Structural variation molecular marker influencing pig abdomen subcutaneous fat weight on pig chromosome 5 and application thereof

Abstract

The invention discloses a structural variation molecular marker influencing pig abdomen subcutaneous fat weight on a pig chromosome 5 and application of the structural variation molecular marker. The structural variation molecular marker is a DNA fragment inserted behind a 66120412bp site on a chromosome 5 of an international pig reference genome version 11.1; the nucleotide sequence of the DNA fragment is as shown in SEQ ID NO: 1. The structural variation molecular marker provided by the invention is remarkably related to the pig abdominal subcutaneous fat weight character, the pig abdominal subcutaneous fat weight character can be identified by identifying the structural variation molecular marker, and the pig abdominal subcutaneous fat weight character can be identified by selecting individuals carrying the structural variation molecular marker in the breeding process. The weight of abdominal subcutaneous fat of offspring pigs can be reduced, and the lean meat percentage of the offspring pigs is improved.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology and relates to a structural variation molecular marker on pig chromosome 5 that affects the weight of subcutaneous fat in the pig abdomen and an application thereof. Background Art

[0002] As one of my country's most important livestock breeds, the pig produces pork and its products, accounting for nearly 50% of global protein consumption and production. Pork and its products play an irreplaceable role in maintaining human nutrition and quality of life. Pork quality and yield are closely related to lean meat percentage and fat deposition, with abdominal subcutaneous fat weight being a key indicator. Generally, lower fat deposition indicates a higher lean meat percentage, which directly impacts the pig's carcass weight, meat yield, and other meat values. Therefore, optimizing abdominal subcutaneous fat weight is crucial for improving pig production performance and market competitiveness.

[0003] Although traditional breeding methods can improve the abdominal subcutaneous fat weight trait of pigs to a certain extent, since this trait is a complex quantitative trait regulated by multiple genes, it leads to limitations such as low improvement efficiency and long selection cycles. In order to accelerate the breeding process, molecular genetic methods such as molecular marker-assisted selection (MAS) technology have become an effective alternative. Through quantitative genetic methods such as genome-wide association study (GWAS), effective molecular markers can be screened with high throughput. Compared with single nucleotide polymorphisms (SNPs), structural variation (SV) contributes more to complex phenotypes and can explain some of the "lost heritability." Summary of the Invention

[0004] The purpose of the present invention is to provide a molecular marker for structural variation on porcine chromosome 5 that affects the weight of subcutaneous fat in the pig abdomen and an application thereof.

[0005] According to one aspect of the present invention, a structural variation that affects the weight of subcutaneous fat in the pig abdomen is provided, which is specifically a 344bp DNA fragment (named chr5:66120412:INS) inserted after the 66120412bp site on chromosome 5 corresponding to the international pig reference genome version 11.1.

[0006] The nucleotide sequence of the structural variation molecular marker provided by the present invention is shown in SEQ ID NO: 1, which is significantly correlated with the abdominal subcutaneous fat weight trait of pigs. Specifically, the abdominal subcutaneous fat weight of pigs carrying chr5:66120412:INS is less than that of pigs not carrying chr5:66120412:INS. By identifying the structural variation molecular marker of the present invention, the abdominal subcutaneous fat weight trait of pigs can be identified. By breeding pigs carrying the structural variation molecular marker of the present invention, the pig breeding process can be accelerated, the abdominal subcutaneous fat weight of offspring pigs can be improved, and the lean meat percentage of offspring pigs can be increased, thereby achieving genetic improvement of pigs.

[0007] Therefore, the structural variation molecular markers on porcine chromosome 5 and associated with abdominal subcutaneous fat weight provided by the present invention can be applied to:

[0008] (1) Identify the abdominal subcutaneous fat weight characteristics of pigs;

[0009] (2) preparing a product for identifying the weight and properties of abdominal subcutaneous fat in pigs;

[0010] (3) Pig genetic improvement, based on breeding pigs carrying molecular markers of structural variation on chromosome 5 that affect the weight of subcutaneous fat in the pig abdomen;

[0011] (4) Prepare a product for assisting pig genetic improvement, which assists pig genetic improvement based on identifying molecular markers of structural variation on pig chromosome 5 that affect the weight of subcutaneous fat in the pig abdomen.

[0012] In some embodiments, the pig is preferably a Duroc-Landrace triple hybrid pig, that is, a commercial pig obtained by hybridizing Duroc, Landrace, and Yorkshire pigs.

[0013] According to another aspect of the present invention, a primer pair is provided that can specifically amplify an amplified fragment containing the nucleotide sequence set forth in SEQ ID NO: 2, wherein the nucleotide sequence of the upstream primer is set forth in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer is set forth in SEQ ID NO: 4. The nucleotide sequence set forth in SEQ ID NO: 2 contains the structural variation molecular marker on porcine chromosome 5 that affects the weight of subcutaneous fat in the pig abdomen, as provided by the present invention.

[0014] According to a third aspect of the present invention, a kit is provided, comprising a primer pair having nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0015] Applications of the primer pairs and kits provided by the present invention include but are not limited to:

[0016] (1) Identify molecular markers of structural variation on porcine chromosome 5 that affect abdominal subcutaneous fat mass in pigs;

[0017] (2) preparing a product for identifying molecular markers of structural variations on porcine chromosome 5 that affect subcutaneous fat weight in the pig abdomen;

[0018] (3) Identifying the abdominal subcutaneous fat weight trait of pigs, based on identifying molecular markers of structural variation on chromosome 5 that affect the abdominal subcutaneous fat weight of pigs;

[0019] (4) preparing a product for identifying the abdominal subcutaneous fat weight trait of pigs, wherein the product is based on identifying molecular markers of structural variation on chromosome 5 that affect the abdominal subcutaneous fat weight of pigs to achieve the identification of the abdominal subcutaneous fat weight trait of pigs;

[0020] (5) Pig genetic improvement, based on breeding pigs carrying molecular markers of structural variation on chromosome 5 that affect the weight of subcutaneous fat in the pig abdomen;

[0021] (6) Prepare a product for assisting pig genetic improvement, which assists pig genetic improvement based on identifying molecular markers of structural variation on pig chromosome 5 that affect the weight of subcutaneous fat in the pig abdomen.

[0022] In some embodiments, the kit provided by the present invention may further include: dNTPs, DNA polymerase, Mg 2+ The components of conventional PCR reaction systems such as PCR reaction buffer and PCR reaction buffer can be directly referenced or used in conventional commercially available PCR amplification kits.

[0023] According to a fourth aspect of the present invention, there is provided a method for genetic improvement of pigs, comprising the following steps:

[0024] (1) Identifying whether the pig to be tested carries the structural variation molecular marker provided by the present invention;

[0025] (2) Select and breed individuals carrying structural variation molecular markers; thereby improving the abdominal subcutaneous fat weight and increasing the lean meat rate of offspring pigs.

[0026] In some embodiments, in step (1), the method for identifying whether the pig to be tested carries the structural variation molecular marker provided by the present invention may include the following steps:

[0027] The whole genome DNA of the pig to be tested is extracted, and PCR amplification is performed using the primer pair with nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4. The amplified product is sequenced, and based on the sequencing results, it is determined whether the pig to be tested carries the structural variation molecular marker provided by the present invention.

[0028] In some embodiments, the pig is a Duroc-Changda hybrid pig.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention provides a structural variation molecular marker located on chromosome 5 of pigs, which is significantly correlated with the abdominal subcutaneous fat weight of pigs, and verifies the effect of the structural variation molecular marker on the abdominal subcutaneous fat weight trait of pigs, which helps to establish a molecular marker-assisted selection breeding technology for rapid improvement of the abdominal subcutaneous fat weight trait of pigs, improve the breeding process of Duchangda three-way hybrid pigs, so as to meet the needs of the breeding pig market, increase the price of breeding pigs, and reduce breeding costs.

[0031] (2) The present invention provides a primer pair that can be used to identify molecular markers of structural variation on pig chromosome 5 that affect the weight of subcutaneous fat in the pig abdomen. Through this primer pair, an efficient and accurate molecular marker-assisted breeding technology can be established to quickly and accurately select traits and accelerate the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a genome-wide association study (GWAS) analysis of the abdominal subcutaneous fat weight trait on chromosome 5 of the Duchang-Changda hybrid pig; wherein: the horizontal axis represents the chromosome number of the pig; the vertical axis represents the -logP value.

[0033] Figure 2 This is an analysis diagram of the expression quantitative trait loci (eQTL) for CCND2 on chromosome 5 of the Duchang-Changda hybrid pig; the horizontal axis represents the distance from the CCND2 transcription start site (TSS); the vertical axis represents the -logP value;

[0034] Figure 3 is the result of co-localization analysis, where the ordinate represents the -logP value of GWAS analysis and the abscissa represents the -logP value of eQTL analysis;

[0035] Figure 4 The overexpression and knockdown of CCND2 in porcine preadipocytes were detected by RT-qPCR at the mRNA level 24 hours and 7 days after transfection (n = 5 biologically independent samples); oe-NC was the blank plasmid transfection group; oe was the CCND2 overexpression plasmid transfection group; si-NC was the negative control siRNA transfection group; si was the CCND2-targeting siRNA transfection group; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001;

[0036] Figure 5 The results of Oil Red O staining show the effects of overexpression and knockdown of CCND2 on lipid droplet deposition in porcine preadipocytes; oe-NC CCND2 represents the blank plasmid transfection group; oe CCND2 represents the CCND2 overexpression plasmid transfection group; si-NC CCND2 represents the negative control siRNA transfection group; and si CCND2 represents the CCND2-targeted siRNA transfection group.

[0037] Figure 6 This is an analysis of the effects of CCND2 overexpression and knockdown on triglyceride synthesis in porcine preadipocytes; oe-NC represents the blank plasmid transfection group; oe represents the CCND2 overexpression plasmid transfection group; si-NC represents the negative control siRNA transfection group; and si represents the CCND2-targeted siRNA transfection group. *** indicates P < 0.001; **** indicates P < 0.0001;

[0038] Figure 7 This is an analysis chart of abdominal subcutaneous fat weight and CCND2 gene expression in pigs with different genotypes. *** Indicates P<0.001. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below with reference to the following embodiments. The examples are provided for illustrative purposes only and are not intended to limit the present invention in any way. Unless otherwise specified, the raw materials and reagents used in the examples are commercially available conventional products. Experimental procedures in the examples where specific conditions are not specified are generally performed in accordance with conventional conditions in the art or the conditions recommended by the manufacturer.

[0040] Example 1 Identification and Validation of Structural Variation Molecular Markers Associated with Pig Abdominal Subcutaneous Fat Weight

[0041] (1) Experimental pig herd

[0042] The experimental pig population used in the present invention is 1,496 Duchangda three-way hybrid pigs from the pig breeding branch of Guangdong Wen's Food Group Co., Ltd., with detailed pedigree records. The pigs were raised under unified feeding standards with free access to feed and water and raised to a body weight of 100±5 kg.

[0043] (2) Method for measuring abdominal subcutaneous fat weight

[0044] After the pigs were raised to a body weight of 100 ± 5 kg, they were slaughtered and placed on a clean laboratory surface. The internal organs were removed and the abdominal area was cleaned. Sterile dissecting instruments were used to longitudinally incise the skin along the midline of the abdomen to expose the subcutaneous fat layer. The subcutaneous fat layer was then completely separated from the muscle tissue, ensuring that no muscle or connective tissue remained in the fat sample. The separated abdominal fat sample was immediately weighed using a balance and the data was recorded to obtain phenotypic data for the pig's abdominal subcutaneous fat weight trait.

[0045] (3) Pig genomic DNA extraction

[0046] Ear tissue was collected from the 1,496 Du-Chang-Dang hybrid pigs described above. Whole-genome DNA was extracted using the standard phenol-chloroform method. DNA quality and concentration were then measured for each sample using a Nanodrop-ND1000 spectrophotometer. DNA samples were considered acceptable if their A260 / 280 ratios were between 1.8 and 2.0, and their A260 / 230 ratios were between 1.7 and 1.9. Finally, qualified DNA samples were uniformly diluted to approximately 50 ng / μL.

[0047] (4) Detection of pig structural variation genotypes

[0048] An SV dataset was constructed based on third-generation nanopore sequencing data (Oxford Nanopore Technologies, ONT) from 140 pigs, including 50 Duroc, 50 Landrace, 30 Large White, and 10 Duroc-Changda commercial pigs. The measured electrical signal files were converted to FASTQ format. SV detection based on third-generation reads was performed using Sniffles 2 software. The detection results were merged using Bcftools 1.19 and Truvari 4.2.1 software to form an SV variant dataset. Using the reference SV set, the second-generation resequencing data from 1496 Duroc-Changda commercial pigs were aligned to the reference SV set using Vg-tools 1.54 software based on the reference genome, resulting in 92,486 structural variant sites. Genotype data were quality controlled using PLINK v1.9. Structural variant markers with a detection rate <90% and a mimor allele frequency (MAF) <5% were removed. Structural variant markers located at unknown positions and on sex chromosomes were also excluded. The remaining 63,623 structural variation markers and 1,496 samples from the final quality control were used for subsequent data analysis.

[0049] (5) Genome-wide association (GWAS) analysis

[0050] Because kinship and population stratification effects may cause false positive results, a kinship matrix needs to be constructed using GCTA software before association analysis. Principal component analysis is also performed using GCTA software. The first five principal components are used as covariates to correct for population structure. The sex, age, field, and batch of individuals are also considered. GWAS analysis is then performed using a univariate mixed model using GCTA software. The present invention refers to the human genome significance threshold and sets the genomic and chromosomal significance thresholds to 5.00E-08 (5.00×10 -8 ) and 1.00E-06(1.00×10 -6 ).

[0051] GWAS analysis results are as follows Figure 1 As shown. Figure 1 It can be seen that in the Duchang-Changda hybrid pig, there is a structural variation site on chromosome 5 that significantly affects the pig abdominal subcutaneous fat weight trait. Its type is structural variation, and the strongest associated structural variation is chr5:66120412:INS (P=1.735×10 -8 ), the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0052] (6) Processing of pig abdominal fat transcriptome sequencing data

[0053] Abdominal fat tissue was collected from 476 Duchangda commercial pigs in the experimental population. Dual-end sequencing was performed on the Illumina NovaSeq 6000 platform using a dUTP library construction method. All RNA sequencing samples were processed using a unified workflow. Fastp was used to cut the adapter sequence and remove low-quality reads. Clean reads were then aligned to the porcine reference genome (Sscrofa11.1) using STAR. Raw read counts of genes were extracted using featureCounts and then normalized to the sum of the exon lengths of each gene and the sequencing depth of the individual to obtain the standardized expression of each gene (i.e., transcripts per million, TPM).

[0054] (7) eQTL location analysis

[0055] Genotypic PCs were calculated based on the filtered variant sites using GCTA, and the top five genotypic PCs were used to account for the sample population structure in the tissue. The expression residual probability estimation method implemented in the PEER R package was also used to estimate a set of potential covariates within each tissue based on the gene expression matrix to account for confounding factors in the RNA-seq samples. The top 10 PEER factors were used as covariates. Gene expression was then normalized using the trimmed mean M value (TMM) method implemented in the edgeR R package, and the TMM was subsequently inverse normalized. e-QTL positioning analysis was performed using the general linear regression model in TensorQTL (v.1.0.9), which took into account the estimated sequencing data covariates and the sex, age, field, and batch of the individuals. The 1MB upstream and downstream of the transcription start site (TSS) of the gene was defined as the cis-acting window of the gene.

[0056] The results of eQTL positioning analysis are as follows Figure 2 The structural variant with the strongest association in the GWAS analysis was chr5:66120412:INS, which is located in the promoter region 5.8 kb upstream of the CCND2 gene. Further eQTL mapping analysis showed that this structural variant is an eQTL site that significantly affects CCND2 gene expression (P = 8.02 × 10 -8 ), indicating that this structural variation may affect fat deposition by regulating the transcription level of CCND2.

[0057] (8) Colocalization analysis

[0058] Colocalization analysis was performed using the R package coloc, based on the P value, standard error, number of individuals analyzed, and minor allele frequency for each locus obtained from the GWAS and eQTL mapping analyses. Five mutually exclusive model hypotheses (H0-H4) were used for colocalization analysis, with H4 indicating a significant association between abdominal subcutaneous fat mass and gene expression at the same causal variant. A posterior probability of H4 (PP.H4) > 90% was used to determine if a variant was a shared key variant between the GWAS and eQTL mapping analyses.

[0059] The colocalization analysis results are as follows Figure 3 As shown, the structural variant chr5:66120412:INS was identified as a shared key variant site between GWAS and eQTL positioning analysis (PP.H4>90%), indicating that this variant site simultaneously regulates abdominal subcutaneous fat weight and CCND2 expression.

[0060] (9) Overexpression plasmid and siRNA construction

[0061] The CCND2 cDNA fragment (nucleotide sequence shown in SEQ ID NO:5) was amplified by PCR using primers with nucleotide sequences shown in SEQ ID NO:6 and SEQ ID NO:7. The CCND2 cDNA fragment was then inserted into the pCMV-HA vector to construct an overexpression plasmid. The oligonucleotide sequences required for the overexpression plasmid were custom synthesized by IGE Biotechnology (Guangzhou, China).

[0062] CCND2 cDNA primer-F:

[0063] 5'-CGCCACCATGGAGCTGCTGTGCTGCGAGGTGGACCCGGTCCG CAGGGCCGTGCCGGACG-3' (SEQ ID NO: 6);

[0064] CCND2 cDNA primer-R:

[0065] 5'-CCTCACAGGTCAATATCCCGCACGTCTGTAGGGGTGCTGGCTTGGTCCAGCTCGTCCTC-3' (SEQ ID NO: 7).

[0066] The oligonucleotide sequences of the CCND2-targeting siRNA (si-CCND2) and the negative control siRNA (si-NC) were designed using an online design tool (https: / / www.thermofiser.com / us / en / home / brands / invitrogen / ambion.html) and chemically synthesized by Generay Biotech (Shanghai, China). The specific nucleotide sequences of the siRNAs are shown in Table 1.

[0067] Table 1 siRNA sequences

[0068]

[0069] (10) RT-qPCR was used to detect the effects of overexpression plasmid and siRNA treatment on the expression level of CCND2 gene in pig preadipocytes.

[0070] Porcine preadipocytes (PIG-iCell-s019, Cellverse, China) were seeded into six-well cell culture plates. Differentiation was induced using F-12 medium containing 2.5 μM dexamethasone, 8.6 μM insulin, 0.1 mM 3-isobutyl-1-methylxanthine, 1% penicillin-streptomycin, and 10% fetal bovine serum. After 4 days of differentiation induction, the medium was replaced with maintenance medium containing 10% FBS and 8.6 μM insulin, and was changed every 3 days until day 9. After preadipocyte differentiation was completed, blank plasmid, CCND2 overexpression plasmid, siRNA targeting CCND2, and negative control siRNA were transfected into differentiated porcine preadipocytes using Lipofectamine 3000. Cells were collected 24 hours and 7 days after transfection, respectively, and total RNA was extracted using the Trizol method. Reverse transcription was performed using the M5 First Strand cDNA Synthesis Kit, and genomic DNA was removed at the same time. RT-qPCR was performed on the Bio-Rad iQ5 Real-Time PCR System using the PerfectStart TM Green qPCR SuperMix was used. A 10 μL RT-qPCR reaction system consisted of 3 μL diluted cDNA, 5 μL 2× ChamQ Universal SYBR qPCR Master Mix, 0.3 μL each of 10 μM forward and reverse primers, and 1.4 μL ddH₂O. The PCR program was as follows: 95°C for 30 s; 95°C for 10 s; 55–60°C for 30 s; 40 cycles. GAPDH was used as an internal control. Specific primer sequences are shown in Table 2.

[0071] Table 2 RT-qPCR primers

[0072]

[0073]

[0074] The results are as follows Figure 4 After transfection of CCND2 overexpression plasmid, the expression level of CCND2 mRNA in porcine preadipocytes was significantly increased (P<0.0001), while siRNA targeting CCND2 significantly knocked down the expression of CCND2 (P<0.01).

[0075] (11) Red O staining and cell triglyceride determination

[0076] Porcine preadipocytes (PIG-iCell-s019, Cellverse, China) were seeded into six-well cell culture plates. Differentiation was induced using F-12 medium containing 2.5 μM dexamethasone, 8.6 μM insulin, 0.1 mM 3-isobutyl-1-methylxanthine, 1% penicillin-streptomycin and 10% fetal bovine serum. After 4 days of differentiation induction, the culture medium was replaced with a maintenance medium containing 10% FBS and 8.6 μM insulin, which was changed every 3 days until the 9th day. After the preadipocyte differentiation was completed, blank plasmid, CCND2 overexpression plasmid, siRNA targeting CCND2 and negative control siRNA were transfected into differentiated porcine preadipocytes using Lipofectamine3000. Six days after transfection, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 30 minutes, and then stained with Oil Red O. After staining, the cells were washed three times with PBS and observed under an inverted microscope. Triglyceride levels were quantified using a triglyceride colorimetric assay kit.

[0077] The experimental results are as follows Figure 5 and Figure 6 As shown, CCND2 expression levels significantly regulate lipid droplet deposition and triglyceride synthesis in porcine preadipocytes. Specifically, in cells overexpressing CCND2, the area of lipid droplet deposition was significantly reduced, as evidenced by a significant decrease in staining intensity, suggesting that high CCND2 expression inhibits lipid accumulation. In contrast, in cells with CCND2 knockdown, the area of lipid droplet deposition increased significantly, with enhanced staining intensity, indicating that low CCND2 expression promotes lipid accumulation. Furthermore, intracellular triglyceride concentrations in the CCND2 overexpression group were significantly lower than in the control group (P < 0.001), further confirming its role in inhibiting lipid synthesis. In contrast, triglyceride concentrations were significantly increased in the CCND2 knockdown group (P < 0.0001).

[0078] Based on the above results, it can be determined that CCND2 is the causal gene affecting the abdominal subcutaneous fat weight trait of pigs, and the structural variation located at 66120412bp on chromosome 5 of the international pig reference genome version 11.1 is the root cause of the change in CCND2 gene expression level.

[0079] (12) Correlation analysis between different genotypes and pig abdominal subcutaneous fat weight phenotype and CCND2 gene expression, verifying the effect of structural variation molecular markers on pig abdominal subcutaneous fat weight traits

[0080] The results are shown in Tables 3 and 4. Figure 7 shown.

[0081] As shown in Table 3, the structural variation molecular marker provided by the present invention is extremely significantly correlated with the abdominal subcutaneous fat weight trait of pigs (P<0.01), indicating that this structural variation molecular marker significantly affects the abdominal subcutaneous fat weight of pigs. By auxiliary selection of this structural variation molecule in pigs, the abdominal subcutaneous fat weight of the group can be changed, thereby improving the economic benefits of pig farming.

[0082] According to Table 3, Figure 7 The results show that the weight of abdominal subcutaneous fat in individuals carrying the structural variation molecular marker of the present invention is small, indicating that the weight of abdominal subcutaneous fat in individuals carrying the structural variation molecular marker is reduced. Selecting individuals carrying the structural variation molecular marker during the breeding process can improve the lean meat rate and meat production performance of the group.

[0083] According to Table 4, Figure 7 The results show that the structural variation molecular marker provided by the present invention is extremely significantly correlated with the expression level of the CCND2 gene (P<0.01), indicating that the structural variation molecular marker is the root cause of the change in the expression level of the CCND2 gene, and CCND2 is the causal gene affecting the abdominal subcutaneous fat weight trait of pigs.

[0084] Table 3 Correlation analysis between genotypes of structural variation molecular markers and abdominal subcutaneous fat weight traits

[0085]

[0086] Table 4 Correlation between genotypes of structural variation molecular markers and CCND2 gene expression

[0087]

[0088] (13) Effect analysis

[0089] The present invention provides a structural variation molecular marker that is significantly correlated with the pig's abdominal subcutaneous fat weight trait. Using the structural variation molecular marker for marker-assisted selection can accelerate the breeding process of the abdominal subcutaneous fat weight of breeding pigs. In the breeding process, if individuals carrying the structural variation molecular marker that affects the pig's abdominal subcutaneous fat weight trait are selected and individuals that do not carry the structural variation molecular marker are eliminated, the abdominal subcutaneous fat weight of each pig can be reduced by about 9.30%. Since the fat deposition trait is negatively correlated with the lean meat rate, the lean meat rate can be increased by reducing the fat deposition trait of the pig, and the body shape improvement of the fat deposition trait can make the pig's body shape more beautiful, more in line with the appearance of the breeding pig selection, and more able to bring benefits to the breeding pig breeding enterprise. Therefore, selecting the breeding pigs carrying the structural variation molecular marker provided by the present invention will bring greater economic benefits to the enterprise.

[0090] Example 2 Genetic Improvement Method for Pigs

[0091] The DNA sequence containing the structural variation molecular marker on porcine chromosome 5 that affects the weight of pig abdominal subcutaneous fat (the specific nucleotide sequence is shown in SEQ ID NO: 1) is used as the amplified sequence (the specific nucleotide sequence is shown in SEQ ID NO: 2), and the upstream and downstream primers for PCR amplification are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0092] Upstream primer primer-F: 5′-GGGGAAAGAGCCAAAGAA-3′ (SEQ ID NO: 3);

[0093] Downstream primer primer-R: 5′-AGTGGACATTTTAAAGAC-3′ (SEQ ID NO: 4).

[0094] The method for genetic improvement of pigs includes the following steps:

[0095] S1. Identify whether the pigs to be tested carry structural variation molecular markers

[0096] (1) Collect pig ear tissue or piglet tail tissue, extract the pig whole genome DNA according to the standard phenol-chloroform method, and then perform quality detection and concentration determination on the extracted DNA.

[0097] (2) PCR amplification

[0098] Prepare a 10 μL system, including: 1 μL DNA sample, 0.3 μL upstream primer, 0.3 μL downstream primer, 5 μL PCR mix, 3.4 μL ddH2O; PCR mix can be commercially available 2× Taq PCR Star Mix (Dye) (manufacturer: GenStar, product number: A012-101), which includes dNTPs, DNA polymerase, MgCl2, dNTPs ... 2+ Components of conventional PCR reaction systems such as PCR reaction buffer.

[0099] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 64°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles, and a final extension at 72°C for 5 min.

[0100] (3) DNA sequence identification

[0101] The PCR amplification product was sequenced, and both forward and reverse reactions of the gene fragment were measured. The measured sequence was compared with the genome sequence of the International Porcine Reference Genome Version 11.1 to confirm whether the corresponding structural variation sequence (SEQ ID NO: 1) was inserted. The sequencing results are shown below:

[0102]

[0103] Note: The underlined portion is the verified 344 bp insert sequence (SEQ ID NO: 1), and the bolded portions at the beginning and end of the sequence are the positions of the designed primer sequences.

[0104] S2. Select pigs carrying structural variation molecular markers as parents for breeding.

[0105] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A molecular marker for structural variation on porcine chromosome 5 that affects the weight of subcutaneous fat in the pig abdomen, characterized in that: The structural variation molecular marker is a DNA fragment inserted after the 66120412bp site on chromosome 5 corresponding to the international porcine reference genome version 11.1; the nucleotide sequence of the DNA fragment is shown in SEQ ID NO:

1.

2. The structural variation molecular marker according to claim 1, characterized in that The structural variation molecular marker is associated with the weight of abdominal subcutaneous fat in pigs. The weight of abdominal subcutaneous fat in pigs carrying the structural variation molecular marker is less than that in pigs not carrying the structural variation molecular marker.

3. The structural variation molecular marker according to claim 1 or 2, characterized in that The pig is a Duchangda triple hybrid pig.

4. The use of a structural variation molecular marker according to any one of claims 1 to 3, characterized in that: The applications include: (1) Identify the abdominal subcutaneous fat weight characteristics of pigs; (2) preparing a product for identifying the weight and properties of abdominal subcutaneous fat in pigs; (3) Pig genetic improvement, based on breeding pigs carrying molecular markers of structural variation on chromosome 5 that affect the weight of subcutaneous fat in the pig abdomen; (4) Prepare a product for assisting pig genetic improvement, wherein the product assists pig genetic improvement based on identifying molecular markers of structural variation on pig chromosome 5 that affect the weight of subcutaneous fat in the pig abdomen.

5. A primer pair, characterized in that The primer pair specifically amplifies an amplified fragment containing the nucleotide sequence shown in SEQ ID NO: 2; the amplified fragment contains the structural variation molecular marker according to claim 1; the nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:

4.

6. The use of the primer pair according to claim 5, characterized in that: The applications include: (1) Identify molecular markers of structural variation on porcine chromosome 5 that affect abdominal subcutaneous fat mass in pigs; (2) preparing a product for identifying molecular markers of structural variations on porcine chromosome 5 that affect subcutaneous fat weight in the pig abdomen; (3) Identifying the abdominal subcutaneous fat weight trait of pigs, based on identifying molecular markers of structural variation on chromosome 5 that affect the abdominal subcutaneous fat weight of pigs; (4) preparing a product for identifying the abdominal subcutaneous fat weight trait of pigs, wherein the product is based on identifying a molecular marker for structural variation on chromosome 5 that affects the abdominal subcutaneous fat weight of pigs to achieve identification of the abdominal subcutaneous fat weight trait of pigs; (5) Pig genetic improvement, based on breeding pigs carrying molecular markers of structural variation on chromosome 5 that affect the weight of subcutaneous fat in the pig abdomen; (6) Prepare a product for assisting pig genetic improvement, wherein the product assists pig genetic improvement based on identifying molecular markers of structural variation on pig chromosome 5 that affect the weight of subcutaneous fat in the pig abdomen.

7. A kit, characterized in that Its composition includes the primer pair described in claim 5.

8. Use of the kit according to claim 7, characterized in that, The applications include: (1) Identify molecular markers of structural variation on porcine chromosome 5 that affect abdominal subcutaneous fat mass in pigs; (2) preparing a product for identifying molecular markers of structural variations on porcine chromosome 5 that affect subcutaneous fat weight in the pig abdomen; (3) Identifying the abdominal subcutaneous fat weight trait of pigs, based on identifying molecular markers of structural variation on chromosome 5 that affect the abdominal subcutaneous fat weight of pigs; (4) preparing a product for identifying the abdominal subcutaneous fat weight trait of pigs, wherein the product is based on identifying a molecular marker for structural variation on chromosome 5 that affects the abdominal subcutaneous fat weight of pigs to achieve identification of the abdominal subcutaneous fat weight trait of pigs; (5) Pig genetic improvement, based on breeding pigs carrying molecular markers of structural variation on chromosome 5 that affect the weight of subcutaneous fat in the pig abdomen; (6) Prepare a product for assisting pig genetic improvement, wherein the product assists pig genetic improvement based on identifying molecular markers of structural variation on pig chromosome 5 that affect the weight of subcutaneous fat in the pig abdomen.

9. A method for genetic improvement of pigs, characterized in that: The steps include: (1) Identifying whether the pig to be tested carries the structural variation molecular marker according to claim 1; (2) Select individuals that carry molecular markers of structural variation.

10. The method for genetic improvement of pigs according to claim 9, characterized in that: In step (1), the method for identifying whether the pig to be tested carries the structural variation molecular marker according to claim 1 comprises the following steps: Extract the whole genome DNA of the pig to be tested, perform PCR amplification using the primer pair according to claim 5, sequence the amplified product, and determine whether the pig to be tested carries the structural variation molecular marker according to claim 1 based on the sequencing results.

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