Molecular marker of structural variation affecting pig abdominal subcutaneous fat weight on pig chromosome 5 and application thereof

Abstract

This invention discloses a structural variation molecular marker on chromosome 5 of pigs that affects the weight of subcutaneous abdominal fat and its application. The structural variation molecular marker is a DNA fragment inserted after the 66120412 bp site on chromosome 5 corresponding to the International Pig Reference Genome Version 11.1; the nucleotide sequence of the DNA fragment is shown in SEQ ID NO:1. The structural variation molecular marker provided by this invention is significantly correlated with the trait of subcutaneous abdominal fat weight in pigs. By identifying this structural variation molecular marker, the trait of subcutaneous abdominal fat weight in pigs can be identified. Furthermore, by selecting individuals carrying the structural variation molecular marker during the breeding process, the subcutaneous abdominal fat weight of offspring pigs can be reduced, and the lean meat percentage of offspring pigs can be increased.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to a structural variation molecular marker on chromosome 5 of pigs that affects the weight of subcutaneous fat in the abdominal region and its application. Background Technology

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

[0003] While traditional breeding methods can improve the abdominal subcutaneous fat weight trait in pigs to some extent, this trait is a complex quantitative trait regulated by multiple genes, leading to limitations such as low improvement efficiency and long selection cycles. To accelerate the breeding process, molecular genetic methods, such as marker-assisted selection (MAS), have become an effective alternative. Quantitative genetic methods, such as genome-wide association studies (GWAS), can screen for effective molecular markers at high throughput. Structural variations (SVs) contribute more to complex phenotypes than single nucleotide polymorphisms (SNPs) and can explain some of the "lost heritability." Summary of the Invention

[0004] The purpose of this invention is to provide a structural variation molecular marker on chromosome 5 of pigs that affects the weight of subcutaneous fat in the abdominal region and its application.

[0005] According to one aspect of the invention, a structural variation affecting the weight of subcutaneous fat in the abdomen of pigs is provided, specifically a 344 bp DNA fragment (named chr5:66120412:INS) inserted after the 66120412 bp 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 this invention is shown in SEQ ID NO:1. It is significantly correlated with the abdominal subcutaneous fat weight trait in pigs. Specifically, pigs carrying chr5:66120412:INS have a lower abdominal subcutaneous fat weight than pigs not carrying chr5:66120412:INS. By identifying the structural variation molecular marker of this invention, the abdominal subcutaneous fat weight trait in pigs can be determined. Furthermore, by selectively breeding pigs carrying the structural variation molecular marker of this invention, the pig breeding process can be accelerated, improving the abdominal subcutaneous fat weight and lean meat percentage of offspring pigs, thereby achieving genetic improvement in pigs.

[0007] Therefore, the structural variation molecular markers on chromosome 5 of pigs that are associated with abdominal subcutaneous fat weight provided by this invention can be applied to:

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

[0009] (2) Prepare products for identifying the weight characteristics of subcutaneous fat in the abdomen of pigs;

[0010] (3) Pig genetic improvement: pigs are genetically improved by selecting pigs that carry molecular markers of structural variations on chromosome 5 that affect the weight of subcutaneous fat in the abdomen.

[0011] (4) Prepare a product for assisting in the genetic improvement of pigs. This product is based on identifying molecular markers of structural variations on chromosome 5 of pigs that affect the weight of subcutaneous fat in the abdomen of pigs to assist in the genetic improvement of pigs.

[0012] In some implementations, the pigs are preferably Duroc-Landrace-Large White crossbred pigs, i.e., commercial pigs obtained by crossing Duroc, Landrace, and Large White 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 shown in SEQ ID NO:2, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO:3 and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:4. The nucleotide sequence shown in SEQ ID NO:2 contains a structural variation molecular marker on chromosome 5 of pigs that affects the weight of subcutaneous fat in the abdominal region, as provided by the present invention.

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

[0015] The applications of the primer pairs and kits provided by this invention include, but are not limited to:

[0016] (1) Identify the molecular markers of structural variations on chromosome 5 of pigs that affect the weight of subcutaneous fat in the abdominal region;

[0017] (2) Prepare products to identify molecular markers of structural variations on chromosome 5 of pigs that affect the weight of subcutaneous fat in the abdominal region;

[0018] (3) Identify the abdominal subcutaneous fat weight trait of pigs. The identification of the abdominal subcutaneous fat weight trait of pigs is achieved by identifying the molecular markers of structural variations on chromosome 5 of pigs that affect the abdominal subcutaneous fat weight.

[0019] (4) Prepare a product for identifying the abdominal subcutaneous fat weight trait of pigs. This product is based on identifying molecular markers of structural variations on chromosome 5 of pigs that affect the abdominal subcutaneous fat weight.

[0020] (5) Pig genetic improvement: pigs are genetically improved by selecting pigs that carry molecular markers of structural variations on chromosome 5 that affect the weight of subcutaneous fat in the abdomen.

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

[0022] In some embodiments, the kit provided by the present invention may further include: dNTPs, DNA polymerase, and Mg. 2+ The components of a standard PCR reaction system, including PCR reaction buffer, can be directly referenced or adopted from the relevant components of commercially available PCR amplification kits.

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

[0024] (1) Identify whether the pigs to be tested carry the structural variation molecular markers provided in this invention;

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

[0026] In some embodiments, 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] Whole-genome DNA was extracted from the pigs to be tested, and PCR amplification was performed using primer pairs with nucleotide sequences as shown in SEQ ID NO:3 and SEQ ID NO:4. The amplification products were sequenced, and the results were used to determine whether the pigs to be tested carried the structural variation molecular markers provided in this invention.

[0028] In some implementations, the pigs are Duroc-Landrace ...Handcross hybrids.

[0029] Compared with the prior art, the beneficial effects of the present invention include:

[0030] (1) This invention provides a structural variant molecular marker located on chromosome 5 of pigs that is significantly associated with the weight of abdominal subcutaneous fat in pigs, and verifies the effect of this structural variant molecular marker on the trait of abdominal subcutaneous fat in pigs. This helps to establish a molecular marker-assisted selection breeding technology for rapid improvement of the trait of abdominal subcutaneous fat in pigs, improve the breeding process of Duroc-Landrace-Large White crossbred pigs, so as to meet the needs of the breeding pig market, increase the price of breeding pigs, and reduce breeding costs.

[0031] (2) This invention provides a primer pair that can be used to identify molecular markers of structural variations on chromosome 5 of pigs that affect the weight of subcutaneous fat in the 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. Attached Figure Description

[0032] Figure 1 This is a genome-wide association (GWAS) diagram of the abdominal subcutaneous fat weight trait on chromosome 5 in Duroc-Landrace-Large White crossbred pigs; where: the horizontal axis represents the chromosome number of the pig; the vertical axis represents the -logP value.

[0033] Figure 2 This is a graph showing the quantitative trait locus (eQTL) analysis of CCND2 expression on chromosome 5 in Duroc-Landrace-Large White crossbred pigs; where: the horizontal axis represents the distance from the CCND2 transcription start site (TSS); and the vertical axis represents the -logP value;

[0034] Figure 3 This is a graph showing the results of the colocalization analysis, where the vertical axis represents the -logP value of the GWAS analysis and the horizontal axis represents the -logP value of the eQTL analysis.

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

[0036] Figure 5 The results of Oil Red O staining show the effects of CCND2 overexpression and knockdown on lipid droplet deposition in porcine preadipocytes; among them: oe-NC CCND2 is the group transfected with blank plasmid; oe CCND2 is the group transfected with CCND2 overexpression plasmid; si-NC CCND2 is the group transfected with negative control siRNA; si CCND2 is the group transfected with siRNA targeting CCND2.

[0037] Figure 6 This is a graph showing the results of the effects of CCND2 overexpression and knockdown on triglyceride synthesis in porcine preadipocytes; where: oe-NC is the group transfected with blank plasmid; oe is the group transfected with CCND2 overexpression plasmid; si-NC is the group transfected with negative control siRNA; si is the group transfected with siRNA targeting CCND2. *** This indicates that P < 0.001; **** This indicates that P < 0.0001;

[0038] Figure 7 This is a graph showing the analysis of subcutaneous fat weight in the abdomen and CCND2 gene expression levels in pigs of different genotypes. *** This indicates that P < 0.001. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the embodiments. The embodiments are for illustrative purposes only and do not limit the invention in any way. Unless otherwise specified, the raw materials and reagents used in the embodiments are conventional products that can be obtained commercially; experimental methods that do not specify specific conditions in the embodiments are generally performed under conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0040] Example 1: Identification and Validation of Structural Variation Molecular Markers Related to Subcutaneous Fat Weight in Pig Abdomen

[0041] (1) Experimental pig herd

[0042] The experimental pig herd used in this invention consisted of 1496 Duroc-Landrace-Large White crossbred pigs from the breeding pig division of Guangdong Wens Foodstuff Group Co., Ltd., with detailed pedigree records. The pigs were raised under standardized feeding conditions, with free access to feed and water, until they reached a body weight of 100±5 kg.

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

[0044] Once pigs reached a weight of 100±5 kg, they were slaughtered and placed on a clean laboratory table. Internal organs were removed, and the abdominal area was cleaned. Using sterile dissecting tools, the skin was longitudinally incised 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 samples were immediately weighed using a balance, and the data were recorded to obtain phenotypic data on the weight characteristics of the pig's abdominal subcutaneous fat.

[0045] (3) Extraction of porcine genomic DNA

[0046] Ear tissue was collected from 1496 Duroc-Landrace-Large White crossbred pigs. Whole-genome DNA was extracted using the standard phenol-chloroform method. Subsequently, the DNA quality and concentration of each sample were determined using a Nanodrop-ND1000 spectrophotometer. An A260 / 280 ratio of 1.8–2.0 and an A260 / 230 ratio of 1.7–1.9 were considered acceptable. Finally, the acceptable DNA samples were uniformly diluted to approximately 50 ng / μL.

[0047] (4) Genotyping of porcine structural variations

[0048] A structural variant (SV) dataset was constructed based on third-generation nanopore sequencing data from 140 pigs (Oxford Nanopore Technologies, ONT), including 50 Duroc, 50 Landrace, 30 Large White, and 10 Duroc-Landrace-Landrace-Commercial pigs. The measured electrical signal files were converted to FASTQ format. SV detection based on third-generation read lengths 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 above reference SV set as a reference genome, second-generation resequencing data from 1496 Duroc-Landrace-Landrace-Commercial pigs were aligned to the reference SV set using Vg-tools 1.54 software, ultimately yielding 92486 structural variant sites. The obtained genotype data underwent quality control using PLINK v1.9, removing structural variant markers with a detection rate <90%, a mimor allelic frequency (MAF) <5%, and those located at unknown positions or on sex chromosomes. 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 analysis (GWAS)

[0050] Because kinship and population stratification effects may cause false positives, a kinship matrix needs to be constructed using GCTA software before association analysis. Principal component analysis is then performed using GCTA software, with the first five principal components used as covariates to correct for population structure. Individual sex, age, session number, and batch number are also considered. Finally, GWAS analysis is performed using a univariate mixture model in GCTA software. This invention references the human genome significance threshold, setting the genomic and chromosomal significance thresholds to 5.00E-08 (5.00 × 10⁻⁸). -8 ) and 1.00E-06 (1.00×10 -6 ).

[0051] GWAS analysis results are as follows Figure 1 As shown. From Figure 1 It was found that in Duroc-Landrace-Large White crossbred pigs, there is a structural variation site on chromosome 5 that significantly affects the weight of subcutaneous fat in the abdomen. The type of variation is structural variation, and the strongest associated structural variation is chr5:66120412:INS (P = 1.735 × 10⁻⁶). -8 Its nucleotide sequence is shown in SEQ ID NO:1.

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

[0053] Abdominal fat tissue was collected from 476 Duroc, Landrace, and Large White commercial pig individuals in the experimental population. DUTP libraries were constructed and paired-end sequencing was performed on the Illumina NovaSeq 6000 platform. All RNA sequencing samples were processed using a standardized workflow. Adapter sequences were spliced ​​using FASTP, and low-quality reads were removed. Clean reads were then aligned to the porcine reference genome (Sscrofa11.1) using STAR. Raw read counts were extracted using featureCounts, and then normalized to obtain the normalized expression (transcripts per million, TPM) for each gene based on the sum of exon lengths and the sequencing depth of the individual.

[0054] (7) eQTL location analysis

[0055] Genotype probabilities (PCs) were calculated using GCTA based on filtered variant sites, and the top five genotype PCs were used to explain the sample population structure within tissues. Simultaneously, the expression residual probability estimation method implemented in the PEER R package was used to estimate a set of latent covariates within each tissue based on the gene expression matrix to explain confounding factors in RNA-seq samples. The top 10 PEER factors were used as covariates. Gene expression was then normalized using the M-value trimmed mean (TMM) method implemented in the edgeR R package, followed by an inverse normal transformation of the TMM. e-QTL localization analysis was performed using a general linear regression model in TensorQTL (v.1.0.9), considering the estimated sequencing data covariates and individual sex, age, batch, and lot number. The 1MB upstream and downstream of the transcription start site (TSS) was defined as the cis-action window for the gene.

[0056] eQTL location analysis results are as follows: Figure 2 As shown in the figure, the structural variant with the strongest association in the GWAS analysis was chr5:66120412:INS, located in the promoter region 5.8 kb upstream of the CCND2 gene. Further eQTL localization analysis showed that this structural variant is an eQTL site that significantly affects CCND2 gene expression (P = 8.02 × 10⁻⁶). -8 This suggests that the structural variation may affect lipid deposition by regulating the transcriptional level of CCND2.

[0057] (8) Colocation Analysis

[0058] Based on the p-value, standard error, number of individuals used in the analysis, and allele frequency of each locus obtained from GWAS and eQTL mapping analyses, co-localization analysis was performed using the R package coloc. The co-localization analysis employed five mutually exclusive model assumptions (H0-H4), where H4 indicates that abdominal subcutaneous fat weight and gene expression are significantly associated with the same causal variant locus. A posterior probability (PP.H4) > 90% under the H4 assumption was used to determine whether the variant locus was a shared key variant between GWAS and eQTL mapping analyses.

[0059] 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 localization analyses (PP.H4>90%). This indicates that this variant site simultaneously regulates abdominal subcutaneous fat weight and CCND2 expression.

[0060] (9) Construction of overexpression plasmids and siRNA

[0061] The cDNA fragment of CCND2 (nucleotide sequence shown in SEQ ID NO:5) was amplified by PCR using primers with nucleotide sequences as 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 its overexpression plasmid. The oligonucleotide sequences required for the overexpression vector 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 below.

[0067] Table 1 siRNA sequences

[0068]

[0069] (10) The effects of overexpression plasmid and siRNA treatment on the expression level of CCND2 gene in porcine preadipocytes were detected by RT-qPCR.

[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 this was repeated every 3 days until day 9. After preadipocyte differentiation was complete, blank plasmid, CCND2 overexpression plasmid, CCND2-targeting siRNA, and negative control siRNA were transfected into differentiated porcine preadipocytes using Lipofectamine 3000. Cells were collected 24 hours and 7 days after transfection, respectively. Total RNA was extracted using the Trizol method and reverse transcribed using the M5 First Strand cDNA Synthesis Kit, while genomic DNA was removed. RT-qPCR was performed on the Bio-Rad iQ5 real-time PCR system using PerfectStart. TM The RT-qPCR reaction was performed using Green qPCR SuperMix. The 10 μL RT-qPCR reaction mixture included 3 μL diluted cDNA, 5 μL 2×ChamQ Universal SYBR qPCR Master Mix, 0.3 μL (10 μM) each of forward and reverse primers, and 1.4 μL ddH2O. The PCR program was: 95℃ for 30 s; 95℃ for 10 s; 55–60℃ for 30 s; 40 cycles. GAPDH was used as an internal control. The specific primer sequences are shown in Table 2 below.

[0071] Table 2 RT-qPCR Primers

[0072]

[0073]

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

[0075] (11) Red O staining and cellular triglyceride assay

[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 medium was replaced with maintenance medium containing 10% FBS and 8.6 μM insulin, and changed every 3 days until day 9. After preadipocyte differentiation was complete, blank plasmid, CCND2 overexpression plasmid, CCND2-targeting siRNA, and negative control siRNA were transfected into differentiated porcine preadipocytes using Lipofectamine 3000. Six days after transfection, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 30 minutes, and then stained with Oil Red O. After staining, cells were washed three times with PBS and observed under an inverted microscope. Triglyceride levels were quantified using a triglyceride colorimetric assay kit.

[0077] Experimental results are as follows Figure 5 and Figure 6 As shown, the expression level of CCND2 significantly regulates lipid droplet deposition and triglyceride synthesis in porcine preadipocytes. Specifically, in cells overexpressing CCND2, the lipid droplet deposition area was significantly reduced, as evidenced by a marked decrease in staining intensity, suggesting that high CCND2 expression inhibits lipid accumulation. Conversely, in cells with CCND2 knockdown, the lipid droplet deposition area was significantly increased, and the staining intensity was enhanced, indicating that low CCND2 expression promotes lipid accumulation. Furthermore, the intracellular triglyceride concentration in the CCND2 overexpression group was significantly lower than that in the control group (P<0.001), further confirming its inhibitory effect on lipid synthesis; while the triglyceride concentration in the CCND2 knockdown group was significantly higher (P<0.0001).

[0078] Based on the above results, it can be determined that CCND2 is a causal gene affecting the weight of subcutaneous fat in the abdomen of pigs, and the structural variation at 66120412 bp on chromosome 5 of the International Pig Reference Genome Version 11.1 is the fundamental reason for the change in the expression level of the CCND2 gene.

[0079] (12) Association analysis between different genotypes and the phenotype of abdominal subcutaneous fat weight in pigs and the expression level of the CCND2 gene was conducted to verify the effect of structural variation molecular markers on the trait of abdominal subcutaneous fat weight in pigs.

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

[0081] As shown in Table 3, the structural variant molecular markers provided by this invention are highly significantly correlated with the abdominal subcutaneous fat weight trait in pigs (P<0.01), indicating that these structural variant molecular markers significantly affect the abdominal subcutaneous fat weight in pigs. By using the auxiliary selection of this structural variant molecule in pigs, the abdominal subcutaneous fat weight of this population can be changed, thereby improving the economic benefits of pig farming.

[0082] According to Table 3, Figure 7 The results showed that individuals carrying the structural variant molecular marker of the present invention had less abdominal subcutaneous fat weight, indicating that individuals carrying the structural variant molecular marker had reduced abdominal subcutaneous fat weight. Selecting individuals carrying the structural variant molecular marker during the breeding process can improve the lean meat percentage and meat production performance of the population.

[0083] According to Table 4, Figure 7 The results showed that the structural variation molecular marker provided by this invention was 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 that CCND2 is a causal gene affecting the weight of subcutaneous fat in the abdomen of pigs.

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

[0085]

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

[0087]

[0088] (13) Effect analysis

[0089] This invention provides a structural variant molecular marker that is significantly correlated with the subcutaneous fat weight trait in pigs. Using this marker for marker-assisted selection can accelerate the breeding process of reducing subcutaneous fat weight in pigs. During the selection process, if individuals carrying the structural variant molecular marker that affects subcutaneous fat weight are selected, and those without the marker are culled, the subcutaneous fat weight per pig can be reduced by approximately 9.30%. Since fat deposition traits have a favorable negative correlation with lean meat percentage, reducing fat deposition can increase lean meat percentage. Furthermore, improved body shape due to fat deposition traits can result in more aesthetically pleasing pigs, better suited for breeding stock selection, and thus more profitable for pig farming enterprises. Therefore, selecting breeding pigs carrying the structural variant molecular marker provided by this invention can bring greater economic benefits to enterprises.

[0090] Example 2: Methods for genetic improvement of pigs

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

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

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

[0094] The genetic improvement methods for pigs include the following steps:

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

[0096] (1) Collect ear tissue from pigs or tail tissue from piglets, extract whole genome DNA from pigs using the standard phenol-chloroform method, and then perform quality testing and concentration determination on the extracted DNA.

[0097] (2) PCR amplification

[0098] Prepare a 10 μL mixture, including: 1 μL DNA sample, 0.3 μL upstream primer, 0.3 μL downstream primer, 5 μL PCR mix, and 3.4 μL ddH2O. The PCR mix can be commercially available 2×Taq PCR Star Mix (Dye) (manufacturer: GenStar, catalog number: A012-101), which includes dNTPs, DNA polymerase, and Mg2+. 2+ Components of conventional PCR reaction systems, such as PCR reaction buffer.

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

[0100] (3) DNA sequence sequencing identification

[0101] The PCR amplification products were sequenced, with gene fragments sequenced in both forward and reverse reactions. The obtained sequences were compared with the genome sequence of the International Swine Reference Genome Version 11.1 to confirm whether the corresponding structural variant sequence (SEQ ID NO:1) was inserted. The sequencing results are shown below:

[0102]

[0103] Note: The underlined part shows the verified 344bp insertion sequence (SEQ ID NO:1), and the bolded part at the beginning and end of the sequence shows the positions of the designed primer sequences.

[0104] S2. Select pigs carrying structurally variable molecular markers as parents for breeding.

[0105] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. The application of primer pairs for detecting structural variation molecular markers on chromosome 5 of pigs that affect the weight of subcutaneous fat in the abdomen in the genetic improvement of the trait of subcutaneous fat weight in pigs, characterized in that, The structural variation molecular markers are located on chromosome 5 corresponding to International Pig Reference Genome Version 11.1; The primer pair specifically amplifies the amplified fragment with the nucleotide sequence shown in SEQ ID NO:2; the amplified fragment contains a structural variation molecular marker with the nucleotide sequence shown in SEQ ID NO: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; The abdominal subcutaneous fat weight of pigs carrying the aforementioned structural variant molecular marker was less than that of pigs not carrying the aforementioned structural variant molecular marker. The genetic improvement of the abdominal subcutaneous fat weight trait in pigs involves selecting individuals carrying structural variant molecular markers to reduce the abdominal subcutaneous fat weight of offspring pigs. The pigs in question are Duroc-Landrace-Large White crossbred pigs.

2. A method for genetically improving the subcutaneous fat weight trait in the abdomen of pigs, characterized in that, Includes the following steps: (1) Extract the whole genome DNA of the pig to be tested, perform PCR amplification using primer pairs, sequence the amplification products, and determine whether the pig to be tested carries a structural variation molecular marker with a nucleotide sequence as shown in SEQ ID NO:1 based on the sequencing results; 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. (2) Select individuals carrying structural variation molecular markers to reduce the subcutaneous fat weight in the abdomen of offspring pigs; The pigs in question are Duroc-Landrace-Large White crossbred pigs.

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