SNP (Single Nucleotide Polymorphism) molecular marker related to porcine reproductive traits in porcine DDR2 (Double Data Rate 2) gene, primer pair and application of SNP molecular marker

Abstract

The invention provides an SNP molecular marker related to porcine reproductive traits in a porcine DDR2 gene, and belongs to the technical field of porcine molecular marker screening, and the reproductive traits are total litter size and live litter size. The SNP molecular marker is located in a pig DDR2 gene sequence, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1 in a sequence table, and a Ggt exists at the 265bp position of the sequence; a basic group mutation; the invention also provides a primer pair for amplifying the molecular marker and a method for detecting and judging porcine reproductive traits. The invention provides a new marker for molecular marker-assisted breeding of the porcine reproductive traits, and the marker can be utilized to realize rapid identification of the porcine reproductive traits so as to improve the efficiency of porcine breeding.

Description

Technical Field

[0001] This invention belongs to the field of porcine molecular marker screening technology, specifically relating to a SNP molecular marker, primer pair and its application in the porcine DDR2 gene that is related to porcine reproductive traits. Background Technology

[0002] Genetic improvement of pig reproductive traits is receiving increasing attention from breeders. Total litter size and live birth number are several important indicators for evaluating pig reproductive traits, and improving these two metrics is significant for improving the overall economic benefits of the pig farming industry. However, because pig reproductive traits have low heritability (around 0.1), improvement through conventional breeding is quite difficult. Therefore, identifying molecular markers that influence reproductive traits and using molecular-assisted breeding is of great importance for improving pig reproduction.

[0003] The DDR2 gene (discoidin domain receptor tyrosine kinase 2, DDR2) is located on chromosome 4 of pigs and is highly expressed in pig ovarian tissue. As a key kinase in the DNA damage response (DDR) pathway, it maintains genomic stability by phosphorylating downstream substrates, initiating cell cycle checkpoints, DNA repair, or inducing apoptosis.

[0004] However, the molecular mechanism by which the DDR2 gene regulates pig reproductive traits has not yet been reported. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention aims to provide a molecular marker, primer pair, and their application for SNPs related to porcine reproductive traits within the porcine DDR2 gene, providing a reference for marker-assisted selection of porcine reproductive traits.

[0006] The specific technical solution of the present invention is as follows: In a first aspect, the present invention discloses SNP molecular markers in the porcine DDR2 gene that are related to porcine reproductive traits, characterized in that the reproductive traits are total litter size and live litter size; the SNP molecular markers are located in the porcine DDR2 gene sequence, and their nucleotide sequence is shown in SEQ ID NO.1, with a G>A base mutation at position 265bp of the sequence.

[0007] In a preferred embodiment of the present invention, the polymorphism of the SNP molecular marker is expressed as three genotypes: GG, AG, or AA, wherein the G allele is the dominant allele.

[0008] In a preferred embodiment of the present invention, the total number of offspring and the number of live offspring of individuals with the GG genotype are significantly higher than those of individuals with the AG genotype and individuals with the AA genotype, and the AG genotype is significantly higher than that of individuals with the AA genotype.

[0009] In a second aspect, the present invention discloses a primer pair for amplifying the molecular marker as described in the first aspect, the primer pair comprising an upstream primer with the sequence shown in SEQ ID NO.2 and a downstream primer with the sequence shown in SEQ ID NO.3.

[0010] Thirdly, the present invention discloses a kit for detecting reproductive traits in pigs, wherein the reproductive traits are total litter size and live litter size, and includes the primer pair described in the second aspect.

[0011] Fourthly, the present invention discloses the application of the SNP molecular marker as described in the first aspect, or the primer pair as described in the second aspect, or the kit as described in the third aspect in detecting or assisting in the detection of reproductive traits in pigs, wherein the reproductive traits are total litter size and live litter size.

[0012] Fifthly, this invention discloses the application of an SNP molecular marker as described in the first aspect, a primer pair as described in the second aspect, or a kit as described in the third aspect in pig breeding, genetic improvement, or marker-assisted selection.

[0013] In a sixth aspect, the present invention discloses the application of an SNP molecular marker as described in the first aspect, or a primer pair as described in the second aspect, or a kit as described in the third aspect, in pig screening.

[0014] Seventhly, the present invention discloses a method for detecting and judging reproductive traits in pigs, wherein the reproductive traits are total litter size and live litter size, comprising the following steps: S1. Using porcine genomic DNA extracted from the sample to be tested as a template, PCR amplification was performed using primers with sequences shown in SEQ ID NO. 2~3, and the PCR products were obtained and purified; S2. Sequencing analysis of the purified PCR products to obtain the genotype; S3. Determine the reproductive traits of pigs based on the genotypes described.

[0015] Eighthly, the present invention discloses a method for pig screening or pig breeding, including the method for detecting and judging pig reproductive traits as described in the seventh aspect.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to discover that a SNP molecular marker (rs327648460) in the porcine DDR2 gene is associated with porcine reproductive traits, specifically total litter size and live litter size. Therefore, this molecular marker can be used to screen for porcine reproductive traits and for breeding, providing a new application of a single SNP molecular marker in the porcine DDR2 gene for marker-assisted breeding of porcine reproductive traits. This enables early screening of porcine reproductive traits, and the screening method is simple and rapid. Attached Figure Description

[0017] Figure 1 This is an agarose gel electrophoresis detection image of the PCR product in Example 1 of the present invention, where lane M is the DL2000 Marker, and lanes 1-3 are the amplified fragments from Large White pigs, with a fragment size of 584 bp. Figure 2 This is the sequencing map of the rs327648460 G>A site in Example 1 of the present invention. Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Unless otherwise specified, all examples were conducted under standard experimental conditions. All reagents and materials used are commercially available unless otherwise specified.

[0020] Example 1: Obtaining SNP fragments from the porcine DDR2 gene and establishing a method for detecting polymorphic sites: 1. Extraction of porcine genomic DNA: The experimental pig breed used in this invention was the Large White pig. Genomic DNA was extracted using the Invitrogen Animal Tissue Genomic DNA Extraction Kit (PureLink™ Pro 96 Genomic DNA Purification Kit, K182104A), following the kit's instructions. The extracted DNA was tested for concentration and quality, and stored at -20°C for later use.

[0021] 2. Obtaining the SNP genetic marker fragment of the porcine DDR2 gene: (1) PCR amplification.

[0022] Based on the SNP genetic marker detection sequence in the porcine DDR2 gene genome sequence (GenBank ID: 100154199), as shown in SEQ ID NO.1, a pair of primers was designed to amplify fragments at polymorphic sites. The primers are as follows: Amplification of the upstream primer of SEQ ID NO.1 sequence: 5'- GCCAAAGTCACAATCATCT-3' (SEQ ID NO.2); The downstream primer for amplifying the sequence SEQ ID NO.1 is: 5'-TCACAGGGTTACTGTAGGG-3' (SEQ ID NO.3).

[0023] Using the above primers and Large White pig genomic DNA as a template, PCR amplification was performed. The PCR reaction system was 50 μL, and the components of the system were: 100 ng genomic DNA, 25 μL PCR mix, 1 μL each of the above upstream and downstream primers, and ddH2O was added to bring the total volume to 50 μL.

[0024] The PCR procedure was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min; storage at 4℃. The PCR products were detected by 1.5% agarose gel electrophoresis, and the results are attached. Figure 1 As shown, lane M is the DL2000 Marker, and lanes 1-3 are the amplified fragments from Large White pigs, with a fragment size of 584bp.

[0025] (2) Purification of PCR products The PCR amplification products were purified using the Gel Extraction Kit from Shanghai Sangon Biotech Co., Ltd. Specific steps are detailed in the kit's instruction manual.

[0026] 3. Molecular markers were detected using direct sequencing of PCR products. The purified PCR products obtained above were directly sent to Beijing Aoke Technology Co., Ltd. for sequencing. The genotype of the locus in the test population was determined based on the sequencing results. Analysis was performed using SeqMan software, and the results are shown in the attached figure. Figure 2 As shown, a G>A allele mutation was found at 265bp in the sequence, namely rs327648460 G>A. This mutation causes polymorphism in the DDR2 gene.

[0027] Example 2: Detection of polymorphic distribution of SNP molecular markers in Large White pigs: In this example, the polymorphic distribution of the rs327648460 G>A site of the porcine DDR2 gene was detected in 507 Large White pigs.

[0028] Table 1. Distribution pattern of polymorphism at the G>A site of rs327648460 in the DDR2 gene: ; The results in Table 1 show that in Large White pigs... DDR2 The gene rs327648460 G>A site exhibits three genotypes: AA, AG, and GG. Among them, the GG genotype is more common, and the frequency of the G allele is 72.9%, therefore the G allele is the dominant allele.

[0029] Example 3: Association analysis of SNP molecular markers and porcine reproductive traits: To determine the relationship between SNP molecular markers and porcine reproductive traits... DDR2 To investigate the correlation between the rs327648460 G>A gene locus and differences in pig reproductive traits, polymorphism detection was performed using the method established in Example 1, and the correlation between different genotypes of this polymorphic locus and the traits of total litter size and live piglet number was analyzed. The GLM model in SAS 19.0 software was used to perform association analysis on the variant loci. The model used was: ; Where: Y ijkG represents the phenotypic value; μ represents the population mean; G represents the population mean. i Indicates genotype effect; A j Indicates the seasonal effect; S k Indicates the paternal effect; e ijk This represents the random residual effect. P A value <0.05 is considered statistically significant. P A value <0.01 is considered highly significant.

[0030] Association analysis was conducted on different genotypes with total litter size and live litter size in Large White pigs. The statistical results are shown in Table 2. Table 2 DDR2 Association analysis between the rs327648460 G>A site and porcine reproductive traits: ; Note: The trait means in the table are expressed as mean ± standard deviation. A, B, and C indicate highly significant differences. P <0.01).

[0031] As shown in Table 2, in Large White pigs, the total litter size and live birth size of individuals with the GG genotype at the rs327648460 G>A locus were significantly higher than those with the AG genotype. P <0.01), the total number of litters and the number of live offspring in individuals with the AG genotype were significantly higher than those with the AA genotype. P <0.01), therefore the G allele is the dominant allele, and individuals carrying this dominant allele should be retained in breeding, which is beneficial to improving the total number of offspring and the number of live offspring in the population.

[0032] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. Pig DDR2 SNP molecular markers within genes that are associated with porcine reproductive traits, characterized by: The reproductive traits are total litter size and live birth size; the SNP molecular markers are located in pigs. DDR2 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and there is a G>A base mutation at 265 bp in the sequence.

2. The SNP molecular marker according to claim 1, characterized in that, The polymorphism of the SNP molecular marker is expressed as three genotypes: GG, AG, or AA, with the G allele being the dominant allele.

3. The SNP molecular marker according to claim 2, characterized in that, The total number of offspring and the number of live offspring were significantly higher in individuals with the GG genotype than in individuals with the AG and AA genotypes, with the AG genotype being significantly higher than the AA genotype.

4. A primer pair for amplifying the molecular marker as described in claim 1 or 2, characterized in that, The primer pair comprises an upstream primer with the sequence shown in SEQ ID NO.2 and a downstream primer with the sequence shown in SEQ ID NO.

3.

5. A kit for detecting reproductive traits in pigs, characterized in that, The reproductive traits are total number of offspring and number of live offspring, and include the primer pair described in claim 4.

6. The application of the SNP molecular marker as described in any one of claims 1-3, the primer pair as described in claim 4, or the kit as described in claim 5 in detecting or assisting in the detection of reproductive traits in pigs, wherein the reproductive traits are total litter size and live litter size.

7. The application of the SNP molecular marker as described in any one of claims 1-3, the primer pair as described in claim 4, or the kit as described in claim 5 in pig breeding, genetic improvement, or molecular marker-assisted selection.

8. The use of the SNP molecular marker as described in any one of claims 1-3, the primer pair as described in claim 4, or the kit as described in claim 5 in pig screening.

9. A method for detecting and judging reproductive traits in pigs, characterized in that, The reproductive traits are total number of offspring and number of live offspring, and include the following steps: S1. Using porcine genomic DNA extracted from the sample to be tested as a template, PCR amplification was performed using primers with sequences shown in SEQ ID NO. 2~3 to obtain PCR products and purify them; S2. Sequencing analysis of the purified PCR products to obtain the genotype; S3. Determine the reproductive traits of pigs based on the genotypes described.

10. A method for pig screening or pig breeding, characterized in that, This includes the method for detecting and determining pig reproductive traits as described in claim 9.

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