A molecular marker related to mutton tenderness and application thereof

Abstract

The application belongs to the technical field of biology, and particularly relates to a molecular marker related to mutton tenderness and application thereof. The application provides that the molecular marker is located at 13672927bp of the 4th chromosome of a sheep reference genome ARS-UI_Ramb_v3.0, and the polymorphic base is A or G. The molecular marker provided by the application is related to mutton tenderness, the mutton shear force of a mutton sheep individual with a genotype of GG corresponding to the molecular marker in genomic DNA is significantly higher than that of individuals with genotypes of AA and AG, and the mutton tenderness of a mutton sheep individual containing the GG allele is poor. A primer and a kit are developed based on the molecular marker, the mutton tenderness in a mutton quality trait can be predicted through simple PCR amplification, a mutton sheep individual with good mutton tenderness is screened, a mutton sheep individual with poor mutton tenderness is eliminated, mutton sheep breeding with a high-tenderness trait is carried out, and the breeding speed of mutton sheep is improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a molecular marker related to the tenderness of mutton and its application. Background Technology

[0002] Meat tenderness is a key standard for measuring the quality of meat products, characterized by the amount of force required to cut the muscle. This characteristic determines the meat's texture and market popularity. The internationally accepted method uses shear force (N / cm) for quantitative assessment, which corresponds to specific sensations in the eating experience, such as how easily it is chewed and how much fiber remains after chewing. The fundamental influencing factors are the composition of proteins in the muscle and the quantity and structure of connective tissue. Meat tenderness has become a decisive factor influencing consumers' purchasing decisions for meat products.

[0003] Single nucleotide polymorphism (SNP) analysis refers to the polymorphism in DNA sequences caused by individual nucleotide differences or small insertions, mutations, or deletions between different alleles at the same locus at the genomic level. SNP markers facilitate rapid and efficient genotyping and are currently the most abundant, widely distributed, and densest polymorphic genetic markers. While there are numerous reports on SNPs related to mutton meat quality traits, reports on SNPs related to meat tenderness are fewer. For example, existing technology CN117887862A discloses molecular markers for SNPs related to mutton meat quality traits and their application in meat quality trait screening. These SNP markers consider both high cooked meat percentage and high palmitoleic acid content in meat quality traits, but do not address tenderness selection. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular marker related to the tenderness of mutton, to predict the tenderness of mutton meat, to screen mutton sheep individuals with good tenderness, to eliminate individuals with poor tenderness, to carry out mutton sheep breeding with high tenderness traits, and to improve the speed of mutton sheep breeding.

[0005] This invention provides a molecular marker associated with the tenderness of mutton. The molecular marker is located at 13672927 bp on chromosome 4 of the sheep reference genome ARS-UI_Ramb_v3.0, and the polymorphic base is A or G.

[0006] This invention provides a DNA fragment related to the tenderness of mutton. Compared with the wild-type PDK4 gene, the DNA fragment has a polymorphism at 33 bp in the 5th intron, and the polymorphic base is A or G.

[0007] The wild-type PDK4 gene has an accession number of ENSOARG00020078476 in Ensembl.

[0008] Preferably, the nucleotide sequence of the DNA fragment is as shown in SEQ ID NO:3, wherein R is a degenerate base.

[0009] This invention provides primers for detecting the molecular markers or DNA fragments described in the above-mentioned technical solutions, including an upstream primer and a downstream primer;

[0010] The nucleotide sequence of the upstream primer is shown in SEQ ID NO:1;

[0011] The nucleotide sequence of the downstream primer is shown in SEQ ID NO:2.

[0012] This invention provides a kit for measuring the tenderness of mutton, the kit comprising the primers described in the above technical solution.

[0013] Preferably, the kit further includes one or more of the following: DNA extraction reagent, 2×ES Taq Master Mix, and positive standard;

[0014] The positive standard includes a DNA sequence with a nucleotide sequence as shown in SEQ ID NO:3 or a complementary DNA sequence to SEQ ID NO:3.

[0015] The present invention provides the use of substances for detecting the molecular markers described in the above-mentioned technical solutions, substances for detecting the DNA fragments described in the above-mentioned technical solutions, primers described in the above-mentioned technical solutions, or kits described in the above-mentioned technical solutions in one or more of the following:

[0016] (1) Assess mutton tenderness; (2) Identify mutton tenderness; (3) Predict mutton tenderness; (4) Screen mutton sheep with high mutton tenderness; (5) Assisted breeding of mutton sheep with high mutton tenderness;

[0017] Meat sheep with the genotype GG corresponding to the molecular marker described in the above technical solution in their genomic DNA exhibit poor meat tenderness.

[0018] Preferably, the tenderness of the mutton includes the tenderness of the longissimus dorsi muscle of the mutton; the breed of the mutton is Shuanggan mutton.

[0019] This invention provides a method for selecting mutton sheep with high tenderness, comprising the following steps:

[0020] The genotypes of meat sheep corresponding to the molecular markers described in the above technical solution were detected in the genomic DNA of meat sheep. Meat sheep individuals with the GG genotype were removed, and meat sheep individuals with the AA or AG genotypes were retained.

[0021] This invention provides a breeding method for sheep with high mutton tenderness, comprising the following steps:

[0022] Detect the genotype of the molecular markers in the genomic DNA of meat sheep corresponding to the above technical solution, remove meat sheep individuals with the GG genotype, retain meat sheep individuals with the AA genotype or AG genotype, and carry out crossbreeding in any of the following methods (1) to (3);

[0023] (1) Using the AA genotype sheep individuals as parents;

[0024] (2) The AA genotype sheep individuals are used as the maternal line and the AG genotype sheep individuals are used as the paternal line;

[0025] (3) The AA genotype sheep individuals are used as the father and the AG genotype sheep individuals are used as the mother.

[0026] Beneficial effects:

[0027] This invention provides a molecular marker and DNA fragment related to the tenderness of mutton. The molecular marker is located at 13672927 bp on chromosome 4 of the sheep reference genome ARS-UI_Ramb_v3.0, with a polymorphic base of A or G. The DNA fragment is compared with the wild type. PDK4 The gene exhibits a polymorphism at 33 bp in intron 5, with the polymorphic base being either A or G. The molecular markers and DNA fragments provided by this invention are related to mutton tenderness. Mutton shear strength in sheep individuals with the genotype GG corresponding to the molecular marker in their genomic DNA is significantly higher than that in individuals with the AA and AG genotypes. Mutton individuals containing the GG allele have poor meat tenderness.

[0028] This invention develops primers and kits based on the aforementioned molecular markers. Through simple PCR amplification, tenderness in the meat quality trait of mutton sheep can be predicted, allowing for the selection of mutton sheep with good tenderness and the elimination of those with poor tenderness. By using AA genotype mutton sheep as parents, or AA genotype mutton sheep as the mother and AG genotype mutton sheep as the father, or AA genotype mutton sheep as the father and AG genotype mutton sheep as the mother, crossbreeding can be carried out to achieve mutton sheep breeding with high tenderness traits, thereby increasing the speed of mutton sheep selection. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0030] Figure 1 Electrophoresis diagram of PCR amplification products of molecular markers provided by the present invention; wherein lanes 1 and 2 are both PCR amplification products;

[0031] Figure 2 This is a diagram showing the site typing results of the molecular markers provided by the present invention. Detailed Implementation

[0032] This invention provides a molecular marker associated with the tenderness of mutton. The molecular marker is located at 13672927 bp on chromosome 4 of the sheep reference genome ARS-UI_Ramb_v3.0, and the polymorphic base is A or G.

[0033] This invention provides a DNA fragment related to the tenderness of mutton. Compared with the wild-type PDK4 gene, the DNA fragment has a polymorphism at 33 bp in the 5th intron, with the polymorphic base being A or G. The wild-type PDK4 gene has the accession number ENSOARG00020078476 in Ensembl.

[0034] As one embodiment, the nucleotide sequence of the DNA fragment of the present invention is shown in SEQ ID NO:3, wherein R is a degenerate base.

[0035] This invention provides a primer for detecting the molecular marker or DNA fragment described in the above-mentioned technical solutions, comprising an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO:1; and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:2.

[0036] This invention provides a kit for measuring the tenderness of mutton, the kit comprising the primers described in the above technical solution.

[0037] In one embodiment, the kit of the present invention further includes one or more of DNA extraction reagents, 2×ES Taq MasterMix, and positive standards; the positive standards include a DNA sequence with a nucleotide sequence as shown in SEQ ID NO:3 or a complementary DNA sequence of SEQ ID NO:3.

[0038] The present invention provides the application of substances for detecting the molecular markers described in the above technical solutions, substances for detecting the DNA fragments described in the above technical solutions, primers described in the above technical solutions, or kits described in the above technical solutions in one or more of the following: (1) assessing the tenderness of mutton; (2) identifying the tenderness of mutton; (3) predicting the tenderness of mutton; (4) screening for mutton sheep with high tenderness; (5) assisting in the breeding of mutton sheep with high tenderness; mutton sheep whose genotype corresponding to the molecular markers described in the above technical solutions in their genomic DNA is GG exhibit poor tenderness of mutton.

[0039] The molecular markers described in this invention can be used as the sole identification target, or they can be used in conjunction with other targets, such as other polymorphic molecular markers (e.g., SSR molecular markers, STR molecular markers, or InDel molecular markers), or other screening methods known in the art to assist in the prediction of mutton tenderness traits.

[0040] In one embodiment, the substances described in this invention include, but are not limited to, one or more of the following: reagents for nucleic acid amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, and reagents for sequencing. In another embodiment, the substances described in this invention include, but are not limited to, one or more of the following: primers, probes, enzymes for nucleic acid amplification reactions, fluorescent labels, buffer reagents, dNTPs, and salts. Depending on the specific detection method, those skilled in the art can select the above-mentioned reagents for nucleic acid amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, and reagents for sequencing based on the methods described in general and more specific textbooks, references, process manuals, product instructions, and standard documents. This invention does not impose any limitations on these selections.

[0041] In one embodiment, the tenderness of the mutton described in this invention includes the tenderness of the longissimus dorsi muscle of the mutton. In another embodiment, the breed of mutton described in this invention is the Shuanggan mutton sheep.

[0042] This invention provides a method for screening mutton sheep with high tenderness, comprising the following steps: detecting the genotype of the molecular markers in the genomic DNA of the mutton sheep corresponding to the above-described technical solution, removing mutton sheep individuals with the GG genotype, and retaining mutton sheep individuals with the AA genotype or the AG genotype.

[0043] In one embodiment, this invention utilizes the primers described in the above-described technical solution to perform PCR amplification on sheep genomic DNA. After obtaining the PCR amplification product, sequencing or genotyping is performed to detect the genotype in the sheep genomic DNA corresponding to the molecular markers described in the above-described technical solution. In one embodiment, the PCR amplification system of this invention, in 20 μL increments, includes: 10 μL of 2×ES Taq Master Mix, 0.5 μL of upstream primer, 0.5 μL of downstream primer, 1 μL of DNA, and 8 μL of ddH2O. In one embodiment, the PCR amplification program of this invention is as follows: 95℃ pre-denaturation for 4 min; 95℃ denaturation for 40 s, 57℃ annealing for 30 s, 72℃ extension for 1 min, 34 cycles; 72℃ extension for 5 min; storage at 4℃.

[0044] This invention provides a breeding method for high-tenderness mutton sheep, comprising the following steps: detecting the genotype of the molecular marker corresponding to the above-mentioned technical solution in the genomic DNA of the mutton sheep, eliminating mutton sheep individuals with the GG genotype, retaining mutton sheep individuals with the AA genotype or AG genotype, and carrying out crossbreeding according to any one of the following (1) to (3): (1) using the AA genotype mutton sheep individuals as parents; (2) using the AA genotype mutton sheep individuals as mothers and the AG genotype mutton sheep individuals as fathers; (3) using the AA genotype mutton sheep individuals as fathers and the AG genotype mutton sheep individuals as mothers.

[0045] The breeding method provided by this invention, by eliminating GG genotype sheep individuals and using AA genotype sheep individuals as parents, or using AA genotype sheep individuals as mothers and AG genotype sheep individuals as fathers, or using AA genotype sheep individuals as fathers and AG genotype sheep individuals as mothers, can increase the gene frequency of allele A in the hybrid offspring, enabling the hybrid offspring to exhibit superior meat tenderness traits and improving the breeding efficiency of sheep individuals with superior meat tenderness.

[0046] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a molecular marker related to the tenderness of mutton and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1

[0048] 1. Sample Collection

[0049] The experimental group in this embodiment consisted of 35 dry-fed sheep raised under the same conditions. Blood was collected from the jugular vein and placed in anticoagulant tubes, which were then stored at 4°C.

[0050] 2. Genomic DNA extraction

[0051] Genomic DNA was extracted using a rapid blood genomic DNA extraction kit (Axygen, AP-MN-BL-GDNA-50), and its concentration and purity were determined using an ultra-micro spectrophotometer. Qualified DNA samples were aliquoted and stored at -20°C.

[0052] 3. Primer design

[0053] According to information published on Ensembl, the meat sheep PDK4 The gene sequence information (ENSOARG00020078476) was used to design primers using Primer Premier 5.0 software. The target fragment length is 1046 bp. The primers were synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and the specific information is as follows:

[0054] Upstream primer: 5'-TTGATTTGTGTAGACGGGTAC-3' (SEQ ID NO:1);

[0055] Downstream primer: 5'-ATCTGGAATGGAAGTGTGGTT-3' (SEQ ID NO:2).

[0056] 4. PCR amplification

[0057] The PCR reaction system consisted of: 10 μL of 2×ES Taq Master Mix, 0.5 μL of upstream primer, 0.5 μL of downstream primer, 1 μL of DNA, and 8 μL of ddH2O.

[0058] The PCR reaction program was as follows: 95℃ pre-denaturation for 4 min, 95℃ denaturation for 40 s, 57℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 34 cycles starting from the second step; 72℃ extension for 5 min; storage at 4℃. The PCR products were detected by 1.5% agarose gel electrophoresis. PCR products with bright bands were sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The PCR amplification electrophoresis image is shown below. Figure 1 As shown, the PCR amplification product is approximately 1046 bp in length, consistent with the expected size. The sequence obtained from sequencing is as follows:

[0059]

[0060] 5. Sequencing and sequence analysis

[0061] The PCR amplification product of the 1046 bp target fragment was recovered from the gel and subjected to Sanger sequencing. DNASTAR 7.1 software was used for sequencing. PDK4 Comparative analysis of the gene's DNA sequence (ENSOARG00020078476) revealed... PDK4 There is a polymorphic site A>G at 33bp in the 5th intron of the gene, with the corresponding chromosome location information as chr.4:13672927 and the reference genome version number as ARS-UI_Ramb_v3.0.

[0062] 6. Meat sheep PDK4 Gene polymorphism analysis

[0063] Based on the chr.4:13672927 locus, for meat sheep PDK4 Gene polymorphisms and genetic indicators were analyzed. The results are as follows: Figure 2 As shown in Table 1, the genotypes at the chr.4:13672927 locus are AA, GA, and GG, respectively; the alleles are A and G, respectively.

[0064] Table 1 Meat Sheep PDK4 Gene polymorphism analysis and genetic indicators

[0065]

[0066] 7. Meat sheep PDK4 Association analysis between genetic variation and meat quality traits

[0067] The cooked meat yield, drip loss rate (24h, 48h, 72h), pressing moisture, meat tenderness, pH value, and protein content were measured in 35 double-dried mutton sheep. Based on the chr.4:13672927 locus, association analysis was performed between the three genotypes and meat quality traits in the double-dried mutton sheep population. The results are shown in Table 2. The chr.4:13672927 locus, except for its correlation with mutton tenderness (shear force), was not significantly correlated with any of the other detected meat quality traits. In this population, the shear force of mutton from individuals with the GG genotype was significantly higher than that from individuals with the AA and GA genotypes, indicating that individuals containing the GG allele have significantly higher shear force than homozygous wild-type (AA) individuals, and that individuals with the GG allele have poorer meat tenderness than homozygous wild-type (AA) individuals.

[0068] Table 2 Meat Sheep PDK4 Gene polymorphism analysis and genetic indicators

[0069]

[0070] As can be seen from the above, the molecular markers provided by this invention are related to the tenderness of mutton, and can be used to predict the tenderness of mutton meat, screen mutton sheep individuals with high tenderness traits, eliminate individuals with poor tenderness, and obtain a breeding method for mutton sheep with high tenderness traits, thereby improving the speed of mutton sheep breeding.

[0071] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of primers or kits for detecting molecular markers in one or more of the following: (1) Assess mutton tenderness; (2) Identify mutton tenderness; (3) Predict mutton tenderness; (4) Screen mutton sheep with high mutton tenderness; (5) Assisted breeding of mutton sheep with high mutton tenderness; Sheep with the genotype GG corresponding to the molecular marker in their genomic DNA exhibit poor mutton tenderness; sheep with the genotype AA or AG corresponding to the molecular marker in their genomic DNA exhibit high mutton tenderness. The breed of sheep mentioned is Shuanggan sheep; The molecular marker is located at 13672927 bp on chromosome 4 of the sheep reference genome ARS-UI_Ramb_v3.0, and the polymorphic base is either A or G.

2. The application according to claim 1, characterized in that, The primers include an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO:1; the nucleotide sequence of the downstream primer is shown in SEQ ID NO:2; The kit includes the primers described above.

3. The application according to claim 2, characterized in that, The kit also includes one or more of the following: DNA extraction reagent, 2×ESTaq Master Mix, and positive standard; The positive standard includes a DNA sequence with a nucleotide sequence as shown in SEQ ID NO:3 or a complementary DNA sequence to SEQ ID NO:

3.

4. A method for selecting mutton sheep with high tenderness, characterized in that, Includes the following steps: The genotypes of corresponding molecular markers in the genomic DNA of meat sheep were detected, and meat sheep individuals with the GG genotype were removed, while meat sheep individuals with the AA or AG genotypes were retained. The molecular marker is located at 13672927 bp on chromosome 4 of the sheep reference genome ARS-UI_Ramb_v3.0, and the polymorphic base is A or G; The breed of sheep in question is Shuanggan sheep.

5. A breeding method for mutton sheep with high meat tenderness, characterized in that, Includes the following steps: Detect the genotype of the corresponding molecular marker in the genomic DNA of meat sheep, remove meat sheep individuals with the GG genotype, retain meat sheep individuals with the AA genotype or AG genotype, and carry out crossbreeding in any of the following methods (1) to (3); (1) Using the AA genotype sheep individuals as parents; (2) The AA genotype sheep individuals are used as the maternal line, and the AG genotype sheep individuals are used as the paternal line; (3) The AA genotype sheep individuals are used as the father and the AG genotype sheep individuals are used as the mother; The molecular marker is located at 13672927 bp on chromosome 4 of the sheep reference genome ARS-UI_Ramb_v3.0, and the polymorphic base is A or G; The breed of sheep in question is Shuanggan sheep.

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