SNP molecular marker related to intramuscular fat content trait of pig and application thereof

Abstract

The application belongs to the technical field of pig molecular marker, and particularly discloses a SNP molecular marker related to a pig intramuscular fat content character and application thereof. Based on SNP molecular marker, whole genome correlation analysis and meta-analysis are carried out, and two SNP molecular markers related to the pig intramuscular fat content character are screened out. The SNP molecular marker is located on chromosome 5 of the pig, and can be used for marker-assisted selection of the pig intramuscular fat content character.

Description

Technical Field

[0001] This invention belongs to the field of porcine molecular marker technology, specifically relating to SNP molecular markers related to the intramuscular fat content trait in pigs and their applications. Background Technology

[0002] Intramuscular fat (IMF) is an important indicator for evaluating pork quality, closely related to flavor, tenderness, juiciness, and consumer acceptance. Studies by Fernandez et al. have shown that moderately increasing IMF levels in pig muscle helps improve the sensory and eating quality of meat (Fernandeze et al. 1999a; Fernandeze et al. 1999b). IMF is a typical complex quantitative trait, regulated by multiple genes and various physiological processes, and has therefore been an important target for the genetic improvement of pork quality (Davolian and Braglia 2007; Wone et al. 2018).

[0003] In recent years, with the development of high-density SNP chips and genome-wide association studies (GWAS) techniques, research on the genetic basis of intramuscular fat content (IMF) in pigs has deepened. Davoli et al. identified multiple SNP loci associated with IMF in Large White pigs and identified candidate genes such as PPP3CA, SCPEP1, and SDK1 (Davoli et al. 2016). Won et al. conducted GWAS analysis in Berkshire pig populations and identified multiple significant IMF-associated loci and candidate genes (Won et al. 2018). Zhuang et al. further discovered multiple genomic segments affecting IMF genetic variation in a large Duroc pig population and identified candidate genes such as BDKRB2, ATG2B, MED6, MAP3K9, and TCF7L2 (Zhuang et al. 2021). Furthermore, Wang et al. detected IMF-related QTLs on chromosome 5 in Suhuai pigs and proposed LRRK2 and PDZRN4 as candidate genes (Wang et al. 2021); Li et al. also detected significant IMF-related SNPs in commercial Duroc-Landrace-Large White crossbred pigs and proposed ABLIM3, DPH5, and DOCK10 as candidate genes (Li et al. 2022). Although various molecular markers related to IMF in pigs have been reported, the existing results show that the significant loci and candidate genes detected are not completely consistent among different breeds and populations, indicating that this trait has strong population specificity and a complex genetic background. Therefore, further exploration of SNP molecular markers that are stably associated with intramuscular fat content in pigs and are easy to detect and apply remains of great significance for molecular-assisted selection and genetic improvement of pork quality traits.

[0004] This invention uses genome-wide association analysis and meta-analysis to screen for SNP molecular markers that are significantly associated with intramuscular fat content in pigs. These markers can be used for molecular-assisted selection, breeding grouping, and early identification of superior individuals for the trait of intramuscular fat content in pigs, providing a new molecular tool for the genetic improvement of pork quality traits. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art, perform genome-wide association analysis on the trait of intramuscular fat content in pigs, and screen out two SNPs that are significantly associated with the trait of intramuscular fat content in pigs, thus providing new SNP molecular marker resources for the improvement of pork quality traits.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: SNP molecular markers significantly associated with intramuscular fat content in pigs were identified through genome-wide association analysis and meta-analysis. The nucleotide sequences of each SNP (100 bp upstream and downstream) were obtained from the pig Sscrofa 11.1 reference genome in the Ensembl database, as follows: The nucleotide sequence containing the SNP1 marker is shown in SEQ ID NO. 1 and 2. The SNP1 marker is located at position 101 of the sequence, corresponding to position 62852955 of chromosome 5 of the pig genome. The accession number in the dbSNP database is rs327084629. The polymorphic site is A or G, and the favorable allele is A. The nucleotide sequences containing the SNP2 marker are shown in SEQ ID NO. 3 and 4. The SNP2 marker is located at position 101 of the sequence, corresponding to position 62857928 on chromosome 5 of the pig genome. Its accession number in the dbSNP database is rs81229353. The polymorphic site is G or A, and the favorable allele is G.

[0007] Compared with the prior art, the present invention has the following beneficial effects: Compared to conventional screening methods, this invention has significant advantages such as high efficiency and precision. Applying it to selective breeding practices for the intramuscular fat content trait in pigs can effectively increase intramuscular fat content. Attached Figure Description

[0008] Figure 1 The Manhattan plot, based on GWAS meta-analysis, visualizes the results of GWAS meta-analysis.

[0009] Figure 2 The QQ plot, based on GWAS meta-analysis, compares the probability distributions of the actual -log10(P) value and the expected -log10(P) value to further determine the reliability of the GWAS meta-analysis results. Detailed Implementation

[0010] Example 1: Genotyping Detection and Data Processing 1. DNA extraction ① Longissimus dorsi muscle tissue was collected from 116 crossbred pigs of Large White, Meishan, and Duroc breeds. The collected tissues were homogenized and placed in a glass homogenizer. An equal volume of cell lysis buffer was added, which consisted of 100 mmol / L Tris-saturated phenol, 500 mmol / L disodium ethylenediaminetetraacetate (EDTA), 20 mmol / L sodium chloride (NaCl), 10% sodium dodecyl sulfate (SDS), and 20 μg / ml trypsin. Then, 10 ng / mL proteinase K was added, mixed well, and placed in a 65°C water bath for 30 min. The centrifuge tubes were then gently vortexed for 15 min and centrifuged at 12,000 rpm for 5 min. The supernatant was then transferred to another centrifuge tube. ② Add equal volumes of phenol, chloroform, and isoamyl alcohol (volume ratio 25:24:1), shake to mix, centrifuge at 12000 rpm for 5 min, and then transfer the supernatant to another centrifuge tube. ③ Add an equal volume of phenol, chloroform, and isoamyl alcohol (volume ratio 25:24:1), shake to mix, and centrifuge at 12000 rpm for 10 min. Transfer the supernatant to another centrifuge tube. ④ Add 2 volumes of pre-cooled anhydrous ethanol, let stand until the ethanol evaporates, then pick out the DNA precipitate and dissolve the DNA in ultrapure water. ⑤ DNA quality was detected using a DNA concentration analyzer and agarose gel electrophoresis.

[0011] 2. Whole genome sequencing After passing quality testing, the extracted DNA was sent to Shanghai Paisenno Biotechnology Co., Ltd. for whole-genome sequencing. Following library construction, paired-end sequencing was performed using the BGI T7 high-throughput sequencing platform, with an average sequencing depth of approximately 30×.

[0012] 3. Sequencing data processing and variant detection ① Quality control and alignment: The raw data was quality controlled using fastp software to remove adapters, polyglycated G / X sequences and low-quality sequences; the data was aligned to the pig reference genome (Sscrofa 11.1) using BWA software, and then converted and sorted using SAMtools to generate BAM files; ② Deduplication and recalibration: The MarkDuplicates module of GATK software is used to remove duplicate sequences, FixMateInformation is used to repair alignment information, and then the BaseRecalibrator and ApplyBQSR modules are used to complete the base quality fraction recalibration (BQSR). ③ Mutation detection: Use the GATK HaplotypeCaller module (-ERC GVCF mode) to perform mutation detection independently for each sample and generate a single-sample GVCF file; ④ Combined Detection and SNP Filtering: GATK CombineGVCFs was used to merge GVCF files of samples by chromosome. Single nucleotide polymorphism (SNP) sites were extracted using GATK SelectVariants. Subsequently, VariantFiltration was used for hard filtering with the following parameters: QD < 2.0, FS > 60.0, MQ < 40.0, SOR > 4.0, MQRankSum < -12.5, ReadPosRankSum < -8.0. Low-quality sites were removed, and the high-quality population SNP dataset marked "PASS" was retained for downstream analysis.

[0013] 4. Chip Data Processing Based on microarray data: The genotypes of 179 Enshi Black pigs were detected using the KPS_PorcineBreeding_plus microarray; the genotypes of 578 Suhuai pigs were detected using the GeneSeek Genomic Profiler (GGP) Porcine 80K microarray; the genotypes of 1,490 Duroc pigs, 453 Lulai Black pigs, and 582 Duroc-Landrace-Large White three-way crossbred pigs were detected using the GeneSeek Genomic Profiler (GGP) Porcine 50K microarray; and the genotypes of 233 Large White pigs were detected using the Illumina PorcineSNP60K microarray. The obtained SNP molecular marker loci were quality controlled using PLINK v1.9 software. SNPs with a missing locus rate greater than 10%, a sample missing rate greater than 10%, or a minimum allele frequency less than 0.01 were removed. Outliers were also removed based on PCA. Using Beagle v5.1 software, with the Large White pig whole genome resequencing data as the reference group, the microarray data was filled, and SNP molecular marker sites with DR2>0.8 and MAF>0.05 were screened for subsequent analysis.

[0014] Example 2: Genome-wide association analysis of intramuscular fat content trait in pigs 1. Phenotypic Correction We collected and processed 3,677 intramuscular fat content records from a total of 3,631 pigs, including 233 Large White pigs, 179 Enshi Black pigs, 578 Suhuai pigs, 1,490 Duroc pigs, 453 Lulai Black pigs, 582 Duroc-Landrace-Large White three-way crossbred pigs, and 116 Large White, Meishan, and Duroc crossbred pigs. Phenotypic data were processed according to the mean ± 3 standard deviations.

[0015] 2. Genome-wide association analysis based on a mixed linear model In this embodiment, the experimental pig herd used for the genome-wide association analysis (GWAS) consisted of 3,615 pigs after excluding outliers. A genome-wide phylogenetic matrix was first constructed using GEMMA software, followed by a GWAS analysis of intramuscular fat content using a linear mixture model (LMM) module, with the first five principal components used as covariates. The specific model is shown below:

[0016] in It is a phenotypic vector; It is a covariate matrix (containing the first 5 principal components and other fixed effects). It is a fixed effects vector; It is a tagged genotype vector. It is the effect size corresponding to the label. It is a random multigene effect vector, which follows ,in It is a genome kinship matrix constructed using GEMMA. It is additive genetic variance; It is a random residual vector that follows ,in It is the residual variance. It is an identity matrix.

[0017] 3. Genome-wide meta-analysis based on multiple populations Using METAL software, a standard error-based inverse variance weighting method was employed to perform a fit analysis of the total variation effect. In the meta-analysis, genomic control was enabled to correct for test statistic inflation caused by population stratification or latent kinship. After the integration analysis, the p-value at the genome-wide level was also calculated as -log[…]. 10 (P), the significance threshold is calculated according to the formula -log 10 (1 / number of SNPs) is calculated, and the SNP label is -log 10 A p-value greater than or equal to 7.282 indicates a significant association between this SNP and the intramuscular fat content trait. Manhattan plots and QQ plots were plotted using the CMplot package in R. Figure 1 , Figure 2 ).

[0018] Table 1: SNP markers associated with porcine intramuscular fat content identified based on GWAS meta-analysis

[0019] Table 2: Genotype frequencies and mean intramuscular fat content of SNP markers in pig populations

[0020] Table 3: Genotype distribution of rs327084629 locus in different populations

[0021] Table 4: Genotype distribution of rs327084629 locus in different populations

[0022] In summary, this invention identified two SNP loci significantly associated with intramuscular fat content in pigs. A polymorphic locus with genotype A or G exists at chromosome 62852955 (reference genome version Sscrofa 11.1) in Duroc, Suhuai, and Large White, Meishan, and Duroc crossbred pigs. This locus has accession number rs327084629 in the dbSNP database. Compared to the G / G genotype, pigs carrying the A / A or G / A genotypes have higher intramuscular fat content, with the favorable allele being A. A polymorphic locus at chromosome 62857928 (reference genome version Sscrofa) in Duroc, Duroc-Landrace-Large White crossbred pigs, Enshi Black, Lulai Black, Suhuai, Large White, and Large White, Meishan, and Duroc crossbred pigs. 11.1) There is a polymorphic site with genotype A or G. The accession number of this site in the dbSNP database is rs81229353. Compared with genotype A / A, pigs carrying genotypes A / G or G / G have higher intramuscular fat content, and the favorable allele is G.

[0023] Main references: [1]Fernandez X, Monin G, Talmant A, Mourot J, Lebret B. Influence ofintramuscular fat content on the quality of pig meat - 1. Composition of thelipid fraction and sensory characteristics of m. longissimus lumborum. MeatScience. 1999;53(1):59-65. [2]Fernandez X, Monin G, Talmant A, Mourot J, Lebret B. Influence ofintramuscular fat content on the quality of pig meat - 2. Consumeracceptability of m. longissimus lumborum. Meat Science. 1999;53(1):67-72. [3]Davoli R, Braglia S. Molecular approaches in pig breeding toimprove meat quality. Briefings in Functional Genomics&Proteomics. 2007;6(4):313-321. [4]Davoli R, Luise D, Mingazzini V, Zambonelli P, Braglia S, Serra A,Russo V. Genome-wide study on intramuscular fat in Italian Large White pigbreed using the PorcineSNP60 BeadChip. Journal of Animal Breeding andGenetics. 2016;133(4):277-282. [5]Wang X, Ding R, Quan J, Yang L, Yang M, Zheng E, Liu D, Cai G, WuZ, Yang J. Genome-wide association analysis reveals genetic loci andcandidate genes associated with intramuscular fat in Duroc pigs. Frontiers ofAgricultural Science and Engineering. 2017;4(3):335-341. [6]Won S, Jung J, Park E, Kim H. Identification of genes related tointramuscular fat content of pigs using genome-wide association study. Asian-Australasian Journal of Animal Sciences. 2018;31(2):157-162. [7]Zhuang Z, Ding R, Qiu Y, Wu J, Zhou S, Quan J, Zheng E, Li Z, WuZ, Yang J. A large-scale genome-wide association analysis reveals QTL andcandidate genes for intramuscular fat content in Duroc pigs. Animal Genetics.2021;52(4):518-522. [8]Wang BB, Hou LM, Zhou WD, Liu H, Tao W, Wu WJ, Niu PP, Zhang ZP,Zhou J, Li Q, Huang RH, Li PH. Genome-wide association study reveals aquantitative trait locus and two candidate genes on Sus scrofa chromosome 5affecting intramuscular fat content in Suhuai pigs. Animal. 2021;15(9):100341. [9]Li H, Xu C, Meng F, Yao Z, Fan Z, Yang Y, Meng X, Zhan Y, Sun Y,Ma F, Yang J, Yang M, Wu Z, Cai G, Zheng E. Genome-Wide Association Studiesfor Flesh Color and Intramuscular Fat in (Duroc × Landrace × Large White)Crossbred Commercial Pigs. Genes. 2022;13(11):2131.

Claims

1. The application of SNP markers in the selection of intramuscular fat content in pigs, characterized in that, Includes at least one of the following SNP tags: Nucleotide sequences containing the SNP1 marker are shown in SEQ ID NO.1 and 2, where the SNP1 marker is located at position 101 and the polymorphic site is A or G; Nucleotide sequences containing the SNP2 marker are shown in SEQ ID NO.3 and 4, where the SNP2 marker is located at position 101 of the sequence and the polymorphic site is G or A.

2. The application according to claim 1, characterized in that: The favorable allele for the SNP1 marker is A, and the favorable allele for the SNP2 marker is G. Samples with the favorable genotype exhibit high intramuscular fat content.

3. The application according to claim 1 or 2, characterized in that, The SNP1 marker applies to Duroc pigs, Suhuai pigs, and crossbred pigs of Large White, Meishan, and Duroc; the SNP2 marker applies to Duroc pigs, Duroc-Landrace-Large White three-way crossbred pigs, Enshi Black pigs, Lulai Black pigs, Suhuai pigs, Large White pigs, and crossbred pigs of Large White, Meishan, and Duroc.

4. The application of the reagent kit in the selection of intramuscular fat content in pigs, characterized in that, The kit contains reagents for detecting polymorphic sites on porcine chromosome 5, wherein the polymorphic sites include at least one of the following: The polymorphic site at position 62852955 of chromosome 5 is either A or G; The polymorphic site at position 62,857,928 of chromosome 5 is either G or A. The reference genome version is Sscrofa 11.1.

Images