chromosome 2 (NC_006089.5) are retained as breeder chickens.

[0027] In a preferred embodiment, individuals with genotype CT at position 129345608 on chicken chromosome 2 (NC_006089.5) are retained as breeder chickens.

[0028] In a preferred embodiment, individuals with genotype AG at position 129338261 and CT at position 129345608 on chicken chromosome 2 (NC_006089.5) are retained as breeder chickens.

[0029] The specific steps include:

[0030] (1) Extracting genomic DNA from the chicken to be tested;

[0031] (2) testing the genotype of the chicken to be tested;

[0032] (3) Based on the test results, determine the genotype of the SNP marker at position 129338261 on chromosome 2 (NC_006089.5) of the chicken reference genome GRCg6a, and / or the T>C mutation at position 129345608 of the chicken to be tested.

[0033] (4) making corresponding selections based on the genotypes detected in step 3) to increase the genotype frequency associated with the pullorum resistance trait at the corresponding locus generation by generation, thereby improving the pullorum resistance trait of the offspring chickens.

[0034] Optionally, individuals with genotype GG at position 129338261 on chicken chromosome 2 (NC_006089.5) are eliminated.

[0035] Optionally, individuals with the CC genotype at position 129345608 on chicken chromosome 2 (NC_006089.5) are eliminated.

[0036] In a preferred embodiment, individuals with genotype AG at position 129338261 on chicken chromosome 2 (NC_006089.5) are retained as breeder chickens.

[0037] In a preferred embodiment, individuals with genotype CT at position 129345608 on chicken chromosome 2 (NC_006089.5) are retained as breeder chickens.

[0038] In a preferred embodiment, individuals with genotype AG at position 129338261 and CT at position 129345608 on chicken chromosome 2 (NC_006089.5) are retained as breeder chickens.

[0039] Compared with the prior art, the present invention has the following advantages and effects:

[0040] First, new molecular markers affecting the pullorum resistance trait in chickens were identified.

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

[0042] Second, the SNP molecular markers determined by the present invention are applied to the genetic improvement of the pullorum resistance trait of breeder chickens, which can improve the disease resistance of the offspring to pullorum, 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 is the genotype distribution frequency of the relevant SNPs in the susceptible group and the disease-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).

[0045] Figure 2 This is the linkage map of the 129338261 and 129345608 sites of the Jingyuan chicken on chromosome 2 (NC_006089.5) of the chicken reference genome GRCg6a. 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 resistance of Jingyuan chicken to pullorum

[0049] The Jingyuan chickens used in this study were all from the Jingyuan Chicken National Breeding Farm (Pengyang, China). First, we conducted preliminary pathological observations on the Jingyuan chickens with symptoms of pullorum. The 126-day-old chickens showed listlessness, drooping wings, closed eyes and lethargy, and diarrhea and excretion of white paste-like loose feces. Some chicks began to have diarrhea and open mouth breathing, often isolated from the group, standing still, and slow to react to the outside world. Some early-stage chicks died. Then we performed autopsies on the dead chickens. The adult dead hens had atrophied and deformed follicles, and oily or watery substances were found inside the follicles. The oviducts were swollen and enlarged, the yolk membrane was thickened, and the liver and spleen were enlarged, with blunt edges and a brittle texture. After the situation of the breeder farm stabilized after a period of drug treatment, 2,500 126-day-old Jingyuan chickens were selected and two blood samples were collected from the wings, each of which was sent to the Jiangsu Poultry Science Research Institute (Jiangsu, China) and Ningxia Haobiao Testing Service Research Institute (Yinchuan, China) for pullorum plate agglutination test. The Jiangsu Poultry Science Research Institute detected a total of 53 strong positive individuals and 48 weak positive individuals; Ningxia Haobiao Testing Service Research Institute detected a total of 103 positive samples and 2,397 negative samples. 50 Jingyuan chickens with typical symptoms of Salmonella pullorum (hepatosplenomegaly, white diarrhea, and death) and positive results of plate agglutination test were selected as the susceptible group. At the same time, 50 Jingyuan chickens without symptoms and negative test results were selected as the disease-resistant group. Blood samples were collected from the subwing veins of 100 Jingyuan chickens for whole genome resequencing. Among the total 100 Jingyuan chicken samples, anticoagulated blood was collected from the subwing veins of 50 susceptible individuals and 50 disease-resistant individuals. 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.

[0050] Example 2 Resequencing and SNP analysis of pullorum resistance trait

[0051] 1. Anticoagulated blood was collected from the subwing vein of 50 susceptible individuals and 50 resistant individuals in a total of 100 samples of Siberian 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, followed by electrophoresis on a 1% agarose gel to determine its quality. Qualified DNA samples were first broken by ultrasound using a Covaris instrument, and then the sample band was concentrated to about 350-500 bp, and the library was constructed using the TruSeq DNA LT Sample Prep kit. The 3′ end of the DNA was repaired, and a base was added to the end of the reaction system. A PCR reaction device was established to amplify the ligation product. Amplification products were screened for fragments using Agencourt AMPure XP Medium, and PCR products were analyzed using an Agilent 2100 bioanalyzer. The circularization reaction system was established after the PCR product was denatured into a single strand, and the final library was generated after digesting the uncircularized linear DNA molecules. The fragment size and concentration of the library were evaluated using an Agilent 2100 bioanalyzer. The obtained DNA nanoballs were added to the mesh of the chip using high-density DNA nanochip technology. The DNBSEQ platform was based on rolling cycle amplification (RCA) linear amplification technology, and sequencing used combined probe anchor polymerization (cPAS) technology. The Illumina HiSeq platform was used to perform whole genome resequencing of the samples used.

[0052] 2 Genome-wide association analysis

[0053] 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.

[0054] y=Xα+Zβ+Wμ+e

[0055] 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).

[0056] 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).

[0057] In this study, a total of 11,731,596 SNPs were used for GWAS analysis, and two SNPs significantly associated with pullorum in Shizuohara chicken were identified on chromosome 2 (Table 1). Linkage disequilibrium analysis showed that there was a strong linkage relationship between the two loci (r 2 =0.99, D'=0.97, Figure 2 ).

[0058] Table 1 SNPs on chromosome 2 significantly associated with pullorum in Shizuohara chickens

[0059]

[0060] Genetic analysis of polymorphic loci of the Jingyuan chicken showed that the dominant genotypes of the loci 2:g.129338261A>G and 2:g.129345608T>C were AG and CT, respectively (Table 2).

[0061] Table 2: Distribution of significant SNP genotypes

[0062]

[0063] To further determine the relationship between SNP locus genotype and disease resistance and susceptibility characteristics, we used SPSS23.0 to determine 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: the AG genotype was chr2_129338261 and the CT genotype was chr2_129345608, which were significantly associated with disease resistance, and these genotypes were considered to be beneficial for disease resistance.

[0064] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A SNP molecular marker affecting chicken resistance to pullorum, characterized in that: The SNP marker site is SNP1 or SNP1, and the SNP1 corresponds to the A>G mutation at position 129338261 on chromosome 2 (NC_006089.5) 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 T>C mutation at position 129345608 on chromosome 2 (NC_006089.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.

2.

2. Use of the SNP molecular marker described in claim 1 in improving the pullorum resistance trait of chickens.

3. Use of the reagent and kit for detecting the SNP molecular marker according to claim 1 or 2 in identifying or assisting in identifying the strength of the chicken's resistance to pullorumia.

4. Use of the reagent and kit for detecting the SNP molecular marker according to claim 1 or 2 in genomic selection breeding for pullorum resistance.

5. A method for identifying or assisting in identifying the strength of a chicken's pullorum resistance trait, comprising the following steps: Detect the genotype at position 129338261 and / or the genotype at position 129345608 on chicken chromosome 2 (NC_006089.5). Individuals with genotype at position 129338261 being GG or genotype at position 129345608 being CC are susceptible individuals, while individuals with genotype at position 129338261 being AG or genotype at position 129345608 being CT are resistant individuals.

6. A method for improving the pullorum resistance of chickens, characterized in that: The method comprises the following steps: detecting the genotype of the nucleotide site 129338261 and / or the genotype of the nucleotide site 129345608 on chromosome 2 (NC_006089.5) of the chicken genome GRCg6a, and eliminating non-resistant individuals according to the detection results, so as to increase the genotype frequency related to the pullorum resistance trait at the corresponding site generation by generation.

7. The method according to claim 6, characterized in that The method for detecting the 129338261st genotype on chromosome 2 (NC_006089.5) of the chicken genome GRCg6a genome of the chicken, and / or the 129345608th genotype comprises the following steps: (1) Extracting genomic DNA from the chicken to be tested; (2) testing 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.

8. The method according to claim 6, characterized in that The chicken population includes Shizuohara chicken and its synthetic strains.

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