Variations in the seventh intron sequence of the porcine SH2D3C gene as molecular markers for meat quality traits and their applications.

Abstract

This application discloses a molecular marker for the seventh intron sequence variation of the porcine SH2D3C gene as a meat quality trait and its application. The molecular marker is a G>A mutation located at 268250812 bp of the porcine SH2D3C genome nucleotide sequence. The seventh intron sequence corresponding to the molecular marker is shown in SEQ ID NO: 1 and SEQ ID NO: 2, and a G>A base mutation exists at 249 bp in both SEQ ID NO: 1 and SEQ ID NO: 2. This molecular marker is closely associated with pork quality traits such as intramuscular fat content and drip loss, and can be applied to molecular-assisted selection breeding of high-quality new pig breeds. Based on this, this application also provides a primer set for detecting the above-mentioned molecular marker, a kit including the primer set, and a method for screening individuals with superior pork quality traits using the molecular marker.

Description

Technical Field

[0001] This application relates to the field of porcine molecular marker technology, specifically to a molecular marker and its application of a variation in the seventh intron sequence of the porcine SH2D3C gene as a meat quality trait. Background Technology

[0002] Meat quality is a core economic trait in pig breeding, directly determining the production efficiency and economic benefits of farms. Therefore, improving pork quality is one of the important goals of pig genetic breeding. The heritability of pork quality traits is known to range from 0.25 to 0.42, with most traits belonging to the moderate heritability category, theoretically possessing certain potential for genetic improvement. However, existing conventional breeding techniques face significant bottlenecks in improving meat quality traits: on the one hand, meat quality traits must be obtained through post-slaughter testing, as live animals cannot be directly and accurately evaluated, leading to long breeding cycles and high testing costs; on the other hand, individuals with excellent meat quality traits are often unable to be selected for breeding due to slaughter testing, severely restricting breeding efficiency and genetic progress. Therefore, finding efficient and accurate breeding aids is crucial to overcoming these bottlenecks.

[0003] However, our understanding of key genes and their functional sites affecting pork quality traits remains very limited, and the number of molecular markers available for practical breeding is scarce, severely restricting the efficiency of breeding high-quality pig breeds. The SH2D3C gene encodes a protein 3C containing an SH2 domain, which can recognize and bind phosphorylated tyrosine residues, playing an important role in cell signal transduction, proliferation, and differentiation. Previous studies have suggested that SH2D3C can interact with DPP3, participating in the regulation of the NRF2-KEAP1 pathway in oxidative stress responses. Since oxidative stress is closely related to physiological mechanisms such as lipid metabolism, this suggests that the SH2D3C gene may have a certain function in the meat formation mechanism. Nevertheless, no studies have yet clearly revealed the association between specific variations in the SH2D3C gene and pork quality traits, and its potential application value in pig breeding remains unexplored.

[0004] Therefore, screening and identifying molecular markers of the SH2D3C gene that affect pork quality traits is of great technical significance for expanding the molecular breeding tool library and improving the efficiency of meat quality genetic improvement. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a molecular marker for the seventh intron sequence variation of the porcine SH2D3C gene as a meat quality trait and its application. The applicant previously analyzed GWAS data from a resource population of pigs with selenium-rich black pork and discovered significant variation sites in the SH2D3C gene region associated with meat quality traits. Therefore, the applicant amplified a portion of the nucleotide sequence of the porcine SH2D3C gene and used this sequence to screen and identify genetic variation sites and conduct association analysis with meat quality traits such as intramuscular fat content, obtaining molecular markers associated with pork quality traits. These molecular markers can be used to assist in the selection of breeding pigs with target traits, improving breeding efficiency.

[0006] To achieve the above objectives, this application provides at least the following technical solutions:

[0007] In one aspect, this application provides a molecular marker for a variation in the seventh intron sequence of the porcine SH2D3C gene as a meat quality trait, wherein the molecular marker is a G>A mutation located at 268250812 bp of the nucleotide sequence of the porcine SH2D3C genome.

[0008] Furthermore, the molecular marker sequence contains a DNA fragment from the seventh intron of the porcine SH2D3C gene, with nucleotide sequences shown in SEQ ID NO: 1 (355 bp) and SEQ ID NO: 2 (355 bp). A G>A base mutation exists at position 249 of both SEQ ID NO: 1 and SEQ ID NO: 2. That is, the aforementioned molecular marker includes single nucleotide polymorphism (SNP) sites with a G>A base mutation at position 249 of SEQ ID NO: 1 or an A>G base mutation at position 249 of SEQ ID NO: 2, exhibiting three genotypes: GG, GA, and AA.

[0009] Secondly, this application provides a primer set for amplifying the sequence of the molecular marker described in the first aspect, the primer set comprising:

[0010] Forward primer: 5'-CAGAACACACGTACAGCATAAA-3' (SEQ ID NO: 3);

[0011] Reverse primer: 5'-GCAGGGAGAGAAAGAGAGAGGA-3' (SEQ ID NO: 4).

[0012] Thirdly, this application provides a kit comprising the primer set described in the second aspect.

[0013] Fourthly, this application provides a method for screening individuals with superior pork quality traits using the molecular markers described in the first aspect, comprising the following steps:

[0014] Extract genomic DNA from the pigs to be tested;

[0015] The genomic DNA was amplified by PCR using the primer set described in the second aspect;

[0016] The PCR amplification products were sequenced; the genotype of the pig at position 268250812 bp of the SH2D3C genome nucleotide sequence was determined based on the sequencing base peak results; and the dominant pig individuals in terms of meat quality traits were identified based on the genotype.

[0017] Furthermore, the genotypes include three types: GG, GA, and AA.

[0018] Furthermore, the pork quality trait is at least one of intramuscular fat content and drip loss.

[0019] Furthermore, the criteria for determining the dominant individual pigs in terms of meat quality traits among the candidate pigs are as follows:

[0020] Individuals with the AA genotype had significantly higher intramuscular fat content than individuals with the GG and GA genotypes; individuals with the GG genotype had significantly lower drip loss than individuals with the GA and AA genotypes.

[0021] Preferably, the breed of the pig to be tested is the Xidu Black Pig.

[0022] Fifthly, this application provides the application of the molecular markers described in the first aspect, the primer sets described in the second aspect, the reagent kits described in the third aspect, and / or the methods described in the fourth aspect in the detection and analysis of pork quality traits, and in the screening of individuals with superior pork quality traits.

[0023] Compared with the prior art, the technical solution of this application has at least the following beneficial effects:

[0024] This application utilizes existing PCR and sequencing technologies to identify a single nucleotide polymorphism (SNP) site with a G>A allele mutation at 249 bp in the seventh intron of the porcine SH2D3C gene. Furthermore, this SNP site was found to be significantly associated with multiple meat quality traits in pigs and can serve as a molecular marker for early selection of meat quality traits. This provides a novel molecular breeding marker for marker-assisted breeding of pork meat quality traits and is helpful for research on pork meat quality traits. Attached Figure Description

[0025] Figure 1 The overall technical flow diagram provided for this application.

[0026] Figure 2The image shows an agarose gel electrophoresis diagram of the PCR amplification product of the seventh intron of the porcine SH2D3C gene provided in the embodiments of this application. Lane M in the diagram is the LD 1500 bp ladder, and lanes 1, 2, and 3 are the amplified specific gene fragments of SEQ ID NO: 1 and SEQ ID NO: 2.

[0027] Figure 3 The sequencing map of the porcine SH2D3C gene G>A mutation provided in this application embodiment shows, from top to bottom, the GG homozygous type, the GA heterozygous type, and the AA homozygous type.

[0028] Figure 4 The image shows a visual representation of the nucleotide sequence of the seventh intron fragment of the porcine SH2D3C gene provided in this application (corresponding to the sequence listings shown in SEQ ID NO: 1 and SEQ ID NO: 2 above). The bolded letters in the box represent mutation sites, which are located at the 249th base of the sequence. The underlined sequences represent primer positions. Detailed Implementation

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The technical solution of this application and the technical effects achieved will be described in detail below through more specific embodiments.

[0034] Example 1: Obtaining the porcine SH2D3C gene fragment and establishing a method for polymorphism detection.

[0035] 1. Extraction of porcine genomic DNA

[0036] The experimental pig breed used in this application embodiment is the Xidu Black Pig, and the samples were obtained from Hubei Huajian Xiyuan Agricultural and Animal Husbandry Technology Co., Ltd. The Xidu Black Pig is a new black pig breed developed over 12 years by the Hubei Academy of Agricultural Sciences Animal Husbandry and Veterinary Research Institute, Hubei Tianzhili High-Quality Pig Breeding Co., Ltd., and other units, based on Enshi Black Pig, Meishan Pig, and Hubei White Pig. Pig genomic DNA was extracted using a genomic DNA kit produced by Beijing Baitek Biotechnology Co., Ltd. (operated according to the kit's instructions). The specific steps are as follows:

[0037] (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;

[0038] (2) Add 20 μL of proteinase K (20 mg / ml), mix thoroughly by vigorous inversion, and digest overnight in a water bath at 55°C;

[0039] (3) Add 200 μL of binding buffer CB (provided with the kit), mix thoroughly by inverting, and incubate at 70°C for 10 min;

[0040] (4) After cooling, add 100 μL of isopropanol and mix thoroughly by vigorous inversion;

[0041] (5) Use a 1 mL pipette tip to draw up 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.

[0042] (6) Add 500 μL of inhibitor removal solution IR (provided with the kit), centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

[0043] (7) Add 700 μL of wash buffer WB (provided with the kit), centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

[0044] (8) Repeat step 7;

[0045] (9) Place the adsorption column AC back into the collection tube and centrifuge at 12000 rpm for 2 min to remove as much of the washing liquid as possible to prevent residual ethanol from inhibiting the downstream reaction;

[0046] (10) Take out the adsorption column AC and put it into a clean centrifuge tube. Add 50-100 μL of elution buffer EB (provided with the kit) to the middle part of the adsorption membrane, place it at room temperature for 3-5 min, centrifuge at 12000 rpm for 1 min, and collect the solution into the centrifuge tube.

[0047] (11) After testing the concentration and quality of the extracted DNA, it was stored at -20℃ for later use.

[0048] The remaining muscle samples were sealed in bags and stored at 4°C. Within 4 hours, they were sent to the Ministry of Agriculture's Swine Quality Supervision and Testing Center (Wuhan) at Huazhong Agricultural University for the determination of meat quality traits such as intramuscular fat content.

[0049] 2. Obtaining the seventh intron fragment of the porcine SH2D3C gene

[0050] (1) PCR amplification

[0051] The following primer set was designed based on the genomic sequence of the porcine SH2D3C gene (GenBank accession number: NC_010443.5):

[0052] Forward primer SH2D3C-F: 5'-CAGAACACACGTACAGCATAAA-3'

[0053] Reverse primer SH2D3C-R: 5'-GCAGGGAGAGAAAGAGAGAGGA-3'

[0054] 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:

[0055] 100 ng template DNA, 10× buffer (containing Mg) 2+ 4 μL, 0.5 μM each of forward and reverse primers, 2.5 μM dNTPs, and 1 U Taq DNA polymerase.

[0056] 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℃. The PCR products were detected by 1.5% agarose gel electrophoresis, and the results are as follows. Figure 2 As shown.

[0057] (2) Purification of PCR products

[0058] The PCR products were purified using the Gel Extraction Kit from Shanghai Sangon Biotech Co., Ltd. (following the kit's instructions). The specific steps are as follows:

[0059] First, cut the gel containing the target fragment from the agarose gel and place it in a 1.5 mL centrifuge tube. Add 400 μL of sol solution and incubate in a 50-60°C water bath until the gel is completely melted. While heating to melt the gel, mix every 2 minutes. Cool to room temperature. Place the centrifuge column in a collection tube and transfer the mixture to the column. Incubate at room temperature for 2 minutes. Centrifuge at 12000 rpm for 1 minute. At this point, the DNA will be adsorbed onto the column. Discard the waste liquid in the collection tube. Place the centrifuge column in the same collection tube and add 700 μL of elution buffer. Centrifuge at 12000 rpm for 1 minute. Discard the waste liquid in the collection tube and centrifuge at 12000 rpm for 1 minute. Place the centrifuge column in a pre-prepared sterile 1.5 mL centrifuge tube and add 40 μL of elution buffer or double-distilled water (pH > 7.0). Incubate at room temperature or 37°C for 2-3 minutes. Centrifuge at 12000 rpm for 1 minute. The liquid in the centrifuge tube is the recovered DNA fragment.

[0060] 3. Identification of the variant site in the seventh intron of the porcine SH2D3C gene

[0061] The recovered DNA fragments were sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing using an ABI 3730XL sequencer. Figure 3 As shown, a single-base mutation site was found, a G>A mutation at 268250812 bp in the SH2D3C genome nucleotide sequence (i.e., the complete sequence) (GenBank NC_010443.5). The mutation sites corresponding to the SH2D3C gene fragment of this application are: a G>A base mutation (i.e., an allelic mutation) at 249 bp in SEQ ID NO: 1; and an A>G base mutation (i.e., an allelic mutation) at 249 bp in SEQ ID NO: 2. Figure 4 As shown.

[0062] 4. Molecular marker genotyping

[0063] Using the DNA sample of the individual to be tested as a template, the seventh intron sequence fragment of the porcine SH2D3C gene was amplified according to the method described in step 2 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 (see reference). Figure 3 ).

[0064] Example 2: Application of molecular markers in association analysis of pork quality traits

[0065] This embodiment examines the association between the molecular markers screened in Example 1 and pork quality traits in a herd of 265 Xidu black pigs (from Hubei Huajian Selenium Garden Agricultural and Animal Husbandry Technology Co., Ltd.). The specific method is as follows:

[0066] Genotyping was performed using the PCR direct sequencing method established in Example 1. Statistical analysis was conducted using SPSS statistical software (Statistical Package for the Social Sciences, Version 26.0) with a general linear model (GLM). The model used was: Y ijklm =μ+G i +A j +X k +S l +e ijklm , where: Y ijklm G represents the phenotypic value of meat quality traits; μ represents the population mean; G i Indicates genotype effect; A j Indicates the seasonal effect; X k Indicates the gender effect; S l Indicates the paternal effect; e ijklm The result represents the random residual effect. Results are expressed as least squares mean ± standard error, and P < 0.01 is considered statistically significant.

[0067] The association analysis results are shown in Table 1. Table 1 shows that the SH2D3C gene g.11845 G>A site significantly affected intramuscular fat content and drip loss traits in pork (P<0.01). Individuals with the AA genotype had significantly higher intramuscular fat content than those with other genotypes (GG, GA), while individuals with the GG genotype had significantly lower drip loss than those with other genotypes (GA, GG).

[0068] Table 1. Association analysis between porcine SH2D3C gene g.11845 G>A mutation and meat quality traits.

[0069] Properties GG genotype (n=133) GA genotype (n=87) AA genotype (n=45) F P Water loss over 48 hours (%) <![CDATA[3.327±0.088 C ]]> <![CDATA[3.569±0.114 B ]]> <![CDATA[4.089±0.155 A ]]> 9.10 0.0002 Intramuscular fat content (%) <![CDATA[3.600±0.096 C ]]> <![CDATA[3.942±0.125 B ]]> <![CDATA[4.554±0.171 A ]]> 12.07 0.0001 Moisture (%) <![CDATA[73.375±0.072 A ]]> <![CDATA[73.154±0.094 B ]]> <![CDATA[72.720±0.128 C ]]> 10.03 0.0001

[0070] Note: Different capital letters in the superscript indicate significant differences between data in the same row, P<0.01.

[0071] In summary, the G>A mutation in the seventh intron of the SH2D3C gene significantly affects multiple meat quality traits. In breeding, if the goal is to select a population with good meat quality (high intramuscular fat content), individuals carrying the AA genotype should be given priority. Molecular marker-assisted selection of replacement gilts can be carried out using the molecular marker of the SH2D3C gene g.11845 G>A mutation.

[0072] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that for those skilled in the art, several improvements and modifications can be made to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A method for screening individuals with superior pork quality traits, wherein the method utilizes a sequence variation in the seventh intron of the porcine SH2D3C gene as a molecular marker for meat quality traits, wherein the sequence of the molecular marker is a DNA fragment of the seventh intron of the porcine SH2D3C gene, and its nucleotide sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2: a G>A base mutation exists at position 249 bp in SEQ ID NO: 1; and an A>G base mutation exists at position 249 bp in SEQ ID NO:

2. The method includes the following steps: Extract genomic DNA from the pigs to be tested; The genomic DNA was amplified by PCR using a primer set comprising: Forward primer: 5'-CAGAACACACGTACAGCATAAA-3'; and Reverse primer: 5'-GCAGGGAGAGAAAGAGAGAGGA-3'; The PCR amplification products were sequenced; the genotype of the DNA fragment in the seventh intron of the porcine SH2D3C gene was determined based on the sequencing base peak results; and the dominant pig individuals in terms of meat quality traits were identified based on the genotypes. The genotypes include three types: GG, GA, and AA; the pork quality traits are at least one of intramuscular fat content and drip loss; the criteria for judging the dominant individual pig in terms of meat quality traits are: the intramuscular fat content of AA genotype individuals is significantly higher than that of GG and GA genotype individuals; the drip loss of GG genotype individuals is significantly lower than that of GA and AA genotype individuals; the breed of the pigs to be tested is Xidu Black Pig.

2. The application of the method described in claim 1 in the detection and analysis of pork quality traits, and in the screening of individuals with superior pork quality traits.

Images