ADIPOQ gene snp molecular marker related to pig backfat thickness character and application thereof

Abstract

The application belongs to the field of molecular biology, and discloses a SNP molecular marker related to a pig backfat thickness character and application thereof. The application screens a SNP site g.348A>G related to the pig backfat thickness character in an intron region of an ADIPOQ gene. The SNP site is located at a 348bp nucleotide downstream of a transcription start site of the ADIPOQ gene, and the polymorphism is A or G. The genotype of the mutation site is used to determine the pig backfat thickness, thereby obtaining a functional gene and a molecular genetic marker related to the pig backfat thickness. Through selection of a dominant allele of the SNP molecular marker and rapid detection of the site, a reference basis can be provided for accurate evaluation of a pig fat deposition capacity regulation related SNP molecular marker assisted breeding, and the application has important significance for pig line or breed improvement.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and relates to an ADIPOQ gene SNP molecular marker associated with the backfat thickness trait in pigs and its application. Background Technology

[0002] Pork accounts for 35% of global meat consumption and is an important part of the human diet. Excessive fat deposition in pigs leads to low feed utilization and makes them unpopular with consumers. It is well known that the percentage of lean meat in a carcass is strongly negatively correlated with backfat thickness. To align production with consumer demand, one of the key objectives of pig breeding programs is to reduce carcass fat and improve growth efficiency and lean meat content. Backfat thickness is a good indirect indicator of body fat content and has high heritability; therefore, breeding efforts place great emphasis on selecting for the backfat thickness trait.

[0003] Single nucleotide polymorphisms (SNPs) play a vital role in gene function research. As high-throughput, high-density genetic markers, SNPs are widely used in modern breeding and genetic analysis. They are crucial molecular markers, densely distributed throughout the genome, providing comprehensive coverage and a wealth of genetic markers for genomic selection, thereby improving breeding precision. Association analysis between SNP markers and traits can yield genotypes that guide breeding practices. Summary of the Invention

[0004] The purpose of this invention is to provide an ADIPOQ gene SNP molecular marker related to the backfat thickness trait in pigs and its application. The SNP molecular marker can be used to conveniently and quickly determine the backfat thickness and lean meat percentage of pig carcasses, thereby providing a basis for breeding high lean meat pig breeds.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] In a first aspect, the present invention seeks protection for the use of a substance for detecting the SNP site g.348A>G polymorphism or genotype in the ADIPOQ gene of the porcine genome in any of the following:

[0007] (a1) Application in identifying or assisting in the identification of backfat thickness in pigs;

[0008] (a2) Application in the preparation of products for identifying or assisting in the identification of backfat thickness in pigs;

[0009] (a3) Application in screening or assisting screening of pig breeds with low backfat thickness;

[0010] (a4) Application in the preparation of products for screening or assisting in the screening of pig breeds with low backfat thickness;

[0011] (a5) Application in assisting the breeding of pig breeds with high lean meat percentage;

[0012] The SNP site g.348A>G is located at the 348th nucleotide downstream of the transcription start site in the SEQ ID NO.2 sequence of the porcine ADIPOQ gene, and its polymorphism is A or G.

[0013] The SNP site screened in this invention is located 348 bp downstream of the transcription start site of the ADIPOQ gene (intron region). The genotype of the SNP site is AA, AG or GG, and the SNP is named g.348A>G.

[0014] The porcine ADIPOQ gene sequence is described in Chromosome 13,NC_010455.5 (124633906..124646237). The region from 2000 bp upstream to 700 bp downstream of the ADIPOQ gene transcription start site is listed in the sequence listing (as shown in SEQ ID NO.1). The first bold underlined nucleotide "A" is the transcription start site, and the second bold underlined nucleotide "A" is the SNP site g.348A>G located 348 bp downstream of the transcription start site. This site is located in the first intron region.

[0015] Furthermore, the substance is (b1) or (b2) or (b3) as follows:

[0016] (b1) PCR primers for amplifying a porcine genomic DNA fragment containing the SNP site g.348A>G in the ADIPOQ gene;

[0017] (b2) PCR reagents containing the PCR primers described in (b1);

[0018] (b2) A kit containing the PCR primers described in (b1) or the PCR reagents described in (b2).

[0019] Furthermore, the PCR primers comprise an upstream primer ADIPOQ-5F and a downstream primer ADIPOQ-5R:

[0020] ADIPOQ-5F: 5'-ACCTTCTCCTCATTTCCATC-3'(SEQ ID NO.3)

[0021] ADIPOQ-5R: 5'-TTCTCACGAACCCTACCC-3' (SEQ ID NO. 4).

[0022] Furthermore, in the above application, the SNP site g.348A>G is genotyped, and the backfat thickness of individuals with the GG genotype is higher than that of individuals with the AG or AA genotypes.

[0023] Secondly, the present invention seeks protection for the use of the aforementioned PCR primers in the preparation of products for detecting the aforementioned SNP site g.348A>G polymorphism or genotype.

[0024] Thirdly, this invention claims protection for a product used to detect the g.348A>G polymorphism or genotype of the aforementioned SNP site, containing the aforementioned substance or the aforementioned PCR primers. This product may be a PCR reagent or a kit.

[0025] Fourthly, the present invention claims protection for a method for identifying or assisting in the identification of backfat thickness in pigs, which involves detecting the SNP site g.348A>G polymorphism or genotype in the pig genome; the backfat thickness of individuals with genotype GG is higher than that of individuals with genotype AG or AA.

[0026] Fifthly, this invention claims protection for a genetic breeding method for a high lean meat percentage pig breed, which involves detecting the SNP site g.348A>G polymorphism or genotype in the pig genome; retaining individuals with homozygous AA genotype and heterozygous AG genotype, eliminating individuals with homozygous GG genotype, and increasing the lean meat percentage of pigs generation by generation.

[0027] Furthermore, in the above method, the method for detecting the SNP site g.348A>G polymorphism or genotype in the ADIPOQ gene on the pig genome is as follows: using the pig genomic DNA to be tested as a template, PCR amplification is performed using the aforementioned PCR primers or the aforementioned products; the amplified fragment is digested and genotyped using the PCR-RFLP-Ssil method, and the digestion products of different genotypes will show different numbers of bands: the GG genotype has two bands of 363bp and 212bp, the AG genotype has three bands of 575bp, 363bp and 212bp, and the AA genotype has one band of 575bp.

[0028] In a specific embodiment of the present invention, the method for detecting the SNP site g.348A>G polymorphism or genotype in the ADIPOQ gene on the pig genome specifically includes the following steps:

[0029] 1) Extract DNA from the pig tissue sample to be tested;

[0030] 2) Use the above-mentioned PCR primers or the above-mentioned product to perform PCR amplification on the DNA obtained in step 1) to obtain the amplified fragment;

[0031] 3) The amplified fragments were digested and genotyped using the PCR-RFLP-SsiI method. Different genotypes showed different numbers of bands in their digestion products: the GG genotype had two bands of 363bp and 212bp, the AG genotype had three bands of 575bp, 363bp and 212bp, and the AA genotype had one band of 575bp.

[0032] 4) When the classification result is GG type, it is determined that its back fat thickness is higher than that of individuals with AG or AA type back fat thickness.

[0033] In a specific embodiment of the present invention, the 25 μL PCR amplification reaction system consists of: 22 μL Es Taq MasterMix (Dye), 1 μL each of forward and reverse primers, and 1 μL of DNA to be detected; the PCR reaction program is as follows: 94 °C for 2 min; 94 °C for 30 s; 54 °C for 10 s annealing; 72 °C for 30 s; 35 cycles; 72 °C for 2 min.

[0034] The reaction system for the PCR-RFLP-SsiI method is as follows: 8 μL of the PCR amplification product to be detected, 0.2 μL of Ssil enzyme, and 10×FastDigest. TM Buffer 1 μL, ddH2O 0.8 μL; reaction conditions: 37℃, 10 min.

[0035] In a sixth aspect, the present invention seeks protection for a molecular marker containing the aforementioned SNP site g.348A>G, the nucleotide sequence of which is shown in SEQ ID NO.2, wherein the SNP site g.348A>G is located at the 348th nucleotide downstream of the transcription start site of the ADIPOQ gene in the sequence of SEQ ID NO.2.

[0036] The room temperature described in this invention is 25±10℃.

[0037] The beneficial effects of this invention are:

[0038] This invention investigates the correlation between the key gene ADIPOQ in lipid metabolism and the backfat thickness trait in pigs. By screening an SNP locus associated with backfat thickness in the intron region, functional genes and molecular genetic markers related to backfat thickness in pigs were obtained. This lays the foundation for further genetic improvement of pig backfat thickness and to assist in increasing the lean meat percentage of pig carcasses. It also allows for direct genotypic selection or marker-assisted selection of pigs, thereby accelerating the breeding of superior pig breeds. Attached Figure Description

[0039] Figure 1 The SNP site g.348A>G of the porcine ADIPOQ gene was discovered during sequencing in this invention.

[0040] Figure 2 This is a schematic diagram showing the genotyping results of the porcine ADIPOQ gene SNP site g.348A>G at three genotypes (GG, AG, and AA) using the PCR-RFLP-Ssil method. Marker: 2000bp DNA molecular weight marker.

[0041] Figure 3 Backfat thickness was determined for individuals with different genotypes at the ADIPOQ gene SNP site g.348A>G in the Duroc-Landrace-Large White crossbred population. Detailed Implementation

[0042] The present invention will be further described below with reference to the embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally carried out in accordance with known means in the art.

[0043] Example 1

[0044] 1. Experimental Materials

[0045] 1.1 Laboratory Animals

[0046] This study used 241 Duroc-Landrace-Landrace-Large White crossbred pigs. The experimental animals were obtained from Jiangsu Changzhou Kang Le Livestock Co., Ltd., and were raised under the same conditions. Liver tissue samples were collected for DNA extraction.

[0047] 1.2 Test Reagents

[0048] (1) Routine genomic DNA purification kit (containing RNase A): includes silica gel membrane centrifuge column, EB, BB2,

[0049] LB2, WB2, Proteinase K, and RNase A were purchased from Beijing TransGen Biotech Co., Ltd., and stored at room temperature.

[0050] (2) 2×Es Taq MasterMix(Dye): Contains 2×Es Taq MasterMix(Dye) and ddH2O, purchased from Nanjing Qingke Biotechnology Co., Ltd., and stored at -20℃.

[0051] (3) Goldview nucleic acid gel dye: purchased from Hunan Aikerui Biotechnology Co., Ltd., and stored at 4℃.

[0052] (4) DL2000 Marker: Purchased from Nanjing Qingke Biotechnology Co., Ltd. Store at -20℃ and after thawing, store at 4℃.

[0053] (5) Anhydrous ethanol AR 500mL: purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.

[0054] (6) FastDigest SsiⅠ restriction endonuclease: Contains FastDigest restriction endonuclease, 10× FastDigest TM Green Buffer and 10×FastDigest TM Buffer. Purchased from Thermo Fisher Scientific Nanjing Co., Ltd., store at -20℃.

[0055] (7) 1×TAE buffer: Prepared in-house. To prepare a 50mL system, you will need 12.1g of Tris base, 2.855mL of glacial acetic acid and 5mL of EDTA (0.5M, pH 8.0).

[0056] (8) Agarose powder: purchased from Nanjing Qingke Biotechnology Co., Ltd.

[0057] 1.3 Instruments and Equipment

[0058] (1) CF1524R benchtop high-speed micro-volume refrigerated centrifuge: purchased from Shanghai SCILOGEX Technology Co., Ltd.

[0059] (2) NanoDrop 2000 micro-volume spectrophotometer: purchased from Thermo Fisher Scientific.

[0060] (3) PCR instrument: purchased from Zhuhai Heima Medical Instrument Co., Ltd.

[0061] (4) Gradient PCR instrument: purchased from Bio-Rad Biomedical Products, Inc., USA

[0062] (5) Horizontal electrophoresis apparatus: purchased from Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd.

[0063] (6) Tanon 3500 gel imaging system: purchased from Shanghai Tianneng Technology Co., Ltd.

[0064] (7) Mini metal bath instrument: purchased from Zhejiang JOANLAB Experimental Instrument Co., Ltd.

[0065] (8) Double-hole constant temperature water bath: purchased from Zhejiang JOANLAB Experimental Instrument Co., Ltd.

[0066] The amplified fragment was sequenced by Qingke (Nanjing) Biotechnology Co., Ltd.

[0067] 2. Experimental Methods

[0068] 2.1 Determination of carcass backfat thickness

[0069] After slaughter, the backfat thickness of pigs is measured using vernier calipers, in centimeters. The measurement sites include the backfat thickness at the lumbosacral junction, the thickest part of the shoulder at the 3rd and 4th ribs, and the average backfat thickness at these three points.

[0070] 2.2 Extraction of DNA from Liver Tissue

[0071] Follow the steps outlined in the "Routine Genomic DNA Purification Kit" instructions.

[0072] (1) Liver tissue was collected from Duroc-Landrace-Large White crossbred pigs. The liver was minced with scissors and forceps, and its weight was controlled to be about 25 mg. The minced liver tissue was placed in a 1.5 mL centrifuge tube. 100 μl L LB2 and 20 μl Proteinase K were added to completely immerse the tissue in the solution. The constant temperature water bath (BHS-2, Qun'an Test Instruments Co., Ltd., Ningbo, Zhejiang) was preheated to 55 °C and incubated for 3 h to completely lyse the tissue sample. The sample was centrifuged at 12000 × g for 5 min at 25 °C. The supernatant was transferred to a new sterile centrifuge tube.

[0073] (2) Add 500 μL of BB2 to the supernatant, shake rapidly for 5 seconds to mix evenly, and incubate at room temperature for 10 min. Transfer all liquid in the tube to a centrifuge column, centrifuge at 12000×g for 30 s at 25°C, and discard the effluent.

[0074] (3) Add 500 μL of solution CB2 with added anhydrous ethanol to the centrifuge column, continue centrifuging at 12000×g for 30s at 25℃ and discard the effluent.

[0075] (4) Next, add 500 μL of WB2 solution with added anhydrous ethanol to the centrifuge column, and centrifuge at 12000×g for 30s at room temperature, then discard the effluent. Repeat this step twice. To completely remove the WB2 residue in the column, centrifuge again at the same speed for 2min.

[0076] (5) After centrifugation, place the centrifuge column into a new, clean centrifuge tube and add 50–200 μL of preheated EB solution (60℃–70℃) or deionized water (pH>7.0). Let it stand at room temperature for 1 min, then centrifuge at 12000×g for 1 min. The liquid in the tube at this point is the eluted DNA. To obtain more DNA, repeat this step. Store the eluted DNA at -20℃ for later use.

[0077] 2.3 DNA Concentration Determination and DNA Sample Mixing

[0078] DNA concentration and purity were determined using a NanoDrop2000 spectrophotometer, and the OD260 / OD280 ratio was calculated to determine the DNA extraction quality. If the OD260 / OD280 ratio of all samples should be between 1.8 and 2.0, the DNA extraction quality is considered acceptable.

[0079] 2.4 Primer Design and Synthesis

[0080] Based on the porcine gene sequence ADIPOQ (Chromosome 13,NC_010455.5(124633906..124646237)) from the GenBank database, five primer pairs were designed for the five segments from the 2000 bp before the transcription start codon to the 700 bp downstream of it in the promoter region: ADIPOQ-1F and ADIPOQ-1R, ADIPOQ-2F and ADIPOQ-2R, ADIPOQ-3F and ADIPOQ-3R, ADIPOQ-4F and ADIPOQ-4R, and ADIPOQ-5F and ADIPOQ-5R. The primer sequences are shown in Table 1.

[0081] Table 1 Primer design information for the porcine ADIPOQ gene

[0082]

[0083] 2.5 PCR-RFLP reaction system and procedure

[0084] The PCR reaction systems used in this experiment are shown in Tables 2 and 3:

[0085] Table 2 PCR reaction system

[0086]

[0087] Table 3. Genotyping Enzyme Digestion Reaction System

[0088]

[0089]

[0090] PCR reaction procedure:

[0091] 94℃, 2 min;

[0092] 94℃, 30s; 54℃, 30s; 72℃, 30s; 35 cycles.

[0093] 72℃, 2min; store at 4℃;

[0094] The amplified PCR products were digested with enzymes according to the system in Table 3. The reaction system was 37℃ for 10 min.

[0095] 2.6 Agarose gel electrophoresis detection

[0096] The amplified and digested PCR products were subjected to electrophoresis on a 2% agarose gel at approximately 120V for about 30 minutes, followed by observation using a gel imaging system. The successfully amplified PCR products were then digested with enzymes. After the digestion reaction, the digested products were subjected to agarose gel electrophoresis. Different genotypes corresponded to different numbers of bands; the genotype of each sample was determined based on the number of bands observed in the electrophoresis results.

[0097] 3. Experimental Results

[0098] 3.1 SNP Screening

[0099] Twenty DNA samples were randomly selected as templates. The promoter region, 5′UTR region, and intron region of the ADIPOQ gene, from 2000 bp upstream to 700 bp downstream of the transcription start site, were amplified using ADIPOQ-1F and ADIPOQ-1R, ADIPOQ-2F and ADIPOQ-2R, ADIPOQ-3F and ADIPOQ-3R, ADIPOQ-4F and ADIPOQ-4R, and ADIPOQ-5F and ADIPOQ-5R, respectively. A fragment of approximately 2700 bp from the porcine ADIPOQ gene was obtained. The PCR products were then sequenced (serviced by Qingke Biotechnology (Nanjing) Co., Ltd.). Sequencing results showed that only one SNP site was found in the fragment amplified by primer pairs ADIPOQ-5F and ADIPOQ-5R, located at 348 bp downstream of the ADIPOQ gene transcription start site, belonging to the first intron region, and identified as either adenine A or guanine G. Figure 1 As shown, this SNP is named g.348A>G.

[0100] 3.2 PCR-RFLP Genotyping

[0101] PCR amplification was performed on the aforementioned SNP sites using primers ADIPOQ-5F and ADIPOQ-5R. Genotypes were identified by digesting the PCR products with SsiI restriction endonuclease. Agarose gel electrophoresis showed different numbers of bands for different genotypes: the GG genotype showed two bands (363 bp and 212 bp), the AG genotype showed three bands (575 bp, 363 bp, and 212 bp), and the AA genotype showed one band (575 bp). Statistical analysis of the 241 Duroc x Large White three-way crossbred individuals revealed 52 individuals with the GG genotype, 77 with the AA genotype, and 112 with the AG heterozygous genotype. Some PCR product digestion results are shown below. Figure 2 .

[0102] 3.3 Correlation between mutation sites and backfat thickness

[0103] The number of individuals with different genotypes was statistically analyzed, and the correlation between SNPs and backfat thickness in pigs was examined. The results showed a correlation between the two. Individuals with the GG genotype had the highest backfat thickness (0.9983±0.1941 cm), followed by individuals with the AG genotype (0.9253±0.1861 cm), and individuals with the AA genotype had the lowest backfat thickness (0.8753±0.2173 cm). The backfat thickness of individuals with the GG genotype was significantly higher than that of individuals with the AG or AA genotypes (P<0.05). Figure 3 .

[0104] 3.4 Prediction of transcription factor binding sites

[0105] Based on the prediction of the transcription factor binding site at the g.348A>G site of the ADIPOQ gene from the JASPAR database website, the prediction results are shown in Table 4.

[0106] Table 4. Prediction results of ADIPOQ gene g.348A>G transcription factor binding sites.

[0107] Base type To bind transcription factors A SPI1 G ——

[0108] 4. Results Analysis

[0109] The mutation sites screened in this invention are significantly correlated with backfat thickness in pigs and can be developed into SNP molecular markers for predicting backfat thickness in live pigs. For the ADIPOQ intron region g.348A>G, individuals with the GG genotype have higher backfat thickness than those with the AG or AA genotypes. When the ADIPOQ gene intron region g.348A>G site is A, the transcription factor SPI1 may bind to it, thereby regulating ADIPOQ gene expression and thus affecting backfat thickness in Duroc-Landrace-Landrace-Great Wall three-way crossbred pigs.

[0110] This invention screened a SNP locus related to backfat thickness in pigs within the ADIPOQ intron region, thereby obtaining the functional gene and molecular genetic marker associated with backfat thickness. The molecular markers provided by this invention can efficiently and accurately identify backfat thickness in pigs, thereby determining their meat production performance. This has significant application value in breeding pigs and provides a reliable reference for the selection of high-quality pig breeds.

[0111] It is understood that the above embodiments are merely exemplary implementations used to illustrate the principles of the invention. However, the present invention is not limited thereto. Those skilled in the art can make various improvements and modifications without departing from the essence of the present invention, and these improvements and modifications also fall within the protection scope of the present invention.

[0112] SEQ ID NO.1

[0113] This sequence represents the region from 2000 bp upstream to 700 bp downstream of the transcription start site in the porcine ADIPOQ gene sequence Chromosome 13,NC_010455.5(124633906..124646237). The yellow highlighted sequence represents the amplification region of ADIPOQ primers ADIPOQ-5F and ADIPOQ-5R; the first bold underlined nucleotide "A" indicates the transcription start site, and the second bold underlined nucleotide "A" indicates the SNP site g.348A>G located 348 bp downstream of the transcription start site, which is situated in the first intron region.

[0114]

[0115]

[0116] SEQ ID NO.2

[0117]

Claims

1. A substance for detecting the SNP site g.348 A>G polymorphism or genotype of ADIPOQ gene in pig genome for any of the following applications: (a1) identifying or assisting in identifying pig backfat thickness; (a2) preparing a product for identifying or assisting in identifying pig backfat thickness; (a3) screening or assisting in screening pig breeds with low backfat thickness; (a4) preparing a product for screening or assisting in screening pig breeds with low backfat thickness; the SNP site g.348 A>G is located at the 348th nucleotide downstream of the transcription start site in the sequence of pig ADIPOQ gene SEQ ID NO. 2, and the polymorphism is A or G, and the genotype of the SNP site g.348 A>G is AA, AG and GG; the pig is Duroc × Landrace × Yorkshire three-way crossbred pig.

2. Use according to claim 1, characterized in that, the substance is (b1) or (b2) or (b3) as follows: (b1) a PCR primer for amplifying a fragment of pig genomic DNA containing the SNP site g.348 A>G; (b2) a PCR reagent containing the PCR primer of (b1); (b2) a kit containing the PCR primer of (b1) or the PCR reagent of (b2).

3. Use according to claim 2, characterized in that, the PCR primer comprises an upstream primer ADIPOQ-5F and a downstream primer ADIPOQ-5R: ADIPOQ-5F: 5'-ACCTTCTCCTCATTTCCATC -3' ADIPOQ-5R: 5'-TTCTCACGAACCCTACCC -3'.

4. Use according to claim 1, characterized in that, Genotyping the SNP site g.348 A>G, the backfat thickness of individuals with genotype GG is higher than that of individuals with genotype AG or AA.

5. A method for identifying or aiding in the identification of pigs with high backfat, characterized in that, detecting the SNP site g.348 A>G polymorphism or genotype of ADIPOQ gene in pig genome; the backfat thickness of individuals with genotype GG is higher than that of individuals with genotype AG or AA; the pig is Duroc × Landrace × Yorkshire three-way crossbred pig.

6. The method of claim 5, wherein, The method for detecting the SNP site g.348 A>G polymorphism or genotype of ADIPOQ gene in pig genome is: using the pig genomic DNA to be tested as a template, and using the PCR primer of claim 3 to perform PCR amplification; performing enzyme digestion typing of the amplified fragment by PCR-RFLP-SsiI method, and different genotypes of enzyme digestion products will present different band numbers: the GG genotype is 363 bp and 212 bp two bands, the AG genotype is 575 bp, 363 bp and 212 bp three bands, and the AA genotype is 575 bp one band.

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