Molecular marker related to pork quality character and application thereof

Abstract

The invention discloses a molecular marker related to pork quality characters and application of the molecular marker. The molecular marker is an SNP (Single Nucleotide Polymorphism) site of nucleotide at the position of 7311198bp of a No.1 chromosome NC010443.5 of a pig reference genome GCF 000003025.6, and the molecular marker is an SNP (Single Nucleotide Polymorphism) site. The molecular marker is closely associated with the characteristics of pig carcass length and intramuscular fat content. The molecular marker can be applied to molecular-assisted selective breeding of high-quality pig carcass and meat quality traits.

Description

[0001] This application claims priority to the invention patent application with the application number of 202510257871.X and the name of "A molecular marker related to pig carcass length and intramuscular fat content traits and its application", which was filed on March 5, 2025. TECHNICAL FIELD

[0002] The present application relates to the technical field of pig molecular markers, in particular to a molecular marker related to pork quality traits and its application. BACKGROUND

[0003] Pork is one of the most important livestock products in China. As the world's largest pork producer and consumer, the stability and development of the pork industry are of great importance in the national economy. Pork quality traits are the core economic traits in pig breeding, and their advantages and disadvantages are directly related to the economic benefits and market competitiveness of breeding production. Therefore, genetic improvement of pork quality traits has always been the key target of breeding work. Studies have shown that pork quality traits are medium heritability traits (about 0.25~0.42), which theoretically have the potential for genetic improvement through selection.

[0004] However, the existing conventional breeding techniques face significant technical bottlenecks in the improvement of meat quality traits. The primary bottleneck is the difficulty of phenotypic determination of meat quality traits: key meat quality indicators such as pig carcass length and intramuscular fat content must be accurately determined after individual slaughter. This leads to a series of problems: (1) long breeding cycle: the breeding value of the parents cannot be evaluated until the offspring are slaughtered, greatly extending the generation interval. (2) high determination cost: slaughtering and determination consume a large amount of manpower, material resources and financial resources; (3) excellent germplasm resources cannot be preserved: individuals that perform well in slaughtering and determination have lost their lives and cannot be directly used as breeding individuals for further reproduction, which severely restricts breeding efficiency and genetic progress.

[0005] In order to break through the above bottleneck, to find a method of assisted breeding which can be used to evaluate the genetics in early stage of living body quickly and accurately, becomes a key problem to be solved urgently in the industry. The molecular marker assisted selection (MAS) technology provides a potential possibility for this. At present, the molecular markers used for assisted selection include microsatellite markers, single nucleotide polymorphism (SNP) markers and the like. Among them, the SNP marker is regarded as an ideal molecular marker type due to its wide distribution in the genome, rich quantity, high stability and easy realization of high-throughput automatic detection and the like. However, although the theory of molecular marker assisted selection has been mature, the core of its successful application lies in finding and verifying the molecular marker which has stable and significant correlation with a specific economic trait (especially the meat quality trait which is difficult to be measured in living body). At present, there is still a lack of specific SNP molecular marker which is verified sufficiently and can be used for early living body selection and closely related to the key meat quality traits such as pig carcass length and intramuscular fat content, resulting in that the existing breeding technology is still difficult to fundamentally solve the problems of difficult measurement, long cycle and high cost of meat quality trait measurement.

[0006] Therefore, there is an urgent need in the art to identify new molecular markers which are closely related to the meat quality traits of pigs, so as to provide a powerful tool for the molecular marker assisted breeding of pigs and accelerate the process of genetic improvement of pigs. SUMMARY

[0007] The protein encoded by the pig SLC22A2 gene (solute carrier family 22 member 2, Gene ID: 396936, located at the position of 7289278..7323418 nt of chromosome 1 NC_010443.5 of the reference genome GCF000003025.6) is an organic cation transporter 2, which belongs to one of the members of the solute carrier family 22. At present, there are few reports directly studying the relationship between the pig SLC22A2 gene and the meat quality traits. Since it is involved in the transport and metabolic balance of substances in the body, it is speculated that this gene may indirectly affect the growth performance indicators such as growth rate and body weight of pigs by affecting the absorption and utilization of nutrients.

[0008] Therefore, the purpose of the present application is to provide a molecular marker related to the meat quality traits of pigs and the application thereof. The present application found that there is a SNP molecular marker related to the meat quality traits in the pig SLC22A2 gene, which is the single nucleotide polymorphism of A / T at the position of 7311198 bp of the nucleotide of chromosome 1 NC_010443.5 of the pig reference genome GCF 000003025.6. The molecular marker is closely related to the meat quality traits such as pig carcass length and intramuscular fat content. The above-mentioned molecular marker can be applied to the molecular assisted selection breeding of low-fat pig lines. For this purpose, the present application discloses at least the following technical solutions:

[0009] In a first aspect, the present application provides a molecular marker related to pork quality traits, which is located at a single nucleotide A>T mutation at 7311198bp of chromosome 1 NC_010443.5 of the pig reference genome GCF 000003025.6.

[0010] Further, the sequence of the molecular marker is a gene fragment of chromosome 1 NC_010443.5 of GCF 000003025.6, the nucleotide sequence of which is shown as SEQ ID NO:1 and SEQ ID NO:2, and there is an A>T base mutation at 251bp of SEQ ID NO:1 and SEQ ID NO:2. That is, the above-mentioned molecular marker includes a single nucleotide polymorphism site (SNP) of A>T base mutation at 251bp of SEQ ID NO:1 or T>A base mutation at 251bp of SEQ ID NO:2, which shows three genotypes of AA, AT and TT.

[0011] In a second aspect, the present application provides a primer set for amplifying the sequence of the molecular marker of the first aspect, which comprises:

[0012] a forward primer: 5'-ATCCCATCGTCATTAATGACCAGG-3' (SEQ ID NO:3);

[0013] a reverse primer: 5'-AGCCTGCGATAAACCAGAGTG-3' (SEQ ID NO:4).

[0014] In a third aspect, the present application provides a kit comprising the primer set of the second aspect.

[0015] In a fourth aspect, the present application provides a method for screening a pig individual with excellent pork quality traits using the molecular marker of the first aspect, which comprises the following steps:

[0016] extracting genomic DNA of a pig to be tested;

[0017] performing PCR amplification on the genomic DNA using the primer set of the second aspect;

[0018] sequencing the PCR amplification product; determining the genotype of the pig at 7311198bp of chromosome 1 NC_010443.5 of the reference genome GCF 000003025.6 according to the sequencing base peak chart result; and determining the superior pig individual in terms of pork quality traits according to the genotype.

[0019] Further, the genotype includes three genotypes of AA, AT and TT.

[0020] Further, the pork quality traits are at least one of carcass length and intramuscular fat content.

[0021] Further, the standard for judging the superior pig individual in the pork quality traits of the pig to be selected is:

[0022] The carcass length of the TT and AT genotype individuals is significantly higher than that of the AA genotype individuals, and the intramuscular fat content of the TT and AT genotype individuals is significantly higher than that of the AA genotype individuals.

[0023] Preferably, the breed of the pig to be tested is the Suidall black pig.

[0024] In a fifth aspect, the present application provides the application of the molecular marker of the first aspect, the primer set of the second aspect, the kit of the third aspect and / or the method of the fourth aspect in the detection and analysis of pork quality traits of pigs, and the screening of excellent individuals in pork quality traits of pigs.

[0025] Compared with the prior art, the technical scheme of the present application has at least the following beneficial effects:

[0026] The present application uses the existing PCR and sequencing technology, and finds that there is an A>T allele mutation single nucleotide polymorphism site at 7311198bp of chromosome 1 of the pig reference genome GCF 000003025.6 NC_010443.5, and the polymorphism site is significantly related to multiple pork quality traits of pigs, which can be used as a molecular marker for early selection of pork quality traits, and provides a new molecular breeding marker for genetic marker assisted breeding of pork quality traits of pigs, and has a certain help for the research of pork quality traits of pigs. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The agarose gel electrophoresis map of the PCR amplification product of the pig SLC22A2 gene fragment provided by the embodiment of the present application.

[0028] Figure 2 The sequencing peak map of the downstream primer sequencing typing of the pig SNP chr1:7311198 provided by the embodiment of the present application, the antisense strand TT genotype corresponds to the sense strand AA genotype, the antisense strand TA genotype corresponds to the sense strand AT genotype, and the antisense strand AA genotype corresponds to the sense strand TT genotype.

[0029] Figure 3 The intuitive diagram of the nucleotide sequence of the pig SNP chr1:7311198 provided by the embodiment of the present application (shown in SEQ ID NO:1 or SEQ ID NO:2), the red box represents the presence of the mutation, and the red sequence represents the primer position. DETAILED DESCRIPTION

[0030] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0031] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before a detailed description of this application, the following definitions are provided to better understand it.

[0033] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0034] The technical solution of this application and the technical effects achieved will be described in detail below through more specific embodiments.

[0035] Example 1: Obtaining the porcine SLC22A2 gene fragment and establishing a method for polymorphism detection.

[0036] 1. Extraction of porcine genomic DNA

[0037] The experimental pig breed used in this application was the Xidu Black Pig, and the samples were obtained from the original breeding pig farm of the Animal Husbandry Institute of Hubei Academy of Agricultural Sciences. Pig genomic DNA was extracted using a genomic DNA kit produced by Beijing Baitek Biotechnology Co., Ltd., following the kit's instructions. The specific steps are as follows:

[0038] Pig genomic DNA was extracted using a genomic DNA kit manufactured by Beijing Biotech Biotechnology Co., Ltd. (the kit was operated according to its instructions). The specific steps are as follows:

[0039] (1) Take the longissimus dorsi muscle tissue from pigs, put it into a 2mL centrifuge tube, add 200μL of lysis buffer TL, and mix it evenly with a pipette tip;

[0040] (2) Add 20 μL of proteinase K (20 mg / ml), mix well, and digest overnight in a 55°C water bath;

[0041] (3) Add 200 μL of binding solution CB (provided in the kit), mix well, and place at 70°C for 10 min;

[0042] (4) After cooling, add 100 μL of isopropanol, mix well, and shake vigorously;

[0043] (5) Use a 1 mL syringe to take the above mixture and add it to the adsorption column AC, centrifuge at 10,000 rpm for 30 s, and discard the waste liquid in the collection tube;

[0044] (6) Add 500 μL of inhibitor removal solution IR (provided in the kit), centrifuge at 12,000 rpm for 30 s, and discard the waste liquid;

[0045] (7) Add 700 μL of rinse solution WB (provided in the kit), centrifuge at 12,000 rpm for 30 s, and discard the waste liquid;

[0046] (8) Repeat step 7;

[0047] (9) Place the adsorption column AC back into the collection tube, centrifuge at 12,000 rpm for 2 min, and try to remove the rinse solution to avoid residual ethanol inhibiting the downstream reaction;

[0048] (10) Take out the adsorption column AC and place it in a clean centrifuge tube, add 50-100 μL of elution buffer EB (provided in the kit) to the middle of the adsorption membrane, and place it at room temperature for 3-5 min, centrifuge at 12,000 rpm for 1 min, and collect the solution into the centrifuge tube;

[0049] (11) After detecting the concentration and quality of the extracted DNA, store it at -20°C for future use.

[0050] The remaining muscle samples were stored in bags at 4°C and sent to the Agricultural Ministry Pig Quality Supervision and Inspection Test Center (Wuhan) of Huazhong Agricultural University within 4 hours for determination of intramuscular fat content and other meat quality traits. The determination of pork traits was performed in accordance with the Technical Specification for Pork Trait Determination of the People's Republic of China Agricultural Industry Standard (Standard No.: NY / T 821-2019).

[0051] 2. Obtaining of the pig SLC22A2 nucleotide sequence

[0052] (1) PCR amplification

[0053] The following primer set was designed according to the pig SLC22A2 gene sequence (Gene ID: 733673 in the GeneBank database):

[0054] Forward primer: 5'-ATCCCATCGTCATTAATGACCAGG-3' (SEQ ID NO: 3);

[0055] Negative primer: 5'-AGCCTGCGATAAACCAGAGTG-3' (SEQ ID NO: 4).

[0056] PCR amplification was performed using the above primers in a pool of 40 mixed genomic DNA samples from Xidu black pigs. The PCR reaction volume (50 μL) was as follows:

[0057] 100ng template DNA, 10× buffer (containing Mg) 2+ 4 μL, 0.5 μM each of positive and negative primers, 2.5 μM dNTPs, and 1 U Taq DNA polymerase.

[0058] The PCR procedure was as follows: preheating at 98℃ for 45 seconds; denaturation at 98℃ for 10 seconds, annealing at 62℃ for 30 seconds, extension at 72℃ for 30 seconds, for a total of 34 cycles; final extension at 72℃ for 10 minutes; storage at 4℃. PCR products were subjected to 1.5% agarose gel electrophoresis. The electrophoresis results are shown below. Figure 1 As shown.

[0059] 3. Obtaining the target fragment variant site containing the target SNP site

[0060] The recovered DNA fragments were sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing using an ABI 3730XL sequencer, which revealed a single-base mutation site. Figure 2 The mutation is an A>T mutation located at 7311198 bp on chromosome 1, NC_010443.5, of the pig reference genome GCF 000003025.6. Figure 3 As shown, there is a single nucleotide with an A base at position 251 bp in SEQ ID NO:1 (i.e., an allelic mutation), and there is a single nucleotide with a T base at position 251 bp in SEQ ID NO:2 (i.e., an allelic mutation).

[0061] 4. Molecular marker genotyping

[0062] Using the DNA sample of the individual to be tested as a template, the target fragment containing the target SNP site was amplified according to the method described in step 1 above. The obtained purified PCR product was directly sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing, and the genotyping results were directly read from the sequencing results. Figure 2 As shown.

[0063] Example 2: Application of molecular markers in association analysis of pork quality traits

[0064] The example 1 screened molecular markers and pork quality traits were detected in 275 selenium all black pig groups (from the original pig breeding field of Hubei Provincial Academy of Animal Science), and the specific method was as follows:

[0065] The PCR direct sequencing method established in example 1 was used for genotyping detection, and the general linear model GLM of SPSS statistical software (Statistical Package for the Social Sciences, Version 26.0) was used for statistical analysis. The model used was: Y ijklm = μ + G i + A j + X k + S l + e ijklm , wherein: Y ijklm represents the phenotype value of pork traits; μ represents the population mean; G i represents the genotype effect; A j represents the annual and seasonal effect; X k represents the gender effect; S l represents the paternal effect; e ijklm represents the random residual effect. The results are expressed as least square mean ± standard error, and P<0.05 is considered to be significantly different.

[0066] The results of association analysis are shown in Table 1, in which different lowercase letters in the shoulder mark represent significant differences between the data in the same row, P<0.05.

[0067] Table 1 Association analysis of mutation at 7311198bp on chromosome 1 of pig genome with carcass length and intramuscular fat content traits

[0068] Traits AA genotype (n=96) AT genotype (n=97) TT genotype (n=82) F P Carcass length (cm) 92.197 ± 0.426 b ]] 93.800 ± 0.251 a ]] 93.762 ± 0.470 a ]] 4.70 0.010 Intramuscular fat content (%) 3.718 ± 0.119 b ]] 3.886 ± 0.117 a ]] 4.196 ± 0.131 a ]] 4.10 0.018

[0069] As can be seen from Table 1, the genotypes at 7311198bp of chromosome 1 of the reference genome GCF 000003025.6 NC_010443.5 include TT, AT and TT. Among them, the carcass length of individuals with TT and AT genotypes is significantly higher than that of AA genotype, and the intramuscular fat content of individuals with TT and AT genotypes is significantly higher than that of AA genotype. Therefore, it is recommended to preferentially select TT genotype and eliminate AA genotype in production, so as to select a population with long body type, high meat yield and good meat quality.

[0070] The application is described in detail above, and the principles and implementation manners of the application are described by applying specific examples. The above description of the examples is only used to help understand the application and the core idea. It should be pointed out that, for ordinary skilled persons in the technical field, some improvements and modifications can be made to the application without departing from the principles of the application, and these improvements and modifications also fall within the protection scope of the claims of the application.

Claims

1. A molecular marker associated with pork quality traits, wherein the molecular marker is a single nucleotide A>T mutation located at 7311198 bp on chromosome 1, NC_010443.5 of the pig reference genome GCF000003025.

6.

2. The molecular marker according to claim 1, wherein the sequence of the molecular marker comprises a fragment of exon 8 of chromosome 1 NC_010443.5 of GCF 000003025.6, and its nucleotide sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2, wherein an A>T base mutation exists at position 251 bp in SEQ ID NO: 1 and SEQ ID NO:

2. That is, the above molecular marker includes single nucleotide polymorphism (SNP) sites with an A>T base mutation at position 251 bp in SEQ ID NO: 1 or a T>A base mutation at position 251 bp in SEQ ID NO: 2, exhibiting three genotypes: AA, AT, and TT.

3. A primer set, said primer set being used to amplify the sequence of the molecular marker according to claim 1 or 2, said primer set comprising: Forward primer: 5'-ATCCCATCGTCATTAATGACCAGG-3'; Negative primer: 5'-AGCCTGCGATAAACCAGAGTG-3'.

4. A kit comprising the primer set of claim 3.

5. A method for screening individuals with superior pork quality traits, comprising the following steps: Extract genomic DNA from the pigs to be tested; The genomic DNA was amplified by PCR using the primer set described in the second aspect; The PCR amplification products were sequenced; The genotype of the pig at 7311198 bp on chromosome 1, NC_010443.5 of the reference genome GCF 000003025.6 was determined based on the sequencing base peak results; the dominant pig individuals in terms of meat quality traits were determined based on the genotype.

6. The method according to claim 5, wherein the genotype includes three genotypes: AA, AT, and TT.

7. The method according to claim 5, wherein the pork quality trait is at least one of carcass length and intramuscular fat content.

8. The method according to claim 5, wherein the criterion for determining the dominant pig individual in terms of meat quality traits is: The carcass length of individuals with the TT and AT genotypes was significantly greater than that of individuals with the AA genotype, and the intramuscular fat content of individuals with the TT and AT genotypes was significantly greater than that of individuals with the AA genotype.

9. The method according to claim 5, wherein the breed of the pig to be tested is the Xidu Black Pig.

10. The application of the molecular marker of claim 1 or 2, the primer set of claim 3, the kit of claim 4, and / or the method of any one of claims 5 to 9 in the detection and analysis of pork quality traits, and in the screening of individuals with superior pork quality traits.

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