SNP (Single Nucleotide Polymorphism) molecular marker related to chicken meat quality traits and application thereof

Abstract

The invention belongs to the technical field of molecular markers, and particularly relates to an SNP (Single Nucleotide Polymorphism) molecular marker related to chicken meat quality traits and application thereof. The SNP molecular marker is a single nucleotide polymorphism site in a chicken PVALB gene, and the single nucleotide polymorphism site comprises one or more of a site 1, a site 2 and a site 3; the site 1 is located at the 51652391bp site of the chromosome 1, and the mutation basic group is A / G; the site 2 is located at the 51659615bp site of the chromosome 1, and the mutant base is C / T; the site 3 is located at the 51659905bp site of the chromosome 1, and the mutant base is C / T. The SNP molecular marker can accurately show traits related to chicken meat quality.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically relating to SNP molecular markers related to chicken meat quality traits and their applications. Background Technology

[0002] Meat quality traits are core indicators for evaluating the economic value and edible quality of livestock and poultry products. Especially in the breeding of high-quality broiler breeders, muscle fat content, meat color, and marbling directly determine the flavor, tenderness, and juiciness of the meat, thus affecting consumer acceptance and market competitiveness. Muscle fat, as the main carrier of flavor substances, can significantly improve meat texture when deposited appropriately; meat color and marbling directly reflect the sensory quality of the meat. Therefore, elucidating the genetic regulatory mechanisms of meat quality traits and developing efficient molecular marker-assisted selection technologies are crucial for accelerating the breeding of high-quality broiler breeds and improving industry efficiency.

[0003] In recent years, molecular genetics research has focused on identifying candidate genes related to meat quality traits, among which the albumin gene (…) PVALB Because it is highly expressed in muscle tissue and acts as a calcium-binding protein involved in regulating muscle contraction and relaxation, it has become a key target for elucidating the genetic mechanisms of meat quality traits. In pig studies, Han Yuqing et al. discovered... PVALB Gene expression levels differed significantly between Tibetan pigs and Large White pigs in skeletal muscle. Upstream regulatory SNP sites (such as G1659C and C1269T) altered transcription factor binding characteristics, affecting muscle growth and development. Xu et al. further identified multiple promoter region SNP sites (such as g.10953632 A>C, g.10953695 G>A, and g.10953713 C>A) in Xidu Black Pigs, which were correlated with muscle fat content, meat color, and marbling score. They also constructed the dominant haplotype GACA, which significantly improved meat quality indicators. However, existing research mainly focuses on pigs and other species, and studies targeting broiler chickens... PVALB The development of functional SNP markers related to genes and meat quality traits is still relatively lacking, and there is a lack of effective genetic markers that can be directly applied to molecular breeding of high-quality broilers.

[0004] In the breeding of high-quality broiler chickens, traditional breeding methods rely on phenotypic selection, which suffers from problems such as long cycles, low efficiency, and insufficient accuracy. Molecular marker technology, particularly specific SNP markers for chicken meat quality traits (especially key indicators such as muscle fat content and muscle fiber density), has not been fully explored and validated, making it difficult to meet the needs of precision breeding. Therefore, there is an urgent need to develop functional SNP molecular markers that are significantly correlated with chicken meat quality traits. Through marker-assisted selection (MAG) technology, individuals carrying advantageous genotypes can be rapidly screened, accelerating the breeding process of high-quality broiler chicken strains and improving meat quality and industry competitiveness. Summary of the Invention

[0005] Based on this, the present invention provides an SNP molecular marker related to chicken meat quality traits and its uses.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: This invention provides, in one aspect, a SNP molecular marker related to chicken meat quality traits, wherein the SNP molecular marker is chicken PVALB Single nucleotide polymorphism (SNP) sites in genes include one or more of sites 1, 2, and 3; site 1 is located at position 51652391 bp on chromosome 1, with a mutated base of A / G; site 2 is located at position 51659615 bp on chromosome 1, with a mutated base of C / T; site 3 is located at position 51659905 bp on chromosome 1, with a mutated base of C / T.

[0007] In another aspect, the present invention provides a primer pair for amplifying the above-mentioned SNP molecular markers.

[0008] Preferably, in the above primer pairs, the primer pair sequences for amplifying the SNP molecular marker containing site 1 are shown in SEQ ID NO:1 and SEQ ID NO:2, the primer pair sequences for amplifying the SNP molecular marker containing site 2 are shown in SEQ ID NO:3 and SEQ ID NO:4, and the primer pair sequences for amplifying the SNP molecular marker containing site 3 are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0009] In another aspect, the present invention provides a detection reagent for detecting SNP molecular markers, the detection reagent comprising the primer pair as described in claim 2 or 3.

[0010] In another aspect, this invention provides the use of the above-mentioned SNP molecular markers related to chicken meat quality traits, or the above-mentioned primers, or the above-mentioned detection reagents, wherein the use is selected from any one of the following 1)-8): 1) prediction of chicken meat quality traits; 2) preparation of reagents for predicting chicken meat quality traits; 3) screening or identifying chicken strains characterized by high intramuscular fat content and / or high muscle fiber density; 4) preparation of reagents for screening or identifying chicken strains characterized by high intramuscular fat content and / or high muscle fiber density; 5) breeding of chicken meat quality traits; 6) marker-assisted breeding related to chicken meat quality traits; 7) breed improvement related to chicken meat quality traits; 8) PVALB Genotyping; for SNP markers containing locus 1, the genotype is AA, corresponding to the traits of high intramuscular fat content and high muscle fiber density; for SNP markers containing locus 2, the genotype is CC, corresponding to the traits of high intramuscular fat content and high muscle fiber density; for SNP markers containing locus 3, the genotype is CC, corresponding to the traits of high intramuscular fat content and high muscle fiber density.

[0011] In another aspect, this invention provides a method for breeding chickens with superior meat quality, the method comprising: selecting individuals with high intramuscular fat content and high muscle fiber density as breeding parents based on SNP molecular markers; the SNP molecular markers are chicken... PVALB Single nucleotide polymorphism (SNP) sites in genes include one or more of sites 1, 2, and 3. Site 1 is located at position 51652391 bp on chromosome 5, with a mutated base of A / G; site 2 is located at position 51659615 bp on chromosome 5, with a mutated base of C / T; site 3 is located at position 51659905 bp on chromosome 5, with a mutated base of C / T. For SNP markers containing site 1, the genotype is AA, corresponding to the traits of high intramuscular fat content and high muscle fiber density; for SNP markers containing site 2, the genotype is CC, corresponding to the traits of high intramuscular fat content and high muscle fiber density; for SNP markers containing site 3, the genotype is CC, corresponding to the traits of high intramuscular fat content and high muscle fiber density.

[0012] The beneficial effects of this invention include: (1) Individuals with the AA genotype at locus g.51652391A>G (locus 1) had significantly higher intramuscular fat content and muscle fiber density in their pectoral muscles than individuals with the AG and GG genotypes (P<0.05), while there were no significant differences in pH value, vitamin B1 content, flesh color and water-holding capacity. (2) Individuals with the CC genotype at locus g.51659615C>T (locus 2) had significantly higher intramuscular fat content and muscle fiber density in their pectoral muscles than individuals with the CT and TT genotypes (P<0.05), while there were no significant differences in pH value, vitamin B1 content, flesh color and water-holding capacity. (3) Individuals with the CC genotype at locus g.51659905C>T (locus 3) had significantly higher intramuscular fat content and muscle fiber density in their pectoral muscles than individuals with the CT and TT genotypes (P<0.05), while there were no significant differences in pH value, vitamin B1 content, flesh color and water-holding capacity. Attached Figure Description

[0013] Figure 1 for PVALB Sanger sequence map of gene g.51652391A>G (site 1); Figure 2 for PVALB Sanger sequence map of gene g.51659615C>T (site 2); Figure 3 for PVALB Sanger sequence map of gene g.51659905C>T (site 3); Figure 4for PVALB Mass spectrometry genotype of gene g.51652391A>G (site 1); Figure 5 for PVALB Mass spectrometry genotype of gene g.51659615C>T (site 2); Figure 6 for PVALB Mass spectrometry genotype of gene g.51659905C>T (site 3). Detailed Implementation

[0014] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0015] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0016] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0017] In the following examples, PVALB The gene sequence is located on chromosome 1, reference genome: bGalGal1.mat.broiler.GRCg7b.

[0018] In the following example, the relevant shape testing methods were as follows: 1 mL of blood was collected from each chicken, and the blood sample was anticoagulated with citrate glucose and frozen at -20℃ for later use; within 1 hour after slaughter, the pectoral muscle of the same side was taken, and samples were taken from the same location with the cut surface perpendicular to the direction of the muscle fibers. The samples were trimmed into tissue blocks with a length of 0.5 cm, a width of 0.5 cm, and a thickness of 0.3 cm. These blocks were first flash-frozen in liquid nitrogen and then transferred to -70℃ for storage to prepare frozen sections; 10 serial sections were made from each frozen tissue block, with a section thickness of 10 µm, and were stained with HE and ATPase (alkaline: pH 10.4), mounted, and observed and measured under a microscope; under 10x magnification, the field of view was selected for observation, and 1-2 complete muscle bundles were selected from each group of sections, with a total of 6-7 muscle bundles taken from each chicken. The area of ​​all muscle fibers was analyzed using Image-ProPlus 4.5 analysis software. The remaining breast muscle samples were sent to the Poultry Quality Supervision and Testing Center (Yangzhou) within 2 hours after slaughter for testing of meat color, tenderness, water-holding capacity, pH value, and intramuscular fat.

[0019] Example 1 This invention uses the first paternal parent (H strain) and terminal paternal parent (S09 strain) of slaughter-grade "Sanhuang" high-quality broiler chickens as research subjects to observe and measure meat color, tenderness, water-holding capacity, pH value, intramuscular fat, and muscle fiber area. 1 mL of blood was collected, and DNA was extracted using the phenol-chloroform method. The H strain and S09 strain were then screened using a Sanger sequencing combined with Sequenom MassARRAY system. PVALB By analyzing the SNP loci of genes, the association between different SNP genotypes and traits such as meat color, tenderness, water-holding capacity, pH value, intramuscular fat, and muscle fiber area of ​​the H and S09 lineages was determined. Dominant genes were screened to provide molecular breeding markers for the breeding of high-quality slaughter-type "three-yellow" broilers.

[0020] (a) Primer design Sanger sequencing primers were based on chicken sequencing data published in GenBank. PVALB The DNA sequence of the gene was analyzed in chickens using Primer 5.0 software. PVALB Four pairs of primers were designed for the gene mRNA region. Primer information is shown in Table 1. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0021] Table 1 PVALB Information on Sanger sequencing primers for gene mRNA regions

[0022] In addition, regarding the results determined by Sanger sequencing PVALBThe two SNP sites of the gene were used to design the MassARRAY SNP primer set using the primer design software Assay Design 3.1 from Sequenom. The primer information is shown in Table 2. The primers were synthesized by Genewiz Biotechnology (Beijing) Co., Ltd.

[0023] Table 2 PVALB Information related to sequencing primers for gene mRNA region SNP sites using mass spectrometry array technology

[0024] (II) PCR amplification, first-generation sequencing and SNP site determination 1 µL of DNA samples from each of 384 individuals was extracted and placed in a 5 ml sterile centrifuge tube. After mixing, the samples were used as DNA templates for PCR amplification using first-generation sequencing primers. The amplification system consisted of 25 µL, including 2.5 µL of 10×PCR buffer (containing Mg2+), 1.0 µL each of 10 µmol / L forward and reverse primers, 2.0 µL of 2 mmol / L dNTPs, 0.5 µL of 5.0 U / µL Taq DNA polymerase, 1.0 µL of 50 ng / µL DNA template, and ultrapure water to a final volume of 25 µL. PCR amplification conditions were as follows: 94 °C pre-denaturation for 5 min; followed by 94 °C denaturation for 30 s, annealing for 15 s (annealing temperatures are shown in Table 1), and extension at 72 °C for 30 s, for 35 cycles; finally, 72 °C extension for 10 min, and storage at 4 °C. After PCR products were detected by 1.5% agarose gel electrophoresis, they were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing using an ABI3730 sequencer. The sequencing results were analyzed using DNAstar software to determine the SNP sites.

[0025] (III) Determination of Sequenom MassARRAY-SNPs Multiplex PCR was used to amplify DNA fragments containing multiple target sites (primers are shown in Table 2). Excess primers and dNTPs in the multiplex PCR reaction system were purified using shrimp alkaline phosphatase (SAP). Single-base extension reactions were performed using dideoxynucleotides (ddNTPs) as substrates with iPLEX enzyme and extension primers designed for each target site (primers are shown in Table 2). The extension products were diluted 3-fold, desalted on resin, and transferred to a 384-well SpectroCHIP chip for matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) on a Sequenom mass spectrometer. Different alleles at the same target site formed different detection peaks due to their different molecular weights. The mass spectrometry peaks were detected using Typer 4.0 software, and the SNP genotype of each site was determined based on the size of the detection peaks.

[0026] The reaction systems are shown below: Multiplex PCR reaction system: HPLC-grade water 972.5 μL, 10×Buffer 331.25 μL, 25 mM MgCl2 172.25 μL, 25 mM dNTP 53 μL, 0.5 μM forward and reverse primer mixture 530 μL, 5 U / μL Taq polymerase 106 μL. After mixing, add 4 μL / well to a 384-well plate, then add 1 μL of 10 ng / μL DNA template to each well. Multiplex PCR amplification program: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 20 s, 56℃ annealing for 30 s, 72℃ extension for 60 s, for a total of 45 cycles of denaturation, annealing, and extension; 72℃ extension for 3 min. SAP digestion system: 810.9 μL HPLC-grade water, 90.1 μL 10×Buffer, 159 μL 1.7 U / μL SAP, mixed and added 24 μL / well to a 384-well plate. SAP reaction program: 37℃ for 40 min, 85℃ for 5 min; Extension reaction system: 400.2 μL HPLC-grade water, 106 μL 10×iPLEX Buffer plus, 106 μL iPLEX stop solution, 426.1 μL 1.0 μM primers, 21.7 μL iPLEX enzyme, mixed and added 2 μL / well to a 384-well plate. Extension reaction program: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 5 s, 1 cycle; 52℃ annealing for 5 s; 80℃ extension for 5 s; annealing and extension for 5 cycles; then denaturation, annealing and extension for another 40 cycles; 72℃ extension for 3 min.

[0027] (iv) Statistical Analysis Using Excel software to statistically analyze the parent lines of the H and S09 series PVALB The gene frequencies, genotype frequencies, heterozygosity (He), polymorphism information content (PIC), and effective allele count (Ne) of the g.51652391, g.51659615, and g.51659905 bp sites were analyzed using the Hardy-Weinberg test with the chi-square test.

[0028] (v) Results (1) Sequencing results Sequencing of the amplification products from 384 samples using primer P1-4 revealed... PVALB The gene has mutations at three sites, such as Figures 1 to 3 As shown, g.51652391 undergoes an A→G mutation, g.51659615 undergoes a C→T mutation, and g.51659905 undergoes a C→T mutation.

[0029] (2) PVALB Mass spectrometry analysis of gene SNP sites There were 192 paternal lineage members from the H lineage and 192 from the S09 lineage, totaling 384 chickens. PVALB Mass genotyping maps of the three SNP loci of the gene, as shown below. Figure 4 , Figure 5 and Figure 6 And as shown in Table 3; as can be seen from Table 3, PVALB The mutation A→G at the g.51652391 site in the whole genome revealed three genotypes in the individuals: AA, AG and GG, with AG being the predominant genotype. The frequency of the G allele was significantly higher than that of the A allele. PVALB The C→T mutation at the g.51659615 site in the whole genome revealed three genotypes: CC, CT, and TT. The CT genotype was the predominant one, and the C allele frequency was significantly higher than the T allele frequency. PVALB The C→T mutation at the g.51659905 site in the whole genome revealed three genotypes: CC, CT, and TT. The CT genotype was the most prevalent, and the frequency of the C allele was significantly higher than that of the T allele.

[0030] Table 3 PVALB Allelic frequencies and genotype frequencies at three SNP loci of a gene

[0031] Individuals with a sum of three genotypes at the same locus of less than 384 are considered undetected.

[0032] (3) PVALB Population genetic analysis of gene SNP loci The Hardy-Weinberg equilibrium test results for the three loci from the H-line paternal line and the S09-line paternal line are shown in Table 4. Table 4 shows that the Hardy-Weinberg equilibrium test indicates that locus g.51652391 is in Hardy-Weinberg disequilibrium (P<0.01); loci g.51659615 and g.51659905 are both in Hardy-Weinberg equilibrium (P>0.05). The expected heterozygosity values ​​of the three SNP loci all fall within the range of 0.25 to 0.5, and the polymorphism information content values ​​of the three SNP loci are also within the range of 0.25 to 0.5. Therefore, these three SNP loci are all moderately polymorphic.

[0033] Table 3: Hardy-Weinberg equilibrium detection and population genetic parameters for three SNP loci

[0034] (4) PVALBAssociation analysis of gene polymorphism with traits such as meat color, tenderness, water-holding capacity, pH value, intramuscular fat, and muscle fiber area. The correlation between SNP sites and traits such as meat color, tenderness, water-holding capacity, pH value, intramuscular fat, and muscle fiber area was analyzed, and the results are shown in Table 5.

[0035] Table 5 PVALB Association analysis of different genotypes at three SNP loci of a gene with meat quality traits

[0036] Note: Within the same locus, the differences in means among those with the same letter epigraph are not significant. P >0.05), while those with different letter subheadings showed significant differences ( P <0.05). Individuals with a sum of three genotypes at the same locus of less than 384 were considered undetectable.

[0037] As shown in Table 5, the intramuscular fat and muscle fiber density of the pectoral muscles of individuals with the AA genotype at locus g.51652391 were significantly higher than those of individuals with the AG and GG genotypes, but there were no significant differences in pH, vitamin B1, flesh color, and water-holding capacity of the pectoral muscles among the three genotypes. The intramuscular fat and muscle fiber density of the pectoral muscles of individuals with the CC genotype at locus g.51659615 were significantly higher than those of individuals with the CT and TT genotypes, but there were no significant differences in pH, vitamin B1, flesh color, and water-holding capacity of the pectoral muscles among the three genotypes. The intramuscular fat and muscle fiber density of the pectoral muscles of individuals with the CC genotype at locus g.51659905 were significantly higher than those of individuals with the CT and TT genotypes, but there were no significant differences in pH, vitamin B1, flesh color, and water-holding capacity of the pectoral muscles among the three genotypes.

[0038] In summary, the AA genotype at locus g.51652391, the CC genotype at locus g.51659615, and the CC genotype at locus g.51659905 can serve as important molecular markers for screening meat quality traits.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A SNP molecular marker associated with chicken meat quality traits, characterized in that, SNP molecular marker for chicken PVALB Single nucleotide polymorphism sites in the gene, the single nucleotide polymorphism sites include one or more of site 1, site 2 and site 3; site 1 is located at 51652391bp of chromosome 1, and the mutation base is A / G; site 2 is located at 51659615bp of chromosome 1, and the mutation base is C / T; and site 3 is located at 51659905bp of chromosome 1, and the mutation base is C / T.

2. A primer pair for amplifying the SNP molecular marker of claim 1.

3. The primer pair according to claim 2, characterized in that, The primer pair sequence for amplifying the SNP molecular marker comprising site 1 is shown as SEQ ID NO: 1 and SEQ ID NO: 2, the primer pair sequence for amplifying the SNP molecular marker comprising site 2 is shown as SEQ ID NO: 3 and SEQ ID NO: 4, and the primer pair sequence for amplifying the SNP molecular marker comprising site 3 is shown as SEQ ID NO: 5 and SEQ ID NO:

6.

4. A detection reagent for detecting a SNP molecular marker, characterized by, The detection reagent comprises the primer pair of claim 2 or 3.

5. Use of the SNP molecular marker associated with chicken meat quality traits of claim 1 or the primer of claim 2 or 3 or the detection reagent of claim 4, the use is selected from any one of the following 1) to 8): 1) chicken meat quality trait prediction; 2) preparation of reagent for chicken meat quality trait prediction; 3) screening or identifying chicken breed, the chicken of which has the characteristics of high intramuscular fat content and / or high muscle fiber density; 4) preparation of reagent for screening or identifying chicken breed, the chicken of which has the characteristics of high intramuscular fat content and / or high muscle fiber density; 5) chicken meat quality trait selection; 6) molecular marker assisted breeding associated with chicken meat quality traits; 7) breed improvement associated with chicken meat quality traits; 8) PVALB genotyping; wherein For the SNP molecular marker comprising site 1, the genotype AA corresponds to the traits of high intramuscular fat content and high muscle fiber density; For the SNP molecular marker comprising site 2, the genotype CC corresponds to the traits of high intramuscular fat content and high muscle fiber density; For the SNP molecular marker comprising site 3, the genotype CC corresponds to the traits of high intramuscular fat content and high muscle fiber density.

6. A method of breeding a chicken with superior meat quality, characterized by, The method comprises: selecting individuals with high intramuscular fat content and high muscle fiber density as breeding parents for mating according to the SNP molecular marker; SNP molecular marker for chicken PVALB a single nucleotide polymorphism site in a gene, the single nucleotide polymorphism site comprising one or more of site 1, site 2 and site 3; site 1 is located at 51652391 bp of chromosome 1, and the mutation base is A / G; site 2 is located at 51659615 bp of chromosome 1, and the mutation base is C / T; and site 3 is located at 51659905 bp of chromosome 1, and the mutation base is C / T. For the SNP molecular marker comprising site 1, the genotype AA corresponds to the traits of high intramuscular fat content and high muscle fiber density; For the SNP molecular marker comprising site 2, the genotype CC corresponds to the traits of high intramuscular fat content and high muscle fiber density; For the SNP molecular marker comprising site 3, the genotype CC corresponds to the traits of high intramuscular fat content and high muscle fiber density.

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