Pls3 gene snp marker for rapid detection of economic traits of tibetan chicken and detection method and application thereof

Abstract

This invention belongs to the field of molecular biology detection, specifically relating to a rapid detection method for economic traits of Tibetan chickens. PLS3 Gene SNP markers, detection methods, and applications. This invention is the first to discover: Tibetan chickens... PLS3 The G / A mutation at position 28469 of the gene and its presence BstZ17 The presence or absence of the I restriction enzyme site is closely related to the main economic traits of Tibetan chickens. Based on this new discovery and the SNP site, this invention provides a method for rapidly detecting this SNP site and screening for economic traits in Tibetan chickens. The method is simple, convenient, fast, low-cost, and provides reliable results.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, specifically relating to the rapid detection of economic traits in Tibetan chickens. PLS3 Gene SNP markers, detection methods, and applications. Background Technology

[0002] Single nucleotide polymorphism (SNP) refers to a variation of a single nucleotide at a specific position in a genomic DNA sequence, caused by base transitions, insertions, deletions, or transversions. In other words, an SNP is a single base variation at a specific site in a chromosomal DNA sequence. This variation exhibits inter-individual differences due to various factors, and when the frequency of such a variation in a population reaches 1% or higher, it is considered polymorphic. SNP markers are third-generation DNA genetic markers. Due to their high specificity, they have become an effective means of detecting gene expression levels in genetic material and are widely used in research on biological genetic diversity and molecular breeding. SNP analysis can quickly screen for target genes and improve the utilization rate of germplasm resources; therefore, SNP detection technology has great potential and application prospects. However, the high cost of SNP detection limits its large-scale application in the animal breeding industry through genome-wide selection. Furthermore, how to accurately infer haplotypes from SNPs and how to estimate the genetic effects of haplotypes and SNPs to provide a basis for accurate breeding value estimation are also challenges in SNP applications.

[0003] Animal molecular breeding combines molecular genetics theory with traditional quantitative genetics theory. It utilizes the combination of molecular and polygenic genetic information to conduct genetic analysis of traits to be improved in livestock, thereby enabling rapid and efficient breeding of livestock and poultry breeds. Molecular breeding, also known as molecular mark-assist selection (MAS), uses DNA molecular markers to select breeding materials, thereby improving the traits of livestock and poultry breeds. MAS combines modern molecular biology with traditional genetic breeding methods for the selection of new breeds. In livestock and poultry breeding, by selecting DNA markers closely related to economic traits, early selection can be achieved, improving selection accuracy and leading to greater genetic progress.

[0004] PLS3 T-plastin, also known as T-filament protein, is a subtype of the actin crosslinking and binding protein family. PLS3Encoding an actin-binding protein with a wide expression pattern, it plays a role in actin binding and binding within the cytoskeleton by inhibiting cofilin-mediated depolymerization of actin filaments. Currently, there is no... PLS3 A study on the relationship between genes and economic traits of Tibetan chickens. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid method for detecting economic traits in Tibetan chickens. PLS3 Gene SNP markers, detection methods, and applications.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is: an SNP molecular marker affecting the traits of Tibetan chickens, wherein the SNP molecular marker is located in the Tibetan chicken... PLS3 At position 28469 of the gene, the mutated base is either G or A. The genotype of the SNP molecular marker is any one of GG, AA, or AG.

[0007] Correspondingly, the SNP molecular markers are used in detecting economic traits of Tibetan chickens or in the breeding of Tibetan chickens.

[0008] Accordingly, kits, test strips, or detection reagents containing the SNP molecular marker. The kits, test strips, or detection reagents include at least a specific primer pair for amplifying the SNP molecular marker. The primer pair includes: an upstream primer as shown in SEQ ID NO: 1; and a downstream primer as shown in SEQ ID NO: 2.

[0009] Accordingly, the application of the kit, test strip, or detection reagent in detecting Tibetan chicken traits. The application includes the following steps:

[0010] (1) Obtain blood from the Tibetan chicken to be tested;

[0011] (2) Extracting genomic DNA from blood;

[0012] (3) Perform PCR amplification on genomic DNA to obtain PCR amplification products;

[0013] (4) The PCR amplification products were digested with enzymes, electrophoresed and analyzed to determine the genotype of the SNP molecular marker in the Tibetan chicken to be tested.

[0014] Accordingly, a method for rapid identification of the SNP molecular marker includes the following steps:

[0015] (1) PCR amplification of Tibetan chicken PLS3 Gene;

[0016] (2) Determine whether the obtained PCR amplification product can be digested by the restriction endonuclease BstZ17 I; if both strands can be digested by BstZ17 I, then it is identified as... PLS3 The genotype at position 28469 of the gene is GG; if neither strand can be cleaved by BstZ17 I, it is identified as... PLS3 The genotype at position 28469 of the gene is AA; if only one strand can be cleaved by BstZ17 I, it is identified as... PLS3 The genotype at position 28469 of the gene is AG.

[0017] Accordingly, a method for rapidly determining the economic traits of Tibetan chickens using the SNP molecular markers includes the following steps:

[0018] (1) PCR amplification of Tibetan chicken PLS3 Gene;

[0019] (2) Determine whether the obtained PCR amplification product can be digested by the restriction endonuclease BstZ17 I; if the result shows that both strands can be digested by BstZ17 I, then the product is considered valid. PLS3 The genotype at position 28469 of the gene is GG; if the result shows that one strand can be digested by BstZ17 I, then the gene is identified as GG. PLS3 The genotype at position 28469 of the gene is AG; if the result shows that neither strand can be digested by BstZ17I, then the identification... PLS3 The genotype at position 28469 of the gene is AA;

[0020] Identification PLS3 The economic traits of Tibetan chickens with genotype GG at gene position 28469 are superior to those with genotype AA, and the economic traits of Tibetan chickens with genotype AA are superior to those with genotype AG.

[0021] This invention has the following beneficial effects: This invention is the first to discover: Tibetan chicken PLS3 The A / G mutation at position 28469 of the gene is closely related to the presence or absence of the BstZ17 I restriction site, and is also closely related to the main economic traits of Tibetan chickens (breast width, live weight, semi-eviscerated weight, leg muscle weight, and leg muscle percentage).

[0022] Based on this new discovery and SNP site, this invention provides a method for rapidly detecting the SNP site and screening for economic traits in Tibetan chickens. The method is simple, convenient, fast, low-cost, and yields reliable results. Attached Figure Description

[0023] Figure 1 for PLS3 Sequencing peak diagram of gene 28469 A > G site;

[0024] Figure 2 for PLS3 Electrophoresis results of enzyme digestion at gene site 28469 A > G. Detailed Implementation

[0025] This invention marks the first discovery of Tibetan chickens. PLS3 The A / G mutation at position 28469 of the gene (located in the intron region) is closely related to the presence or absence of the BstZ17 I restriction site in the gene, and is also closely related to the main economic traits of Tibetan chickens (breast width, live weight, semi-eviscerated weight, leg muscle weight, and leg muscle percentage).

[0026] If no mutation occurs at this site and the genotype is AA, then neither strand has the BstZ17 I restriction site, and the main economic traits of Tibetan chickens (breast width, live weight, semi-eviscerated weight, leg muscle weight, and leg muscle percentage) are moderate.

[0027] If the mutation at this site results in the AG genotype, a chain forms the BstZ17 I restriction site, and the main economic traits of Tibetan chickens (breast width, live weight, semi-eviscerated weight, leg muscle weight, and leg muscle percentage) decrease compared to the AA genotype.

[0028] If the mutation at this site results in the genotype GG, then both strands will have the BstZ17 I restriction site, and the main economic traits of Tibetan chickens (breast width, leg muscle weight, and leg muscle rate) will be significantly increased compared to the AA genotype.

[0029] Based on this new discovery, the present invention provides a primer pair for rapid detection of Tibetan chickens, the primer pair being based on SNP sites, i.e., Tibetan chickens. PLS3 The design was based on position 28469 of the gene to accurately amplify the DNA fragment containing the SNP site.

[0030] An optional scheme is that the primer pairs include:

[0031] Upstream primer: 5'-TGGGGTGGCTTTTTGTCAGT-3' (SEQ NO ID:1)

[0032] Downstream primer: 5'-CAGGGGGAAGGAAGGCAAAT-3' (SEQ NO ID:2)

[0033] This invention also provides a method for rapid detection of Tibetan chickens using the primer pair: PCR amplification of Tibetan chickens using the primer pair. PLS3A partial gene fragment (preferably amplified from position 28297 to 28809, with a total fragment length of 531 bp) was amplified by PCR. The DNA fragment product was analyzed by Sanger sequencing. The PCR amplification product was digested with the restriction endonuclease BstZ17 I, followed by agarose gel electrophoresis. Identification was performed based on the agarose gel electrophoresis results. PLS3 Genotype of the SNP locus at position 28469 of the gene.

[0034] If the result shows that both strands can be digested by BstZ17 I, then the identification... PLS3 The genotype at position 28469 of the gene is GG, and this indicates that the Tibetan chicken exhibits excellent economic traits. If the result shows that one strand can be digested by BstZ17 I, then the identification... PLS3 The genotype at position 28469 of the gene is AG, and this indicates poor economic traits in this Tibetan chicken. If the result shows that neither strand can be digested by BstZ17 I, then the identification... PLS3 The genotype at position 28469 of the gene is AA, and the economic trait of this Tibetan chicken is identified as ordinary.

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0036] Example 1: Tibetan Chicken PLS3 Gene DNA sequence amplification and SNP detection

[0037] 1. Tibetan chicken sample collection: In this embodiment, three strains of Tibetan chickens were collected as the test subjects. The specific collection details are shown in Table 1.

[0038] Table 1. Collection of Tibetan chicken samples

[0039]

[0040] 2. DNA extraction, purity testing, and DNA pool construction from Tibetan chicken blood samples

[0041] DNA was extracted from blood samples of 180 Tibetan chickens using the high-efficiency animal genomic DNA extraction kit from Beijing Qingke Biotechnology Co., Ltd., according to the instructions. Nucleic acid concentration and OD were then analyzed in the DNA samples. 260 / 280 Determination of OD value. 260 / 280 If the value is in the range of 1.8 to 2.0, it indicates that the purity of the DNA sample is relatively high and can be used for subsequent experiments.

[0042] 180 qualified DNA samples were diluted to a concentration of 50 ng / μL with dd H2O, and 10 samples were randomly selected from each strain, for a total of 30 samples. 15 μL of each of these 30 samples was placed in a 1.5 mL centrifuge tube to construct a DNA mixing chamber. After vortex mixing, the mixture was stored at -20°C for later use.

[0043] 3. Primer design

[0044] According to the NCBI database (http: / / www.ncbi.nlm.nih.gov / ), chickens PLS3 The DNA sequence of gene (NC_052535.1) is the reference sequence (https: / / www.ncbi.nlm.nih.gov / nuccore / NC_052535.1?report=genbank&from=2906836&to=2951321). Primer pair XP3 was designed using Primer 5.0, and its sequence is as follows:

[0045] Upstream primer: 5'-TGGGGTGGCTTTTTGTCAGT-3' (SEQ NO ID: 1);

[0046] Downstream primer: 5'-CAGGGGGAAGGAAGGCAAAT-3' (SEQ NO ID:2).

[0047] The primer sequence is located in Tibetan chicken. PLS3 The second intron region of the gene.

[0048] 4. PCR amplification and sequencing

[0049] PCR amplification of DNA pool samples was performed using gradient temperatures (57℃, 58℃, 59℃, 60℃, 61℃, 62℃). The PCR reaction system is shown in Table 2, and the PCR reaction program is shown in Table 3.

[0050] Table 2 PCR reaction system

[0051]

[0052] Table 3 PCR reaction procedure

[0053]

[0054] The PCR amplification products were subjected to agarose gel electrophoresis. Based on the electrophoretic band results, the PCR amplification product with the singlest and brightest temperature band was selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing. The results are as follows: Figure 1 As shown in the diagram. Sequencing peak analysis revealed the following: Figure 1 The AG double peak in the SNP locus is located in Tibetan chicken. PLS3 The 28469th position of the gene. Figure 1 The part highlighted in the middle is the mutation site at position 28469.

[0055] Example 2: Tibetan Chicken PLS3 PCR-RFLP detection of gene SNP loci and analysis of population genetic indicators

[0056] 1. PCR-RFLP detection of SNP sites

[0057] PCR amplification: 180 DNA samples were amplified by PCR at a specified temperature (56℃). The PCR reaction system and procedure were the same as in Example 1. The amplification products were subjected to 2% agarose gel electrophoresis. The results showed that a 513bp band was clearly visible on the electrophoresis pattern of all 180 samples, proving that primer pair XP3 could amplify the target fragment containing this SNP site.

[0058] PCR-RFLP detection: The PCR amplification products corresponding to 180 samples were digested with restriction endonuclease BstZ17 I at 37℃ for 1 h, followed by 3% agarose gel electrophoresis. The digestion system is shown in Table 4.

[0059] Table 4 Enzyme digestion system

[0060]

[0061] Electrophoresis was performed at 90V for 40 minutes. After DNA fragments of different sizes were clearly separated, gel imaging was used to photograph and analyze the samples, and the genotype of each sample was recorded. Results are as follows: Figure 2 As shown.

[0062] Figure 2 In the middle, lane 7 is Marker I, with six bands from bottom to top: 100bp, 200bp, 300bp, 400bp, 500bp, and 600bp. The results showed that the band patterns clearly distinguished the three genotypes (AA, GG, and AG). The AA genotype had one band of 513bp (108 bands); the GG genotype had two bands of 340bp and 173bp (3 bands); and the AG genotype had three bands of 513bp, 340bp, and 173bp (69 bands).

[0063] 2. PLS3 Population genetic marker analysis of gene locus 28469 A > G

[0064] The definitions and formulas used in population genetic index analysis are as follows:

[0065] (1) Genotype frequency: refers to the proportion of individuals with a certain genotype in a population.

[0066] (2) Allele frequency: refers to the ratio of the number of a certain allele on a specific chromosome to the total number of all alleles at that locus in a population. It is intended to reflect the genetic diversity in the population. The ratio ranges from 0 to 1.

[0067] (3) Genetic homozygosity: refers to the degree of allele binding at a specific gene locus on a chromosome within a population. The calculation formula is as follows:

[0068] ,

[0069] n represents the number of alleles, i represents the number of alleles, and Pi represents the allele frequency.

[0070] (4) Genetic heterozygosity: refers to the proportion of heterozygous genotypes at a specific gene locus on a chromosome in a population. The calculation formula is as follows:

[0071] ,

[0072] n represents the number of alleles, i represents the number of alleles, and Pi represents the allele frequency.

[0073] (5) Effective allele count: This refers to the number of alleles required for a given locus in a population to produce the same homozygosity as in the actual population under ideal conditions. It is essentially the reciprocal of genetic homozygosity. The calculation formula is as follows:

[0074] ,

[0075] n represents the number of alleles, i represents the number of alleles, and Pi represents the allele frequency.

[0076] (6) Polymorphism Information Content: This refers to important markers used to assess population genetic polymorphism, reflecting the degree of locus polymorphism. The polymorphism of genetic markers provides a measure of information content in linkage analysis. The calculation formula is as follows:

[0077] ,

[0078] Where n represents the number of alleles, i and j represent the i-th and j-th alleles respectively, and Pi and Pj represent the frequencies of the i-th and j-th alleles respectively.

[0079] (7) Genetic effect analysis of SNP loci: Preliminary statistical analysis of the data was performed using Excel software, and genetic diversity analysis was conducted on each SNP locus. Subsequently, association analysis between the genotype of each SNP locus and growth and slaughter traits was performed using a general linear model in SPSS software. The linear model used was:

[0080] ,

[0081] Where Yijk represents the phenotypic values ​​of growth and slaughter traits; u represents the population mean of growth and slaughter traits; Ai represents the i-th strain (i=1, 2, 3); Bj represents the sex effect (j=1, 2, representing roosters and hens respectively); Ck represents the k-th genotype effect (k corresponds to the genotype at each point); and Dijk represents the random residual effect.

[0082] Tibetan chicken PLS3 The population genetic indicators of gene locus 28469 G>A are shown in Table 5. Among them, PIC > 0.5 indicates high polymorphism, 0.25 < PIC < 0.5 indicates moderate polymorphism, and PIC < 0.25 indicates low polymorphism; χ² 2 The coefficients for Hardy-Weinberg equilibrium test; the numbers in parentheses represent the number of genotypes.

[0083] Table 5 Tibetan Chicken PLS3 Population genetic marker at gene locus 28469 A>G

[0084]

[0085] 3. Tibetan Chicken PLS3 Association analysis of gene locus 28469 A>G with economic traits

[0086] The main economic traits measured for Tibetan chickens included: breast width, live weight, semi-eviscerated weight, leg muscle weight, and leg muscle percentage. One-way ANOVA was performed using SPSS software. PLS3 Association analysis was performed between the genotype at gene locus 28469 A>G and the economic traits of Tibetan chickens. PLS3 The association analysis between the genotype at gene locus 28469 A>G and the economic traits of Tibetan chickens is shown in Table 6. For the same trait, different letters represent significant differences; abc represents P < 0.05.

[0087] Table 6 Tibetan Chicken PLS3 Association analysis of gene locus 28469 G > A with economic traits of Tibetan chickens

[0088]

[0089] As can be seen from Table 6: Tibetan chicken PLS3The A>G locus at gene 28469 was significantly associated with economic traits, and the three genotypes showed significant differences in chest width, live weight, semi-eviscerated weight, leg muscle weight, and leg muscle percentage in Tibetan chickens. The GG genotype significantly increased chest width, live weight, semi-eviscerated weight, leg muscle weight, and leg muscle percentage in Tibetan chickens, indicating that... PLS3 Mutation at gene site 28469 A > G can serve as a candidate molecular marker to improve the economic traits of Tibetan chickens.

[0090] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of SNPs in detecting economic traits of Tibetan chickens or in the breeding of Tibetan chickens, characterized by: The SNP is located in the Tibetan chicken. PLS3 At the 28469th base of the gene, the... PLS3 The gene reference sequence is NC_052535.1, and the mutated base is G or A; the application is to detect the chest width, live weight, semi-eviscerated weight, leg muscle weight or leg muscle rate of the Azang 1, Azang 2 or Azang 3 lineage.

2. The application of a kit or reagent for detecting SNPs in the detection of traits in Tibetan chickens, characterized in that: The Tibetan chicken traits refer to the chest width, live weight, semi-eviscerated weight, leg muscle weight, or leg muscle percentage traits of the Azang 1, Azang 2, or Azang 3 strains; the SNPs are located in Tibetan chickens. PLS3 At position 28469 of the gene, a mutated base is either G or A. PLS3 The gene reference sequence is NC_052535.

1.

3. The application according to claim 2, characterized in that: Includes the following steps: (1) Obtain blood from the Tibetan chicken to be tested; (2) Extracting genomic DNA from blood; (3) Perform PCR amplification on genomic DNA to obtain PCR amplification products; (4) The PCR amplification products are digested with enzymes, electrophoresed and analyzed to determine the genotype of the SNP of the Tibetan chicken to be tested; the enzyme digestion is performed using restriction endonuclease BstZ17 I.

4. The application according to claim 3, characterized in that: The kit or detection reagent includes at least one specific primer pair for amplifying the SNP.

5. The application according to claim 4, characterized in that: The primer pair includes: an upstream primer as shown in SEQ ID NO: 1; and a downstream primer as shown in SEQ ID NO:

2.

6. A method for rapidly determining the economic traits of Tibetan chickens using SNPs, characterized in that: The SNP is located in the Tibetan chicken. PLS3 At the 28469th base of the gene, the... PLS3 The gene reference sequence is NC_052535.1, and the mutant base is G or A; the economic traits are the chest width, live weight, semi-eviscerated weight, leg muscle weight or leg muscle percentage of the Azang 1, Azang 2 or Azang 3 strains. The method includes the following steps: (1) PCR amplification of Tibetan chicken using primer pairs PLS3 The gene, wherein the primer pair comprises: an upstream primer as shown in SEQ ID NO: 1; and a downstream primer as shown in SEQ ID NO: 2; (2) Determine whether the obtained PCR amplification product can be restricted by restriction endonucleases. BstZ17 I Enzyme digestion; if the result is that both strands can be digested... BstZ17 I Enzyme digestion is used for identification. PLS3 The genotype at position 28469 of the gene is GG; if the result is that one strand can be... BstZ17 I Enzyme digestion is used for identification. PLS3 The genotype at position 28469 of the gene is AG; if the result is that neither strand can be... BstZ17 I Enzyme digestion is used for identification. PLS3 The genotype at position 28469 of the gene is AA; Identification PLS3 The economic traits of Tibetan chickens with genotype GG at gene position 28469 are superior to those with genotype AA, and the economic traits of Tibetan chickens with genotype AA are superior to those with genotype AG.

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