SNP molecular marker of il-15 gene associated with low backfat thickness and high intramuscular fat content in pigs and application thereof

Abstract

The application discloses an SNP molecular marker related to low back fat thickness and high intramuscular fat content of pigs on an IL-15 gene and application thereof, and belongs to the field of molecular biology. The application screens an SNP site, which is located at 334bp upstream of a start codon of an IL-15 gene and is specifically G / A mutation. Research finds that the SNP site genotype is closely related to back fat thickness and intramuscular fat content; the intramuscular fat content of individuals with GG genotype and GA genotype is significantly higher than that of individuals with AA genotype, and the back fat thickness of individuals with GG genotype is significantly lower than that of individuals with AA genotype. The application provides an important molecular marker for improving and cultivating high-yield and high-quality pig breeds by using the SNP of the IL-15 gene, and provides certain help for the research on pork body traits and meat quality traits.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, and specifically relates to a molecular marker on the IL-15 gene related to low backfat and high intramuscular fat content in pigs and application thereof. BACKGROUND

[0002] With the increase of pork consumption, improving meat quality has become an important goal of the pig industry. Among them, intramuscular fat content is a key factor affecting pork quality. Studies have shown that intramuscular fat not only affects the taste and flavor of meat, but its nutritional value also has an important impact on consumer health. Backfat thickness is directly related to the production efficiency and economic benefits of pigs.

[0003] In recent years, the rapid development of genomics has provided a new perspective for the genetic improvement of pigs. Single nucleotide polymorphism (SNP) as the most common form of genetic variation plays an important role in trait inheritance. In this study, we found that the SNP of IL-15 gene was significantly associated with intramuscular fat content and backfat thickness in pigs. Specifically, the G / A mutation upstream of the start codon of the IL-15 gene may affect the carcass performance and meat quality characteristics of pigs by regulating the biological pathways related to fat metabolism.

[0004] Through the analysis of different genotypes (such as GG, AG and AA types), we can better understand how these variations affect meat quality traits, thereby providing a scientific basis for breeding selection. In view of this, the development of a detection method based on the SNP of IL-15 gene will provide a new tool for genetic evaluation and selection of pigs, and will help improve the overall quality of meat and market competitiveness. SUMMARY

[0005] The purpose of the present application is to provide a molecular marker on the IL-15 gene related to both backfat thickness and intramuscular fat content in pigs and application thereof. In the present application, we found that a SNP site of IL-15 (Interleukin-15) gene was significantly associated with both backfat thickness and intramuscular fat content in pigs. Therefore, it can be used as a molecular marker to improve breeding efficiency. Using this molecular marker, we can quickly predict the differences in backfat thickness and intramuscular fat content between different genotypes of pigs, which can be used as a reliable marker for breeding pigs with low backfat thickness and high intramuscular fat content in breeding work.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] In a first aspect, the present application claims the application of a substance for detecting the polymorphism or genotype of the SNP site on the IL-15 gene in the pig genome in any of the following:

[0008] (a1) application in identifying or assisting in identifying backfat thickness and intramuscular fat content in pigs;

[0009] (a2) the use of the product for identifying or assisting in detecting the backfat thickness and intramuscular fat content of pigs;

[0010] (a3) the use of the product for assisting in breeding pigs with low backfat thickness and high intramuscular fat content;

[0011] The SNP site is located at 334 bp upstream of the start codon of the IL-15 gene, and the polymorphism thereof is G or A, and the genotype of the SNP site is GG, AG or AA. Therefore, the SNP site is named c.-334G>A.

[0012] The IL-15 gene sequence is shown in Chromosome 8, NC_010450.4 (85740980-85666963). The region from 2208 bp upstream of the start codon to 298 bp downstream of the start codon of the IL-15 gene is listed in the sequence table (as shown in SEQ ID NO. 1), wherein the sequence marked with a wavy line is the amplification region of the IL-15 primers IL-15-4F and IL-15-4R; the first bold underlined nucleotide "G" is the SNP site c.334G>A at 334 bp upstream of the start codon. The second bold underlined nucleotide "ATG" is the start codon.

[0013] Further, the substance is (b1) or (b2) or (b3) as follows:

[0014] (b1) a PCR primer for amplifying a fragment of the pig genomic DNA containing the SNP site;

[0015] (b2) a PCR reagent containing the PCR primer of (b1);

[0016] (b2) a kit containing the PCR primer of (b1) or the PCR reagent of (b2).

[0017] Further, the PCR primer comprises an upstream primer IL-15-4F and a downstream primer IL-15-4R:

[0018] IL-15-4F: 5'-TCAATAACAAAGGGAATCAGA-3' (SEQ ID NO. 3)

[0019] IL-15-4R: 5'-ACAACTTCAGGGAGTAGCG-3' (SEQ ID NO. 4).

[0020] Further, the application is used for genotyping the SNP site, and the intramuscular fat content of GG and GA type individuals is significantly higher than that of AA type individuals, and the back fat thickness of GG type individuals is significantly lower than that of AA type individuals.

[0021] In the second aspect, the application claims the use of the aforementioned PCR primer in the preparation of a product for detecting the polymorphism or genotype of the aforementioned SNP site.

[0022] In the third aspect, the application claims a product for genotyping the aforementioned SNP site, which contains the aforementioned substance for detecting the polymorphism or genotype of the SNP site of the IL-15 gene in the pig genome or the aforementioned PCR primer. In the specific embodiment of the application, the product is a kit.

[0023] In the fourth aspect, the application claims a method for identifying or assisting in identifying the back fat thickness and intramuscular fat content of pigs, which detects the polymorphism or genotype of the SNP site of the IL-15 gene in the pig genome; the intramuscular fat content of GG and GA type individuals is significantly higher than that of AA type individuals, and the back fat thickness of GG type individuals is significantly lower than that of AA type individuals.

[0024] In the fifth aspect, the application claims a method for breeding pig individuals with low back fat thickness and high intramuscular fat content, which detects the polymorphism or genotype of the SNP site of the IL-15 gene in the pig genome; retains GG homozygous genotype individuals, eliminates GA heterozygous genotype individuals and AA homozygous genotype individuals, and breeds pig individuals with low back fat thickness and high intramuscular fat content generation by generation.

[0025] Further, in the aforementioned method, the method for detecting the polymorphism or genotype of the SNP site of the IL-15 gene in the pig genome is as follows: taking the pig genomic DNA to be detected as a template, using the aforementioned PCR primer or the aforementioned product to perform PCR amplification; performing enzyme digestion typing on the amplified fragment by PCR-RFLP-SsiI method, and different genotypes of enzyme digestion products will present different band numbers: AA genotype is one band of 910 bp; GG genotype is two bands of 213 and 697 bp; and GA genotype is three bands of 213, 697 and 910 bp.

[0026] In the specific embodiment of the application, the method specifically includes the following steps:

[0027] 1) Extracting DNA of a pig tissue sample to be detected;

[0028] 2) Using the aforementioned PCR primer pair or the aforementioned product (such as a kit) to perform PCR amplification on the DNA obtained in step 1) to obtain an amplified fragment;

[0029] 3) The amplified fragments are subjected to enzyme digestion typing by PCR-RFLP-SsiI method, and the enzyme digestion products of different genotypes will present different band numbers: the GG genotype has two bands of 213 and 697 bp, the GA genotype has three bands of 213, 697 and 910 bp, and the AA genotype has one band of 910 bp;

[0030] 4) When the typing result is GG type and GA type, it is judged that the intramuscular fat content is higher than that of the AA type individual, and in terms of backfat thickness, the GG type individual is significantly lower than the AA type individual.

[0031] Further preferably, in step 2), the PCR amplification reaction system is 25 μL: 2 × Es Taq MasterMix (Dye) 12.5 μL, upper and lower primers 1 μL each, DNA to be detected 1 μL, ddH2O 9.5 μL; the PCR reaction program is: 94℃, 2 min; 94℃, 30 s; 54℃, 30 s annealing; 72℃, 30 s; 35 cycles; 72℃, 2 min.

[0032] Further preferably, the reaction system of the PCR-RFLP-SsiI method is: PCR amplified product to be detected 8 μL, SsiI enzyme 0.2 μL, 10 × FastDigest Buffer 1 μL, ddH2O 0.8 μL; the reaction condition is: 37℃, 10 min. TM

[0033] In a sixth aspect, the application claims to protect a molecular marker containing the above-mentioned SNP site c.-334G>A, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO. 2, and the SNP site c.-334G>A is located at 334 bp upstream of the start codon of the IL-15 gene in the sequence of SEQ ID NO. 2.

[0034] The room temperature in the application is 25±10℃, but is not limited thereto.

[0035] The application has the following beneficial effects:

[0036] The application screens a SNP site related to the backfat thickness and intramuscular fat content of pigs in the promoter region by studying the correlation between the important growth factor IL-15 in the organism and the backfat thickness and intramuscular fat content of pigs, thereby obtaining a functional gene and a molecular genetic marker related to the backfat thickness and intramuscular fat content of pigs, laying a foundation for further genetic improvement of the backfat thickness and intramuscular fat content of pigs, and directly selecting the genotype of pigs or marker-assisted selection, so as to accelerate the improvement and cultivation of high-yield and high-quality pig breeds. BRIEF DESCRIPTION OF DRAWINGS

[0037] ​Figure 1 G>A mutation found in the pig IL-15 gene sequencing in the present application.

[0038] Figure 2 Electrophoresis result schematic diagram of three genotypes (GG, AG or AA) of SNP site c.-334G>A of pig IL-15 gene detected by PCR-RFLP-SsiI method. Marker: 2000bp DNA molecular weight marker.

[0039] Figure 3 Correlation analysis of the genotype of SNP molecular marker and the backfat thickness and intramuscular fat content traits of Duroc Large White pigs. DETAILED DESCRIPTION

[0040] The present application is further described below in conjunction with examples. The experimental methods not specified in the following examples are generally according to the known means in the art.

[0041] Example 1

[0042] 1. Experimental materials

[0043] 1.1 Experimental animals

[0044] In the present study, 211 Duroc Large White three-way crossbred pigs were used, and the experimental animals were obtained from Jiangsu Changzhou Kangle Animal Husbandry Co., Ltd. and were raised under the same conditions. The liver tissue samples were collected for DNA extraction.

[0045] 1.2 Test reagents

[0046] (1) Conventional genomic DNA purification kit (containing RNase A): containing silica gel membrane centrifugal column, EB, BB2, LB2, WB2, Proteinase K and RNase A, purchased from Beijing Zengshi Gold Biotechnology Co., Ltd., stored at room temperature.

[0047] (2) 2×Es Taq MasterMix (Dye): containing 2×Es Taq MasterMix (Dye) and ddH2O, purchased from Nanjing Qikexin Biological Technology Co., Ltd., stored at -20℃.

[0048] (3) Goldview nucleic acid gel dye: purchased from Hunan Aikewei Biological Engineering Co., Ltd., stored at 4℃.

[0049] (4) DL2000 Marker: purchased from Nanjing Qikexin Biological Technology Co., Ltd., stored at -20℃, and after melting, stored at 4℃.

[0050] (5) Anhydrous ethanol AR 500mL: purchased from Shanghai Reagent Corporation

[0051] (6) FastDigest Ssi I restriction endonuclease: contains FastDigest restriction endonuclease, 10x FastDigest Green Buffer and 10x FastDigest TM Green Buffer and 10x FastDigest TM Buffer. Purchased from Nanjing Thermo Fisher Scientific, stored at -20 °C.

[0052] (7) 1x TAE buffer: self-made. To prepare 50 mL system, Tris base 12.1 g, glacial acetic acid 2.855 mL and EDTA (0.5 M, pH 8.0) 5 mL are needed.

[0053] (8) Agarose powder: purchased from Nanjing Qikeng Biological Co., Ltd.

[0054] 1.3 Instruments and equipment

[0055] (1) CF1524R desktop high-speed microfuge: purchased from Shanghai SCILOGEX Technology Co., Ltd.

[0056] (2) NanoDrop 2000 ultramicro spectrophotometer: purchased from Thermo Fisher Scientific

[0057] (3) PCR instrument: purchased from Zhuhai Haima Medical Instrument Co., Ltd.

[0058] (4) Gradient PCR instrument: purchased from Bio-Rad Life Science Co., Ltd.

[0059] (5) Horizontal electrophoresis instrument: purchased from Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd.

[0060] (6) Tanon 3500 gel imaging system: purchased from Shanghai Tian Neng Technology Co., Ltd.

[0061] (7) Mini metal bath instrument: purchased from Zhejiang Qunan Experimental Instrument (JOANLAB) Co., Ltd.

[0062] (8) Double-hole constant-temperature water bath: purchased from Zhejiang Qunan Experimental Instrument (JOANLAB) Co., Ltd.

[0063] The amplified fragments were sequenced by Qikeng (Nanjing) Biological Co., Ltd.

[0064] 2. Experimental methods

[0065] 2.1 Backfat thickness and intramuscular fat content traits of carcass determination

[0066] 2.1.1 Determination of backfat thickness of carcass

[0067] After slaughter, the backfat thickness of the pigs was measured with a vernier caliper, in cm. The measurement sites included the lumbar-sacral joint, the thickest part of the shoulder, the backfat thickness at the 3rd and 4th rib, and the average backfat thickness at three points.

[0068] 2.1.2 Determination of intramuscular fat content of longissimus dorsi muscle

[0069] (1) Disperse 9 cm of medium-speed quantitative filter paper in a clean enamel tray and bake at 105°C for 2 h. Weigh using a millionth scale, and record the weight of the filter paper as Wl;

[0070] (2) Weigh 2-3 g of longissimus dorsi muscle sample after chopping and wrap with dried filter paper, and record the wet weight of the paper package as W2;

[0071] (3) Spread the paper package on the enamel tray, taking care not to touch it, and bake in the oven at 65°C for 15 h. After drying, weigh the paper package and record it as W3, which is the constant weight after baking at 65°C for 2 h;

[0072] (4) After drying, place the paper package in a Soxhlet extractor and add anhydrous ether to reflux once. Soak the paper package again overnight, and the next day, start the extraction by turning on the water bath at 70°C for 9 h or more;

[0073] (5) Take out the paper package and air-dry it in the enamel tray for 30 min to allow the ether to fully volatilize. Bake at 105°C for 2 h and repeat several times until the weight is constant. Record the weight of the paper package as W4;

[0074] 6) To eliminate errors caused by different water contents during the processing of each sample, express the relative content of intramuscular fat as the percentage of the total amount of extracted fat to the final constant weight of the sample. The calculation formula is:

[0075]

[0076] 2.2 Extraction of liver tissue DNA

[0077] (1) Before starting the test, prepare a 55°C water bath 30 minutes in advance.

[0078] (2) Place chopped liver tissue ≤25 mg (the size of a mung bean) in a sterile 1.5 ml centrifuge tube.

[0079] (3) Add 100 ul of LB2 solution and 20 ul of Proteinase K (to ensure that the tissue sample is fully infiltrated with the liquid).

[0080] (4) Incubate at 55°C until complete lysis (about 3 hours, which may vary depending on the tissue), and the lysis solution will be viscous.

[0081] (5) If RNA removal is required, add 20 ul of RNase A to the sample and incubate at room temperature for 2 minutes.

[0082] (6) Place the centrifuge tube into a high speed centrifuge and centrifuge at 12,000 x g for 5 minutes. Immediately after centrifugation, transfer the supernatant to a sterile centrifuge tube.

[0083] (7) Add 500 ul of BB2 solution to the isolated supernatant and vortex immediately for 5 seconds. Confirm that the liquid is clear and free of visible particulate matter. Incubate at room temperature for 10 minutes.

[0084] (8) Transfer the entire solution to a spin column and centrifuge at 12,000 x g for 30 seconds. Discard the flow-through.

[0085] (9) Add 500 ul of CB2 solution (with 88 ml of absolute ethanol added prior to first use) to the spin column. Centrifuge at 12,000 x g for 30 seconds. Discard the flow-through.

[0086] (10) Add 500 ul of WB2 solution (with 88 ml of absolute ethanol added prior to first use) to the spin column. Centrifuge at 12,000 x g for 30 seconds. Discard the flow-through.

[0087] (11) Repeat step (10) once.

[0088] (12) Centrifuge at 12,000 x g for 2 minutes to remove any residual WB2 solution from the spin column.

[0089] (13) Place the spin column in a clean centrifuge tube and add 50-200 ul of pre-warmed EB solution (60°C-70°C) or deionized water (pH > 7.0) to the center of the column.

[0090] (60°C-70°C) or deionized water (pH > 7.0), room temperature for 1 minute, and centrifuge at 12,000 x g for 1 minute to elute the DNA.

[0091] (14) To obtain more DNA, repeat step (13) for a second elution.

[0092] (15) Measure the concentration of the newly extracted DNA using a spectrophotometer. Store the qualified DNA at -20°C.

[0093] 2.3 Primer design and synthesis

[0094] The Sus scrofa pig IL-15 full sequence gene information ( > NC_010450.4: c85740980-85665963 Sus scrofa isolate TJ Tabasco breed Duroc chromosome 8, Sscrofa11.1, whole genome shotgun sequence) was searched on the website NCBI (https: / / www.ncbi.nlm.nih.gov / ), and the region from 2208bp upstream of the start codon to 298bp downstream of the start codon was taken as the target sequence. The primer was designed using Premier 5.0, and the designed primer sequence was synthesized by General Biological (Anhui) Co., Ltd.

[0095] The above region was divided into four segments, and four pairs of primers IL-15-1, IL-15-2, IL-15-3, and IL-15-4 were designed for amplification, respectively. The primer sequence information is shown in Table 1.

[0096] Table 1 Pig IL-15 gene primer design information

[0097]

[0098] 2.4 PCR-RFLP reaction system and procedure

[0099] The PCR reaction system used in the experiment is shown in Table 2 and Table 3:

[0100] Table 2 PCR reaction system

[0101]

[0102] Table 3 Genotyping enzyme digestion reaction system

[0103]

[0104] PCR reaction procedure:

[0105] 94℃, 2min;

[0106] 94℃, 30s, 54℃, 30s, 72℃, 30s, 35 cycles;

[0107] 72℃, 2min; 4℃ preservation;

[0108] The amplified PCR product was digested according to the system in Table 3, and the reaction system was: 37℃, 10min.

[0109] 2.6 Agarose gel electrophoresis detection

[0110] The amplification product and the enzyme digestion product are subjected to electrophoresis on a 2% agarose gel, at a voltage of about 120 V, for about 30 min, and then observed by using a gel imaging system. The PCR product obtained by successful amplification is subjected to enzyme digestion. After the enzyme digestion reaction is completed, the enzyme digestion product is subjected to agarose gel electrophoresis. Different genotypes correspond to different numbers of bands, and the genotype of each sample is determined according to the number of bands in the electrophoretic result.

[0111] 3. Experimental results

[0112] 3.1 SNP screening

[0113] In a large sample, samples with extreme IMF content are selected, and 14 samples are selected for each of the high and low groups. Each DNA template is diluted to a concentration of 100 ng / μL. The DNA of the high group samples is mixed into a high H group DNA pool, and the DNA of the low group samples is mixed into a low L group DNA pool. The regions from 2208 bp upstream of the start codon to 298 bp downstream of the start codon of the IL-15 gene (as shown in SEQ ID NO. 1) are amplified using IL-15-1, IL-15-2, IL-15-3, and IL-15-4, respectively, to obtain a segment of about 2500 bp in the pig IL-15 gene, and then the PCR product is sequenced (served by Jingke Biotechnology (Nanjing) Co., Ltd.). The sequencing result shows that there is a SNP site in the segment (as shown in SEQ ID NO. 2) amplified by the primer pair IL-15-4, which is located at 334 bp upstream of the start codon of the IL-15 gene, and is respectively a guanine G or an adenine A, as shown in SEQ ID NO. 2. This SNP is named c.-334G>A. Figure 1

[0114] 3.2 PCR-RFLP genotyping

[0115] For the above SNP site, the primer IL-15-4 is used for PCR amplification, and the genotypes are identified by using SsiⅠ restriction enzyme to digest the PCR product. Through agarose gel electrophoresis, the enzyme digestion products of different genotypes will present different band numbers: the GG genotype has two bands of 213 and 697 bp, the GA genotype has three bands of 213, 697, and 910 bp, and the AA genotype has one band of 910 bp. According to statistics, in a 211 Duroc × Landrace × Yorkshire crossbreeding population, there are 100 individuals of the GG genotype, 21 individuals of the AA genotype, and 90 individuals of the AG heterozygote, and part of the PCR product enzyme digestion results are shown in Figure 2 .

[0116] 3.3 Correlation between the mutation site and backfat thickness and intramuscular fat content

[0117] ​The correlation between the SNP genotypes and the backfat thickness and intramuscular fat content of the pigs is analyzed, and the results show that the genotypes are significantly correlated with the backfat thickness and the intramuscular fat content. The backfat thickness of the AA type individual (0.9882±0.2154 cm) is the highest, the backfat thickness of the GA type individual (0.8949±0.1874 cm) is the second, and the backfat thickness of the GG type individual (0.8881±0.1804 cm) is the lowest. The backfat thickness of the AA type individual is significantly higher than that of the GG type individual (P<0.05), and there is no significant difference between the backfat thickness of the GA type individual and that of the GG type individual. In terms of the intramuscular fat content, the intramuscular fat content of the GG type and GA type individuals is significantly higher than that of the AG type individual (P<0.05), for example, Figure 3 .

[0118] 4. Result analysis

[0119] The mutation site screened in the application is significantly correlated with the backfat thickness and the intramuscular fat content of the pigs, and can be developed into a SNP molecular marker and used for predicting the difference in the backfat thickness and the intramuscular fat content between the pigs of different genotypes.

[0120] The application screens a SNP site related to the backfat thickness and the intramuscular fat content of the pigs at the upstream of the start codon of the IL-15 gene, thereby obtaining a functional gene and a molecular genetic marker related to the backfat thickness and the intramuscular fat content of the pigs. Through the molecular marker provided by the application, the backfat thickness and the intramuscular fat content of the pigs can be simultaneously identified efficiently and accurately, and the meat production performance of the pigs can be judged, which has a significant application value in the selection of breeding pigs and provides a reliable reference for the improvement and selection of high-yield and high-quality pig breeds.

[0121] It can be known that the above embodiment is only an exemplary embodiment adopted for illustrating the principles of the application, however, the application is not limited to this, and those skilled in the art can make various improvements and changes without departing from the essence of the application, and these improvements and changes also belong to the protection scope of the application.

[0122] SEQ ID NO. 1

[0123]

[0124]

[0125] SEQ ID NO.2

[0126]

[0127]

Claims

1. The use of a substance for detecting a SNP molecular marker on IL-15 gene in pig genome in any of the following: (a1) identifying or assisting in identifying pig backfat thickness and intramuscular fat content; (a2) preparing a product for identifying or assisting in identifying pig backfat thickness and intramuscular fat content; (a3) assisting in breeding pig individuals with low backfat thickness and high intramuscular fat content traits. The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO. 2, and the SNP molecular marker is located at position 213 of SEQ ID NO. 2, the base is G / A, and the genotype is GG, GA or AA.

2. Use according to claim 1, characterized in that, The substance is (b1) or (b2) or (b3) as follows: (b1) PCR primers for amplifying a pig genomic DNA fragment containing the SNP molecular marker; (b2) PCR reagents containing the PCR primers of (b1); (b2) a kit containing the PCR primers of (b1) or the PCR reagents of (b2).

3. Use according to claim 2, characterized in that, The PCR primers comprise an upstream primer IL-15-4F and a downstream primer IL-15-4R: IL-15-4F: 5'-TCAATAACAAAGGGAATCAGA-3' IL-15-4R: 5'-ACAACTTCAGGGAGTAGCG-3'.

4. Use according to claim 2 or 3, characterized in that, Genotyping the SNP molecular marker, the intramuscular fat content of individuals of GG type and GA type is significantly higher than that of individuals of AA type, and the backfat thickness of individuals of GG type is significantly lower than that of individuals of AA type.

5. A method for identifying or aiding in the identification of backfat and intramuscular fat content in swine, characterized in that, Detecting the SNP molecular marker on IL-15 gene in pig genome as claimed in claim 1; the intramuscular fat content of individuals of GG type and GA type is significantly higher than that of individuals of AA type, and the backfat thickness of individuals of GG type is significantly lower than that of individuals of AA type.

6. A method for selecting a pig individual for low backfat, high intramuscular fat content traits, characterized in that, Detecting the SNP molecular marker on IL-15 gene in pig genome as claimed in claim 1; retaining individuals with homozygous genotype of GG, eliminating individuals with heterozygous genotype of GA and homozygous genotype of AA, and breeding pig individuals with low backfat thickness and high intramuscular fat content traits generation by generation.

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