Product for detecting SNP (Single Nucleotide Polymorphism) molecular marker related to porcine nipple number character and application of product

Abstract

The invention relates to a product for detecting a pig nipple number character related SNP molecular marker and application thereof, and the SNP molecular marker is located in a PRPF18 gene, and is located at a 48144674 nucleic acid site of a 10th chromosome of an international pig genome Sscrofa 11.1 version, A > G base mutation is located at the 48144674 nucleic acid site of the 10th chromosome of the international pig genome Sscrofa 11.1 version; the product at least comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is as shown in SEQ ID No.3, and the nucleotide sequence of the downstream primer is as shown in SEQ ID No.4. The product of the SNP molecular marker provided by the invention is used for breeding and breeding of multi-variety sows with high reproductive performance, and the breeding and breeding efficiency of the sows can be improved.

Description

Technical Field

[0001] This invention relates to an SNP molecular marker technology, specifically to products and applications for detecting SNP molecular markers related to the number of pig nipples. Background Technology

[0002] The number of teats in sows is a crucial indicator for assessing sow lactation performance. A low number of teats in actual production can lead to insufficient nutrient intake in some piglets during lactation, thus reducing piglet survival rates. A sufficient number of teats is essential to meet the increasing demands of sows for higher litter sizes, higher survival rates, and higher weaning rates. Improving the total number of teats in sows is not only an urgent breeding task but also closely related to the economic benefits of core pig farms. The teat number trait is typically an economically important trait with moderate heritability, regulated not only by major genes but also by many minor genes. Therefore, further research is needed to identify candidate genes and effector loci for the teat number trait before its full application in practical breeding. Identifying more key variant loci related to the total number of teats through genetic methods is a significant challenge in improving sow lactation performance and reproductive efficiency.

[0003] The number of teats in pigs is also an important trait closely related to economic traits such as body length and number of ribs. Sows with fewer ribs or shorter body lengths will have fewer teats at birth. In contrast, larger pig breeds with longer body lengths can accommodate more teats, resulting in stronger lactation performance and increased weaning piglet numbers and litter weight. In today's intensive farming environment, more teats mean lower labor costs and higher economic benefits. Therefore, the number of teats is one of the important economic traits worthy of attention from breeders, and exploring the genetic mechanisms of teat number using genomic resources is particularly important for subsequent molecular marker breeding of sows with high teat numbers.

[0004] Previous genome-wide association studies have identified several candidate genes associated with the porcine nipple number trait. For example, the VRTN gene was reported to be associated with the porcine nipple number trait as early as 2014. This gene is also associated with the number of ribs in porcines; some studies have shown that knocking out this gene in mice significantly reduces the number of ribs. However, no more detailed mechanistic analysis has been found regarding the nipple number trait. Because the functions of genes associated with the porcine nipple number trait are limited at the molecular level, key effector loci of the porcine nipple number trait need to be validated in real-world breeding data before being applied in breeding programs. This validation will demonstrate the effectiveness of the effector loci and provide a more comprehensive set of molecular marker-assisted target selections for the porcine nipple number trait.

[0005] CN119614720A reports a SNP marker related to the number and shape of teats in Large White pigs, located on the FRMD4A gene on chromosome 10, exhibiting C / T polymorphism. However, the validation in this Large White population was singular and small-scale, lacking replication validation in independent populations. Therefore, developing SNP markers significantly associated with teat number is of significant practical importance for early selection of sow reproductive traits, improving breeding efficiency, and promoting genetic progress in the pig industry. Especially in high-performing populations, this marker can identify individuals with higher genetic potential, thereby accelerating the accumulation and spread of superior genes and improving reproductive efficiency. With the development of genomic selection technology, the precise application of SNP markers can not only produce significant benefits in low-performing populations but also provide a stable genetic basis for the continuous improvement of high-yielding and high-quality populations, driving the livestock industry towards a more efficient and sustainable direction. Summary of the Invention

[0006] The first technical problem to be solved by this invention is to provide a product for detecting SNP molecular markers related to the number of pig nipples.

[0007] The second technical problem to be solved by the present invention is to provide a product for detecting SNP molecular markers related to the number of piglet teats in piglets and its application in the breeding of sows for the number of piglet teats at birth.

[0008] The third technical problem to be solved by this invention is to provide a product for detecting SNP molecular markers related to the number of pig nipples and its application in breeding sow breeds with high-yielding piglet nipple count.

[0009] The fourth technical problem to be solved by this invention is to provide a product for detecting SNP molecular markers related to the number of piglet teats in piglets and its application in improving the number of piglet teats in sow populations.

[0010] The technical solution adopted by the present invention to solve its first technical problem is a product for detecting SNP molecular markers related to the number of pig teats, wherein the SNP molecular marker is an A>G base mutation located at the 48144674 nucleic acid site on chromosome 10 of the international pig genome Sscrofa 11.1 within the PRPF18 gene.

[0011] Specifically, by detecting the genotype at locus 48144674 on chromosome 10, an individual's genetic potential can be directly determined. When this locus contains the G allele, it indicates the presence of a dominant genotype associated with a high number of nipples; when the locus contains the A allele, the number of nipples is significantly reduced. The detection product specifically identifies this mutation site, enabling early screening at the DNA level. Molecular detection based on this marker can accurately distinguish between GG, AG, and AA genotypes, providing a genetic basis for breeding decisions and avoiding reliance on later phenotypic observations.

[0012] Furthermore, the nucleotide sequence before the SNP molecular marker mutation is shown in SEQ ID No. 1; the nucleotide sequence after the mutation is shown in SEQ ID No. 2;

[0013] The product includes at least an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID No. 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 4.

[0014] The technical solution adopted by the present invention to solve its second technical problem is the application of products that detect SNP molecular markers related to the number of piglet teats in the selection of sows for the number of piglet teats at birth.

[0015] Furthermore, the sow's teat count trait includes one or a combination of traits such as total number of piglets, live number of piglets, or healthy number of piglets.

[0016] Furthermore, the method of application includes the following steps:

[0017] 1) Detect the SNP molecular markers in replacement breeding sows;

[0018] 2) Select individuals with the GG or AG genotype at the 48144674 nucleotide site on chromosome 10 of the international pig genome Sscrofa 11.1 version obtained in step 1) to effectively increase the number of teats in sows.

[0019] Among them, the SNP molecular marker refers to the A>G base mutation located at the 48144674th nucleic acid site on chromosome 10 within the PRPF18 gene. Specifically, it can be achieved by PCR amplification combined with sequencing or probe hybridization technology. This marker is genetically associated with the number of teats per litter of sows.

[0020] In some specific embodiments, during the selection of sow reproductive traits, genomic DNA is first extracted from the individuals to be tested. A detection product containing specific primer pairs is used to amplify the target SNP locus, and the allele genotype of the 48,144,674 locus is determined through genotyping. When the test result is a GG or AG genotype, it indicates that the individual carries the dominant allele G, which is associated with a high teat number trait, and such individuals are preferentially selected for the breeding population. Through continuous screening over multiple generations, the distribution frequency of the dominant allele in the population is gradually increased, thereby stably increasing the number of teats per litter at the genetic level.

[0021] The technical solution adopted by this invention to solve its third technical problem is the application of products that detect SNP molecular markers related to the number of pig nipples in the breeding of sow breeds with high-yielding piglet nipple count.

[0022] Furthermore, the method of application includes the following steps:

[0023] 1) Detect the SNP molecular markers in replacement breeding sows;

[0024] 2) Select individuals with the GG or AG genotype at the 48144674 nucleotide site on chromosome 10 of the international pig genome Sscrofa 11.1 version obtained in step 1) as breeding sows, and mate these breeding sows;

[0025] 3) Test the SNP molecular markers of the sows born from mating in step 2), retain individuals with the GG or AG genotype at the 48144674 nucleotide site, and cull individuals with the AA genotype at the 48144674 nucleotide site. Then breed them to cultivate a high-reproductive-performance sow breed with a high number of teats.

[0026] By detecting specific SNP sites within the PRPF18 gene, it is possible to directly determine whether sows carry the G allele associated with a high number of teats. During the detection process, specific primers are used to amplify the target region, and genotyping is performed using probe hybridization or sequencing technology to ensure accuracy. The screening step, based on dominant inheritance effects, retains individuals carrying at least one G allele, thereby increasing the frequency of dominant alleles at the population level. This method, by targeting key gene sites controlling embryonic development, avoids the interference of environmental factors on low-heritability traits in traditional breeding, achieving early and precise genotype-based selection.

[0027] The technical solution adopted by the present invention to solve its fourth technical problem is the application of products that detect SNP molecular markers related to the number of piglet teats in improving the number of piglet teats in sow herds.

[0028] Furthermore, the method of application includes the following steps:

[0029] The method of application includes the following steps:

[0030] 1) Detect the SNP molecular markers in replacement breeding sows;

[0031] 2) Select individuals with the GG or AG genotype at the 48144674 nucleotide site on chromosome 10 of the international pig genome Sscrofa 11.1 version obtained in step 1) as breeding sows, and mate these breeding sows;

[0032] 3) Test the SNP molecular markers of the sows born from mating in step 2), retain individuals with the GG or AG genotype at the 48144674 nucleotide locus, and cull individuals with the AA genotype at the 48144674 nucleotide locus. Breed these individuals to increase the frequency of the dominant allele G at the 48144674 nucleotide locus generation by generation, thereby improving the number of teats for farrowing piglets in the sow population and increasing the number of teats for farrowing piglets in the sow population.

[0033] Among them, the dominant allele G refers to the genotype that carries the G base at this SNP site. Specifically, individuals with the GG or AG genotype can be screened out through genotyping. The enrichment of this allele can promote the genetic improvement of the teat number trait in sow populations.

[0034] Among them, the population genetic improvement strategy refers to continuously screening individuals with superior genotypes over multiple generations. Specifically, it can be achieved by combining breeding with offspring genotype testing to gradually increase the frequency of superior alleles in the population.

[0035] Specifically, by detecting the genotype of this SNP molecular marker, replacement sows carrying the GG or AG genotype are preferentially selected for breeding. Genotype testing is continuously performed on the offspring, eliminating individuals carrying the AA genotype and retaining those with the dominant genotype for the next breeding cycle. Through the cyclical implementation of this strategy, the distribution frequency of the dominant allele G in the population shows a progressively increasing trend across generations, thereby achieving targeted improvement of the teat number trait in sows at the genetic level.

[0036] In this invention, the product refers to a reagent or kit. The reagent or kit amplifies the sequence containing the molecular genetic marker nucleotide site using primers, and then obtains its genotype through sequencing.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] This invention identifies molecular genetic markers associated with the number of teats in sows at birth, facilitating targeted improvement of this performance. Utilizing these molecular genetic markers, the performance of sows at birth can be efficiently assessed, improving the accuracy and convenience of testing. Through genotypic selection, these markers can optimize breeding strategies, improving the birth quality and quantity of piglets, providing technical support to core breeding farms, and contributing to more efficient and precise genetic improvement. This invention, through the discovery and application of molecular genetic markers, provides a novel method for improving the performance of sows at birth across multiple breeds, contributing to enhanced overall economic efficiency and scientific rigor in pig farming. Attached Figure Description

[0039] Figure 1 Manhattan plot for genome-wide association analysis of the number of teats in Large White pigs.

[0040] Figure 2 QQ plot for genome-wide association analysis of the number of teats in Large White pigs.

[0041] Figure 3 This is a linkage analysis diagram of SNP sites.

[0042] Figure 4 This is a violin diagram for population validation of SNP loci in Example 1.

[0043] Figure 5 This is a violin diagram for population validation of SNP loci in Example 3. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0045] Many specific details of the invention are set forth in the following description to enable those skilled in the art to implement and fully understand the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make various modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0047] Unless otherwise specified, the reagents and other materials used in the following examples are all commercially available products.

[0048] Example 1: Screening of SNP molecular markers related to the number of teats in piglets born to sows

[0049] 1. Material screening and phenotypic recording

[0050] We collected 1661 purebred Large White pigs from Hunan Xinwufeng Co., Ltd., including 1661 growth measurement records and 3139 pedigree records from 2022 to 2025. We also compiled the total teat count phenotype data, with a minimum of 12, a maximum of 20, a mean of 15.43, a standard deviation of 1.12, and a coefficient of variation of 7.24%. We also collected and compiled sow breed, farm-year-season, and sex information, using the total teat count phenotype as the input phenotype for genome-wide association analysis.

[0051] 2. SNP chip detection and genotyping

[0052] DNA was extracted from tissue samples using a magnetic bead method. The concentration of DNA samples was detected using a Qubit real-time fluorescence analyzer; the integrity of DNA samples was detected by 1% agarose gel electrophoresis, and samples that passed quality control were used for library preparation.

[0053] 2.2 SNP Chip Detection and Merging

[0054] SNP genotyping was performed on 1661 Large White pigs using the "HuaXin Pig Pro" solid-phase chip from Suzhou Lasso Biochip Technology Co., Ltd. This chip contains 73,715 SNP loci, integrating genetic differences from multiple pig breeds and covering the entire pig genome. The biochip system mainly consists of the chip, scanner, and analysis software. Genome Studio analysis software was used to visualize the chip data and obtain genotype data. Genotype file quality control was performed using Plink 1.9 software with the command "--geno 0.1 --maf 0.01 --hwe 1e-6", retaining 60,235 SNP loci for subsequent analysis.

[0055] 3. Genome-wide association analysis to locate target SNP loci.

[0056] 3.1 Genome-wide association analysis

[0057] Genome-wide association analysis (GWA) was performed on the material data from chip detection using GAPIT3 (Wang J and Zhang Z 2021). Since nipple number is a moderately heritable trait with a complex genetic regulatory mechanism, its genetic background is jointly regulated by major genes and minor genes. Conventional linear GWA models often underestimate the effects of key loci. Therefore, a Bayesian information model with a linkage disequilibrium iterative nested keyway model (BLINK) was used for GWA, and principal components were added as covariates to the model for correction. GWA was performed on the total nipple number phenotypic trait collected, and a Manhattan plot was generated (see [link to relevant documentation]). Figure 1 ) and QQ pictures (see Figure 2The significance threshold is calculated by taking the logarithm of the Bonferroni-corrected P-value (p=0.05 / N, where N is the number of SNPs) minus log10, and then calculating the significantly associated SNP sites.

[0058] 3.2 Discovering significant marker sites

[0059] Based on genome-wide association analysis and literature review, two significant marker loci were identified in the total nipple count trait. The top significant SNP locus is chr7_97614602, located only 105 bp downstream of the VRTN gene, which has been previously identified as a functional gene closely related to the number of pig nipples. The newly identified significant SNP locus in genome-wide association analysis is rs81333862, located at chr10_48144674 in the genome. This SNP locus falls within the PRPF18 gene (see [link to PRPF18 gene]). Figure 3 A search of the NCBI database revealed that the PRPF18 gene had the highest expression level in ovarian tissue. It is a gene closely related to reproduction, and studies have shown that PRPF18 is involved in meiosis in human and porcine oocytes. Furthermore, this gene regulates human skin pigmentation and the skin barrier, suggesting its involvement in epidermal differentiation, which is closely related to nipple formation. Approximately 95 kb upstream of this significant locus, the FRMD4A gene was annotated. This gene was previously detected in a genome-wide association study of the porcine nipple number trait and is considered a candidate gene for this trait. The GWAS results for SNP chr10_48144674, calculated using the BLINK statistical model, showed a p-value of 1.61 × 10⁻⁶. -7 The beta effect value was 0.16, and the direction was positive. Genotyping statistics were performed at this locus within this population. The average total number of teats in AA-type Large White sows was 15.08; the average total number of teats in AG-type Large White sows was 15.46; and the average total number of teats in GG-type Large White sows was 15.82 (see [link to relevant documentation]). Figure 4 Among them, the total number of teats in AA-type Large White sows was significantly different from that in AG-type sows (P=1.9×10). -9 The results also showed significant differences compared to the GG type (P=2.22×10⁻⁶). -16 There were also significant differences between the AG and GG types (P=6×10). -7 Therefore, the AG and GG genotypes are favorable alleles, and individuals with the mutant allele G have a significantly increased total number of nipples.

[0060] After obtaining significant SNP markers from genome-wide association analysis, 1000 bp sequences before and after the SNP were extracted (the nucleotide sequences are shown in SEQ ID No. 1). The reference genome was Sus Scrofa Build11.1. The nucleotide sequence before the mutation is shown in SEQ ID No. 1, and the nucleotide sequence after the mutation is shown in SEQ ID No. 2.

[0061] SNP nucleotide sequence:

[0062]

[0063] Example 2: Detection of SNP molecular markers related to the number of teats in sows at birth

[0064] This example demonstrates the design of upstream and downstream primers for detecting the A / G nucleotide site at 48,144,674 bp on chromosome 10 of the SNP locus (International Swine Genome Sscrofa version 11.1) obtained in Example 1. Sequencing primers were designed using BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ) and synthesized by Invitrogen for later material validation.

[0065] The sequencing primers are as follows:

[0066] Upstream primer: 5'- TGATTTGGGTTGAGGGAAG-3' (SEQ ID No. 3)

[0067] Downstream primer: 5'-TAGAATGTGGGTGAGCTGG-3' (SEQ ID No. 4)

[0068] 2. Large White pig genome extraction

[0069] 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.

[0070] 3. PCR amplification of SNP loci associated with the total number of teats in Large White pigs.

[0071] Using the extracted DNA as a template, PCR amplification was performed using the primers described above.

[0072] 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.

[0073] Amplification procedure:

[0074] Pre-denaturation at 95℃ for 15 minutes;

[0075] Denaturation at 95°C for 20 seconds; annealing at 58°C for 20 seconds; extension at 72°C for 20 seconds; repeat 40 cycles; extension at 72°C for 5 minutes.

[0076] After amplification, the temperature was maintained at 4°C to preserve the product and terminate the reaction.

[0077] 4. Sequencing and analysis of amplified products

[0078] The PCR products were detected by agarose gel electrophoresis, and the amplified products were then sequenced to obtain their sequences. The sequencing results of these amplified products were compared and analyzed with relevant gene fragments from Large White pigs in GenBank to determine whether the target SNPs related to the total number of nipples in this sequence contained genotypes AG and GG that increase the total number of nipples.

[0079] Example 3: Validation of SNP molecular markers

[0080] To further validate the identified SNP locus chr 10_48144674, which is significantly associated with the total number of teats, a large-scale validation was conducted using a population with no genetic exchange with the GWAS population. Ear tissue samples were collected from 1324 Large White sows at the Jiangxi Dajia Core Breeding Farm. The target SNP marker was detected, and the Wilcoxon test was used to compare the number of teats among different genotypes.

[0081] The results showed that three genotypes (AA, AG, and GG) existed at the chr 10_48144674 locus in a population of 1324 Large White pigs. The average total number of teats in AA-type Large White sows was 14.27; in AG-type Large White sows, it was 14.42; and in GG-type Large White sows, it was 14.48 (see [link to relevant documentation]). Figure 5 Among them, the total number of teats in AA-type Large White sows was significantly different from that in AG-type sows (P=0.0026) and also significantly different from that in GG-type sows (P=0.00047).

[0082] Example 4: A method for genetic improvement of sows with a high number of teats for high-yielding piglets.

[0083] The specific steps in this embodiment are as follows:

[0084] 1) SNP molecular marker detection related to the number of teats in litter was performed on replacement breeding pigs, genomic DNA was extracted, and the primers and methods described in Example 2 above were used for detection;

[0085] 2) Select individuals with the genotype GG or AG as breeding pigs, and mate the selected boars and sows;

[0086] 3) Perform SNP molecular marker testing on piglets born after mating, retain individuals with the genotype GG or AG, and cull individuals with the genotype AA, thereby cultivating a high-fertility, high-teacupuncture pig breed.

[0087] Furthermore, steps (2) and (3) can be repeated based on step (3) to select breeding pigs with genotypes GG or AG 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 teats for offspring pigs.

[0088] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A product for detecting SNP molecular markers related to the number of pig teats, characterized in that, The SNP molecular markers are in PRPF18 Within the gene, there is an A>G base mutation located at the nucleotide site 48144674 on chromosome 10 of the international pig genome Sscrofa version 11.

1.

2. The product according to claim 1, characterized in that, The product includes at least an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID No. 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No.

4.

3. The application of the product for detecting SNP molecular markers related to the number of pig teats as described in claim 1 or 2 in the breeding of sows with the number of teats trait.

4. The application according to claim 3, characterized in that, The method of application includes the following steps: 1) Detect the SNP molecular markers in replacement breeding sows; 2) Select individuals with the GG or AG genotype at the 48144674 nucleotide site on chromosome 10 of the international pig genome Sscrofa 11.1 version obtained in step 1) to effectively increase the number of teats in sows.

5. The application of the product for detecting SNP molecular markers related to the number of pig teats as described in claim 1 in the breeding of sow breeds with high teat counts.

6. The application according to claim 5, characterized in that, The method of application includes the following steps: 1) Detect the SNP molecular markers in replacement breeding sows; 2) Select individuals with the GG or AG genotype at the 48144674 nucleotide site on chromosome 10 of the international pig genome Sscrofa 11.1 version obtained in step 1) as breeding sows, and mate these breeding sows; 3) Test the SNP molecular markers of the sows born from mating in step 2), retain individuals with the GG or AG genotype at the 48144674 nucleotide site, and cull individuals with the AA genotype at the 48144674 nucleotide site. Then breed them to cultivate a high-reproductive-performance sow breed with a high number of teats.

7. The application of the product for detecting SNP molecular markers related to the number of pig teats as described in claim 1 in improving the number of teats in sow populations.

8. The application according to claim 7, characterized in that, The method of application includes the following steps: 1) Detect the SNP molecular markers in replacement breeding sows; 2) Select individuals with the GG or AG genotype at the 48144674 nucleotide site on chromosome 10 of the international pig genome Sscrofa 11.1 version obtained in step 1) as breeding sows, and mate these breeding sows; 3) Test the SNP molecular markers of the sows born from mating in step 2), retain individuals with the GG or AG genotype at the 48144674 nucleotide locus, and cull individuals with the AA genotype at the 48144674 nucleotide locus. Breed these individuals to increase the frequency of the dominant allele G at the 48144674 nucleotide locus generation by generation, thereby improving the number of teats for farrowing piglets in the sow population and increasing the number of teats for farrowing piglets in the sow population.

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