Pig immunity-related SNP (Single Nucleotide Polymorphism) site molecular marker and application thereof

Abstract

The invention discloses a pig immunity-related SNP (Single Nucleotide Polymorphism) site molecular marker and application, and belongs to the related field of pig stress resistance. The marker has three single nucleotide polymorphic sites in total, the three single nucleotide polymorphic sites are respectively positioned at nucleic acid sites at 48950343, 48950346 and 48950523 of a chromosome 2 of a pig reference genome Sscrofa11.1, are respectively subjected to A48950343G, G48950346C and A48950523G single base mutation, are contained at an LYVE1 gene exon and are subjected to non-synonymous mutation, and a nucleotide sequence is shown as SEQ ID NO.1. The invention further discloses a detection method of the single nucleotide polymorphic sites of the pig reference genome Sscrofa11.1 and a detection method of the single nucleotide polymorphic sites of the pig reference genome Sscrofa11.1. The identified sites of the SNP related to the immune index characters can be used as molecular markers for pig excellent character breeding, and the genetic progress of pig disease-resistant breeding is facilitated.

Description

Technical Field

[0001] The invention belongs to the field related to pig stress resistance, and specifically relates to pig immunity-related SNP site molecular markers and applications. Background Art

[0002] In recent years, infectious diseases of pigs have caused great harm to my country's pig industry, such as African swine fever, foot-and-mouth disease and blue ear disease. Therefore, breeding new varieties that are resistant to diseases and stress has become an urgent task to ensure the sustainable development of the pig industry. Through breeding pigs for stress resistance, some excellent traits that can resist adverse environments can be preserved. The bred stress-resistant strains can still maintain their economic traits and stable inheritance in adverse environments. This is of great significance to protecting and developing local pig breeds in my country and maintaining the sustainable development of agricultural and animal husbandry production.

[0003] With the development of omics technology and molecular biology, pig breeding has entered the era of molecular breeding from the initial phenotypic selection breeding. Molecular breeding mainly includes molecular marker-assisted breeding and genomic selection. Molecular breeding has the characteristics of high selection accuracy and short cycle. Researchers have made many achievements in stress resistance. Among them, the LYVE1 (lymphatic vessel endothelial hyaluronan receptor 1) gene encodes a type I integral membrane glycoprotein. The encoded protein acts as a receptor and binds to soluble and immobilized hyaluronic acid. This protein can act as a hyaluronic acid transporter, mediating its catabolism in lymphatic endothelial cells, or transporting it into the lumen of lymphatic vessels, and then reabsorbed and degraded in the lymph nodes. This process also provides theoretical support for the potential role of the LYVE1 gene in immune response.

[0004] With the development of sequencing technology and the demand for lowering sequencing costs, the second generation sequencing technology (Next generation sequencing, NGS) characterized by high throughput came into being and gradually matured. Based on the second generation sequencing technology, whole genome sequencing has been widely used, the whole genomes of a large number of organisms have been successfully sequenced, and the genome plans of a large number of species have been completed. Through selection and elimination analysis, a large number of single nucleotide polymorphisms (SNPs) have been discovered, which has pointed out the direction for the study of some excellent traits of crops and livestock and complex human diseases, and has made new progress in this research field. Summary of the invention

[0005] The purpose of the present invention is to provide a molecular marker of SNP sites related to pig immunity and its application, and to apply the molecular marker to screen individuals with stronger disease resistance, optimize the stress resistance of breeding pigs through rapid evaluation of individual genotypes, and provide technical support for the genetic improvement of pig high-quality traits.

[0006] The object of the present invention is achieved by the following technical solutions: A porcine immune-related SNP site molecular marker, the marker has a total of 3 single nucleotide polymorphism sites, which are located at nucleic acid sites 48950343, 48950346 and 48950523bp on chromosome 2 of the porcine reference genome Sscrofa11.1, respectively, and have single base mutations A48950343G, G48950346C and A48950523G, respectively, contained in the exon of the LYVE1 gene, which are non-synonymous mutations. The nucleotide sequence is shown in SEQ ID NO.1, and the SNP sites to which they belong are the 31st base, the 34th base and the 211th base of the sequence, respectively, and the single nucleotides at the corresponding sites are A base mutation to C, G base mutation to C, and A base mutation to G, respectively, which leads to differences in immune capabilities among individual pigs.

[0007] The present invention also provides an application of a pig immunity-related SNP site molecular marker, and the application of the pig immunity-related SNP site molecular marker in pig genetic breeding.

[0008] The present invention also provides a method for pig disease resistance breeding or assisted breeding using pig immunity-related SNP site molecular markers, which uses the pig immunity-related SNP site molecular markers, including the following steps: collecting pig ear tissue samples or blood samples, extracting genomic DNA, detecting the nucleotide sequences at 48950343, 48950346 and 48950523bp of chromosome 2, respectively A or G, C or G, A or G, determining that the genotypes of the pigs to be tested are AA or GA type, CC or CG type, and then selecting pigs with GA genotype or CG genotype according to production needs for the next step of seed selection and / or breeding. The results show that the immune ability of GA genotype pigs is higher than that of AA type, and the immune ability of CG genotype pigs is higher than that of CC genotype.

[0009] The present invention also provides a method for identifying the above-mentioned pig immunity-related SNP site molecular markers, comprising the following steps: S1. Collect and obtain pig blood, and detect immune indicators in serum including hemoglobin, interleukin, and IFNγ; S2. Collect pig ear tissue samples or blood samples, extract pig genomic DNA, build a library and perform genome resequencing; S3. Use Trimmomatic software to filter the measured raw library data to reduce the impact of base errors on the results; S4. Use BWA-mem software to align the genome sequence to the pig reference genome Sscrofa11.1; S5. Use Samtools software to sort and index the results; S6. Downstream analysis was performed using GATK software; S7. Vcftools software was used to filter the SNPs in the variant according to the population variation distribution. The filtering thresholds included: (1) only retaining sites with two alleles, (2) SNP missing rate exceeding 10%, (3) minimum allele frequency less than 0.05; S8, SNPs that passed the filter were used for selection and elimination analysis; S9. Use any one of Vcftools, XPCLR, RectChr or their combination software to perform selection and elimination analysis on high and low expression IgG pig populations to identify key candidate regions, genes and sites that affect immune traits.

[0010] Preferably, the filtering criteria of step S3 are as follows: (1) remove sequences with adapters; (2) remove sequences whose base information cannot be determined; (3) bases with a quality value less than 3 at the beginning and end will be cut off; (4) use a sliding window for quality trimming with a window size of 4 bases. If the average quality score in the window is less than 15, trim the sequence from this position; (5) if the length of the trimmed sequence is less than 36 bases, remove the sequence.

[0011] Preferably, the downstream analysis of step S6 includes the following steps: first, using GATK MarkDuplicates to remove sequence duplications caused by PCR; then, using GATK HaplotypeCaller to search for variations across the entire genome; finally, using GATK VariantFiltration to perform hard filtering on variant sites; wherein the threshold for SNP filtering is set to "QD < 2.0, MQ < 40.0, FS > 60.0, SOR > 3.0, MQRankSum < -12.5, ReadPosRankSum < -8.0".

[0012] The present invention also provides a primer pair for identifying the above-mentioned pig immunity-related SNP site molecular markers, the primer pair comprises an upstream primer F1 and a downstream primer R1, and the nucleotide sequences of the primer pair are shown in SEQ ID NO: 2 and SEQ ID NO: 3 respectively.

[0013] The present invention also provides an application of a primer pair for molecular markers of SNP sites related to pig immunity, and the application of the primer pair in breeding or assisting in breeding pigs with high / low immunity.

[0014] The present invention also provides a kit for detecting pig immunity-related SNP molecular markers, and the kit contains the above-mentioned primer pair.

[0015] The beneficial effects of the present invention are as follows: the present invention has discovered for the first time the loci of the LYVE1 gene associated with pig immunity, providing an effective candidate gene for breeding pig individuals with excellent traits. At the same time, the present invention uses a selection elimination analysis method to identify that three SNPs (g.48950343A>G, g.48950346G>C, g.48950523A>G) in the LYVE1 gene are significantly associated with pig immunity. The above loci can be applied to immune-related analysis, providing a new genetic marker resource for molecular marker-assisted selection of pig disease resistance.

[0016] The present invention uses a method based on selection elimination analysis to screen and identify pig immune-related genes. For the identified candidate genes, the present invention further uses the candidate gene method strategy to determine whether there are sites associated with pig immune performance in the studied genes, thereby further verifying the relationship between the candidate genes and immune indicators. In addition, through the candidate gene method, the sites of SNPs associated with immune indicator traits identified can be used as molecular markers for the selection of excellent pig traits, which is helpful for the genetic progress of pig disease-resistant breeding.

[0017] The present invention has the characteristics of simple operation, short time consumption, high accuracy of selected markers, etc., and also provides technical support for the genetic analysis of other important economic traits of livestock and poultry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Based on F ST , PI and XPCLR selection clearance analysis to obtain the selected region where the LYVE1 gene is located; Figure 2 To further base on F ST and PI methods to conduct selective clearance analysis of the region; Figure 3 is the haplotype expression pattern in different groups in this region; Figure 4 The different allele frequencies of the three SNP molecular marker sites mentioned above; Figure 5 This is an analysis of the differences in IgG expression levels between different allele types of the three SNP molecular marker sites mentioned above. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. The described embodiments are part of the embodiments of the present invention, rather than all the embodiments.

[0020] Example 1: Screening method for pig immunity-related SNP site molecular markers 1. Selection of high and low expression groups of pigs A sample size of 56 Licha black pigs was selected, all of which were three to four months old. After blood was collected from the jugular vein, immune indicators such as IgG were detected, and individuals were selected for the detected indicators, and 15 individuals with high expression and 15 individuals with low expression were selected as high expression groups and low expression groups, respectively. In this example, the expression levels of IgG in the serum of the two groups of pigs were analyzed by T test (Table 1).

[0021] Table 1

[0022] 2. Whole genome resequencing, alignment analysis and quality control After collecting ear tissues from the above 56 individuals, whole-genome tissue resequencing was performed. The raw data were quality controlled using Trimmomatic software, and then sequence alignment was performed using BWA-mem software. The reference genome was Sscrofa11.1, and the alignment results were sorted and indexed using Samtools software. GATK MarkDuplicates was then used to remove sequence duplications caused by PCR; subsequently, GATK HaplotypeCaller was used to retrieve variations across the genome; finally, GATK VariantFiltration and Vcftools were used to filter variant sites, and the filtering conditions were as described above. In the end, a total of 19,627,493 high-quality SNP sites were obtained, and SNP information of 30 individuals with high and low expression was extracted from the last filtered SNP file for subsequent analysis.

[0023] 3. Choose to clear analysis and annotations Vcftools software was used to calculate FST and PI, and XPCLR software was used to calculate XPCLR. The top 1% of the common regions of each method were taken, and then the top 5 regions in the common regions of each method were taken as candidate selected regions ( Figure 1 We found that the signal value of the selected region of chromosome 2, 48920001-49010000, was strong, and its haplotype also had different expression patterns in the two populations ( Figure 2 , Figure 3). Then, snpEff software was used to annotate genes and determine the variant sites and types in this region. The LYVE1 gene was annotated in this selected region, and sites 48950343, 48950346, and 48950523 were non-synonymous mutations (Table 2).

[0024] Table 2

[0025] 4. Genotype frequency and analysis of differences among different genotypes The hiblup software was used to calculate the genotype frequencies of the three loci, and it was found that the genotype frequencies in different groups were quite different. The Kruskal-Wallis method was first used to test whether there were differences between the genotypes in each group, and then the Dunn method was used to test the differences between each two groups. It was found that the IgG expression levels of the 48950343 GA genotype, 48950346 CG genotype, and 48950523 GA genotype groups were higher than those of the AA, CC, and AA genotype groups, respectively. Figure 4 , Figure 5 ) The genotype frequencies of the three loci of the LYVE1 gene in different populations are shown in Table 3, and the differences in IgG expression levels of different genotypes at the three loci of the LYVE1 gene are shown in Table 4.

[0026] Table 3

[0027] Table 4

[0028] Note: ** indicates that the birth weights of different genotypes are significantly different (P ≤ 0.01); ns indicates that comparison is impossible or the difference is not significant.

[0029] The analysis results showed that at the marker site g.48950343A>G, the IgG of the GA genotype of pigs was 491.155.58μg / mL, which was extremely significantly higher than that of the AA genotype (442.436.62μg / mL); at the marker site g.48950346G>C, the IgG of the GC genotype of pigs was 491.155.58μg / mL, which was extremely significantly higher than that of the GG genotype (442.436.62μg / mL); at the marker site g.48950523A>G, the IgG of the GA genotype of pigs was 494.5±55.44μg / mL, which was extremely significantly higher than that of the AA genotype (443.2±37.13μg / mL). Therefore, the above molecular markers g.48950343A>G, g.48950346G>C, and g.48950523A>G can be used for pig immunity-related molecular marker-assisted selection, among which individuals with the GA, GC, and GA genotypes at these three loci can be selected as objects for the next step of selection or breeding to improve the disease resistance of pigs.

[0030] Example 2: Application of pig immunity-related SNP molecular markers in breeding pigs for disease resistance This example is the design of upstream and downstream primers and amplification results for the detection of nucleic acid sites at 48950343bp, 48950346bp and 48950523bp on chromosome 2 of the SNP sites obtained in Example 1.

[0031] 1. Primer design of target gene sequence According to the target gene sequence, the optimized amplification primers were designed using primer design software. The primer pair sequences are as follows: Upstream primer F1: 5'-CCCCACCAATGATCCCACAT-3' (as shown in SEQ ID NO: 2) Downstream primer R1: 5'-TTTATTTGTATTTGGCAGGCCC-3' (as shown in SEQ ID NO: 3) 2. Pig genome extraction Collect ear tissue samples or blood samples from the pigs to be tested, extract genomic DNA, and store them at -20℃ for future use after quality and concentration testing.

[0032] 3. PCR amplification of pig immunity-related SNP sites The extracted DNA was used as a template and PCR amplification was performed using the above primers.

[0033] PCR amplification system (total volume is 20.0 μL): 10.0 μL of 2xTaq PCR Master Mix, 0.5 μL of upstream primer (10 μmol / L), 0.5 μL of downstream primer (10 μmol / L), 1.0 μL of genomic DNA template, and 8.0 μL of ddH2O.

[0034] The PCR amplification procedure is: Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, for a total of 30 cycles; extension at 72°C for 5 min.

[0035] The PCR products were detected by 1.5% agarose gel electrophoresis and then subjected to first-generation Sanger sequencing.

[0036] 4. Sequencing and analysis of amplified products The PCR product was detected by agarose gel electrophoresis, and the size of the amplified target fragment was 410 bp (as shown in SEQ ID NO: 10). The amplified product was sent for sequencing, and the sequence of the amplified product was obtained by sequencing. The sequencing result was compared and analyzed with the relevant gene fragments of pigs in GenBank (the AA genotype of the 48950343 site is shown in SEQ ID NO: 4, the GG genotype of the 48950343 site is shown in SEQ ID NO: 5, the GG genotype of the 48950346 site is shown in SEQ ID NO: 6, the CC genotype of the 48950346 site is shown in SEQ ID NO: 7, the AA genotype of the 48950523 site is shown in SEQ ID NO: 8, and the GG genotype of the 48950523 site is shown in SEQ ID NO: 9) to determine whether the genotype of the immune-related target SNP site of the sequence is AA or GA type, CC or CG type.

[0037] According to the above results, pigs with high or low immunity are selected. If pigs with high immunity are selected, pigs with GA and CG genotypes are selected; if pigs with low immunity are selected, pigs with AA and CC genotypes are selected.

[0038] Example 3 It is easy for a person skilled in the art to refer to other primers designed according to the aforementioned SNP sites of the present invention or probes for identifying the molecular markers, so as to detect the genetic markers, for example, by PCR amplification to obtain the molecular genetic markers, and then by cloning and sequencing to obtain the corresponding sequences, or by RFLP polymorphism detection. Therefore, the present invention also includes other primers for amplifying the molecular genetic markers or probes for identifying the molecular genetic markers, and a kit containing the primers or probes.

[0039] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A porcine immune-related SNP site molecular marker, characterized by: This marker has a total of three single nucleotide polymorphism sites, which are located at the nucleic acid sites 48950343bp, 48950346bp and 48950523bp on chromosome 2 of the pig reference genome Sscrofa11.1, respectively. There are single base mutations A48950343G, G48950346C and A48950523G, respectively, which are contained in the exon of the LYVE1 gene and are non-synonymous mutations. The nucleotide sequence is as shown in SEQ ID NO.

1.

2. An application of a pig immune-related SNP site molecular marker, characterized in that: Application of the porcine immune-related SNP locus molecular marker described in claim 1 in porcine genetic breeding.

3. A method for pig disease resistance breeding or assisted breeding using pig immunity-related SNP site molecular markers, characterized in that: The method of using the pig immune-related SNP site molecular markers described in claim 1 comprises the following steps: collecting pig ear tissue samples or blood samples, extracting genomic DNA, detecting that the nucleotide sequences at 48950343bp, 48950346bp and 48950523bp of chromosome 2 are A or G, C or G, A or G, respectively, determining that the genotypes of the pigs to be tested are AA or GA, CC or CG, respectively, and then selecting pigs with GA genotype or CG genotype according to production needs for the next step of seed selection and / or breeding.

4. The method for pig disease resistance breeding or assisted breeding using pig immunity-related SNP locus molecular markers according to claim 3, characterized in that: The immune ability of GA genotype pigs is higher than that of AA genotype, and the immune ability of CG genotype pigs is higher than that of CC genotype.

5. A method for identifying the porcine immune-related SNP site molecular marker according to claim 1, characterized in that: The following steps are involved: S1. Collect and obtain pig blood, and detect immune indicators in serum including hemoglobin, interleukin, and IFNγ; S2. Collect pig ear tissue samples or blood samples, extract pig genomic DNA, build a library and perform genome resequencing; S3. Use Trimmomatic software to filter the measured raw library data to reduce the impact of base errors on the results; S4. Use BWA-mem software to align the genome sequence to the pig reference genome Sscrofa11.1; S5. Use Samtools software to sort and index the results; S6. Downstream analysis was performed using GATK software; S7. Vcftools software was used to filter the SNPs in the variant according to the population variation distribution. The filtering thresholds included: (1) only retaining sites with 2 alleles, (2) SNP missing rate exceeding 10%, (3) minimum allele frequency less than 0.05; S8, SNPs that passed the filter were used for selection and elimination analysis; S9. Use any one of Vcftools, XPCLR, RectChr or their combination software to perform selection and elimination analysis on high and low expression IgG pig populations to identify key candidate regions, genes and sites that affect immune traits.

6. The method according to claim 5, characterized in that The filtering criteria of step S3 are as follows: (1) remove sequences with adapters; (2) remove sequences whose base information cannot be determined; (3) bases with a quality value less than 3 at the beginning and end will be cut off; (4) use a sliding window for quality trimming with a window size of 4 bases. If the average quality score in the window is less than 15, the sequence is trimmed from this position; (5) if the length of the trimmed sequence is less than 36 bases, the sequence is removed.

7. The method according to claim 5, characterized in that The downstream analysis of step S6 includes the following steps: first, using GATK MarkDuplicates to remove sequence duplications caused by PCR; then, using GATK HaplotypeCaller to search for variations across the entire genome; finally, using GATK VariantFiltration to perform hard filtering on variant sites; wherein the threshold for SNP filtering is set to "QD < 2.0, MQ < 40.0, FS > 60.0, SOR > 3.0, MQRankSum < -12.5, ReadPosRankSum < -8.0".

8. A primer pair for identifying the porcine immune-related SNP site molecular marker as claimed in claim 1, characterized in that: The primer pair comprises an upstream primer F1 and a downstream primer R1, and the nucleotide sequences of the primer pair are shown in SEQ ID NO: 2 and SEQ ID NO: 3 respectively.

9. Application of a primer pair for molecular markers of SNP sites related to pig immunity, characterized in that: Use of the primer pair described in claim 8 in breeding or assisting in breeding pigs with high / low immunity.

10. A kit for detecting the porcine immune-related SNP molecular marker according to claim 1, characterized in that: The kit contains the primer pair according to claim 8.

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