Pig chromosome 1 snp molecular marker related to multi-rib trait and application thereof

Abstract

This invention discloses a chromosome 1 SNP molecular marker associated with the multirib trait in pigs and its application, belonging to the field of pig molecular breeding technology. The SNP molecular marker is located at base position 161,373,061 of pig chromosome 1, reference genome Sscrofa11.1, chromosome number NC_010443.5. This locus exhibits a G / A single nucleotide polymorphism, where the G allele is the dominant multirib allele. This locus is located within the CCBE1 gene, and Fisher's exact test shows a p-value of 5.347 × 10⁻⁶. ‑8 The marker effect was nearly significant across the entire genome, with an odds ratio (OR) of 65.44. A larger population of 49 samples validated the robustness of the marker effect. Quadruple selection signal analysis further supported positive selection in the CCBE1 region. This invention can be used for early molecular identification and assisted selection of the multirib trait in pigs, shortening the breeding cycle and improving selection efficiency.

Description

Technical Field

[0001] This invention belongs to the field of pig molecular breeding technology, specifically relating to a molecular marker of chromosome 1 SNP related to the multirib trait in pigs and its application. Background Technology

[0002] The number of ribs in pigs is an important economic trait affecting carcass length and meat yield. More ribs result in a longer carcass and higher meat yield, thus having significant economic value for the pig industry. Among local pig breeds in my country, the Shandong Lichai Black Pig is renowned for its multiple ribs (≥16 pairs), making it a valuable local genetic resource.

[0003] Currently, the main reported genes associated with polyribs in pigs include the VRTN gene on chromosome 7 and the NR6A1 gene on chromosome 1. However, these known genes cannot fully explain the population-level variation in rib number, indicating the existence of other undiscovered genetic factors.

[0004] Marker-assisted selection (MAS) can predict multi-ribbed tendency in piglets early in their development through genotyping, eliminating the need to wait until slaughter to obtain phenotype information. This effectively shortens the breeding cycle and improves selection efficiency. Therefore, discovering new and significant multi-ribbed related molecular markers has important breeding application value. Summary of the Invention

[0005] This invention provides a novel SNP molecular marker located in the CCBE1 gene on chromosome 1 for early identification and assisted selection of the multirib trait in pigs. This enables early selection, shortens the breeding cycle, and accelerates the process of population improvement.

[0006] The objective of this invention is achieved through the following technical solutions: A molecular marker for a polyribatic trait on chromosome 1 in pigs, wherein the SNP is located at base 161,373,061 on chromosome 1, reference genome Sscrofa11.1, chromosome number NC_010443.5, and the site exhibits a G / A single nucleotide polymorphism, wherein the G allele is the dominant polyribatic allele.

[0007] The present invention also provides the following technical solutions: The above-mentioned SNP molecular markers on chromosome 1 are used in the selection and / or assisted selection of multiribbed pigs.

[0008] This invention also provides a method for breeding and / or assisting in the breeding of multiribbed pigs using the aforementioned chromosome 1 SNP molecular markers, comprising the following steps: (1) Extracting porcine genomic DNA; (2) Genotyping was performed on the 161,373,061st locus on chromosome 1 to determine whether the base at this locus was A or G, thereby determining whether the genotype of the pig to be tested was AA, GA or GG. (3) Judging the tendency of polyribs based on genotype: individuals with GG and GA genotypes have a higher probability of polyribs, while individuals with AA genotypes have a lower probability of polyribs.

[0009] This invention also provides a method for breeding and / or assisting in the breeding of multi-ribbed pig breeds using the aforementioned chromosome 1 SNP molecular markers, comprising the following steps: 1) Test the SNP molecular markers described above on replacement breeding pigs to determine whether the base at the site is A or G, thereby determining whether the genotype of the pig to be tested is AA, GA or GG; 2) Individuals homozygous for the G allele (GG) or heterozygous for the G allele (GA) were selected as breeding pig candidates; 3) Mating the boars and sows retained for breeding in step 2) and continuing to perform genotyping screening on their offspring to gradually increase the frequency of the G allele and cultivate a stable hereditary multi-ribbed pig breed.

[0010] The present invention also provides a primer pair for identifying the above-mentioned chromosome 1 SNP molecular marker, wherein the primer pair is capable of specifically amplifying a DNA fragment containing nucleotides at position 161,373,061 of chromosome 1.

[0011] Preferably, the primer pair consists of an upstream primer with a sequence as shown in SEQ ID NO: 1 and a downstream primer with a sequence as shown in SEQ ID NO: 2.

[0012] The present invention also provides a kit for detecting the above-mentioned chromosome 1 SNP molecular marker, the kit containing primer pairs or primer pairs with the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 2, which are capable of specifically amplifying a DNA fragment containing nucleotides at position 161,373,061 of porcine chromosome 1.

[0013] The present invention also provides the application of the above-mentioned primer pairs or kits in the selection and / or assisted selection of multi-ribbed pigs.

[0014] Preferably, in the above applications, the pig is a Licha Black Pig or a hybrid population with Licha Black Pigs as parents.

[0015] Beneficial effects The SNP molecular marker is located within the functional gene CCBE1, which encodes a collagen-binding protein with a plausible biological mechanism—CCBE1's collagen-binding function participates in extracellular matrix remodeling and may regulate rib number by influencing somnolence differentiation and rib primordia development. The CCBE1 (Collagen and Calcium Binding EGF Domains 1) gene encodes collagen and calcium-binding EGF domain protein 1, a gene previously never reported to be associated with rib number or multi-rib trait in pigs. CCBE1's collagen-binding function provides a novel molecular mechanism explanation for the multi-rib trait. This invention can be used for early molecular identification and assisted selection of multi-rib trait in pigs, shortening the breeding cycle and improving selection efficiency. Specific details are as follows: 1. Genome-wide association analysis (GWAS) revealed a highly significant association between base position 161,373,061 on pig chromosome 1 and the multirib trait, Fisher's exact test (P = 5.347 × 10⁻⁶). -8 This is the strongest association signal on chromosome 1, and there is sufficient statistical evidence to support it.

[0016] 2. This SNP locus exhibits G / A polymorphism, with the G allele being the dominant allele in the multi-ribbed group. In 30 Licha Black pigs, the frequency of the G allele was 0.9688 in the multi-ribbed group (N=16) and 0.3214 in the control group (N=14), with an allele frequency difference ΔAF=0.6473.

[0017] 3. The odds ratio (OR) was 65.44, indicating that G allele carriers have a highly significant multirib advantage.

[0018] 4. Quadruple selection signal analysis (FST / π-ratio / XP-EHH / iHS) further validated that the CCBE1 gene region was positively selected, supporting the biological reliability of the marker.

[0019] 5. A sample of 49 pigs validated and confirmed that the marker effect was robust (P<0.0001, OR=8.60, ΔAF=0.443). It has strong labeling ability. Attached Figure Description

[0020] Figure 1 Diagram showing the structure of the CCBE1 gene and the location of its SNP sites; Figure 2 Manhattan plot of genome-wide GWAS association analysis; Figure 3 Comparison of G / A allele frequencies at Chr1:161,373,061 loci; Figure 4Genotype distribution map of Chr1:161,373,061 loci; Figure 5 CCBE1 Region Quadruple Selection Signal Analysis. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0022] Example 1 This embodiment provides a chromosome 1 SNP molecular marker associated with the multirib trait in pigs. The SNP molecular marker is located at base 161,373,061 of chromosome 1, with reference genome Sscrofa11.1 and chromosome number NC_010443.5. This site has a G / A single nucleotide polymorphism, in which the G allele is the dominant multirib allele.

[0023] Example 2 A method for obtaining the chromosome 1 SNP molecular marker described in Example 1 includes the following steps: 1. Sample Source Thirty Lichai black pigs were included, with 16 pigs in the multi-rib group (≥16 pairs of ribs) and 14 pigs in the control group (≤15 pairs of ribs). All samples were from the Lichai black pig breeding farm in Shandong Province, and the number of ribs was confirmed by phenotypic identification.

[0024] 2. Genotype Acquisition Whole-genome resequencing was performed on 30 pigs to obtain whole-genome SNP variation data. The BGI T7 platform was used for whole-genome resequencing of the 30 individuals, with a sequencing depth of 30×. Raw read quality control was performed using FastP and Trimmomatic software, and clean reads were aligned to the reference genome using BWA-MEM software. Sus scrofa Sscrofa11.1 (GCF_000003025.6). SNP and InDel detection and hard filtering were performed using the GATK Best Practices workflow. After quality control (MAF>0.05, Miss<0.1, Hardy-Weinberg P>1e-6), with MAF≥0.05 and a deletion rate≤10%, a total of 20,695,766 high-quality SNP loci were obtained. 19,501,542 SNPs were extracted from 18 autosomes for subsequent analysis.

[0025] 3. Association Analysis Methods Association analysis was performed using PLINK v1.90 software. Fisher's exact test (--fisher) was used to statistically analyze the differences in allele frequencies at each SNP locus to eliminate the chi-square test (χ²) under small sample conditions.2 The asymptotic bias of the test was assessed. Simultaneously, a logistic regression model of PLINK (-logistic) was used for association analysis, with the first two principal components of principal component analysis (PCA) as covariates to control for potential confounding factors such as population stratification. The genomic significance threshold was set at P < 5 × 10⁻⁶. -8 See genome-wide association analysis. Figure 2 The results of the significance test for allele frequency differences are shown in Table 1.

[0026] Table 1. Results of the significance test for allele frequency differences

[0027] Analysis showed that base position 161,373,061 on chromosome 1 (NC_010443.5) was highly significantly associated with the multiribbed trait, Fisher's exact test p = 5.347 × 10⁻⁶. -8 The odds ratio (OR) was 65.44. The frequency of the G allele in the multirib group was 96.88%, while the frequency of the G allele in the control group was 32.14%, with a difference in allele frequency ΔAF = 64.74%.

[0028] 4. Genotype distribution verification Genotypes were extracted from 30 pigs at the Chr1:161,373,061 locus. Genotypes are shown in Table 2. The comparison of G / A allele frequencies at the Chr1:161,373,061 locus is shown in [Table 2]. Figure 3 Genotype distribution is shown in Figure 4 .

[0029] Table 2 Genotype Distribution

[0030] The results showed that in the multiribbed group, there were 15 individuals with the GG allele, 1 with the GA allele, and 0 with the AA allele; in the control group, there was 1 individual with the GG allele, 7 with the GA allele, and 6 with the AA allele. These allele frequencies were completely consistent with the GWAS output results, with the G allele showing a significantly higher frequency in the multiribbed group than in the control group. Two independent analysis methods, including population allele frequency statistics based on PLINK and direct genotype counting based on VCF individuals, yielded the same results, confirming that this association signal was not a systematic error caused by the analysis process, but rather reflects a genuine biological association, demonstrating strong robustness and reliability.

[0031] Example 3: Expanded Sample Validation 1. Population Sample A sample of 49 Licha Black Pigs (25 multi-ribbed pigs and 24 controls) was used for expanded validation of the SSC15:125,651,010 locus. 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.

[0032] The two sets of data were merged for analysis after standardized processing to ensure consistency in genotype interpretation. Fisher's exact test was used to test the significance of differences in allele frequencies at this locus in the 49 samples, and the results are shown in Table 3.

[0033] Table 3 Group Validation

[0034] The results showed that at the Chr1:161,373,061 locus in the expanded population of 49 samples, the frequency of the G allele in the multi-ribbed group (86.0%) was significantly higher than that in the control group (41.7%), with an allele frequency difference ΔAF = 44.3%. Fisher's exact test showed P < 0.0001, and the odds ratio (OR) was 8.60. The association direction was completely consistent with the 30-sample finding set. This indicates that the G / A polymorphism at the Chr1:161,373,061 locus is robust to the multi-ribbed trait in Lichai Black Pigs and has the reliability to serve as a molecular marker-assisted selection marker for the multi-ribbed trait in pigs.

[0035] Genotype distribution Genotypes were extracted from 49 pigs at the Chr1:161,373,061 locus. The genotypes are shown in Table 4.

[0036] Table 4 Genotype Distribution

[0037] The genotype distribution of the 49 samples further supports the association between this locus and the multiribbed trait. In the multiribbed group, the dominant genotype was GG homozygote, accounting for 84.0% (21 / 25) of multiribbed individuals, while AA homozygote accounted for only 12.0% (3 / 25). In contrast, in the control group, AA homozygote accounted for 45.8% (11 / 24), and GG homozygote accounted for only 29.2% (7 / 24), showing a clear separation in genotype distribution between the two groups.

[0038] Example 4: Selective Signal Verification To verify the genetic differentiation characteristics of the CCBE1 gene region, a quadruple selection signal analysis was performed on the multiribbed group and the control group. Specific procedures are shown in Table 5, and the results are as follows: Figure 5 .

[0039] Table 5. Results of Selected Signal Analysis

[0040] The results of quadruple selection signal analysis showed that the CCBE1 gene region (Chr1:161.3-161.6Mb) exhibited significant genetic differentiation between the multirib group and the control group (FST highest 0.3538), and this region was positively selected in the multirib group (XP-EHH=2.15, iHS=2.26), which was highly consistent with GWAS signals, further supporting the biological reliability of this marker.

[0041] Example 5: Application of Molecular Marker-Assisted Selection 1. KASP primer design Primers were designed to target the G / A polymorphism at Chr1:161,373,061 sites: Forward primer A (G allele specific): 5'-GCACCCCAAGCATTAGGACT-3' (e.g., SEQ ID NO: 1). Reverse primer B (A allele specific): 5'-GGTTGAAGTTGGGCGTTCAC-3' (e.g., SEQ ID NO: 2).

[0042] 2. Molecular marker-assisted selection process (1) Collect ear tissue or blood samples from piglets and extract genomic DNA; (2) Genotyping of Chr1 loci was performed using KASP primers; (3) Make selection decisions based on genotype results: - GG genotype: 93.8% probability of multiple ribs (30 samples), preferred for breeding; - GA genotype: moderate probability of multiple ribs, can be considered as an alternative; - AA genotype: The probability of multiple ribs is extremely low (0% in 30 samples), and elimination is recommended.

[0043] 3. Scope of application for varieties This patent mark applies to the Lichai Black Pig breed and hybrid populations with Lichai Black Pigs as parents.

[0044] Example 6 A method for selecting and / or assisting in the selection of multiribbed pigs using the SNP molecular markers on chromosome 1 obtained in Example 2 includes the following steps: (1) Extracting porcine genomic DNA; (2) Genotyping was performed on the 161,373,061st locus on chromosome 1 to determine whether the base at this locus was A or G, thereby determining whether the genotype of the pig to be tested was AA, GA or GG. (3) Judging the tendency of polyribs based on genotype: individuals with GG and GA genotypes have a higher probability of polyribs, while individuals with AA genotypes have a lower probability of polyribs.

[0045] Example 7 A method for breeding and / or assisting in the breeding of multiribbed pig breeds using the SNP molecular markers on chromosome 1 obtained in Example 2 includes the following steps: 1) Test the SNP molecular markers described above on replacement breeding pigs to determine whether the base at the site is A or G, thereby determining whether the genotype of the pig to be tested is AA, GA or GG; 2) Individuals homozygous for the G allele (GG) or heterozygous for the G allele (GA) were selected as breeding pig candidates; 3) Mating the boars and sows retained for breeding in step 2) and continuing to perform genotyping screening on their offspring to gradually increase the frequency of the G allele and cultivate a stable hereditary multi-ribbed pig breed.

[0046] Example 8 This embodiment provides a kit for detecting the SNP molecular markers described in Example 1. The kit includes the primer pairs described in Example 6.

Claims

1. A molecular marker for a porcine SNP on chromosome 1 associated with the multirib trait, characterized in that, The SNP molecular marker is located at base 161,373,061 on chromosome 1, reference genome Sscrofa11.1, chromosome number NC_010443.

5. This site has a G / A single nucleotide polymorphism, in which the G allele is a multirib dominant allele.

2. The application of the chromosome 1 SNP molecular marker as described in claim 1 in the selection and / or assisted selection of multiribbed pigs.

3. A method for breeding and / or assisting in the breeding of multi-ribbed pigs using the SNP molecular marker on chromosome 1 as described in claim 1, characterized in that, Includes the following steps: (1) Extracting porcine genomic DNA; (2) Genotyping was performed on the 161,373,061st locus on chromosome 1 to determine whether the base at this locus was A or G, thereby determining whether the genotype of the pig to be tested was AA, GA or GG. (3) Judging the tendency of polyribs based on genotype: individuals with GG and GA genotypes have a higher probability of polyribs, while individuals with AA genotypes have a lower probability of polyribs.

4. A method for breeding and / or assisting in the breeding of multi-ribbed pig breeds using the SNP molecular markers on chromosome 1 as described in claim 1, characterized in that, Includes the following steps: 1) Test the SNP molecular markers described above on replacement breeding pigs to determine whether the base at the site is A or G, thereby determining whether the genotype of the pig to be tested is AA, GA or GG; 2) Individuals homozygous for the G allele (GG) or heterozygous for the G allele (GA) were selected as breeding pig candidates; 3) Mating the boars and sows retained for breeding in step 2) and continuing to perform genotyping screening on their offspring to gradually increase the frequency of the G allele and cultivate a stable hereditary multi-ribbed pig breed.

5. A primer pair for identifying the chromosome 1 SNP molecular marker as described in claim 1, characterized in that, The primer pair is capable of specifically amplifying a DNA fragment containing nucleotide 161,373,061 of chromosome 1.

6. The primer pair according to claim 5, characterized in that, The upstream primer with the sequence shown in SEQ ID NO: 1 and the downstream primer with the sequence shown in SEQ ID NO:

2.

7. A kit for detecting the molecular marker of chromosome 1 SNP as described in claim 1, characterized in that, The kit contains primer pairs or primer pairs with the sequence shown in SEQ ID NO: 1 for the upstream primer and the sequence shown in SEQ ID NO: 2 for the downstream primer, which are capable of specifically amplifying a DNA fragment containing nucleotides at position 161,373,061 of porcine chromosome 1.

8. The use of a primer pair as described in any one of claims 5-6 or a kit as described in claim 7 in the selection and / or assisted selection of multi-ribbed pigs.

9. The application according to claim 2 or claim 8, characterized in that, The pigs mentioned are Lichai Black Pigs or hybrid groups with Lichai Black Pigs as parents.

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