SNP (Single Nucleotide Polymorphism) molecular marker related to local chicken body type character and application thereof

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to a local chicken body type character, the SNP molecular marker is located on a chicken chromosome 4, the number of the SNP molecular marker is Chr475876885, and the genome version is GRCg6a; the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, the SNP molecular marker is located at the 401bp of the nucleotide sequence of the SEQ ID NO.1 and has C / T polymorphism, and the genotype of the site of the polymorphism is that TT corresponds to a large-size individual, and the genotype of CC corresponds to a small-size individual. According to the SNP molecular marker related to the body type character of the local chicken, individuals are directly bred on the genome level, phenotypic information is not depended on, the selection efficiency can be remarkably improved for characters which are low in heritability and difficult to measure, and the breeding process can be accelerated.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a SNP molecular marker related to the body shape trait of a local chicken and its application. Background Technology

[0002] In the broiler industry, body size is one of the most important economic traits. Increased body size translates to increased chicken yield, and the rate of growth also affects chicken quality. In poultry breeding, chicken body size is directly determined by bone and muscle development; therefore, assessing body size by measuring weight and body dimensions provides an objective reflection of poultry growth and skeletal development. Local chicken breeds possess unique meat flavors and are of significant value in the broiler industry; however, their growth rate is relatively slow, and body size varies between different breeds and even within the same breed. Therefore, selective breeding and improvement targeting the body size traits of local chickens can enhance economic efficiency.

[0003] With the advent of high-density whole-genome chips for livestock and poultry, and the continuous deepening of methods, influencing factors, and breeding programs for genomic selection, genomic selection is gradually becoming a research hotspot in the field of livestock and poultry breeding. Among them, SNP markers, with their accurate, efficient, and simple and rapid genotyping methods, have become the most important molecular genetic markers for the mining of genetic mechanisms across the livestock and poultry genome and in breeding practices.

[0004] Body size, as a complex quantitative trait in poultry, has been the subject of numerous studies and candidate genes related to chicken body size, thanks to advancements in genetics and genomics. Our applicant previously developed the first Guangxi high-quality broiler gene breeding chip, "GuiXin-1," based on a batch of candidate genes and molecular markers for important economic traits in high-quality broilers. Using "GuiXin-1," we located SNP loci related to body size traits in local chickens via GWAS, designed primers suitable for identifying these SNP loci, and performed PCR amplification, enzyme digestion, and agarose gel electrophoresis. The results of agarose gel electrophoresis were then used to identify different genotypes of chicken body size traits. This method significantly improves the efficiency and accuracy of genotype determination, is simple, has a short typing time, requires no sequencing or special instruments, is low-cost, and easily promoted and popularized.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a SNP molecular marker related to the body shape trait of local chickens, which can easily and quickly identify chicken body shape trait genes and shorten breeding time.

[0007] Another object of the present invention is to provide an application of SNP molecular markers related to the body type trait of local chickens.

[0008] To achieve the above objectives, this invention provides molecular markers significantly associated with local and body size traits, identified through genotyping using the "Guixin-1" microarray. A local chicken body size trait-related SNP molecular marker is located on chicken chromosome 4. The SNP molecular marker is numbered Chr4_75876885, and its genomic version is GRCg6a. Its nucleotide sequence is shown in SEQ ID NO.1. The SNP molecular marker is located at 401 bp of the nucleotide sequence in SEQ ID NO.1 and exhibits C / T polymorphism. The genotype at this polymorphic site is TT, corresponding to large-body individuals, and CC, corresponding to small-body individuals. In the nucleotide sequence SEQ ID NO.1, Y represents C / T.

[0009] As mentioned above, SNP molecular markers indicate that the body type traits of local chickens include shank circumference and claw weight.

[0010] Primer pairs for amplifying the above-mentioned SNP molecular markers are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0011] The application of a primer pair for detecting the above-mentioned SNP molecular markers or a kit containing the primer pair in identifying the body type trait of local chickens; the upstream primer sequence of the primer pair is shown in SEQ ID NO.2, and the downstream primer sequence is shown in SEQ ID NO.3.

[0012] The application of SNP molecular markers or primer combinations described above in molecular marker-assisted breeding of local chickens.

[0013] Application of the above-mentioned SNP molecular markers in assisted breeding of local chicken body type traits.

[0014] A method for detecting the genotype of local chickens using molecular biology techniques includes the following steps: primers are designed for the nucleotide sequences flanking the above-mentioned SNP molecular marker sites, and the primers are used to perform genotyping on the local chicken materials to be tested, thereby identifying the genotype of the local chicken to be tested.

[0015] The present invention also provides the application of the SNP molecular marker in the preparation of detection kits.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The SNP molecular markers related to the body shape trait of local chickens provided by the present invention can directly select individuals at the genomic level without relying on phenotypic information. For traits with low heritability and difficult to measure, the selection efficiency can be significantly improved and the breeding process can be accelerated.

[0018] (2) This invention designs primers suitable for molecular markers related to body type traits in local chickens. This method can greatly improve the efficiency and accuracy of genotyping, and is simple, quick to genotype, does not require sequencing or special instruments, is low in cost, and is easy to promote and popularize. The method established by this invention can quickly identify the genotype of the individual to be tested, shortening the breeding time. Attached Figure Description

[0019] Figure 1 This is a genome-wide association analysis of tibia circumference in a 120-day-old population of Guangxi Ma chickens in Example 1 of this invention: A is the QQ plot; B is the Manhattan plot.

[0020] Figure 2 This is a genome-wide association analysis of the claw weight of a 120-day-old Guangxi Ma chicken population in Example 1 of the present invention: A is the QQ plot; B is the Manhattan plot.

[0021] Figure 3 This is an electrophoresis image of the PCR amplification and enzyme digestion results of SNP in the Nandan Yao chicken population in Example 2 of the present invention. Detailed Implementation

[0022] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0023] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0024] Unless otherwise specified, all raw materials and reagents used in the examples are commercially available. Example

[0025] Molecular markers related to body size in local chickens were screened using genotyping based on the "Guixin-1" chip.

[0026] 287 roosters and 850 hens of Guangxi Ma chickens aged 120 days were randomly selected. The steps for screening molecular markers are as follows.

[0027] (1) Phenotypic data collection: Phenotypic data of body weight and body size traits (including body length, keel length, chest depth, chest width, hip width, tibia length, tibia circumference, etc.) were determined with reference to the agricultural industry standard of the People's Republic of China NY / T 823-2004.

[0028] (2) DNA extraction and sequencing: Blood from experimental chickens was collected and stored at -20℃. Genomic DNA was extracted using the phenol-formaldehyde method as described in *Molecular Cloning: A Laboratory Manual IV*. The purity of the DNA was tested using a UV spectrophotometer, and after passing the test, chip sequencing was performed. A DNBSEQ-T7 sequencer with a PE150 sequencing frequency was used for sequencing. The sequencing data was filtered to obtain valid data.

[0029] (3) Data processing and quality control: SNP variant information files were generated using GATK-HaplotypeCaller variant detection. Target loci were then extracted and genotyped, retaining loci with a sequencing depth greater than 10×. To reduce false positives in GWAS, PLINK software was used for further quality control of SNPs and genotyped individuals. Filtering criteria included: lower allele frequency >0.05, deletion rate per individual <0.1%, deletion rate per SNP <0.1%, and Hardy-Weinberg equilibrium >1e-6. Principal component analysis (PCA) was used to examine the population structure of the experimental population. This analysis was performed using the --pca parameter in PLINK software.

[0030] (4) Genome-wide association analysis (GWAS): Phenotypes and genotypes are associated using a mixed linear model based on EMMAX (efficient mixed-model association expedited). First, a kinship matrix is ​​constructed using SNP markers. Then, EMMAX is used to perform GWAS on the phenotypic, genotypic, and kinship data. The statistical model formula is:

[0031]

[0032] y is a vector representing the quantitative phenotypic values, X corresponds to the fixed-effects correlation matrix, β is a vector composed of fixed effects, μ is a vector composed of individual additive genetic random effects, Z corresponds to the random-effects correlation matrix, i.e., the SNP effect matrix, and e is a vector composed of all random errors. Gender is added as a covariate to the model to correct for phenotypic differences.

[0033] (5) Screening and extracting molecular markers related to the body size of local chickens: Loci that reached a significant association level were extracted using R software, and then the most significant SNP molecular marker locus (Chr4_75876885) was selected. The locus was significantly associated with the shank circumference and claw weight of Guangxi Ma chicken, with significance levels of 1.17E-06 and 2.68E-13, respectively. This locus was annotated to the LCORL gene, and in recent years, the LCORL gene has been repeatedly confirmed to be related to the body size of livestock and poultry. Therefore, this locus can be used for the breeding of body size traits of local chickens.

[0034] The SNP molecular marker is located on chicken chromosome 4, with the marker number Chr4_75876885 and genomic version GRCg6a. The sequences of the base fragments before and after the SNP marker are shown in SEQ ID NO.1. The SNP marker site is located at 401 bp of the nucleotide sequence in SEQ ID NO.1 and exhibits C / T polymorphism. The genotype at this polymorphic site is TT, corresponding to large individuals, and CC, corresponding to small individuals. In the nucleotide sequence SEQ ID NO.1, Y represents C / T. Example

[0035] Genotyping of SNP causal mutations in Nandan Yao chicken population

[0036] Six chickens of the Guangxi Nandan Yao Chicken breed were randomly selected. The identification method is as follows:

[0037] I. Genomic DNA extraction from the blood of the individual chicken to be tested:

[0038] Blood was collected from individual chickens and stored at -20°C. Genomic DNA was extracted using the phenol-formaldehyde method described in Molecular Cloning, Laboratory Manual IV. The purity of the extracted DNA was then determined using a UV spectrophotometer.

[0039] II. Specific PCR amplification:

[0040] (1) Primer design and dilution: Primers are designed as shown in SEQ ID NO.2-SEQ ID NO.3 and diluted to 10 μM with ddH2O;

[0041] SEQ ID NO.2, upstream primer SNP-F: 5'-TGCATAATGCTAAGGCAAGCG-3'

[0042] SEQ ID NO.3, Downstream primer SNP-R: 5'-GGGGTTCTGTTACTTTCCCCA-3'

[0043] (2) Preparation of PCR reaction system: 20.0 μL total reaction system, including 10.0 μL 2×SanTap PCR MasterMix, 7.5 μL ddH2O, 50 ng template DNA, and 8.0 pmol / L each of upstream and downstream primers.

[0044] (3) The PCR reaction program is as follows: pre-denaturation 94 ℃, 5 min; denaturation 94 ℃, 30 s; annealing temperature 50-60 ℃ for 30 s; extension at 72 ℃, 60 s / kb; final extension at 72 ℃ for 10 min, for a total of 35 cycles.

[0045] III. Single enzyme digestion experiment:

[0046] The obtained PCR product needs further processing. The enzyme digestion system is as follows: 2.33 μL of PCR product, 0.33 μL of restriction endonuclease (Box I), 0.67 μL of 10× Buffer, 6.67 μL of ultrapure water, and enzyme digestion reaction at 37℃ for 0.5 h.

[0047] IV. Gel electrophoresis and imaging for judgment:

[0048] (1) Gel preparation: 20 ml of 2% agarose gel;

[0049] (2) Sample loading: Add 6 μL of each sample in the order of Marker and enzyme digestion product;

[0050] (3) Electrophoresis: agarose gel electrophoresis at 180V for 15min.

[0051] Result Interpretation: The electrophoresis pattern of the sample in this embodiment is shown below. Figure 1 Compare with Marker (M) to determine: From Figure 3 As can be seen from the electrophoresis results of samples 1-2, there is one band at around 500bp, indicating that the SNP is a homozygous mutant genotype (TT); samples 5-6 show two bands at around 250bp and 100bp respectively, indicating that the SNP is a homozygous wild type (CC); samples 3-4 show three bands at around 500bp, 250bp and 100bp respectively, indicating that the SNP is a heterozygous mutant type (TC).

[0052] As can be seen from the above, the identification primers for chicken body size trait-related molecular markers designed by the identification method of the present invention can effectively perform genotyping of SNP causal mutations, thereby facilitating rapid identification and screening of larger individuals in chicken farms for application in production.

[0053] This invention utilizes primers designed and synthesized for PCR amplification, and identifies the genotype of the tested individual by detecting the PCR amplification products using agarose gel electrophoresis. The identification primers designed in this invention possess independent characteristics, effectively recognizing SNP causal mutation sites. This identification method can rapidly and accurately identify body size-related genotypes. Therefore, this causal mutation can serve as an effective molecular genetic marker for chicken body size traits, effectively promoting the breeding of superior chicken breeds.

[0054] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A SNP molecular marker associated with body size traits of local chicken, characterized in that, The SNP molecular marker is located on chicken chromosome 4, and the SNP molecular marker is numbered as: Chr4_75876885, the genome version is: GRCg6a; the nucleotide sequence is shown as SEQ ID NO. 1, the SNP molecular marker is located at 401bp of the nucleotide sequence shown as SEQ ID NO. 1, has C / T polymorphism, the genotype of the site of the polymorphism is TT corresponding to a large body type individual, and the genotype is CC corresponding to a small body type individual.

2. The SNP molecular marker of claim 1, wherein, The body size trait of the local chicken includes tibial circumference and claw weight of the local chicken.

3. A primer pair amplifying the SNP molecular marker of claim 1, characterized in that, The sequence of the upstream primer of the primer pair is shown as SEQ ID NO. 2, and the sequence of the downstream primer is shown as SEQ ID NO.

3.

4. Application of the primer pair for detecting the SNP molecular marker in claim 1 or a kit containing the primer pair in identifying the body size trait of the local chicken; The sequence of the upstream primer of the primer combination is shown as SEQ ID NO. 2, and the sequence of the downstream primer is shown as SEQ ID NO.

3.

5. Application of the SNP molecular marker in claim 1 or the primer combination in claim 2 in local chicken molecular marker assisted breeding.

6. Application of the SNP molecular marker in claim 1 in local chicken body size trait assisted breeding.

7. A method for detecting the genotype of a local chicken using molecular biology techniques, characterized by, The method comprises the following steps: According to the SNP molecular marker site in claim 1, the nucleotide sequences of the flanking sequences are designed into primers, the primers are used for genotyping of the local chicken material to be tested, and the genotype of the local chicken to be tested is identified.

8. Application of the SNP molecular marker in claim 1 in preparing a detection kit.

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