A SNP molecular marker related to backfat thickness on pig chromosome 13 and application thereof

Abstract

The application belongs to the technical field of molecular biology and molecular marker, and particularly relates to a SNP molecular marker related to backfat thickness on a pig chromosome 13 and application. The SNP molecular marker related to backfat thickness on the pig chromosome 13 has a SNP site corresponding to a C>T mutation at position 160685982 on a chromosome 13 of an international pig reference genome 11.1 version, and the polymorphism of the site base influences the backfat thickness. The application can increase the frequency of the dominant allele of the SNP, improve the backfat thickness of pigs, accelerate the genetic improvement progress of pigs, and effectively improve the economic benefits of pig breeding.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biotechnology and molecular marker technology, specifically relating to a SNP molecular marker located on pig chromosome 13 that is associated with backfat thickness and its application. Background Technology

[0002] Backfat thickness typically refers to the thickness of the fat layer between the skin on a pig's back and the sixth rib, reflecting the pig's lean meat percentage. A lower backfat thickness indicates a higher lean meat percentage and thus higher economic benefits per pig. Breeding pigs with low backfat thickness can bring greater economic benefits to pig farms. Since Duroc pigs are commonly used as terminal sires and can directly influence the production performance of Duroc-Landrace-Landrace-Large White crossbred commercial pigs, they play a crucial role in improving important economic traits in pigs, thereby affecting the economic benefits of farms. Accelerating the genetic improvement of Duroc pig backfat thickness would bring significant benefits to pig farming enterprises.

[0003] Currently, linkage analysis and genome-wide association studies (GWAS) have identified numerous QTLs and single nucleotide polymorphisms (SNPs) associated with backfat thickness in the pig genome. However, GWAS based on genotyping chips has relatively poor detection efficiency and locates large QTL regions, which is not conducive to fine-grained localization of important traits. Furthermore, whole-genome sequencing of large breeding populations remains costly; therefore, genotype imputation technology has been proposed.

[0004] Genotype imputation technology can infer the genotype of unobserved polymorphic loci by utilizing linkage disequilibrium, thus overcoming the resolution limitation of SNP genotyping chips by utilizing their low density. Combining genotype imputation technology with genome-wide association analysis (GWAS) can more accurately identify factors influencing the association between genotype and phenotype, thereby improving the genetic improvement of average backfat thickness and ultimately reducing the average backfat thickness of offspring commercial pigs, thus increasing economic benefits. Summary of the Invention

[0005] To overcome the shortcomings and disadvantages of the existing technology, the primary objective of this invention is to provide an SNP molecular marker located on chromosome 13 of pigs that is associated with backfat thickness. This molecular marker can be applied to the genetic improvement of breeding pigs to increase backfat thickness, thereby increasing the backfat thickness of offspring pigs, improving enterprise profits, and enhancing core competitiveness.

[0006] Another object of the present invention is to provide applications of the above-mentioned SNP molecular markers.

[0007] Another object of the present invention is to provide a primer pair for identifying the above-mentioned SNP molecular markers.

[0008] A fourth objective of this invention is to provide applications of the aforementioned primer pairs.

[0009] The fifth objective of this invention is to provide a method for genetic improvement of pigs.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] A molecular marker of a SNP located on chromosome 13 of pigs that is associated with backfat thickness. The SNP site corresponds to the C>T mutation at position 160685982 on chromosome 13 of the International Pig Reference Genome Version 11.1. The polymorphism of the base at this site affects the backfat thickness trait.

[0012] The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO: 1, where M in the sequence is C or T, leading to significant differences in the backfat thickness trait of pigs;

[0013] The SNP site of the SNP molecular marker is the C68-T68 single nucleotide mutation at position 68 of the sequence marked in SEQ ID NO:1 (corresponding to position 160685982 on chromosome 13 in International Pig Reference Genome Version 11.1, named: NC_010442.4:g.160685982C>T);

[0014] The aforementioned SNP molecular markers are used in identifying traits related to backfat thickness in pigs and in pig genetic breeding.

[0015] A method for screening pig breeds with low backfat thickness using the above-mentioned SNP molecular markers includes the following steps:

[0016] Detect the above-mentioned SNP molecular markers on pig chromosome 13, and determine whether the single nucleotide at the SNP site of the molecular marker is C or T, discarding C and retaining T;

[0017] The pigs mentioned are Duroc pigs and their synthetic lines;

[0018] The preferred pigs are the S21 Duroc strain and its synthetic strains;

[0019] A primer pair for identifying the above-mentioned SNP molecular markers, comprising primers P001-F and P001-R, has the following nucleotide sequence:

[0020] Primer P001-F: 5'-TAAGCCTTGGTCCCTAAGCA-3';

[0021] Primer P001-R: 5'-GGCATAGTTACCAAATCTCCCC-3';

[0022] A kit for identifying the above-mentioned SNP molecular markers, comprising the above-mentioned primer pairs;

[0023] The application of the primer pairs or kits described herein in identifying the influence of backfat thickness in pigs;

[0024] Application of the primer pairs or kits in reducing backfat thickness in pigs;

[0025] Application of the primer pairs or kits in marker-assisted breeding of pigs;

[0026] A method for reducing backfat thickness in pigs includes the following steps:

[0027] The genotype at position 160685982 on chromosome 13 of the International Pig Reference Genome 11.1 was detected, and individuals with the TT or TC genotype at position 160685982 were selected as breeding pigs.

[0028] The method for detecting the genotype at position 160,685,982 on chromosome 13 of the International Pig Reference Genome 11.1 includes the following steps:

[0029] (1) Extract genomic DNA from the pigs to be tested;

[0030] (2) Using the primer pairs mentioned above or the primer pairs in the kit mentioned above as amplification primers, and using the genomic DNA of the pig to be tested obtained in step (1) as template DNA, PCR amplification is performed to obtain PCR amplification products.

[0031] (3) Sequencing the PCR amplification products to obtain sequencing results;

[0032] (4) Based on the sequencing results, determine the genotype of the SNP molecular marker;

[0033] The pigs mentioned are Duroc pigs and their synthetic lines;

[0034] The preferred pigs are the S21 Duroc strain and its synthetic strains;

[0035] A method for genetic improvement of pigs, comprising the following steps:

[0036] The sites of the above-mentioned SNP molecular markers in the core breeding pig population were determined, and corresponding selections were made based on the molecular markers: in the breeding pig succession population, breeding pig individuals with the TT or TC genotype at locus 160685982 on chromosome 13 of the International Swine Reference Genome Version 11.1 were selected, and breeding pig individuals with the CC genotype at this locus were culled, so as to increase the frequency of the T allele at this locus in each generation, thereby increasing the backfat thickness of the offspring pigs;

[0037] The pigs mentioned are Duroc pigs and their synthetic lines;

[0038] The preferred pigs are the S21 Duroc strain and its synthetic strains;

[0039] The present invention has the following advantages and effects compared with the prior art:

[0040] (1) This invention uses GWAS analysis to study and identify SNPs that significantly affect backfat thickness in pigs, verifying their effect on the backfat thickness trait. These SNPs are then used in marker-assisted selection to establish an efficient and accurate marker-assisted breeding technology. Genotypes that are beneficial for increasing average backfat thickness are selected for breeding, thereby progressively increasing the gene frequency of dominant alleles, accelerating the process of breeding and improving pigs, and bringing significant economic benefits to pig farming.

[0041] (2) This invention provides a primer pair and kit for identifying the above-mentioned SNP molecular markers located on chromosome 13 of pigs that are related to backfat thickness. With this primer pair and kit, an efficient and accurate molecular marker-assisted breeding technology can be established, and traits can be selected quickly and accurately, accelerating the breeding process. It can be applied to the genetic improvement of backfat thickness-related traits, thereby reducing the backfat thickness of pigs, thereby increasing enterprise profits and enhancing core competitiveness. Attached Figure Description

[0042] Figure 1 This is a Manhattan plot of genome-wide association analysis (GWAS) of backfat thickness on chromosome 13 in S21 Duroc pigs; where: the horizontal axis represents the chromosome number of the pig; the vertical axis represents the -log P value.

[0043] Figure 2 This is a phenotypic comparison of backfat thickness in pigs of different genotypes, where 0.026, 0.00055, and 0.96 are P-values. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0045] In this embodiment, the S21 Duroc pig is an American Duroc pig.

[0046] Example 1

[0047] (1) Laboratory animals

[0048] The experimental pig population used in this invention consisted of American Duroc pigs from the breeding pig division of Wens Foodstuff Group Co., Ltd., representing the core population of the division, with detailed pedigree records. A total of 3760 S21 Duroc pigs from this resource population were selected for this experiment. During the rearing process, the pigs were allowed free access to feed and water, and the feeding methods and conditions remained consistent throughout, following conventional methods.

[0049] When all experimental pigs reached a weight of 100±5 kg, backfat thickness (BF) was measured at the 10th and 11th intercostal spaces using an Aloka 500V SSD ultrasound scanner (Corometrics Medical Systems, USA). The phenotypic values ​​were calculated as follows:

[0050] BF (mm) = Measured BF value * A / [A + B * (Measured weight - 100kg)]

[0051] Note: A and B are different for boars and sows. The values ​​represented by A and B are:

[0052] Boar: A = 13.47, B = 0.1115;

[0053] Sow: A = 15.65, B = 0.156.

[0054] (2) Sample collection

[0055] Ear tissues from the above-mentioned S21 Duroc pigs were collected, soaked in a 75% ethanol solution, and stored at -20°C for later use.

[0056] (3) Pig genome 50K SNP genotyping

[0057] DNA Sample Preparation: Ear tissue was collected from each of the 3760 S21 Duroc pigs selected from the above resource population. Whole-genome DNA was extracted using the standard phenol-chloroform method. The concentration and OD ratio (OD260 / 280, OD260 / 230) of each sample were accurately determined using a NanoDrop 2000 / 2000C nucleic acid and protein analyzer. DNA samples that passed the NanoDrop 2000 / 2000C nucleic acid and protein analyzer were diluted to approximately 50 ng / μL. 6 μL of the extracted DNA sample was then mixed with 2 μL of loading buffer and loaded onto a 1% (w / v) agarose gel. Electrophoresis was performed at 150V for 25 min. The DNA integrity was observed and photographed using a UV spectrophotometer and gel imaging device.

[0058] Genotyping and imputation of 50K SNPs in the whole pig genome: DNA samples were sent to Neogenet Biotechnology (Shanghai) Co., Ltd., and genotyping of the 50K SNP microarray (Illumina, USA) was performed on the Illumina Beadstration platform according to the company's standard procedures. To increase the SNP marker density, the SWIM website (https: / / quantgenet.msu.edu / swim / ) was used to imput the pig genotype data. PLINK v1.9 was used for quality control of the imputed genotype data, removing SNPs with an individual detection rate below 90%, a family Mendelian error rate above 0.1, a minimum allele frequency below 0.05, and a Hardy-Weinberg equilibrium significance level above 10⁻⁶. A total of 7,442,761 SNPs were obtained for subsequent analysis.

[0059] (4) Genome-wide association analysis (GWAS)

[0060] To eliminate population stratification effects, this invention employs a linear mixture model in GEMMA software for GWAS analysis, incorporating the first five principal components from PCA analysis to correct for population structure. Because the Bonferroni correction method is overly stringent and may produce false negatives, this invention selects a more reasonable significance level of 1×10⁻⁶ at the genome-wide significance level. -5 The power is used as a threshold to avoid problems caused by overly strict calibration.

[0061] GWAS analysis results are as follows Figure 1 As shown. From Figure 1 It is known that there is a site on chromosome 13 in the S21 Duroc pig genome that significantly affects backfat thickness. The most strongly associated SNP is NC_010442.4:g.160685982C>T (P=1.51e-7) (C>T mutation at position 160685982 on chromosome 13 in International Swine Reference Genome Version 11.1).

[0062] (5) Association analysis between different genotypes and backfat thickness phenotype

[0063] Table 1 shows that the SNP site NC_010442.4:g.160685982C>T of the molecular marker is highly significantly correlated with backfat thickness (P<0.001), indicating that this molecular marker significantly affects the backfat thickness trait in pigs. Assisted selection at this SNP site in pigs can reduce backfat thickness in the population, thereby accelerating the breeding process. Furthermore, according to Table 1 and... Figure 2It is known that the TC and TT types have lower backfat thickness than the CC type, indicating that allele C is the inferior allele for the backfat thickness trait in pigs. Therefore, in pig genetic breeding, CC type breeding pigs can be gradually eliminated while retaining TT and TC type breeding pigs to increase the frequency of allele T at this locus generation by generation. This can reduce backfat thickness, increase lean meat percentage, and ultimately improve the economic benefits of commercial pigs and increase the profits of enterprises.

[0064] Table 1. Correlation between SNP sites of molecular markers NC_010442.4:g.160685982C>T and backfat thickness.

[0065]

[0066] Note: ① Backfat thickness results at 100kg are expressed as mean ± standard deviation (SD); ②*** indicates that the variance analysis results for genotype and backfat thickness traits are highly significant (P<0.01).

[0067] Example 2: Target DNA Sequence Amplification and Sequencing

[0068] (1) Primer design

[0069] The DNA sequence of SEQ ID NO:1 on pig chromosome 13 was downloaded from the Ensembl website (http: / / asia.ensembl.org / index.html). Primers were designed using Primer Premier 6.0 software, and the DNA sequences of the designed primers are shown below:

[0070] Primer P001-F: 5'-TAAGCCTTGGTCCCTAAGCA-3';

[0071] Primer P002-R: 5'-GGCATAGTTACCAAATCTCCCC-3';

[0072] (2) PCR amplification

[0073] To a 10 μL reaction mixture, add 1 μL of DNA template, 3.4 μL of double-distilled water, 5 μL of 2×Tag PCR StanMix with Loading Dye, and 0.3 μL each of primers P001-F and P002-R. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 45 s, for 35 cycles; and a final extension at 72℃ for 5 min.

[0074] (3) DNA sequencing

[0075] DNA sequencing and identification were performed at BGI Genomics Co., Ltd. in Shenzhen, with two sequencing reactions (positive and negative). The obtained sequences were compared with the NCBI genome sequence to identify mutations at corresponding SNP sites. The sequencing results are shown below:

[0076]

[0077] Note: M marked in the sequence listing is the mutation site, indicated by an underline (the mutated base in parentheses represents the allele mutation). The positions of the primer sequences are indicated by bolding at the beginning and end of the sequence.

[0078] Example 3: SNP site analysis of molecular marker NC_010442.4:g.160685982C>T effect.

[0079] This invention provides a SNP marker that can significantly increase backfat thickness in Duroc pigs. Using this SNP for marker-assisted selection can greatly accelerate the lean meat percentage breeding process in Duroc pigs. Through marker-assisted selection, if this invention eliminates all CC-type individuals (individuals with the molecular markers affecting backfat thickness) and retains only TT and TC-type individuals, the backfat thickness of each pig in a pig farm will decrease by at least 0.1 mm, and the lean meat percentage will increase by 0.08%. For a 100kg pig, this translates to an increase of 0.08kg of lean meat; for a 10,000-head pig farm, this translates to an increase of 1.6 tons of lean meat per 200kg slaughter pig. This can produce more high-quality lean-type commercial pigs to meet the demand for high-quality pork, thereby bringing significant economic benefits to enterprises.

[0080] This invention utilizes the detection of the C>T mutation site at position 160685982 in the SEQ ID NO:1 sequence to conduct preliminary association analysis between its genotype and the backfat thickness trait in pigs, providing a new molecular marker for marker-assisted selection in pigs.

[0081] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a SNP molecular marker located on pig chromosome 13 that is associated with backfat thickness in identifying the backfat thickness trait in pigs and in pig backfat thickness genetic breeding, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO: 1, where M in the sequence is C or T; wherein, the backfat thickness of pigs with TC and TT genotypes is lower than that of pigs with CC genotype. The pigs mentioned are Duroc strain S21 and its synthetic strains.

2. A method for screening a pig breed with low backfat thickness using a SNP molecular marker located on pig chromosome 13 associated with backfat thickness, characterized in that It includes the following steps: The SNP molecular marker described in claim 1 is detected on chromosome 13 of pigs, wherein the single nucleotide at the SNP site of the molecular marker is C or T, and C is eliminated and T is retained; wherein the backfat thickness of pigs with TC genotype and TT genotype at the molecular marker is lower than that of pigs with CC genotype. The pigs mentioned are Duroc strain S21 and its synthetic strains.

3. The application of a primer pair or kit for identifying SNP molecular markers associated with backfat thickness on porcine chromosome 13 in the identification of the backfat thickness trait in pigs, characterized in that: The primer pair comprises primers P001-F and P001-R, whose nucleotide sequences are as follows: Primer P001-F: 5'-TAAGCCTTGGTCCCTAAGCA-3'; Primer P001-R: 5'-GGCATAGTTACCAAATCTCCCC-3'; The kit described above contains the primer pairs described above; The SNP molecular marker is the SNP molecular marker described in claim 1; wherein, the backfat thickness of pigs with TC and TT genotypes at this molecular marker is lower than that of pigs with CC genotype. The pigs mentioned are Duroc strain S21 and its synthetic strains.

4. The application of a primer pair or kit for identifying SNP molecular markers associated with backfat thickness on pig chromosome 13 in marker-assisted breeding of pig backfat thickness, characterized in that: The primer pair comprises primers P001-F and P001-R, whose nucleotide sequences are as follows: Primer P001-F: 5'-TAAGCCTTGGTCCCTAAGCA-3'; Primer P001-R: 5'-GGCATAGTTACCAAATCTCCCC-3'; The kit described above contains the primer pairs described above; The SNP molecular marker is the SNP molecular marker described in claim 1; wherein, the backfat thickness of pigs with TC and TT genotypes at this molecular marker is lower than that of pigs with CC genotype. The pigs mentioned are Duroc strain S21 and its synthetic strains.

5. A method of genetic improvement of swine, characterized in that It includes the following steps: Identify the SNP molecular marker loci of the breeding pigs in the core breeding pig herd as described in claim 1, and make corresponding selections based on the molecular markers: select breeding pig individuals with the SNP molecular markers of claim 1 as TT or TC in the breeding pig succession herd, and eliminate breeding pig individuals with the CC genotype, so as to increase the frequency of the allele T at this locus generation by generation, thereby increasing the backfat thickness of the offspring pigs. The pigs mentioned are Duroc strain S21 and its synthetic strains.

Images