SNP molecular marker related to backfat thickness on pig chromosome 10 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 pig chromosome 10 and application thereof. The SNP molecular marker related to backfat thickness on pig chromosome 10, wherein the SNP site corresponds to T insertion / deletion at position 29505908 on chromosome 10 of the international pig reference genome 11.1 version. The molecular marker is obtained by whole genome association analysis, and can significantly affect the backfat thickness of pigs at 100 kg body weight. The application further provides a primer pair for identifying the molecular marker. The molecular marker and the primer pair can be used to establish a high-efficiency and accurate molecular marker assisted breeding technology, and the technology is applied to genetic improvement of the backfat thickness of pigs, so that the backfat thickness of pigs is reduced, enterprise profit is increased, and core competitiveness is increased.

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 10 that is associated with backfat thickness and its application. Background Technology

[0002] Pork, as an important source of protein, is widely favored by consumers worldwide. With rising living standards, the preference for lean meat is increasing, leading to a generally higher price for lean meat compared to fatty meat. Therefore, the lean meat percentage of pigs has become a crucial factor affecting the economic benefits of pig farming enterprises. The selection and breeding of high-quality lean-type pigs has become a top priority in breeding work, and global breeding goals are gradually shifting from fat-type pigs to lean-type pigs. Studies have shown a significant negative correlation between backfat thickness and lean meat percentage; therefore, backfat thickness is often used as one of the important indicators of lean meat percentage. Breeders are therefore trying to increase lean meat percentage by reducing backfat thickness while minimizing the impact on pork taste. This improvement in backfat thickness will also bring significant economic benefits to pig farming enterprises.

[0003] For decades, breeders have strived to reduce backfat thickness in pigs using traditional selection methods. However, because this trait is a complex quantitative trait regulated by multiple genes, progress in improvement has been slow and time-consuming. Traditionally, common methods for elucidating the genetic mechanisms of quantitative traits in pigs include the candidate gene approach and quantitative trait locus (QTL) mapping. However, while the candidate gene approach is simple and easy to operate, it can only target genes with known biological functions; the confidence intervals for candidate loci identified by QTL mapping are also relatively large, which greatly limits the application of molecular markers for complex traits in livestock genetics and breeding. With the development of high-throughput sequencing technology and the advent of whole-genome microarrays, genome-wide association studies (GWAS) have gradually been explored by researchers. The confidence intervals for QTLs influencing complex traits identified by GWAS are generally smaller than those of QTL mapping methods. GWAS is gradually replacing QTL mapping as the preferred method for studying the genetic mechanisms of economic traits in pigs.

[0004] Base deletion refers to the loss of one or more bases (such as adenine, thymine, guanine, or cytosine) in a DNA molecule. DNA, as the molecule that stores genetic information, is composed of a specific sequence of bases. This deletion, usually denoted by "del," can sometimes cause serious harm, leading to functional impairment in an organism. However, in some cases, small genetic variations such as base deletion can be introduced to alter the genome of an organism, thereby affecting the species' productivity and agricultural efficiency, and consequently, the profitability of a farm. Therefore, base deletion can cause mutations in specific genes, thereby altering the organism's traits, such as growth rate, disease resistance, and adaptability.

[0005] Duroc pigs, as an important commercial pig breed, directly impact the economic benefits of farms through their production performance. Therefore, in recent years, breeders have focused on improving the backfat thickness trait of core Duroc pig herds in order to effectively pass on these improved advantages to offspring, enhancing the competitiveness of commercial pigs and ultimately improving the economic benefits of farms. Summary of the Invention

[0006] In order to overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide an SNP molecular marker located on chromosome 10 of pigs that is associated with backfat thickness.

[0007] Another object of the present invention is to provide applications of the above-mentioned SNP molecular markers.

[0008] Another object of the present invention is to provide a primer pair for identifying the above-mentioned SNP molecular markers.

[0009] A fourth objective of this invention is to provide applications of the aforementioned primer pairs.

[0010] The fifth objective of this invention is to provide a method for genetic improvement of pigs.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] A molecular marker of a SNP located on pig chromosome 10 that is associated with backfat thickness, the SNP site of which corresponds to a T insertion / deletion at position 29505908 on chromosome 10 in International Pig Reference Genome Version 11.1;

[0013] The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO:1, where M in the sequence represents T insertion or T deletion, resulting in differences in backfat thickness in pigs.

[0014] The SNP molecular marker is defined as the insertion / deletion of nucleotides T185-185 at position 185 of the sequence marked in SEQ ID NO:1.

[0015] The application of the aforementioned SNP molecular markers in identifying traits related to backfat thickness in pigs and in pig genetic breeding;

[0016] A method for detecting the backfat thickness trait in pigs includes the following steps:

[0017] Detect the above-mentioned SNP molecular markers on porcine chromosome 10, and determine whether the SNP sites of the molecular markers are missing single nucleotide T.

[0018] The pigs mentioned are Duroc pigs and their synthetic lines;

[0019] The preferred pigs are the S21 Duroc pigs and their synthetic lines;

[0020] A primer pair for identifying the above-mentioned SNP molecular markers, comprising primers primer-F and primer-R, has the following nucleotide sequence:

[0021] Upstream primer-F: 5'-CCCACCCCATTTCTTCCAGT-3';

[0022] Downstream primer-R: 5'-AAGCACCAGTCTCATCCTCC-3';

[0023] A kit for detecting the above-mentioned SNP molecular markers, comprising the above-mentioned primer pairs;

[0024] Application of the primer pairs or kits described herein in identifying traits affecting backfat thickness in breeding pigs;

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

[0026] The application of the primer pairs or kits described therein in reducing backfat thickness in pigs;

[0027] A method for screening pig breeds with low backfat thickness includes the following steps:

[0028] The genotype at position 29505908 bp on chromosome 10 of the International Pig Reference Genome 11.1 was detected, and homozygous individuals with the T genotype at position 29505908 bp were selected as breeding pigs.

[0029] The pigs mentioned are Duroc pigs and their synthetic lines;

[0030] The preferred pigs are the S21 Duroc pigs and their synthetic lines;

[0031] The method for detecting the genotype at position 29505908 bp on chromosome 10 of the International Swine Reference Genome 11.1 includes the following steps:

[0032] (1) Extract genomic DNA from the pigs to be tested;

[0033] (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 as template DNA, PCR amplification was performed to obtain PCR amplification products.

[0034] (3) Sequencing the PCR amplification products to obtain sequencing results;

[0035] (4) Based on the sequencing results, determine the genotype of the SNP molecular markers;

[0036] A method for genetic improvement of pigs, comprising the following steps:

[0037] The above-mentioned SNP molecular markers of breeding pigs in the core breeding pig population were identified, and corresponding selections were made based on the molecular markers: the homozygous individuals with the T genotype at position 29505908 bp on chromosome 10 of the International Swine Reference Genome 11.1 were selected as breeding pigs, and breeding pigs lacking the T genotype at this locus were culled, so as to increase the frequency of the T allele at this locus in each generation, thereby reducing the backfat thickness of the offspring pigs;

[0038] The pigs mentioned are Duroc pigs and their synthetic lines;

[0039] The preferred pigs are the S21 Duroc pigs and their synthetic lines;

[0040] The present invention has the following advantages and effects compared with the prior art:

[0041] (1) This invention studies and identifies the SNP molecular marker affecting backfat thickness in pigs, which is located on the nucleotide sequence of chromosome 10 of the pig. This molecular marker is an InDel molecular marker of the single-base insertion / deletion type. This invention verifies its effect on the backfat thickness trait in pigs, ultimately establishing an efficient and accurate molecular marker-assisted breeding technology. This technology is applied to the genetic improvement of reducing backfat thickness in breeding pigs, thereby increasing lean meat percentage, production efficiency, enterprise economic profits, and core competitiveness. Furthermore, by optimizing the dominant allele of this SNP, the frequency of the dominant allele can be increased generation by generation, reducing backfat thickness in pigs and accelerating the progress of pig genetic improvement, thus effectively improving the economic benefits of breeding pigs.

[0042] (2) This invention provides a primer pair for identifying the aforementioned SNP molecular markers located on chromosome 10 of pigs that are associated with backfat thickness. This primer pair enables the establishment of an efficient and precise molecular marker-assisted breeding technology. This technology can quickly and accurately improve traits through selection, accelerating the breeding process. Applying this technology to the genetic improvement of backfat thickness in breeding pigs will help reduce backfat thickness, thereby increasing enterprise profits and enhancing core competitiveness. Attached Figure Description

[0043] Figure 1 This is a Manhattan plot of genome-wide association (GWAS) analysis of backfat thickness at 100 kg body weight in S21 Duroc pigs on chromosome 10; where: the horizontal axis represents the chromosome number of the pig; the vertical axis represents the -logP value.

[0044] Figure 2 This is an analysis chart showing the backfat thickness of pigs with different genotypes at a weight of 100kg. Detailed Implementation

[0045] 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.

[0046] Example 1

[0047] (1) Laboratory animals

[0048] The experimental pig population used in this invention consists of 3,769 purebred S21 Duroc pigs from the breeding pig division of Wens Foodstuff Group Co., Ltd., which is the core group of the breeding pig division.

[0049] This experiment selected Duroc pigs of the S21 strain from this resource group. The pigs had free access to feed and water, and the feeding method and rearing conditions remained consistent throughout the experiment, which was a conventional method.

[0050] The backfat thickness is measured by taking the vertical distance from the skin of the pig's back to the longest dorsal muscle membrane, located 5 cm from the midline of the back, between the 3rd and 4th ribs from the bottom. The specific procedure involves: measuring the weight of all S21 Duroc pigs at approximately 100 kg (100 ± 5 kg); using a backfat measuring instrument or vernier calipers; and measuring the thickness of the fat layer from the skin surface to below the sixth rib on the pig's back. Measurements can be taken at multiple locations, and the average value is taken as the backfat thickness result.

[0051] (2) Sample collection

[0052] The collected tail and ear tissues from the above-mentioned breeding pigs were soaked in a 75% ethanol solution and stored at -20°C for later use.

[0053] (3) Pig genome 50K SNP genotyping

[0054] Ear or tail tissues were collected from each of the 3769 S21 Duroc pigs selected from the aforementioned 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 test 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.

[0055] DNA samples were sent to Neogene Biotech (Shanghai) Co., Ltd. for genotyping using a 50K SNP microarray (Illumina, USA) on the Illumina Beadstration platform according to the company's standard procedures. The genotyping data from all samples were quality controlled using the checkmarker function in the GenABEL package of the R language, removing samples 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 The SNPs were analyzed, and ultimately, 38,769 valid genotype data for SNPs were obtained.

[0056] (4) Genome-wide association analysis (GWAS)

[0057] To eliminate the population stratification effect, this invention employs a linear mixed model with single-point regression analysis combined with GWAS analysis using the GenABEL software package in R. The analysis model utilizes the similarity of genomes among individuals to correct for the stratification effect. The Bonferrini method is used to determine the significance threshold for the association between SNPs and backfat thickness. The genomic significance threshold is 0.05 divided by the number of effective SNP loci, i.e., the genomic significance threshold is 1.29e-6, or 0.05 / 38769 (number of effective SNPs). The chromosomal significance threshold is 1 divided by the number of effective SNP loci, i.e., the chromosomal significance threshold is 2.58e-5, or 1 / 38769 (number of effective SNPs).

[0058] GWAS analysis results are as follows Figure 1 As shown. From Figure 1It is known that in the S21 Duroc pig line, there is a locus on chromosome 10 that significantly affects the backfat thickness at 100kg, with the strongest association SNP being g.29505908delT (P value 6.61e-08).

[0059] (5) Association analysis between different genotypes and backfat thickness phenotype

[0060] As shown in Table 1, the SNP site g.29505908delT (nucleotide 185 in SEQ NO:1, corresponding to the insertion / deletion of T at position 29505908 on chromosome 10 in International Swine Reference Genome Version 11.1) is associated with backfat thickness trait, indicating that this molecular marker significantly affects the backfat thickness trait in pigs. Assisted selection at this SNP site in pigs can improve backfat thickness in the population, thereby accelerating the breeding process.

[0061] In addition, according to Table 1 and Figure 2 It was found that individuals with the TT genotype (homozygous T insertion) had the thinnest backfat. Specifically, the average backfat thickness phenotype of the TT genotype was 0.06 mm smaller than that of the -T genotype (deleted one T), and 0.49 mm smaller than that of the -- genotype (homozygous T deletion). Therefore, gradually retaining homozygous individuals without the T deletion genotype as breeding pigs to increase the frequency of the T allele at that locus through successive generations can significantly reduce backfat thickness in pigs, bringing greater economic benefits to pig farms.

[0062] Table 1. Correlation analysis of SNP sites of molecular markers g.29505908 delT with lean meat percentage.

[0063]

[0064] Note: ①- indicates T deletion; ②The total number of samples was 3769, of which 32 samples had no genotype data.

[0065] Example 2: Target DNA Sequence Amplification and Sequencing

[0066] (1) Primer design

[0067] The DNA sequence of SEQ ID NO:1 on pig chromosome 10 was downloaded from the Ensembl website (http: / / asia.ensembl.org / index.html). Primers were designed using the primer design software Primer Premier 6.0. The DNA sequences of the designed primers are shown below:

[0068] Upstream primer-F: 5'-CCCACCCCATTTCTTCCAGT-3';

[0069] Downstream primer-R: 5'-AAGCACCAGTCTCATCCTCC-3';

[0070] (2) PCR amplification

[0071] Prepare a 10 μL system, 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 conditions are as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 64℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 35 cycles; and a final extension at 72℃ for 5 min.

[0072] (3) DNA sequencing

[0073] DNA sequence sequencing and identification: 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:

[0074]

[0075] 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.

[0076] Example 3: SNP site effect analysis of molecular markers

[0077] This invention provides a SNP molecular marker that can significantly reduce backfat thickness and increase lean meat percentage in Duroc breeding pigs. Using this SNP molecular marker for marker-assisted selection can greatly accelerate the breeding process for backfat thickness in Duroc pigs. If this invention selects all individuals at the location of the molecular marker affecting backfat thickness into homozygous individuals retaining the T genotype, backfat thickness can be reduced, and the lean meat percentage of the herd can be increased, thereby effectively improving the economic benefits of pig breeding. Therefore, the potential for reducing backfat thickness to provide revenue for the pig industry is enormous. In individuals with this SNP molecular marker, by retaining the dominant allele (T) of this SNP in the Duroc breeder, the economic benefits of commercial pigs can ultimately be improved, thereby increasing the profits of enterprises.

[0078] 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 associated with backfat thickness in the identification of backfat thickness traits in pigs or in the genetic breeding of backfat thickness traits, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO:1, where M in the sequence represents T insertion or T deletion, which leads to differences in backfat thickness in pigs, with individuals of the TT genotype having the thinnest backfat. The pigs mentioned are the S21 Duroc pigs and their synthetic lines.

2. A method for detecting the thickness of pig backfat, characterized in that... It includes the following steps: The detection method involves examining the SNP molecular marker described in claim 1, specifically whether the SNP site of the molecular marker is missing a single nucleotide T; individuals with the TT genotype have the thinnest backfat. The pigs mentioned are the S21 Duroc pigs and their synthetic lines.

3. The application of a primer pair for detecting SNP molecular markers associated with backfat thickness in identifying the backfat thickness trait in pigs or in marker-assisted breeding of the backfat thickness trait in pigs, characterized in that: The primer pair comprises primers primer-F and primer-R, and their nucleotide sequences are shown below: Upstream primer-F: 5'-CCCACCCCATTTCTTCCAGT-3'; Downstream primer-R: 5'-AAGCACCAGTCTCATCCTCC-3'; The SNP molecular marker mentioned is the SNP molecular marker described in claim 1; individuals with the TT genotype have the thinnest backfat. The pigs mentioned are the S21 Duroc pigs and their synthetic lines.

4. A kit for detecting SNP molecular markers associated with backfat thickness in the identification of backfat thickness traits in pigs or in marker-assisted breeding of backfat thickness traits in pigs, characterized in that: The kit comprises the primer pair as described in claim 3; The SNP molecular marker mentioned is the SNP molecular marker described in claim 1; individuals with the TT genotype have the thinnest backfat. The pigs mentioned are the S21 Duroc pigs and their synthetic lines.

5. A method for genetic improvement of pigs, characterized in that... It includes the following steps: Identify the SNP molecular markers described in claim 1 for the breeding pigs in the core breeding pig herd, and make corresponding selections based on the molecular markers: select homozygous individuals with the T genotype of the SNP molecular marker as breeding pigs in the successive breeding of breeding pigs, and eliminate breeding pigs lacking the T genotype, so as to increase the frequency of the allele T at this locus in each generation, thereby reducing the backfat thickness of the offspring pigs. The pigs mentioned are the S21 Duroc pigs and their synthetic lines.

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