A SNP molecular marker associated with pig litter size and its application

Abstract

This invention relates to the field of molecular marker technology, and discloses a SNP molecular marker related to pig litter size and its application. The SNP molecular marker is located at a 34,977,540 bp site on chromosome 2 of the international pig reference genome Sscrofa 11.1 version. The nucleotide sequence of this site is G or A, and it is located within the exon region of the LUZP2 gene. This invention also discloses the application of this SNP molecular marker in the selection of breeding pigs for litter size, and proposes a method for selecting or assisting in the selection of high / low litter size breeding pigs based on this marker. This invention also provides specific primers for identifying this SNP site. By applying these technologies, this invention can achieve accurate prediction and efficient screening of Large White pig litter size, significantly improve the breeding efficiency of superior breeding pigs, and accelerate the process of herd improvement.

Description

Technical Field

[0001] This invention relates to the fields of molecular biotechnology and molecular marker technology, and in particular to a SNP molecular marker related to pig litter size and its application. Background Technology

[0002] The Large White pig is an important meat pig breed in the global livestock industry, possessing excellent characteristics such as rapid growth, high lean meat percentage, strong reproductive capacity, and wide adaptability. It has a high litter size, high piglet survival rate, good maternal instincts, and demonstrates strong adaptability to various climatic conditions. Large White meat has a fine texture and even fat distribution, and is often used for crossbreeding with other breeds to improve the growth performance and reproductive capacity of offspring, making it a key breed in pig breeding and meat production.

[0003] Litter size, as a core indicator of reproductive performance, directly impacts aquaculture efficiency. Molecular marker-assisted breeding (MMR) can precisely locate genes or genetic loci associated with high litter size, accelerating the screening and promotion of superior genotypes, shortening the breeding cycle, reducing breeding costs, and significantly improving reproductive efficiency. Therefore, conducting MMR based on litter size is of great significance. By identifying individuals with superior genetic traits in advance, not only can the productive performance of the population be improved, but genetic stability can also be enhanced, leading to a comprehensive improvement in aquaculture efficiency. Summary of the Invention

[0004] To address the aforementioned problems, this invention proposes a SNP molecular marker associated with pig litter size and its application. This marker can be used for the selection or assisted selection of high-yielding pigs. Through the application of this molecular marker, early screening for high-quality pig breeding can be achieved, effectively shortening the breeding cycle, improving selection efficiency, and promoting genetic improvement and performance optimization of the population.

[0005] To address the above problems, this application provides the following technical solution:

[0006] This study is the first to locate a single nucleotide polymorphism (SNP) molecular marker significantly associated with litter size in Large White pigs at 34,977,540 bp on chromosome 2 of the pig reference genome Sscrofa11.1. This site is located in the exon region of the LUZP2 gene, with a nucleotide sequence of either G or A. The discovery of this molecular marker provides potential candidate genes for improving the reproductive performance of Large White pigs.

[0007] The LUZP2 gene (Leucine Zipper Protein 2) encodes a protein synthesized during embryonic development and primarily expressed in the adult brain and spinal cord. Studies have shown that aberrant expression of the LUZP2 gene is closely associated with complex syndromes such as tumor formation, reproductive organ developmental abnormalities, and mental disorders. Based on its important role in maintaining bodily functions, this study identified the LUZP2 gene as a candidate gene affecting litter size in Large White pigs.

[0008] Identifying and utilizing this novel SNP molecular marker located in the exon region of the LUZP2 gene can assist in screening for high-producing Large White pig populations. Rapid assessment of individual genotypes helps optimize the reproductive potential of breeding pigs, significantly shortens the breeding cycle, improves the efficiency of genetic improvement, thereby creating higher economic benefits for the livestock industry and providing the market with high-quality pork products.

[0009] Furthermore, this study provides the sequence information for 100 bp upstream and downstream of this SNP marker (total length 201 bp), and its detailed nucleotide sequence is recorded in SEQ ID NO:1. The SNP site is located at base R at position 101 of the sequence, exhibiting a single-base mutation of A>G. The sequence polymorphism caused by this mutation provides a molecular basis for assessing pig farrowing performance and further promotes the improvement of pig breeding efficiency.

[0010] Secondly, this application also provides specific applications of the aforementioned SNP molecular markers related to pig litter size in breeding pigs. Based on the significant association between this marker and litter size, the genotype at this locus can be detected to analyze pig litter performance and be used for the selection and breeding of breeding pigs.

[0011] This molecular marker helps to achieve accurate assessment of reproductive performance in pig breeding, providing important technical support and theoretical basis for the breeding of Large White pigs.

[0012] Thirdly, this patent further discloses a method for pig herd screening and breed development using SNP molecular markers related to pig litter size traits, specifically including the following:

[0013] Screening and assisted breeding methods

[0014] (1) Detect the SNP molecular marker at the 34977540 bp site on chromosome 2 of the pig genome, determine whether the base at this site is G or A, and determine whether the genotype of the target pig is GG, AG or AA;

[0015] (2) Select individuals with the appropriate genotype according to the breeding objectives:

[0016] If the goal is to increase the number of piglets born, individuals with the genotype GG or GG and AG can be selected as breeding pigs, while individuals with the AA genotype can be culled.

[0017] If the goal is to select individuals with low litter size, then pigs with the AA genotype can be selected.

[0018] Methods for breeding high-livestock pig breeds

[0019] (1) Detect SNP molecular markers related to litter size in replacement breeding pigs to determine their genotype;

[0020] (2) Select boars and sows with genotypes GG or GA as breeding stock and mate them;

[0021] (3) Test the SNP genotype of piglets born after mating, retain individuals with genotype GG or GA, and eliminate individuals with AA type, thereby cultivating a high-fertility pig breed with high reproductive performance.

[0022] Optional operations:

[0023] The above method can be used to continuously select breeding pigs with genotypes GG or GA by repeating steps (2) and (3) over multiple generations, while eliminating individuals with AA genotype. As the number of breeding generations increases, the frequency of allele G in the pig herd gradually increases, further enhancing the reproductive capacity of offspring pigs.

[0024] This method enables efficient screening of breeding pigs and breeding of superior strains based on molecular markers, providing technical support and practical approaches for improving the reproductive performance of pigs.

[0025] Fourthly, this application also provides a primer pair and related kit for identifying the aforementioned SNP molecular markers related to pig litter size traits. The primer pair includes: an upstream primer 5'-AGTGAATCGACTGACAAGGGA-3' (SEQ ID NO: 2) and a downstream primer 5'-CCTTTCATGTGTTTCTCAGCCA-3' (SEQ ID NO: 3). Furthermore, this patent also provides a kit containing the above primer pair for rapid detection of the SNP molecular marker, facilitating the analysis and screening of target individuals related to pig reproductive performance.

[0026] Fifthly, this application also provides the application of the aforementioned primer pairs or the aforementioned kits in screening or identifying SNP molecular markers related to pig litter size traits and in breeding high-yielding pigs.

[0027] Sixthly, this application also provides a method for obtaining SNP molecular markers related to the total number of piglets born in Large White pigs, wherein the method is as follows:

[0028] (1) In order to eliminate the impact of possible problems during farrowing of primiparous sows on the total number of piglets born, the total number of piglets born in the second litter of the test group and related data were statistically analyzed, including various influencing factors such as number of litters, year, season, and age of farrowing, which were used as covariates; the phenotypic data were analyzed for quality control by removing outliers with a number of piglets born of 0.

[0029] (2) Collect pig tissue samples, extract genomic DNA and test its quality, perform genotyping and quality control on the genotyping data;

[0030] The quality control method is as follows: First, extract chromosome data from chromosomes 1 to 18; then filter out SNP sites with a deletion rate higher than 5%; subsequently filter out individuals with a deletion rate higher than 5%; remove sites with a minimum allelefrequency (MAF) less than 0.01; finally, perform a Hardy-Weinberg equilibrium (HWE) test on each SNP and remove sites with a p-value less than 10. -6 The SNP sites.

[0031] (3) Then, using Gemma software, the kinship matrix and the number of births, years, seasons, and maternal ages were used as covariates in genome-wide association analysis, with a p-value of <10. -4 This is the significance threshold.

[0032] The above method was used to screen for the SNP molecular markers related to the aforementioned pig litter size trait. The site is located at the 34977540bp site on chromosome 2 and is an A>G base mutation.

[0033] This invention has the following advantages and innovations:

[0034] 1. For the first time, a SNP locus (g. 34977540 A>G) significantly associated with litter size in Large White pigs was identified in the LUZP2 gene through genome-wide association analysis (GWAS). The study showed that the genotype at this locus was highly correlated with litter size, with the GG genotype resulting in the highest litter size, followed by the GA genotype, and the AA genotype the lowest. This locus can serve as a genetic marker to aid in marker-assisted breeding and genetic improvement of high litter size in Large White pigs.

[0035] 2. For the aforementioned SNP sites, this invention designs a molecular marker identification primer and kit, and develops an efficient molecular marker-assisted breeding method. This method allows for the rapid screening of breeding pigs with high reproductive performance, accelerating the breeding process and improving the reproductive capacity of offspring. This technology has significant economic value and application potential.

[0036] 3. Gene chip-based genotyping technology performs excellently in non-diagnostic evaluations. Compared with traditional PCR-RFLP technology, gene chip detection is simpler, more efficient, and has higher sensitivity and specificity, providing a convenient and reliable tool for improving the breeding efficiency of Large White pigs.

[0037] 4. This invention also establishes a GWAS-based SNP molecular marker screening method and further clarifies its relationship with the reproductive performance of Large White pigs through functional validation. The screened SNP sites are accurate and comprehensive, significantly improving the efficiency of genetic improvement and accelerating the improvement of reproductive performance in Large White pigs. Attached Figure Description

[0038] Figure 1 This is a Manhattan plot of genome-wide association analysis of the present invention. The red markers are the SNP molecular markers screened by the present invention, which are located on chromosome 2 of Large White pigs. Figure 2 QQ plot for genome-wide association analysis of litter size in Large White pigs; Figure 3 SNP density distribution map used in genome-wide association analysis of litter size in Large White pigs; Figure 4 PCA diagram of the genomes of 1000 Large White pigs; Figure 5 A distribution of total litter size trait among individuals with different genotypes related to litter size in Large White pigs. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0040] Example 1: Screening of SNP molecular markers related to litter size in Large White pigs

[0041] 1. Statistical analysis of the test population and phenotypic data

[0042] To eliminate the impact of potential problems during farrowing on the total number of piglets born, the total number of piglets born in the second litter and related data of the test population were statistically analyzed. A Large White pig breed was selected from a large domestic breeding farm, with a sample size of 1000 pigs. The number of piglets born was weighed and recorded immediately after birth, along with environmental factors (influencing factors) affecting the number of piglets, including farm number, year, season, and age at farrowing.

[0043] The phenotypic data underwent quality control analysis, and outliers with a litter size of 0 were removed, ultimately retaining 961 litter size records. The average litter size of the Large White pig population used in this embodiment was 13.27 ± 3.679 piglets, with a minimum of 2 and a maximum of 24 piglets.

[0044] 2. Data Acquisition and Quality Control

[0045] Large White pig tissue samples were collected for DNA extraction. The extracted DNA was genotyped using the "Zhongxin-1" 50K SNP chip for pigs, yielding approximately 56,000 SNP loci for subsequent genome-wide association studies (GWAS).

[0046] First, vcftools-0.1.16 was used to extract chromosome data from 1 to 18. Then, PLINK v1.9 was used for quality control of the genotype data. First, SNPs with a deletion rate higher than 5% were filtered out; then, individuals with a deletion rate higher than 5% were filtered out; sites with a minimum allele frequency (MAF) less than 0.01 were removed; finally, a Hardy-Weinberg equilibrium (HWE) test was performed on each SNP, eliminating sites with p-values ​​less than 10. -6 SNP sites.

[0047] 4. Genome-wide association analysis

[0048] This embodiment uses a mixed linear model for analysis, incorporating the population kinship matrix and significant influencing factors on litter size (including farm number, year, season, and age) as covariates to screen for SNP loci significantly associated with litter size in Large White pigs (results are shown in [link to results]). Figure 1 )

[0049] After analysis, a total of 6 SNP loci significantly associated with the number of piglets born in Large White pigs were obtained (see Table 1 for details). Based on the information of the pig reference genome (Sscrofa11.1 assembly), the LUZP2 gene was further located as a candidate gene.

[0050] Among them, CNC10020728 is a candidate marker related to litter size. This marker is located at 34977540 bp on chromosome 2 and is a mutation of adenine A to guanine G, represented as g. 34977540 A>G.

[0051] Table 1. SNP markers and candidate genes related to litter size in Large White pigs

[0052] Marker site chromosome Site information (bp) Alleles p-value Intralocutionary genes CNC10061439 6 68230725 G / A 1.11E-05 CAMTA1 CNCB10004570 6 69062870 A / G 2.71E-05 CNC10061440 6 68267328 T / C 3.24E-05 CAMTA1 CNC10032538 3 129637449 C / T 5.97E-05 CNC10020728 2 34977540 A / G 7.65E-05 LUZP2 CNCB10005022 6 150297695 C / T 9.33E-05 PATJ

[0053] 6. Application and Validation of Molecular Markers

[0054] To further analyze the relationship between different genotypes (AA, AG, GG) of the candidate marker locus g.34977540A>G and the number of piglets born in Large White pigs, analysis of variance (ANOVA) was used to assess the number of piglets born in the second litter of 961 Large White pigs, and the significance of differences among different genotypes was compared using the Tukey HSD test (see Table 2 for details). In addition, violin plots were created using the R package ggplot2 to visually display the distribution of piglets born under different genotypes, and significance markers were added to highlight the differences (as shown in Figure 5).

[0055] Table 2. Analysis of differences in litter size among different Large White pig genotypes with molecular marker g. 34977540 A>G

[0056] genotype Number of individuals Genotype frequency (%) Number of piglets born (heads) Multiple comparison results GG 247 41.31 14.1± 3.39 GG vs. AA: *** GA 445 46.99 13.2± 3.73 GG vs. GA: * AA 269 11.70 12.7± 3.73 GA vs. AA: *

[0057] Note: ** indicates that the difference in litter size between different genotypes is extremely significant (P ≤ 0.01); * indicates that the difference in litter size between different genotypes is significant (P ≤ 0.05).

[0058] Analysis showed that the marker site g.34977540 A>G was significantly associated with litter size in Large White pigs. Individuals with the GG genotype had a litter size of 14.1 ± 3.39 piglets, significantly higher than the GA genotype (13.2 ± 3.73 piglets), and extremely significantly higher than the AA genotype (12.7 ± 3.73 piglets). This site is located at 34977540 bp on chromosome 2, where adenine (A) is mutated to guanine (G), corresponding to nucleotide position 101 of SEQ ID NO:1. SEQ ID NO:1 provides nucleotide sequences of 100 bp upstream and downstream, totaling 201 bp. The A / G allele mutation at position 101 caused the polymorphism in this sequence. As a SNP molecular marker closely related to the total number of piglets born in Large White pigs, CNC10020728 can be used for marker-assisted selection. Individuals with the GG genotype are suitable as preferred candidates for further selection or breeding to improve the reproductive performance of Large White pigs.

[0059] Example 2: Design of primers for identifying SNP molecular markers related to litter size in Large White pigs and their application.

[0060] This example demonstrates the design of upstream and downstream primers for detecting the G / A nucleic acid site at 34977540 bp on chromosome 2, the SNP site obtained in Example 1.

[0061] 1. Primer design for the target gene sequence

[0062] Based on the target gene sequence, optimized amplification primers were designed using primer design software. The primer pair sequences are as follows:

[0063] Upstream primer: 5'- AGTGAATCGACTGACAAGGGA-3' (e.g., SEQ ID NO: 2)

[0064] Downstream primer: 5'- CCTTTCATGTGTTTCTCAGCCA-3' (e.g., SEQ ID NO: 3)

[0065] 2. Large White pig genome extraction

[0066] Tissue samples were collected from the Large White pigs to be tested, genomic DNA was extracted, and after quality and concentration testing, the samples were stored at -20℃ for later use.

[0067] 3. PCR amplification of SNP loci associated with litter size in Large White pigs

[0068] Using the extracted DNA as a template, PCR amplification was performed using the primers described above.

[0069] Amplification system: 10 μL of 2× SuperReal PreMix Plus, 8 μL of ddH2O, 0.5 μL each of upstream and downstream primers (10 μM concentration), and 1 μL of cDNA template.

[0070] Amplification procedure:

[0071] Pre-denaturation at 95°C for 15 minutes;

[0072] Cure at 95°C for 20 seconds; anneal at 58°C for 20 seconds; extend at 72°C for 20 seconds; repeat 40 cycles.

[0073] Finally, extend the heat to 72°C for 5 minutes.

[0074] After amplification, the temperature was maintained at 4°C to preserve the product and terminate the reaction.

[0075] 4. Sequencing and analysis of amplified products

[0076] The PCR products were detected by agarose gel electrophoresis, and the amplified products were then sequenced. The sequence of the amplified product was obtained, as shown in SEQ ID NO: 3. The sequencing results of the amplified product were compared and analyzed with the relevant gene fragments of Large White pigs in GenBank (as shown in SEQ ID NO: 1) to determine whether the genotype of the target SNP locus related to litter size was GG, AG, or AA.

[0077] Based on the above results, select Large White pigs with different litter sizes. If selecting Large White pigs with high litter sizes, choose Large White pigs with the GG and AG genotypes; if selecting Large White pigs with low litter sizes, choose Large White pigs with the AA genotype.

[0078] Those skilled in the art can design other primers for amplifying the molecular genetic marker or probes for identifying the molecular genetic marker based on the molecular genetic marker described above, thereby enabling the detection of the genetic marker. For example, the molecular genetic marker can be obtained by PCR amplification, followed by cloning and sequencing to obtain the corresponding sequence, or detected by Bsm-RFLP polymorphism. Therefore, the present invention also includes primers for amplifying the molecular genetic marker or probes for identifying the molecular genetic marker, and kits containing the primers or probes.

[0079] Example 3: Application of the molecular genetic markers of this application in Large White pig breeding

[0080] This embodiment provides a method for applying the molecular genetic markers of this application to Large White pig breeding or assisted breeding, the specific steps of which are as follows:

[0081] 1) Extract genomic DNA from Large White pigs and detect the deoxynucleotide at position 34977540 on chromosome 2 to determine whether the deoxynucleotide at this position is A or G;

[0082] 2) Based on the genotype of the locus (AA, AG or GG), select the genotype that is suitable for the breeding needs for the next step of selection and / or breeding.

[0083] Specifically, if the goal is to breed high-producing Large White pigs, AA-type Large White sows are culled while GG-type Large White sows are retained to increase the frequency of the G allele across generations. Studies have shown that GG-type Large White pigs have a higher total litter size than AA-type sows, with each GG-type Large White pig producing 1.4 more piglets than the AA-type. Assuming 10,000 breeding Large White sows, each litter producing 14,000 piglets, these piglets, after fattening and assuming an 85% slaughter rate, could provide an additional 1,190 tons of Large White pork. Therefore, the molecular genetic markers proposed in this application have promising application prospects and industrial value in Large White pig breeding.

[0084] Example 4: A method for genetic improvement of Large White pigs with high litter size.

[0085] This embodiment provides a method for genetic improvement of Large White pigs with a high litter size trait. The specific steps are as follows:

[0086] 1) SNP molecular marker detection related to litter size trait was performed on replacement breeding pigs, genomic DNA was extracted, and the primers and methods described in Example 2 above were used for detection;

[0087] 2) Select individuals with the genotype GG or GA as breeding pigs, and mate the selected boars and sows;

[0088] 3) Perform SNP molecular marker testing on piglets born after mating, retain individuals with genotype GG or GA, and cull individuals with genotype AA, thereby cultivating a high-fertility pig breed with high reproductive performance.

[0089] Furthermore, steps (2) and (3) can be repeated based on step (3) to select breeding pigs with genotypes GG or GA generation by generation, eliminate individuals with genotype AA, and gradually increase the frequency of the allele G at this SNP locus in the pig population, thereby increasing the number of piglets born in the offspring.

[0090] This invention can be replaced or modified according to the technical solution, and all such modifications or replacements should fall within the protection scope of this invention.

Claims

1. The application of a pig litter size-related SNP molecular marker in the selection of high-yielding pig breeds, characterized in that, The SNP molecular marker is located at position r at the 101st position of the sequence shown in SEQ ID NO:1, with an A>G single base mutation. Individuals with the SNP molecular marker GG genotype have a significantly higher litter size than those with the GA and AA genotypes.

2. A method for breeding high-producing piglets using SNP molecular markers related to litter size as described in claim 1, characterized in that, Includes the following steps: 1) Detect the SNP molecular marker as described in claim 1, determine whether the base at the site is G or A, and thus determine whether the genotype of the Large White pig to be tested is GG, AG or AA; 2) Select individuals with genotype GG or AG as breeding pigs, and cull individuals with genotype AA.

3. The application of the pig litter size-related SNP molecular markers as described in claim 1 in the breeding of high-livestock-size pig breeds, characterized in that, The application includes the following steps: (1) Detection of the SNP molecular markers as described in claim 1 in replacement breeding pigs; (2) Select individuals with the genotype GG or GA obtained in step (1) and use them as breeding pigs, and mate the breeding boars and sows. (3) Test the SNP molecular markers as described in claim 1 on the piglets born from mating in step (2), retain individuals with genotype GG or GA, eliminate individuals with genotype AA, and cultivate a high-fertility pig breed with a high number of piglets.

4. The application according to claim 3, characterized in that, The application also includes: repeating the operations of steps (2) to (3) with the individuals bred in step (3) to select individuals with genotype GG or AG in the offspring and eliminate individuals with genotype AA, so as to increase the frequency of allele G in the offspring pig population generation by generation and increase the number of piglets born in the offspring pigs.

5. The application of a primer pair for identifying SNP molecular markers related to pig litter size as described in claim 1 in the breeding of high-yielding piglets, characterized in that, The upstream primer sequence of the primer pair is shown in SEQ ID NO: 2, and the downstream primer sequence is shown in SEQ ID NO:

3. The SNP molecular marker is located at position r at the 101st position of the sequence shown in SEQ ID NO: 1, with an A>G single base mutation. The litter size of individuals with the SNP molecular marker GG genotype is significantly higher than that of individuals with the GA and AA genotypes.

6. The application of a kit for identifying SNP molecular markers related to pig litter size as described in claim 1 in the breeding of high-producing piglets, characterized in that, The kit contains primer pairs, the upstream primer sequence of which is shown in SEQ ID NO: 2 and the downstream primer sequence of which is shown in SEQ ID NO:

3. The SNP molecular marker is located at position r at the 101st position of the sequence shown in SEQ ID NO: 1, with an A>G single base mutation. Individuals with the SNP molecular marker GG genotype have significantly higher litter numbers than those with the GA and AA genotypes.

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