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

Abstract

The invention provides SNP (Single Nucleotide Polymorphism) loci related to chicken pullorum disease resistance on a chicken Z chromosome, and the loci of the SNP marker are eight SNP loci on the chicken Z chromosome (NC006127.5). 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 Z chromosome related to resistance to pullorum and an application thereof. Background Art

[0002] In the context of globalization, the livestock and poultry farming industry is facing unprecedented challenges and opportunities. In recent years, with the rapid development of biotechnology and information technology, the chicken breed industry is undergoing a profound transformation from traditional breeding mode to intelligent and precise breeding. In this process, genetic improvement and disease prevention and control have become the two core issues in the development of the chicken breed 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 breed 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] The prevalence of the disease has led to problems such as slow resistance to pullorum, decreased production performance, and increased mortality in the chickens, which has 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 chickens through breeding, but the infection of pullorum, especially chronic infection, often leads to the inability to fully demonstrate the genetic potential of the chickens, 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 the Z chromosome (NC_006127.5) of the chicken genome reference sequence.

[0008] The SNP site of the molecular marker corresponds to any molecular marker including SNP1 to SNP8 on the Z chromosome (NC_006127.5) of the chicken GRCg6a genome.

[0009] The SNP1 corresponds to the C>G mutation at position 27712775 on chromosome Z (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.1;

[0010] The SNP2 corresponds to the molecular marker of the A>G mutation at position 27712923 on the Z chromosome (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.2;

[0011] The SNP3 corresponds to the molecular marker of the C>T mutation at position 27718418 on the Z chromosome (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.3;

[0012] The SNP4 corresponds to the molecular marker of the G>T mutation at position 27718468 on the Z chromosome (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.4;

[0013] The SNP5 corresponds to the molecular marker of T>A mutation at position 27718765 on chromosome Z (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.5;

[0014] The SNP6 corresponds to the molecular marker of the T>A mutation at position 27718769 on the Z chromosome (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.6;

[0015] The SNP7 corresponds to the molecular marker of the C>T mutation at position 27718793 on the Z chromosome (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.7;

[0016] The SNP8 corresponds to the molecular marker of the G>A mutation at position 27718912 on chromosome Z (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.8.

[0017] The sequence of the SNP marker is specifically shown in the following table

[0018]

[0019]

[0020]

[0021] Another object of the present invention is to provide the use of the above molecular markers in screening chicken individuals with pullorum resistance traits. Specifically, the genotype of the above SNP molecular marker site on the chicken Z chromosome (NC_006127.5) is detected, and non-resistant individuals are eliminated 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.

[0022] Optionally, individuals whose genotypes of SNP1 to SNP8 are GG, GG, TT, TT, AA, AA, TT or AA, respectively, are eliminated.

[0023] Preferably, individuals whose genotypes of SNP1 to SNP8 are CC, AA, CC, GG, TT, TT, CC or GG, respectively, are retained.

[0024] In a preferred embodiment, individuals whose genotypes of SNP1 to SNP8 are CC, AA, CC, GG, TT, TT, CC and GG respectively are retained as breeding chickens.

[0025] 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 above reagent or kit is used to detect the genotype of the SNP molecular marker as claimed in claim 1 on the chicken Z chromosome (NC_006127.5). Individuals whose genotypes of SNP1 to SNP8 are GG, GG, TT, TT, AA, AA, TT or AA are susceptible individuals, and individuals whose genotypes of SNP1 to SNP8 are CC, AA, CC, GG, TT, TT, CC or GG are resistant individuals.

[0026] In a preferred embodiment, individuals whose genotypes of SNP1 to SNP8 are CC, AA, CC, GG, TT, TT, CC and GG respectively are disease-resistant individuals.

[0027] Another object of the present invention is to provide a reagent or kit for detecting the above molecular markers for use in chicken genomic selection breeding. Specifically, the reagent or kit is used to detect the genotype of the above SNP molecular marker site on the chicken Z chromosome (NC_006127.5), and non-resistant individuals are eliminated 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.

[0028] Optionally, individuals whose genotypes of SNP1 to SNP8 are GG, GG, TT, TT, AA, AA, TT or AA, respectively, are eliminated.

[0029] Preferably, individuals whose genotypes of SNP1 to SNP8 are CC, AA, CC, GG, TT, TT, CC or GG, respectively, are retained.

[0030] In a preferred embodiment, individuals whose genotypes of SNP1 to SNP8 are CC, AA, CC, GG, TT, TT, CC and GG respectively are retained as breeding chickens.

[0031] Another object of the present invention is to provide a method for genetic improvement of chickens, the method comprising: detecting the genotype of the molecular marker loci affecting the excellent resistance to pullorum in chickens in the breeder chicken resource population, eliminating non-resistant individuals according to the detection results, so as to increase the genotype frequency related to the resistance to pullorum trait at the corresponding loci generation by generation. The chicken population includes Jingyuan chicken and its synthetic line.

[0032] Optionally, individuals whose genotypes of SNP1 to SNP8 are GG, GG, TT, TT, AA, AA, TT or AA, respectively, are eliminated.

[0033] Preferably, individuals whose genotypes of SNP1 to SNP8 are CC, AA, CC, GG, TT, TT, CC or GG, respectively, are retained.

[0034] In a preferred embodiment, individuals whose genotypes of SNP1 to SNP8 are CC, AA, CC, GG, TT, TT, CC and GG respectively are retained as breeding chickens.

[0035] The specific steps include:

[0036] (1) Extracting genomic DNA from the chicken to be tested;

[0037] (2) testing the genotype of the chicken to be tested;

[0038] (3) Based on the test results, the genotype of the chicken to be tested at the aforementioned SNP molecular marker site on chromosome Z (NC_006127.5) of the chicken reference genome GRCg6a is determined.

[0039] (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.

[0040] Optionally, individuals whose genotypes of SNP1 to SNP8 are GG, GG, TT, TT, AA, AA, TT or AA, respectively, are eliminated.

[0041] Preferably, individuals whose genotypes of SNP1 to SNP8 are CC, AA, CC, GG, TT, TT, CC or GG, respectively, are retained.

[0042] In a preferred embodiment, individuals whose genotypes of SNP1 to SNP8 are CC, AA, CC, GG, TT, TT, CC and GG respectively are retained as breeding chickens.

[0043] Compared with the prior art, the present invention has the following advantages and effects:

[0044] First, new molecular markers affecting the pullorum resistance trait in chickens were identified.

[0045] Second, it was determined that there was a strong linkage relationship between these molecular markers.

[0046] 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

[0047] 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.

[0048] 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).

[0049] Figure 2 This is the linkage map of the SNP loci of the Jingyuan chicken on chromosome Z of the chicken reference genome GRCg6a. DETAILED DESCRIPTION

[0050] 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.

[0051] 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.

[0052] Example 1 Statistical analysis of resistance of Jingyuan chicken to pullorum

[0053] 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, respectively, and sent to Jiangsu Poultry Science Research Institute (Jiangsu, China) and Ningxia Hui Autonomous Region Haobiao Testing Service Research Institute (Yinchuan, China) for pullorum plate agglutination test. Jiangsu Poultry Science Research Institute detected a total of 53 strong positive individuals and 48 weak positive individuals; Ningxia Hui Autonomous Region 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 wing veins 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.

[0054] Example 2 Resequencing and SNP analysis of pullorum resistance trait

[0055] 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.

[0056] 2 Genome-wide association analysis

[0057] 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.

[0058] y=Xα+Zβ+Wμ+e

[0059] 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).

[0060] 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).

[0061] In this study, a total of 11,731,596 SNPs were used for GWAS analysis, and 8 SNPs significantly associated with pullorum in Shizuohara chicken were identified on chromosome Z (Table 1). The 5.99kb region on chromosome Z is a significant SNP peak significantly associated with pullorum in Shizuohara chicken. The SNP sites in this region form a haplotype block, indicating that there is linkage disequilibrium between these sites. LD analysis showed that these SNPs showed a strong LD (r2 = 0.99) relationship ( Figure 2 ), the most significant 8 SNPs were in strong linkage disequilibrium (D'>=0.97).

[0062]

[0063]

[0064] Genetic analysis of polymorphic loci of Jingyuan chicken showed that the dominant genotype of Z:g.27712775C>G and Z:g.27712923A>G was GG; the dominant genotype of Z:g.27718418C>T, Z:g.27718468G>T and Z:g.27718793C>T was TT, and the dominant genotype of Z:g.27718765T>A, Z:g.27718769T>A and Z:g.27718912G>A was AA (Table 2).

[0065] Table 2: Distribution of significant SNP genotypes

[0066]

[0067] 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 GG genotypes were chrZ_27718468 and chrZ_27718912, the CC genotypes were chrZ_27712775, chrZ_27718418 and chrZ_27718793, the AA genotype was chrZ_27712923, and the TT genotypes were chrZ_27718765 and chrZ_27718769 were significantly associated with disease resistance, and these genotypes were considered to be beneficial for disease resistance.

[0068] 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 molecular marker is SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 or SNP8, The SNP1 corresponds to the C>G mutation at position 27712775 on chromosome Z (NC_006127.5) 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>G mutation at position 27712923 on the Z chromosome (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.2; The SNP3 corresponds to the molecular marker of the C>T mutation at position 27718418 on the Z chromosome (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.3; The SNP4 corresponds to the molecular marker of the G>T mutation at position 27718468 on the Z chromosome (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.4; The SNP5 corresponds to the molecular marker of T>A mutation at position 27718765 on chromosome Z (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.5; The SNP6 corresponds to the molecular marker of the T>A mutation at position 27718769 on the Z chromosome (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.6; The SNP7 corresponds to the molecular marker of the C>T mutation at position 27718793 on the Z chromosome (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.7; The SNP8 corresponds to the molecular marker of the G>A mutation at position 27718912 on chromosome Z (NC_006127.5) of the chicken genome GRCg6a genome, and its nucleotide sequence is shown in SEQ ID NO.

8.

2. A SNP molecular marker group affecting chicken resistance to pullorum, characterized in that: The SNP marker group comprises all the SNP molecular markers described in claim 1.

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

4. 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.

5. 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.

6. A method for identifying or assisting in identifying the strength of a chicken's pullorum resistance trait, comprising the following steps: The genotype of the SNP molecular marker as described in claim 1 on the chicken Z chromosome (NC_006127.5) is detected. Individuals whose genotypes of SNP1 to SNP8 are GG, GG, TT, TT, AA, AA, TT or AA are susceptible individuals, and individuals whose genotypes of SNP1 to SNP8 are CC, AA, CC, GG, TT, TT, CC or GG are resistant individuals.

7. A method for improving the pullorum resistance of chickens, characterized in that: The method comprises the following steps: detecting the genotype of the SNP molecular marker as claimed in claim 1, and eliminating non-resistant individuals according to the detection result, so as to increase the genotype frequency related to the pullorum resistance trait at the corresponding site generation by generation.

8. The method according to claim 7, characterized in that The method for detecting the genotype of the SNP molecular marker according to claim 1 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, determine the genotype of the SNP molecular marker of the chicken to be tested as described in claim 1.

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

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