Pig anti-diarrhea related snp molecular marker combination, detection reagent and application thereof

Abstract

This invention discloses a combination of SNP molecular markers related to antidiarrheal properties in pigs, detection reagents, and their applications, belonging to the field of molecular genetics technology. This invention originates from pigs... BCAP31 Two SNP loci significantly associated with PEDV resistance were identified through internal gene screening and used as molecular markers for auxiliary identification of PEDV resistance or susceptibility in pigs. Compared with traditional methods that rely on clinical symptoms, viral load, diarrhea scores, or challenge tests to evaluate PEDV resistance, this invention enables rapid screening of PEDV-resistant individuals in pigs at an early stage through genotyping. The detection results are stable, the operation is simple, and it is suitable for large-scale application.

Description

Technical Field

[0001] This invention relates to the field of molecular genetics, specifically to combinations of SNP molecular markers related to antidiarrheal properties in pigs, detection reagents, and their applications. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) can cause watery diarrhea, dehydration, and growth retardation in piglets, especially newborn piglets, and is one of the important viral pathogens affecting the healthy production of large-scale pig farming. Because PEDV strains continue to mutate, and different pig herds and individuals have varying susceptibility to PEDV infection, simply relying on vaccination, environmental disinfection, and feeding management is insufficient to address the genetic basis of susceptibility in pig herds.

[0003] With the development of porcine genomics and molecular breeding technologies, the use of molecular markers such as single nucleotide polymorphisms (SNPs), insertion / deletion polymorphisms (InDels), and copy number variations (CNVs) for assisted selection of important economic traits, disease resistance traits, and adaptive traits in pigs has become an important technical means to improve breeding efficiency. Compared with traditional phenotypic selection, molecular marker-assisted selection can identify genotypes in early individuals, at growth stages, and even in the pre-disease state, and has advantages such as fast detection speed, stable results, less susceptibility to environmental influences, and suitability for large-scale herd screening. For PEDV, an acute and highly transmissible enterovirus disease, if host gene molecular markers significantly associated with PEDV resistance or susceptibility can be screened and used for early identification and genetic selection of breeding pigs, it will help to build more disease-resistant pig herds and reduce the risk of PEDV infection and farming losses from the source.

[0004] BCAP31 proteins are important endoplasmic reticulum (ER) membrane-associated proteins with chaperone functions, participating in the transport of secretory and transmembrane proteins from the ER to the Golgi apparatus, recognition of misfolded proteins, ER-related degradation, and apoptosis. Some studies have reported that BCAP31 proteins may act as potential receptors for PEDV, participating in the regulation of Bcl-2-mediated apoptosis. However, for pigs… BCAP31 There are still few studies on the association between specific gene polymorphism sites and PEDV infection phenotype. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a combination of SNP molecular markers related to antidiarrheal treatment in pigs, detection reagents, and their applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a combination of SNP molecular markers related to antidiarrheal properties in pigs, the combination of SNP molecular markers consisting of the following two SNP molecular markers: The SNP1 molecular marker, whose nucleotide sequence is shown in SEQ ID NO.1, has an SNP site at position 297 from the 5' end, with a polymorphism of A or T; details are as follows: CAGTCAGTCAAAGATGGGCTACAAACCAATGATCTGGCACTAACAGTTCAAAGCAACGCAAACAAGCTGGCATTTATTTGATCAGGCAATCGGAACTAGGCATGAGCTCAGGCTTTGGCCACCTAGAACCCATGCCAACTCGGGCCAG TCCATCTTCTCTTGGGCTGCCCTTTTTCATTTACAAAAGCCCATAGAATTATGAAGGGTTATTAAAGTCCTTACTGACTTCAAACAGAACCAGAATCCAAACAGCGCTCGCGCGCGCTCTCTCTCTCTCTCTCTCTCTCTCTCTC A / T CACACACACACACACACACACACACACACACACACACAGGCAAAGCCACATGGCTATTTAGTCTTCAAGAGAAGCCCTTACTTTAACAGGTGAAAACATTCAGGGCCAGAGGCAGAGCTGCTTATCCAAAGCCACAGAGATGCTGAAGTGGCGGCACTGGCCTGGGGACTCCAGTCGCCTGACTTCTGATTCCTCATCCTTCTTCCTATG CTGCCCACCCTCCTAAGAGCAGACACCTAGGCTGTGTGGAGGTGTGCTGCTGATCTGCACCCCATGACGTTCCAGGAACTCCTCCTGTGTAACTGCCATGGAGGGTGGTCTCCCCGCAACTCTACCTCATCCATCCTGCAGCGGCACTGACTCATGTACAGTAACGCTCAAAGCTTTGTTGAGCTAAGCTCCATGAAACCAAACTTGACCGAG Note: The nucleotides in bold italics in the sequence are SNP sites, which are represented in the sequence listing as the corresponding degenerate base "W".

[0007] The SNP1 molecular marker is significantly associated with porcine PEDV resistance, with the dominant allele being T; the TT genotype corresponds to higher PEDV resistance.

[0008] The SNP2 molecular marker has the nucleotide sequence shown in SEQ ID NO.2. The 71st base from the 5' end is the SNP site, and its polymorphism is either A or G. Details are as follows: GGCTACGACATCTTGGGAAATTCGAGAAACAGATCCTATGTGAGCAACTGGTGAATCCATACTGATCCTC A / G GGGTCACTGTTTTCCTAAGAAGTCACACGAAAAGACTTTTTTTTTGATTAGCTACTAGACAGGGACATGGGAGGGAGTGGCGGGCACAGCAAGGCCTCTTTCAGCTAAGATGGAGCGGTCACAGATGTGTGGTGTTACCCTACTCTGCATCTGCCAAACACTCACAGAACTAGATGCTGATGTACACTCTAAGTCAATTTCTAAAATCAACTGC AGGAACTAGGCTGGAGCACAGAGCCCCACAGGGCCGCACAGAGGACTGAGCTGAGCACACTCACTGTCTGGTAGGCCTGGCTACCCGCTGTCCCTTGGCCCGGTCAAATAAGACCTGCCCTGGAGGAGATCACAAAAGCAGAGCATTCACGAGAGTAAGACTTCGAGACCACAGCACATGGGAAAAGACACCACTCCATTTGCCACCACTGCGA.

[0009] Note: The nucleotides in bold italics in the sequence are SNP sites, which are represented in the sequence listing by the corresponding degenerate base "R".

[0010] The SNP2 molecular marker is significantly associated with porcine PEDV resistance, and its dominant allele is A; the AA genotype corresponds to higher PEDV resistance.

[0011] pig BCAP31 The gene is located in the region Chr.X:124456999-124484923 on the pig X chromosome. This region involves a relatively long genetic domain and exhibits a different inheritance pattern than autosomal genes. Differences exist between male and female individuals in gene dosage, genotype interpretation, and statistical models at this locus, which makes... BCAP31The association analysis between gene SNPs and PEDV resistance is more complex. This invention uses pigs... BCAP31 Two SNPs that are significantly associated with PEDV resistance and can be used for genotyping were screened from the gene region (Chr.X:124456999-124484923), which is of great significance for pig resistance breeding.

[0012] A second aspect of the present invention provides the application of the above-described SNP molecular marker combination in (1) or (2) below: (1) Screening for pigs resistant to porcine epidemic diarrhea virus infection; (2) Breed pig breeds with PEDV resistance.

[0013] A third aspect of the present invention provides a detection reagent comprising: a primer pair for detecting the above-mentioned SNP molecular marker combination, wherein the primer pair consists of primer pair 1 for detecting SNP1 molecular marker and primer pair 2 for detecting SNP2 molecular marker; The nucleotide sequences of primer pair 1 are shown in SEQ ID NO. 3 and SEQ ID NO. 4; the nucleotide sequences of primer pair 2 are shown in SEQ ID NO. 5 and SEQ ID NO. 6. Specifically: Primer pair 1: 205-F: 5'-CAGTCAGTCAAAGATGGGCTAC-3'; (SEQ ID NO.3) 937-R: 5'-CTCGGTCAAGTTTGGTTTTCA-3'. (SEQ ID NO.4) Primer pair 2: 431-F: 5'-GGCTACGACATCTTGGGAAAT-3'; (SEQ ID NO.5) 929-R: 5'-TCCGCAGTGGTGGCAAATGGA-3'. (SEQ ID NO. 6).

[0014] Preferably, the detection reagent is a PCR sequencing kit or a KASP typing kit.

[0015] A fourth aspect of the present invention provides the use of the above-described detection reagent in the following (1) or (2): (1) Screening for pigs resistant to porcine epidemic diarrhea virus infection; (2) Breed pig breeds with PEDV resistance.

[0016] In the above applications, the method for screening pigs resistant to porcine epidemic diarrhea virus infection is as follows: Using the genomic DNA of the pigs to be tested as a template, PCR amplification was performed using primer pairs 1 to 2 shown in SEQ ID NO.3-SEQ ID NO.6, respectively, to obtain two amplification products. The two amplification products were sequenced, and the anti-PEDV trait of the pigs was identified based on the sequencing results, and pigs resistant to porcine epidemic diarrhea virus infection were screened.

[0017] Specifically, if the sequencing result of the amplification product of primer pair 1 corresponds to the TT genotype at the 297th base of the sequence shown in SEQ ID NO.1, and the sequencing result of the amplification product of primer pair 2 corresponds to the AA genotype at the 71st base of the sequence shown in SEQ ID NO.2, then it is identified as having the PEDV resistance trait.

[0018] Preferably, the PCR amplification system is as follows: 12.5 μL of Taq DNA polymerase (5 U / μL), 1 μL each of upstream and downstream primers (10 μmol / L), 1 μL of template DNA (1500 ng / μL), and 9.5 μL of ddH2O.

[0019] The beneficial effects of this invention are: This invention is from pigs BCAP31 Two SNP loci significantly associated with PEDV resistance were identified through screening of the gene region and used as molecular markers for auxiliary identification of PEDV resistance or susceptibility in pigs. Compared with traditional methods that rely on clinical symptoms, viral load, diarrhea scores, or challenge tests to evaluate PEDV resistance, this invention enables rapid screening of PEDV-resistant individuals in pigs at an early stage through genotyping. The detection results are stable, the operation is simple, and it is suitable for large-scale application.

[0020] Furthermore, the two SNP sites provided by this invention can be used alone or in combination, which can improve the accuracy and reliability of PEDV resistance assessment, and help in the selection of breeding pigs, the construction of disease-resistant core groups and the cultivation of PEDV-resistant pig herds, thereby reducing production losses caused by PEDV infection and having good industrial application value. Attached Figure Description

[0021] Figure 1 Gel electrophoresis images of the amplified fragments corresponding to SNP1 molecular markers to SNP2 molecular markers; In the figure, A is the gel electrophoresis of the amplified fragment of SNP1 molecular marker; B is the gel electrophoresis of the amplified fragment of SNP2 molecular marker; M: Marker; Lanes 1-4 represent duplicate samples.

[0022] Figure 2Sanger sequencing peak diagrams of PCR amplification products of two target SNP sites; in the figure, (A) is the sequencing peak diagram of the SNP site in the SNP1 molecular marker; (B) is the sequencing peak diagram of the SNP site in the SNP2 molecular marker. Detailed Implementation

[0023] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0025] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein: The PEDV used in this invention is the classic strain CV777 of porcine epidemic diarrhea virus (PEDV), which has the GenBank accession number AF353511.1.

[0026] Example 1: Screening and identification of core SNP sites affecting diarrheal traits in PEDV-resistant infection 1. Test method: One hundred and ten 21-day-old weaned Landrace × Large White crossbred piglets (PEDV negative) from a pig farm in Dezhou City, Shandong Province, were used as experimental subjects. Upon arrival at the pig farm, the piglets were challenged with PEDV and injected with 10... 11 CV777 copies (10 mL) were administered free access to feed and water. PEDV testing was performed 2 days after infection, and the results were positive. Total RNA was extracted from piglet fecal samples using a fecal RNA extraction kit manufactured by Omega Bio-tek. All pigs were housed in a uniform environment, and fecal morphology was recorded during the observation period. Fecal samples were categorized into solid, semi-solid, and liquid types, and assigned scores of 1, 2, and 3. This fecal scoring system was used to record the diarrhea phenotype.

[0027] Phenotypic classification was categorized into diarrhea and non-diarrhea. Diarrhea individuals were those exhibiting significant liquid feces during the observation period (fecal score recorded as 3), while non-diarrhea individuals were those who did not produce liquid feces throughout the observation period (fecal score recorded as ≤2). After phenotypic recording, samples were collected from all piglets' intestines, intestinal contents, ear edges, liver, and spleen for subsequent genomic DNA extraction, genome resequencing, SNP genotyping, and association analysis.

[0028] The collected ear tissue samples were sent to Hangzhou Lianchuan Biomedical Technology Co., Ltd., where whole-genome resequencing was performed on the BGI sequencing platform with a sequencing depth ≥1×.

[0029] Bioinformatics software was used to process the low-depth sequencing data. BWA v0.7.17 (Li and Durbin, 2009) was used to align quality-controlled clean reads to the pig reference genome (Sscrofa11.1). Samtools v1.11 (Li et al., 2009) was used to align and sort the resulting BAM files. Picard tools (Institute, 2019) were used to remove PCR repeats of the same short sequence during sequencing to avoid interference from repetitive sequences. After removing PCR repeats, the X chromosome was first filled with Stitch to fill the low-depth sequencing data. The resulting VCF file was then filtered with parameters set to INFO_SCORE > 0.4, MAF ≥ 0.00001, and deletion rate ≤ 80%. The filtered VCF file was then filled with Beagle to fill in the remaining deletion sites to obtain the X chromosome genotyping data.

[0030] The detected SNP sites were further quality controlled using PLINK v1.90 software. The quality control criteria were to remove individuals with a deletion rate >5%, remove SNP sites with a deletion rate >5%, and remove SNP sites with a minimum allele frequency (MAF) <0.05. Finally, a high-quality SNP dataset of 323,184 samples from 110 samples was retained.

[0031] extract BCAP31 All SNP sites within 2kb upstream and downstream of the gene were analyzed for linkage disequilibrium (LD) using PLINK v1.90, with r 2 A threshold of ≥0.8 was used to select TagSNPs for population genetic association analysis (allele chi-square test), and the P-values ​​of the association between these loci and the diarrheal trait of pigs infected with PEDV were calculated.

[0032] 2. Test Results: By analyzing the diarrheal traits of 110 Large White crossbred piglets infected with PEDV and... BCAP31 Association analysis of gene regions, in BCAP31 Two independent TagSNPs were screened within a 2kb region upstream and downstream of the gene and were significantly associated with diarrheal traits in piglets infected with PEDV. P＜0.05 ), as shown in Table 1.

[0033] Table 1: Information on two SNP sites associated with diarrheal traits in pigs infected with PEDV Note: The physical location was determined based on Sscrofa11.1 (GCF_000003025.6) as the reference genome; A1 is the effect allele; F_A is the frequency of A1 in individuals with diarrhea; F_U is the frequency of A1 in individuals without diarrhea; A2 is the other alleles; CHISQ represents the chi-square statistic; OR represents the odds ratio of A1 alleles.

[0034] Example 2: Design and application validation of SNP molecular marker combinations and detection reagents related to diarrhea resistance in pigs. 1. Design of SNP molecular marker combinations and detection reagents related to diarrhea resistance traits in pigs: Based on the two SNP sites screened in Example 1, this example designs a combination of SNP molecular markers associated with the antidiarrheal trait in pigs, consisting of the following two SNP molecular markers: The SNP1 molecular marker has the nucleotide sequence shown in SEQ ID NO.1. The 297th base from the 5' end of the sequence is the SNP site, and its base polymorphism is A or T. The SNP2 molecular marker has a nucleotide sequence as shown in SEQ ID NO.2. The 71st base from the 5' end of the sequence is the SNP site, and its base polymorphism is A or G.

[0035] A detection reagent was designed based on the above-mentioned SNP molecular marker combinations. The detection reagent is a PCR sequencing kit containing primer pairs for detecting the above-mentioned SNP1 to SNP2 molecular markers. The primer pairs consist of primer pair 1 for detecting SNP1 and primer pair 2 for detecting SNP2. The primer pair design is based on the flanking sequences of the two core SNP sites, each extending approximately 500 bp upstream and downstream. Specific PCR primers were designed using Primer 5.0 software, adhering to strict principles: the amplified fragment length was controlled to be above 500 bp to ensure the target SNP site was located in the middle region of the amplified fragment; the primer Tm value was within the range of 50-65℃; the primers were free of hairpin structures, primer dimers, and non-specific binding sites to ensure the specificity and effectiveness of PCR amplification. The specific nucleotide sequences are shown in Table 2. Table 2: Primer pairs used to detect the above SNP1~2 molecular markers 2. Application Validation Another 22 large crossbred weaned piglets with diarrhea phenotypes were selected as experimental subjects. Genomic DNA was extracted from the subjects (jejunal tissue extraction). The extracted genomic DNA was diluted to an approximate concentration (1500 ng / μL) and mixed in a pool. PCR amplification was performed using the detection primer pairs in Table 2 above. The amplification system was as follows: 12.5 μL Taq DNA polymerase (5 U / μL), 9.5 μL ddH2O, 1 μL each of forward and reverse primers (10 μmol / L), and 1 μL template DNA (1500 ng / μL).

[0036] The PCR amplification program is as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, annealing temperature (adjusted according to primer Tm value, 45-65℃) for 30 s, extension at 72℃ (according to the length of the target fragment, 60 s / kb), 35 cycles; final extension at 72℃ for 2 min.

[0037] The obtained PCR amplification products were subjected to agarose gel electrophoresis. The gel electrophoresis images of the amplified fragments corresponding to the SNP1 molecular marker to the SNP2 molecular marker are shown below. Figure 1 As shown; the bands are clear and without tails, meeting the experimental requirements. Observe whether the size of the electrophoretic bands is consistent with the expected size of the PCR amplification products. Send the qualified PCR products to Sanger sequencing company for first-generation sequencing to obtain the sequencing peak diagram and nucleotide sequence data of each amplified fragment. Compare the sequenced nucleotide sequences with the pig reference genome (Sscrofa11.1) to analyze the genotype polymorphism of the target SNP sites. If a bimodal characteristic is observed at the target SNP site, it indicates that the SNP site has good polymorphism. After Sanger sequencing of the PCR amplification products of the two target SNP sites, the sequencing peak diagrams are clearly distinguishable, without interference from extraneous peaks. The comparison results of the nucleotide sequences with the reference genome sequence show that the base mutation types of the SNP sites are completely consistent with the screening results of the association analysis. Figure 2 ).

[0038] The diarrhea trait in piglets was identified based on the base mutation type of the target SNP site in the sequencing results. If the sequencing result of the amplification product of primer pair 1 (205-F / 937-R) corresponds to the TT genotype at the 297th base of the sequence shown in SEQ ID NO.1, and the sequencing result of the amplification product of primer pair 2 (431-F / 929-R) corresponds to the AA genotype at the 71st base of the sequence shown in SEQ ID NO.2, then the piglets were identified as having the PEDV resistance trait.

[0039] If the sequencing result of the amplification product of primer pair 1 (205-F / 937-R) corresponds to the AA genotype at the 297th base of the sequence shown in SEQ ID NO.1, and the sequencing result of the amplification product of primer pair 2 (431-F / 929-R) corresponds to the TT genotype at the 71st base of the sequence shown in SEQ ID NO.2, then the individual is identified as having the PEDV susceptibility trait.

[0040] The identification results based on molecular markers are compared with actual diarrhea phenotype records. If the results are consistent, it indicates that the SNP molecular marker combination and detection reagent of the present invention can be used for screening pig breeds with excellent resistance to PEDV infection diarrhea traits.

[0041] The results showed that the two results were consistent, indicating that the SNP molecular marker combination and detection reagent of the present invention can be used to screen pigs with excellent resistance to PEDV-infected diarrhea, and the prediction results are accurate and reliable.

[0042] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A combination of SNP molecular markers associated with antidiarrheal properties in pigs, characterized in that, The SNP molecular marker combination consists of the following two SNP molecular markers. composition: The SNP1 molecular marker has the nucleotide sequence shown in SEQ ID NO.

1. The 297th base from the 5' end of the sequence is the SNP site, and its base polymorphism is A or T. The SNP2 molecular marker has a nucleotide sequence as shown in SEQ ID NO.

2. The 71st base from the 5' end of the sequence is the SNP site, and its base polymorphism is A or G.

2. The SNP molecular marker combination according to claim 1, characterized in that, The SNP site in the SNP1 molecular marker is the TT genotype, and the SNP site in the SNP2 molecular marker is the AA genotype, corresponding to the diarrhea resistance trait in pigs.

3. The application of the SNP molecular marker combination according to claim 1 in either (1) or (2): (1) Screening for pigs resistant to porcine epidemic diarrhea virus infection; (2) Breed pig breeds with PEDV resistance.

4. A detection reagent, characterized in that, It comprises: a primer pair for detecting the SNP molecular marker combination of claim 1, wherein the primer pair consists of primer pair 1 for detecting SNP1 molecular marker and primer pair 2 for detecting SNP2 molecular marker; The nucleotide sequences of primer pair 1 are shown in SEQ ID NO.3 and SEQ ID NO.4; the nucleotide sequences of primer pair 2 are shown in SEQ ID NO.5 and SEQ ID NO.

6.

5. The detection reagent according to claim 4, characterized in that, The detection reagent is a PCR sequencing kit or a KASP typing kit.

6. The use of the detection reagent according to claim 4 in either (1) or (2): (1) Screening for pigs resistant to porcine epidemic diarrhea virus infection; (2) Breed pig breeds with PEDV resistance.

7. The application according to claim 6, characterized in that, The method for screening pigs against porcine epidemic diarrhea virus infection is as follows: Using the genomic DNA of the pigs to be tested as a template, PCR amplification was performed using primer pairs 1 to 2 shown in SEQ ID NO.3-SEQ ID NO.6, respectively, to obtain two amplification products. The two amplification products were sequenced, and the anti-PEDV trait of the pigs was identified based on the sequencing results, and pigs resistant to porcine epidemic diarrhea virus infection were screened.

8. The application according to claim 7, characterized in that, If the sequencing result of the amplification product of primer pair 1 corresponds to the TT genotype at the 297th base of the sequence shown in SEQ ID NO.1, and the sequencing result of the amplification product of primer pair 2 corresponds to the AA genotype at the 71st base of the sequence shown in SEQ ID NO.2, then it is identified as having the PEDV resistance trait.

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