Molecular markers, primers, kits, methods and applications in the seventh exon of the porcine SLC22A1 gene
By detecting mutations at the seventh exon 7351159bp of the pig SLC22A1 gene, specific primers were designed for PCR amplification, which solved the problem that the existing technology was difficult to improve the pig growth quality and meat quality at the same time, and achieved rapid and economical pork quality detection and breeding guidance.
Patent Information
- Application Number
- CN202410821021.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-24
- Publication Date
- 2025-04-01
- Estimated Expiration
- 2044-06-24
AI Technical Summary
It is difficult for existing pig breeding technology to improve the growth quality and meat quality of pigs at the same time, resulting in the improvement of meat quality being limited by the improvement of growth quality.
By detecting the single nucleotide mutation C>T at the seventh exon 7351159bp of the pig SLC22A1 gene, specific primers were designed for PCR amplification, and using this molecular marker to distinguish the meat quality differences between pig individuals, providing kits and methods for pork quality detection and breeding.
It has achieved the identification of the polymorphism of the SLC22A1 gene in a short period of time and detected the difference in pork quality, providing guidance on the breeding of pork varieties, and reducing the detection cost and sample demand.
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Figure CN118685534B_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of pig breeding, and in particular to molecular markers, primers, kits, methods and applications in the seventh exon of the pig SLC22A1 gene. Background Art
[0002] In the past few decades, pig farming companies have focused on growth quality such as growth rate, lean meat rate, feed conversion rate, etc., mainly to meet consumer demand in terms of quantity. However, pig growth quality is often negatively correlated with pork quality. While improving growth quality, meat quality will inevitably decline. However, people are more pursuing the taste and flavor of meat. Therefore, the improvement of meat quality has also become a key research work for various breeding companies.
[0003] With the development of disciplines such as genomics, molecular biology and bioinformatics, the widespread application of high-throughput sequencing technology, genotyping technology and gene editing technology, as well as the implementation and completion of pig whole genome sequencing and haplotype planning, have greatly promoted the identification of genes with great breeding value in pigs. Molecular markers used for auxiliary selection include protein markers, microsatellite markers, single nucleotide polymorphism (SNP) markers, etc. SNP markers refer to the polymorphism of DNA sequences caused by single nucleotide variations in the genome. They have the characteristics of large number, high accuracy and high polymorphism. In breeding practice, SNP can be used to locate certain excellent genes, and the association between markers and specific qualities can be determined in combination with phenotypes. Molecular markers can also be verified in populations and applied to molecular breeding. Summary of the invention
[0004] Solute carrierfamily 22member 1, referred to as SLC22A1 gene, encodes SLC22A1 (organic cation transporter 1), a multi-specific membrane transporter, one of the most abundant transporters in human liver. This transporter plays a key role in regulating liver energy status and lipid content, mediating the uptake of vitamin B1, cationic drugs and exogenous substances by hepatocytes. However, there are no reports on the role of this gene in pork quality.
[0005] The present application, through GWAS analysis of the black pork quality resource population in Selenium City, found that there are signal sites significantly associated with pork quality in the SLC22A1 gene region. Therefore, the present application designs specific primers based on the single nucleotide mutation site on the seventh exon of the SLC22A1 gene and performs amplification, and distinguishes the meat quality differences between individual pigs based on the polymorphism detection results of the amplified products. Primers are designed using the molecular markers, and PCR amplification is performed on the nucleotide sequence containing the SNP site, which can identify the polymorphism of the SLC22A1 gene in a relatively short period of time, thereby detecting the difference in pork quality, and providing guidance for the selection and breeding of pork varieties. In addition, the primers provided in the present application can specifically amplify the SNP site sequence contained in the gene intron of SLC22A1, and the detection method has low cost. It can be detected by PCR amplification alone, and the difference in meat quality between different pigs can be compared without a large amount of group-scale sampling and measurement.
[0006] To this end, the embodiments of the present application disclose at least the following technical solutions:
[0007] (1): Molecular markers of pork quality, including the nucleotide sequence formed by a single nucleotide mutation C>T at 7351159 bp in the seventh exon of the pig SLC22A1 gene.
[0008] (2): Molecular marker primers for pork quality, including a DNA molecule as shown in SEQ ID NO:3 and a DNA molecule as shown in SEQ ID NO:4.
[0009] (3): A nucleic acid molecule, which is amplified by PCR using the molecular marker primers described in (2), and the genotype of the nucleic acid molecule is associated with the quality of the pork.
[0010] (4): A kit comprising the molecular marker primers described in (2) and other reagents required for PCR amplification.
[0011] (5): Methods for testing pork quality, including:
[0012] Obtaining the pig genomic DNA to be tested;
[0013] Perform PCR amplification using the molecular marker primers described in (2);
[0014] The genotype of the 7351159 bp portion of the pig chromosome 1 was detected based on the nucleotide sequence of the amplified product;
[0015] The pork quality is determined according to the genotype.
[0016] (6): A pig screening method including the detection method described in (5).
[0017] (7): The use of the molecular marker described in (1), the molecular marker primer described in (2), the nucleic acid molecule described in (3), or the kit described in (4), wherein the use is selected from any one of the following:
[0018] 1) Detection and analysis of pork quality, wherein the pork quality is selected from at least one of muscle color value L24, drip loss 48h and intramuscular fat;
[0019] 2) Screening and breeding of pigs. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 It is the overall technical flow chart of this application.
[0021] Figure 2 This is the sequencing map of the C>T mutation at 7351159bp in the seventh exon of the pig SLC22A1 gene. The red box in the figure is the SNP site, and from top to bottom are the CC genotype, CT genotype and TT genotype.
[0022] Figure 3 It is a visual diagram of the nucleotide sequence of the seventh exon of the porcine SLC22A1 gene (shown in SEQ ID NO: 1 or SEQ ID NO: 2). The red frame represents the location of the mutation, and the mutation position is located at the 137th base of the sequence. The underlined sequence represents the position of the amplification primer. DETAILED DESCRIPTION
[0023] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.
[0024] The present application example sequenced the mutation sites of a partial nucleotide fragment of the SLC22A1 gene and found that the genotype at 7351159 bp of the seventh exon of the SLC22A1 gene was associated with pork quality.
[0025] To this end, the present application discloses a molecular marker for pork quality, including a nucleotide sequence formed by a single nucleotide mutation C>T at 7351159bp of the seventh exon of the pig SLC22A1 gene. Among them, the pig SLC22A1 gene refers to Gene ID: 6580 in the GeneBank database, assembly number GRCh38.p14 (GCF_000001405.40), and position information: NC_000006.12 (160121815..160158718).
[0026] In some embodiments, the pork quality is selected from at least one of muscle color value L24, drip loss 48h and intramuscular fat.
[0027] In some embodiments, at 7351159bp of the seventh exon of the pig SLC22A1 gene, the muscle color value L24 of the TT genotype individual is significantly higher than that of the CC genotype individual, and there is a trend of TT genotype>CT genotype>CC genotype.
[0028] In some embodiments, at 7351159bp of the seventh exon of the pig SLC22A1 gene, the drip loss at 48h in individuals with TT genotype was significantly higher than that in individuals with CC genotype, and there was a trend of TT genotype>CT genotype>CC genotype.
[0029] In some embodiments, at 7351159 bp of the seventh exon of the pig SLC22A1 gene, the intramuscular fat of individuals with TT genotype is significantly higher than that of individuals with CC genotype, and there is a trend of TT genotype>CT genotype>CC genotype.
[0030] Based on this, according to the genotype at 7351159bp of the seventh exon of the pig SLC22A1 gene, the pork quality of individual pigs can be molecularly marked, so as to facilitate the selection of pig breeds with excellent meat quality.
[0031] Therefore, specific primers were designed with reference to the seventh exon of the porcine SLC22A1 gene in the GeneBank database (the sequences of the primers are shown in SEQ ID NO:3 and SEQ ID NO:4 in the sequence listing), PCR amplification was performed using porcine genomic DNA as a template, the amplified products were recovered and sequenced, and gene fragments as shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence listing were obtained.
[0032] Based on this, the present application also discloses molecular marker primers for pork quality, including a DNA molecule as shown in SEQ ID NO: 3 and a DNA molecule as shown in SEQ ID NO: 4. The "molecular marker primers" are used to amplify molecular markers containing pork quality linkage, such as nucleotide sequences of SNPs, to analyze the genotype of the molecular markers, thereby obtaining pork quality.
[0033] On the other hand, the embodiment of the present application also discloses a nucleic acid molecule, which is amplified by PCR using the molecular marker primers, and the genotype of the nucleic acid molecule is associated with the pork quality. Further, the nucleic acid molecule is shown in SEQ ID NO: 1-2.
[0034] On the other hand, the embodiments of the present application also disclose a kit, including the molecular marker primers and other reagents required for PCR amplification.
[0035] Based on this, the present application also discloses a method for detecting pork quality, comprising: obtaining genomic DNA of a pig to be tested; performing PCR amplification using the molecular marker primers; detecting the genotype at 7351159bp of the seventh exon of the pig SLC22A1 gene according to the nucleotide sequence of the amplified product; and determining the pork quality according to the genotype. For example, the muscle color value L24, drip loss 48h and intramuscular fat of the pig are determined according to the genotype.
[0036] In some embodiments, the detection method further comprises sequencing the amplified product, and determining the genotype at 7351159 bp of the seventh exon of the pig SLC22A1 gene according to the sequencing result.
[0037] Based on this, the embodiments of the present application also disclose a method for screening pigs, including the detection method described above, to determine the pig muscle color value L24, drip loss 48h and intramuscular fat, and then screen pig breeds to obtain excellent pig breeds.
[0038] Based on this, the present application also discloses the application of the molecular marker, the molecular marker primer, the nucleic acid molecule or the kit, and the application is selected from any of the following:
[0039] 1) Detection and analysis of pork quality, wherein the pork quality is selected from at least one of muscle color value L24, drip loss 48h and intramuscular fat;
[0040] 2) Screening and breeding of pigs.
[0041] The present application is now further described in conjunction with specific examples. The following examples are only intended to explain the present application, but do not constitute a limitation to the present application. The test samples and test processes used in the following examples include the following (if the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, if not otherwise specified, can all be obtained from commercial sources).
[0042] 1. Extraction of porcine genomic DNA
[0043] The experimental pig breed of this application is Selenium City Black Pig, and the samples come from Hubei Huajian Selenium Garden Agriculture and Animal Husbandry Technology Co., Ltd. The genomic DNA of the pig was extracted using the genomic DNA kit produced by Beijing Biotech Biotechnology Co., Ltd. The extraction was performed according to the instructions of the kit. The concentration and quality of the extracted DNA were tested and stored at -20°C for later use. The remaining muscle samples were sealed in bags and stored at 4°C, and sent to the Agricultural Pig Quality Supervision and Inspection and Testing Center of the Ministry of Agriculture of Huazhong Agricultural University (Wuhan) within 4 hours to measure the quality of pork in accordance with the Agricultural Industry Standard of the People's Republic of China "Technical Specifications for Pork Quality Determination" (Standard No.: NY / T 821-2019).
[0044] 2. Obtaining the porcine SLC22A1 gene fragment and detecting the SLC22A1 gene SNP site
[0045] 1. Obtaining the seventh exon fragment of the porcine SLC22A1 gene
[0046] (1) PCR amplification
[0047] The following primer pairs were designed based on the porcine SLC22A1 gene sequence (Gene ID: 6580 in the GeneBank database):
[0048] Forward primer SLC22A1-F: 5'-ATGCCTCGTCCTTGATTTCCCC-3', SEQ ID NO: 3
[0049] Reverse primer SLC22A1-R: 5'-AATGATGCCCGTTTCCCTCCCC-3', SEQ ID NO: 4
[0050] The above primers were used to perform PCR amplification in a mixed genomic DNA pool of 40 Sedu Black Pigs. The PCR reaction system was 50 μL, and the concentrations of each component in the system were 100 ng template DNA, 10× buffer (containing Mg 2+ )4 μL, 0.5 μM each of the upstream and downstream primers mentioned above, 2.5 μM dNTPs, and 1 U Taq DNA polymerase.
[0051] The PCR operation program was as follows: preheating at 98°C for 45s; denaturation at 98°C for 10s, annealing at 56°C for 30s, extension at 72°C for 30s, for a total of 34 cycles; extension at 72°C for 10min; storage at 4°C. PCR products were electrophoresed using 1.5% agarose gel.
[0052] (2) PCR product purification
[0053] The above PCR products were purified using the Gel Extraction Kit of Shanghai Shenggong Biotechnology Co., Ltd. (according to the instructions of the kit), and the specific steps are as follows: first, the gel containing the target fragment was cut from the agarose gel, placed in a 1.5mL centrifuge tube, 400μL of sol solution was added, and the gel was completely melted in a 50-60℃ water bath. When heating the melted gel, it was mixed every 2 minutes and cooled to room temperature; the centrifuge column was placed in a collection tube, the mixed solution was transferred to the centrifuge column, and it was placed at room temperature for 2 minutes; centrifuged at 12000r / min for 1 minute, at which time the DNA was adsorbed on the column; discarded Collect the waste liquid in the tube, put the centrifuge column into the same collection tube, add 700μL of elution buffer, and centrifuge at 12000r / min for 1min; pour out the waste liquid in the collection tube, and centrifuge at 12000r / min for 1min; put the centrifuge column into a pre-prepared sterilized 1.5mL centrifuge tube, add 40μL of elution buffer or double distilled water (Ph>7.0), and place it at room temperature or 37℃ for 2-3min; centrifuge at 12000r / min for 1min, and the liquid in the centrifuge tube is the recovered DNA fragments.
[0054] 2. Obtaining the mutation site of the seventh exon fragment of the porcine SLC22A1 gene
[0055] The DNA fragments recovered above were sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing using an ABI3730XL sequencer, and a single-base mutation site was found ( Figure 2 ), a C>T mutation located at 7351159bp of the SLC22A1 genomic nucleotide sequence (i.e., the full sequence) (Gene ID: 6580 in the GeneBank database), and the mutation sites corresponding to the SLC22A1 gene fragment of the present application are: a C>T base mutation (i.e., allele mutation) at 137bp in SEQ ID NO:1; and a T>C base mutation (i.e., allele mutation) at 137bp in SEQ ID NO:2.
[0056] 3. Molecular marker genotyping
[0057] The DNA sample of the individual to be tested was used as a template, and the sequence fragment of the seventh exon of the porcine SLC22A1 gene was amplified according to the method described in step 1 above. The obtained PCR purified product was directly sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing, and the genotyping results were directly read from the sequencing results, such as Figure 2 shown.
[0058] 3. Genetic diversity detection and analysis of its association with quality
[0059] Using the method provided in the examples of this application, the genetic diversity of 274 Selenium City black pigs (from Hubei Huajian Selenium Garden Agriculture and Animal Husbandry Technology Co., Ltd.) was detected and the correlation analysis with quality was performed. SPSS statistical software (Statistical Package for the Social Sciences, Version 26.0) was used for statistical analysis using the general linear model GLM. The model used was: Y ijklm =μ+G i +A j +X k +S l +e ijklm , where Yijklm represents the pork quality phenotypic value; μ represents the population mean; G i represents genotype effect; A j represents the year-season effect; X k represents the gender effect; S l represents the paternal effect; e ijklm The results are expressed as the least squares mean ± standard error, and P < 0.05 was considered significant.
[0060] The results of association analysis are shown in Table 1. It was found that the C>T site at 7351159bp of chromosome 1 of the pig genome significantly affected the muscle color value L24, drip loss 48h and intramuscular fat of pigs. The muscle color value L24, drip loss 48h and intramuscular fat content of individuals with TT genotype were significantly higher than those of individuals with CC genotype, and there was a trend of TT genotype>CT genotype>CC genotype. Therefore, in the process of pig breeding, the C>T marker at 7351159bp of chromosome 1 of the pig genome can be used to assist in the early molecular marker selection of high-quality individuals in the reserve pig herd, and individuals carrying the TT genotype are preferred.
[0061] Table 1 Analysis of the association between the C>T mutation at bp 7351159 of chromosome 1 of the pig genome and pork quality
[0062]
[0063] Note: Shoulder marks are significant difference marks of the same quality in different genotypes
[0064] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.
Claims
1. The detection method of the quality of Selenium City Black Pig pork includes: Obtaining the pig genomic DNA to be tested; Performing PCR amplification on the pig genomic DNA using molecular marker primers; Detecting the genotype of the 137 bp site in SEQ ID NO: 1 or SEQ ID NO: 2 according to the nucleotide sequence of the amplified product; determining the pork quality according to the genotype; Wherein, determining the pork quality according to the genotype comprises: The muscle color value L24 of TT genotype individuals was significantly higher than that of CC genotype individuals; The drip loss of individuals with TT genotype at 48h was significantly higher than that of individuals with CC genotype; The intramuscular fat of individuals with TT genotype was significantly higher than that of individuals with CC genotype.
2. The screening method of Selenium City Black Pig includes: The detection method according to claim 1; as well as The individuals with the TT genotype are screened.
3. Application of the molecular marker primer or kit, the application is selected from any of the following: 1) Detection and analysis of the quality of Xidu black pig meat; 2) Screening and breeding of Xidu black pigs, with the traits of screening and breeding being pork quality; in, The pork quality is selected from at least one of muscle color value L24, drip loss 48h and intramuscular fat, and the molecular marker primers include a DNA molecule as shown in SEQ ID NO:3 and a DNA molecule as shown in SEQ ID NO:4; The kit includes the molecular marker primers and other reagents required for PCR amplification, the molecular marker primers detect the genotype of the 137bp site in SEQ ID NO: 1 or SEQ ID NO: 2, and the pork quality is determined according to the genotype; Wherein, determining the pork quality according to the genotype comprises: The muscle color value L24 of TT genotype individuals was significantly higher than that of CC genotype individuals; The drip loss of individuals with TT genotype at 48h was significantly higher than that of individuals with CC genotype; The intramuscular fat of individuals with TT genotype was significantly higher than that of individuals with CC genotype.