A snp molecular marker related to a pig multi-rib trait and application thereof

Abstract

The application belongs to the technical field of animal molecular breeding, and discloses a SNP molecular marker related to a pig multi-rib trait and application thereof. The molecular marker is located at a nucleotide site at 125,651,010 bp of chromosome 15 of a pig reference genome Sscrofa11.1 version 15, and a C / G polymorphism exists at the site, wherein a C allele is a dominant protective allele of the multi-rib trait, and a G allele is a non-dominant risk allele. A C frequency of a multi-rib group is 0.938, a C frequency of a control group is 0.250, and an allele frequency difference ΔAF is 0.688. GWAS analysis shows that the marker is significantly associated with the pig multi-rib trait, a Fisher exact test P value is 2.68*10 ‑8 , a genome-wide significance threshold is reached, and an odds ratio (OR) is 45.00. The molecular marker can be used for molecular marker selection and / or assisted selection breeding of the pig multi-rib trait.

Description

Technical Field

[0001] This invention belongs to the field of animal molecular breeding technology, specifically relating to a SNP molecular marker associated with the multiribbed trait in pigs and its application in marker-assisted selection breeding. Background Technology

[0002] The number of ribs in pigs is an important economic trait affecting meat yield. Studies have shown that for every additional pair of ribs, the carcass length of a pig can increase by approximately 1.3-1.5 thoracic vertebrae, directly increasing meat yield. The Lichai Black Pig is a famous local pig breed in my country, renowned for its unique multi-rib trait (16 pairs of ribs), making it an excellent model for studying the genetic mechanisms of rib number in pigs.

[0003] Early selection of multi-ribbed traits using marker-assisted selection (MAS) technology can significantly shorten the breeding cycle and improve the efficiency of genetic improvement, which is of great significance for the protection and utilization of local pig breeds in my country.

[0004] Currently, the genes reported in the literature related to the number of ribs in pigs mainly include VRTN and NR6A1, which are widely considered to be the major genes for variation in the number of ribs in pigs. However, the predictive effectiveness of existing markers varies among different pig populations. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a SNP molecular marker associated with the multi-ribbed trait in pigs and its application. This molecular marker can be used to select or assist in the selection of multi-ribbed pigs, enabling early selection, shortening the selection cycle, and accelerating the process of population improvement.

[0006] The objective of this invention is achieved through the following technical solutions: A molecular marker for the multi-ribbed trait in pigs, wherein the molecular marker is located at nucleotide position 125,651,010 bp on chromosome 15 of the pig reference genome Sscrofa 11.1, and the site exhibits C / G polymorphism, wherein the C allele is the dominant protective allele for the multi-ribbed trait, and the G allele is the non-dominant risk allele.

[0007] Preferably, the SNP molecular marker is located in the intergenic region between the AP1S3 gene and the WDFY1 gene, wherein the AP1S3 gene is located at nucleotides 125,561,513 to 125,637,215 on chromosome 15, the WDFY1 gene is located at nucleotides 125,674,142 to 125,720,187 on chromosome 15, and the SNP molecular marker is located in the region between the two genes, approximately 13.8 kb from the end of the AP1S3 gene and approximately 23.1 kb from the beginning of the WDFY1 gene.

[0008] The present invention also provides the following technical solutions: The above-mentioned SNP molecular markers are used in the selection and / or assisted selection of multi-ribbed pigs.

[0009] The present invention also provides the following technical solutions: A method for breeding multiribbed pigs using the above-mentioned SNP molecular markers includes the following steps: (1) Detect the SNP molecular markers as described above in the pigs to be tested, determine whether the base at the site is C or G, and thus determine whether the genotype of the pigs to be tested is CC, CG or GG; (2) Based on the genotype, determine the tendency of the pig to have the multi-ribbed trait. Individuals with the C / C homozygous type are predicted to have the multi-ribbed trait and are selected as breeding pigs.

[0010] The present invention also provides the following technical solutions: A method for breeding multi-ribbed pig breeds using the above-mentioned SNP molecular markers includes the following steps: (1) Test the replacement breeding pigs with the SNP molecular markers as described above, determine whether the base at the locus is C or G, and thus determine whether the genotype of the pig to be tested is CC, CG or GG; (2) Select individuals with the genotype C / C obtained in step (1) as breeding pigs; (3) Mating the boars and sows retained in step (2) and continuing to screen their offspring by genotyping to gradually increase the frequency of C alleles and cultivate a stable multi-ribbed pig breed.

[0011] The present invention also provides the following technical solutions: A primer pair for identifying the aforementioned SNP molecular marker, said primer pair being capable of specifically amplifying a DNA fragment containing nucleotide 125,651,010 of porcine chromosome 15.

[0012] Preferably, the upstream primer has the sequence shown in SEQ ID NO: 1 and the downstream primer has the sequence shown in SEQ ID NO: 2.

[0013] The present invention also provides the following technical solutions: A kit for detecting the above-mentioned SNP molecular markers, the kit containing the above-mentioned primer pairs.

[0014] The present invention also provides the following technical solutions: The application of a primer pair or kit as described above in the selection and / or assisted selection of multiribbed pigs.

[0015] Preferably, the application is for Licha Black Pig.

[0016] Compared with the prior art, the technical effects of the present invention are as follows: This invention is the first to identify and validate, in a genome-wide association analysis, a significant association between the SNP locus of SSC15: 125,651,010 bp (located in the intergenic region of AP1S3-WDFY1) and the multi-ribbed trait in Licha Black Pigs, which can be used for molecular marker-assisted breeding of multi-ribbed traits in pigs.

[0017] This invention provides a SNP molecular marker associated with the multiribbed trait in pigs.

[0018] This SNP molecular marker corresponds to a nucleotide site at position 125,651,010 bp on chromosome 15 of the porcine reference genome Sscrofa11.1 version, with a base of either C or G. The C allele is the dominant protective allele for the multi-ribbed trait in pigs, while the G allele is a non-dominant risk allele. This SNP is located in the intergenic region between the AP1S3 and WDFY1 genes.

[0019] This invention provides the application of the above-mentioned SNP molecular markers in marker-assisted selection breeding of pigs.

[0020] The AP1S3 (Adaptor-related protein complex 1 subunit sigma 3) gene encodes a subunit of the AP-1 complex, involved in intracellular protein transport. The WDFY1 (WD repeat and FYVE domain-containing 1) gene encodes a protein containing a WD repeat sequence and an FYVE domain, involved in protein-protein interactions and membrane transport. These two genes are located flanking the SNP site SSC15:125,651,010 (AP1S3 upstream, WDFY1 downstream). This SNP may affect the expression of related genes by regulating long-range enhancer activity, chromatin conformational changes, or non-coding RNA regulation.

[0021] In marker-assisted breeding, this SNP molecular marker can be used to select and / or assist in screening multi-ribbed pig populations. C / C homozygous individuals are predicted to have the multi-ribbed trait (≥16 pairs of ribs), while G / G homozygous individuals are predicted to have the control trait (≤15 pairs of ribs). The application of this molecular marker can significantly shorten the breeding cycle, increase the rate of genetic improvement, and ultimately bring higher economic benefits to the livestock industry. Attached Figure Description

[0022] Figure 1 The Manhattan plot for the genome-wide association analysis of this invention is shown, with the core SNP site SSC15:125,651,010 marked with a red asterisk (P=2.68×10⁻⁶). -8 ); Figure 2 This is a comparison of allele frequencies at this SNP marker between the multiribbed group and the control group. Figure 3 Genotype distribution of this SNP marker in the multirib group and the control group. Detailed Implementation

[0023] The technical solution of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0024] Example 1 A molecular marker for the multi-ribbed trait in pigs, wherein the molecular marker is located at nucleotide position 125,651,010 bp on chromosome 15 of the pig reference genome Sscrofa 11.1, and the site exhibits C / G polymorphism, wherein the C allele is the dominant protective allele for the multi-ribbed trait, and the G allele is the non-dominant risk allele.

[0025] In this preferred embodiment, the SNP molecular marker is located in the intergenic region between the AP1S3 gene and the WDFY1 gene. The AP1S3 gene is located at nucleotides 125,561,513 to 125,637,215 on chromosome 15, and the WDFY1 gene is located at nucleotides 125,674,142 to 125,720,187 on chromosome 15. The SNP molecular marker is located in the region between the two genes, approximately 13.8 kb from the end of the AP1S3 gene and approximately 23.1 kb from the beginning of the WDFY1 gene.

[0026] Example 2 A method for obtaining the SNP molecular marker described in Example 1 includes the following steps: 1. Experimental group Thirty purebred Lichai black pigs were selected and divided into two groups: Multi-rib group: 16 heads, with ≥16 pairs of ribs (sample number LC-01-LC-16). Control group: 14 heads, with ≤15 pairs of ribs (sample number LC-17-LC-30).

[0027] 2. Whole genome resequencing Whole genome resequencing was performed on 30 individuals using the BGI T7 platform, with an average sequencing depth of 30×. The reference genome was Sus scrofa Sscrofa11.1 (GCF_000003025.6).

[0028] 3. Data Analysis Process (1) Data quality control: Use FastP software to perform quality control on raw reads; (2) Sequence alignment: Clean reads were aligned to the reference genome using BWA-MEM software; (3) Mutation detection: SNP / InDel detection and hard filtering were performed using the GATK Best Practices workflow; (4) VCF population filtering: MAF≥0.05, missing rate≤10%, finally obtained 20,695,766 high-quality SNPs; (5) GWAS: Fisher exact test was performed on 19,501,542 SNPs on 18 autosomes; (6) Selective signal analysis: Calculate FST, π ratio, XP-EHH, and iHS; (7) Candidate marker screening: Multi-evidence level comprehensive scoring to screen the optimal diagnostic marker.

[0029] 4. Results Genome-wide association analysis (GWAS) showed that the 124.5–126.5 Mb region on chromosome 15 was the most significant association peak, with a minimum p-value of 2.68 × 10⁻⁶. -8 It reached a genome-wide significance level (P<5×10⁻⁶). -8 The SNP marker located at SSC15:125,651,010 bp in the AP1S3-WDFY1 intergenic region was selected as a representative diagnostic marker. The allele frequency distribution of this SNP site in 30 Licha Black pigs is shown in Table 1. Allele frequency comparisons are shown in [Table 1]. Figure 2 .

[0030] Table 1. Allelic frequencies of SNP marker SSC15:125,651,010 in Licha Black Pigs

[0031] Precisely verified by Fisher (see) Figure 1 The SNP locus showed a highly significant difference between the multirib group and the control group (P=2.68×10). -8 The odds ratio (Odds Ratio) was 45.00, indicating that individuals carrying the C allele were significantly more likely to have the multirib trait than individuals carrying the G allele.

[0032] The genotypic distribution of this SNP locus in 30 Licha Black pigs is shown in Table 2. Figure 3 .

[0033] Table 2 Genotypic distribution of SNP marker SSC15:125,651,010 in Licha Black Pigs

[0034] 5. Select signal analysis Selection signal analysis was performed on the candidate interval of SSC15:124.5-126.5Mb, and the results showed that there was a significant selection signal in this interval (as shown in Table 3).

[0035] Table 3. Statistics of candidate interval selection signals for SSC15: 124.5-126.5Mb

[0036] The results show that the GWAS correlation analysis and selection signal analysis results corroborate each other, both pointing to the SSC15:124.5-126.5Mb interval as a key candidate region for the multi-ribbed trait in Licha Black Pigs.

[0037] Example 3 This embodiment provides a method for breeding and / or assisting in the breeding of multi-ribbed pigs using the SNP molecular markers obtained in Example 2, comprising the following steps: Includes the following steps: (1) Extract the genomic DNA of the pig to be tested, which can be extracted through ear tissue or blood sample; detect the SNP molecular marker described in Example 1 of the pig to be tested, determine whether the base of the site is C or G, and thus determine whether the genotype of the pig to be tested is CC, CG or GG; (2) Based on the genotype, determine the tendency of the pig to have the multi-ribbed trait. Individuals with the C / C type predicted to have the multi-ribbed trait should be given priority for breeding; individuals with the G / G type predicted to have the control trait can be culled or used for other purposes; individuals with the C / G type are intermediate and can be further judged by combining other markers or phenotypes.

[0038] In this embodiment, the discrimination accuracy reached 87.5% (26 / 30) in the training set (30 individuals), of which all 14 individuals of the multi-ribbed C / C type were correctly identified.

[0039] Example 4 This embodiment provides a method for breeding and / or assisting in the breeding of multi-ribbed pig lines using the SNP molecular markers obtained in Example 2, including the following steps: (1) Test the SNP molecular marker described in Example 1 on the replacement breeding pigs to determine whether the base at the site is C or G, thereby determining whether the genotype of the pig to be tested is CC, CG or GG; (2) Select individuals with the genotype C / C obtained in step (1) as breeding pigs; (3) Mating the boars and sows retained in step (2) and continuing to screen their offspring by genotyping to gradually increase the frequency of C alleles and cultivate a stable multi-ribbed pig breed.

[0040] Example 5 This embodiment provides primer pairs for identifying the SNP molecular markers described in Example 1. Based on the porcine reference genome Sscrofa11.1 (SSC15:125,650,793-125,651,246 region), the following specific primer pairs can be designed: Upstream primer: 5'-CCTGAACAAAATGGCACCCAG-3' (e.g., SEQ ID NO: 1). Downstream primer: 5'-AGTAGTCACACTCGTTGCCTG-3' (e.g., SEQ ID NO: 2). Alternatively, the TaqMan probe method can be used to perform real-time fluorescent PCR genotyping using specific probes containing the SSC15:125,651,010 loci.

[0041] Example 6 This embodiment provides a kit for detecting the SNP molecular markers described in Example 1. The kit includes the primer pair described in Example 5.

[0042] In a preferred embodiment, the reagent kit further includes: (1) PCR reaction mixture (containing Taq DNA polymerase, dNTPs, buffer, etc.); (2) Positive control DNA (C / C type) and negative control DNA (G / G type); (3) Instructions for use, including genotype interpretation criteria.

[0043] This kit can be used for routine genotyping in pig breeding farms. It is easy to operate and provides intuitive interpretation.

[0044] Example 7 Further validation of the SSC15:125,651,010 locus was performed using a sample of 49 Licha Black pigs (25 multi-ribbed and 24 controls). This sample consisted of two parts: (1) The whole genome resequencing data of the above 30 Licha Black Pigs (30×), of which LC-01~LC-16 are 16 multi-ribbed individuals and LC-17~LC-30 are 14 control individuals; (2) Whole genome resequencing data of 19 Licha Black Pigs (10×) were obtained from the NCBI database (project number: PRJNA1009598), sample numbers LC-31 to LC-49, including 9 multi-ribbed individuals and 10 control individuals.

[0045] The two sets of data were merged for analysis after undergoing standardized processing to ensure consistency in genotype interpretation. The results are shown in Table 4. Table 4. Validation results of the SNP marker SSC15:125,651,010 in 49 Licha Black Pigs

[0046] Expanded sample validation results showed that the C allele frequency at the SSC15:125,651,010 locus was significantly higher in the multirib group (0.720) than in the control group (0.312), with a gene frequency difference of 0.407 and a Fisher P value of 1.0 × 10⁻⁶. -4 Consistent with the results of the original discovery set, this further confirms the association between this SNP molecular marker and the multi-ribbed trait in Licha Black Pig.

Claims

1. The application of a SNP associated with the multi-ribbed trait of Licha Black Pig in the selection and / or assisted selection of multi-ribbed Licha Black Pigs, characterized in that, The SNP associated with the multi-ribbed trait in Licha Black Pig is located at nucleotide position 125,651,010 bp on chromosome 15 of the pig reference genome Sscrofa 11.

1. This site exhibits C / G polymorphism, where the C allele is the dominant allele for the multi-ribbed trait, and the G allele is the non-dominant allele for the multi-ribbed trait.

2. A method for breeding multi-ribbed black pigs using the application as described in claim 1, characterized in that, Includes the following steps: (1) Detect the SNPs related to the multi-ribbed trait of Licha Black Pig in the pig to be tested, determine whether the base of the locus is C or G, and thus determine whether the genotype of the pig to be tested is CC, CG or GG. (2) Based on the genotype, determine the tendency of the pig to have the multi-ribbed trait. Individuals with the C / C homozygous type are predicted to have the multi-ribbed trait and are selected as breeding pigs.

3. A method for breeding the Duolailicha Black Pig strain using the application described in claim 1, characterized in that, Includes the following steps: (1) Test the replacement breeding pigs for SNPs that are related to the multi-ribbed trait of Licha Black Pig, and determine whether the base of the locus is C or G, so as to determine whether the genotype of the pig to be tested is CC, CG or GG. (2) Select individuals with the genotype C / C obtained in step (1) as breeding pigs; (3) Mating the boars and sows retained in step (2) and continuing to screen their offspring by genotyping to gradually increase the frequency of C alleles and cultivate a stable multi-ribbed pig breed.

4. A primer pair for identifying SNPs associated with the multi-ribbed trait of Licha Black Pig as described in claim 1, characterized in that, The primer pair consists of an upstream primer and a downstream primer. The upstream primer is shown in the sequence of SEQ ID NO: 1, and the downstream primer is shown in the sequence of SEQ ID NO:

2.

5. A kit for detecting SNPs associated with the multi-ribbed trait of Licha Black Pig as described in claim 1, characterized in that, The kit contains the primer pair as described in claim 4.

6. The use of a primer pair as described in claim 4 or a kit as described in claim 5 in the breeding and / or assisted breeding of the Dorrench Black Pig.

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