A SNP molecular marker related to pig birth weight and its application

Abstract

The present invention belongs to the field of molecular biotechnology and molecular marker technology, and discloses a SNP molecular marker related to pig birth weight and its application. The SNP molecular marker related to pig birth weight corresponds to the nucleic acid site at 5379864 bp on chromosome 16 of the pig reference genome Sscrofa11.1 version sequence. The base of the site is C or T and is contained in the intron of the MARCHF11 gene. The present invention also discloses the application of the SNP molecular marker related to pig birth weight in the genetic breeding of Landrace pigs, and provides a method for selecting or assisting in the selection of pigs with high / low birth weight using the SNP molecular marker and a primer for identifying the SNP site. The above method can achieve accurate prediction and efficient selection of Landrace pig birth weight, promote the cultivation of excellent breeding pigs, accelerate the population improvement process, and provide strong support for the sustainable development of the pig farming industry.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology and molecular marker technology, and in particular to a SNP molecular marker associated with pig birth weight and an application thereof. Background Art

[0002] The Landrace pig, a widely used lean meat breed, holds a key position in the global swine industry due to its excellent growth performance, high lean meat percentage, and adaptability. Birth weight is a crucial trait affecting individual growth, development, production performance, and health, directly impacting the economic benefits of pig farming. Low birth weight leads to slow early growth, frequent health problems, and low feed conversion rates in piglets, ultimately impacting market weight and slaughter rate. Therefore, molecular marker-assisted breeding based on birth weight is crucial for improving birth weight and optimizing production performance in the Landrace pig population.

[0003] However, no effective SNP molecular markers related to birth weight of Landrace pigs have been found so far, so the existing technology needs to be further improved. Summary of the Invention

[0004] In response to the above problems, the present invention provides a SNP molecular marker related to pig birth weight and its application. The SNP molecular marker related to pig birth weight can be used in the selection or auxiliary selection of pigs with high / low birth weight, and can be used for the early selection of high-quality pig breeding, shortening the breeding cycle, and accelerating the population improvement process.

[0005] To solve the above problems, this application provides the following technical solutions:

[0006] In the first aspect, the present application provides a SNP molecular marker related to pig birth weight, the SNP site corresponds to the nucleic acid site at 5379864 bp on chromosome 16 of the pig reference genome Sscrofa11.1 version sequence, the base of the site is C or T, and is contained in the intron of the MARCHF11 gene.

[0007] As a member of the MARCH family, the MARCHF11 (Membrane Associated Ring-CH-Type Finger 11) gene plays multiple roles in cell growth, differentiation, apoptosis, and immune regulation. MARCHF11 encodes a protein with E3 ubiquitin ligase activity, which can attach ubiquitin tags to specific target proteins, thereby regulating the degradation, localization, and signal transduction of target proteins. The ubiquitination process plays an indispensable role in key biological processes such as early embryonic cell proliferation and differentiation, and may play a potential role in birth weight and embryonic development. This is also consistent with the above-mentioned SNP sites related to pig birth weight within the MARCHF11 gene screened in this application.

[0008] In marker-assisted selection (MAS), molecular markers improve breeding accuracy and efficiency by identifying genetic variants closely associated with target traits. Molecular markers based on the MARCHF11 gene can assist in the screening of Landrace pig populations with higher birth weights, optimizing the birth weight and growth performance potential of breeding pigs through rapid assessment of individual genotypes. The application of this molecular marker can significantly shorten the breeding cycle, increase the rate of genetic improvement, and ultimately bring higher economic benefits and a higher-quality supply of pork products to the breeding industry. Therefore, the MARCHF11 gene has high application potential in Landrace pig birth weight, and its development and application as a molecular marker is expected to provide a new approach for Landrace pig breeding.

[0009] This application is the first to identify a single nucleotide polymorphism (SNP) (g. 5379864 C>T) in the MARCHF11 gene that is significantly associated with birth weight in Landrace pigs through genome-wide association analysis. This SNP can be applied to genetic improvement of growth and reproductive traits in Landrace pigs, providing a new genetic marker resource for marker-assisted selection of Landrace birth weight. The identified SNP associated with birth weight can be used as a molecular marker for selection of superior traits in Landrace pigs, helping to accelerate the genetic improvement of production performance in Landrace pigs.

[0010] In a second aspect, the present application also provides the application of the above-mentioned pig birth weight-related SNP molecular markers in pig genetic breeding.

[0011] Specifically, the upstream and downstream nucleotide sequences of the SNP molecular marker related to pig birth weight are shown in SEQ ID NO: 1. The SNP site is the 32nd base of the sequence. The single nucleotide at this site is a T base mutation to a C, which leads to differences in the birth weight of Landrace pigs.

[0012] The aforementioned SNP molecular markers associated with pig birth weight can be used for the selection or auxiliary selection of breeding pigs with high / low birth weight, which will help accelerate the progress of genetic improvement of the production performance of Landrace pigs.

[0013] In a third aspect, the present application further provides a method for breeding or assisting in breeding pigs with high / low birth weight using the above-mentioned pig birth weight-related SNP molecular markers, which comprises the following steps:

[0014] The aforementioned pig birth weight-related SNP molecular marker at 5379864 bp on chromosome 16 of the pig genome is detected. The base at this site is C or T, thereby determining whether the genotype of the pig to be tested is CC, CT or TT. Pigs with TT genotype or CC genotype are then selected for the next step of breeding and / or breeding according to production needs.

[0015] Optionally, in the method of using the aforementioned pig birth weight-related SNP molecular markers to select or assist in the selection of pigs with high / low birth weight, if high birth weight pigs are selected, CC genotype Landrace pigs are selected; if low birth weight pigs are selected, TT genotype pigs are selected.

[0016] The birth weight of the TT genotype Landrace pig is higher than that of the CT genotype Landrace pig, and the birth weight of the CT genotype Landrace pig is higher than that of the CC genotype Landrace pig.

[0017] Optionally, in the application of the above-mentioned SNP molecular marker in pig genetic breeding or in the method of using the above-mentioned SNP molecular marker in breeding or assisting breeding of pigs with high / low birth weight, the pig is a Landrace pig.

[0018] In a fourth aspect, the present application further provides a primer pair for the aforementioned pig birth weight-related SNP molecular marker, the primer pair comprising: an upstream primer F1 having a sequence as shown in SEQ ID NO: 2 and a downstream primer R1 having a sequence as shown in SEQ ID NO: 3.

[0019] In a fifth aspect, the present application also provides the use of the above primer pairs in the selection or auxiliary selection of pigs with high / low birth weight.

[0020] In a sixth aspect, the present application also provides a kit for detecting the above-mentioned SNP molecular markers, which contains the above-mentioned primer pair.

[0021] In a seventh aspect, the present application further provides an application of the above-mentioned SNP molecular marker in breeding high birth weight pig strains, the application comprising the following steps:

[0022] (1) Detecting the aforementioned SNP molecular markers related to pig birth weight in reserve breeding pigs;

[0023] (2) Selecting individuals with the TT genotype detected in step (1) as breeding pigs, and mating the breeding boars and sows;

[0024] (3) The piglets born from mating in step (2) are tested for the aforementioned SNP molecular markers, individuals with the genotype of the site being TT are retained, and individuals with the genotype being CC are eliminated, thereby breeding a pig breed with a high birth weight.

[0025] Optionally, the application further comprises: repeating the operations of steps (2) to (3) with the individuals bred in step (3), thereby selecting breeding pig individuals with a molecular marker genotype of TT in the offspring and eliminating individuals with a CC genotype, so as to increase the frequency of the allele T in the offspring pig population generation by generation and increase the birth weight of the offspring pigs.

[0026] In an eighth aspect, the present application further provides a method for screening SNP molecular markers associated with pig birth weight, comprising the following steps:

[0027] (1) Record and count the birth weight of each individual in the pig population, including various influencing factors (such as farm, year, season, parity, sex, etc.), and use the least square mean (LSM) hypothesis test and multiple comparison method to test whether the influencing factors reach a significant level (P ≤ 0.05);

[0028] (2) Collect pig tissue or blood samples, extract genomic DNA and test its quality, and perform genotyping using the pig "SMIC No. 1" 50K chip; then perform quality control on the genotyping data, filter out SNP sites that do not meet the requirements, and exclude individuals with a sample detection rate of less than 95% from the sample;

[0029] (3) Fixed and random model circulating probability unification (FarmCPU) was used to perform genome-wide association analysis to screen SNPs, key candidate genes, and molecular markers associated with pig birth weight.

[0030] Optionally, in the method for screening SNP molecular markers associated with pig birth weight, the SNP sites that do not meet the requirements are: SNP sites with a call rate (CR) less than 95%, a minimum gene frequency (MAF) less than 0.05, and no map information.

[0031] Preferably, the pig is a Landrace pig.

[0032] The present invention has the following beneficial effects:

[0033] 1. This study, for the first time, discovered a locus in the MARCHF11 gene associated with birth weight in Landrace pigs, providing an effective candidate gene for improving the pig's superior growth and reproductive traits. Furthermore, using genome-wide association analysis, the present invention identified a single nucleotide polymorphism (SNP) (g.5379864 T>C) in the MARCHF11 gene that is significantly associated with birth weight in Landrace pigs. It was found that Landrace pigs with the TT genotype at this locus have higher birth weight than those with the CT genotype, which in turn has higher birth weight than those with the CC genotype. Therefore, this locus can be applied to genetically improve growth and reproductive traits in Landrace pigs, providing a new genetic marker resource for molecular marker-assisted selection of Landrace pigs for birth weight.

[0034] 2. Based on the discovery of the aforementioned SNP molecular marker associated with Landrace birth weight, this application also provides a method for utilizing this SNP molecular marker associated with Landrace birth weight to select or assist in the breeding of Landrace pigs with high or low birth weights, thereby accelerating the genetic improvement of Landrace pig production performance. This method aims to achieve accurate prediction and efficient selection of Landrace pig birth weight, enable early selection of high-quality breeding pigs, promote the cultivation of superior breeding pigs, shorten the breeding cycle, and provide strong support for the sustainable development of the pig farming industry.

[0035] 3. The invention also provides a method for screening candidate markers and genes associated with birth weight in Landrace pigs using a genome-wide association analysis approach. The SNPs identified in this screening can be used as molecular markers for selecting for superior traits in Landrace pigs, helping to accelerate the genetic improvement of production performance. This screening method is characterized by its ease of use, comprehensive coverage, and high accuracy of selected markers, and also provides technical support for the genetic analysis of other important economic traits in livestock and poultry. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 To screen the genome-wide association analysis Manhattan plot of MARCHF11 gene and SNP sites associated with birth weight in Landrace pigs;

[0037] Figure 2 This is the QQ plot of the genome-wide association analysis of Landrace pig birth weight;

[0038] Figure 3 Genomic distribution map of SNP loci used in the whole-gene association analysis of birth weight in Landrace pigs;

[0039] Figure 4 This is the principal component analysis (PCA) diagram of the Landrace pig test group;

[0040] Figure 5 Analysis of birth weight differences among different genotypes of the SNP molecular marker g. 5379864 T>C (CNC10160101). DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work 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 this field. The methods in the following embodiments, unless otherwise specified, are conventional methods in this field.

[0042] Example 1 Screening Method for Candidate Genes and SNP Molecular Markers Related to Birth Weight of Landrace Pigs

[0043] 1. Statistical analysis of the test population and phenotypic data

[0044] A large domestic breeding farm of Landrace pigs was selected for testing, with a sample size of 1,145 pigs. The birth weight of Landrace pigs was weighed and recorded immediately after birth. Environmental factors (influencing factors) affecting birth weight, including farm number, year, season, parity, and sex, were also recorded. Quality control analysis of phenotypic data was performed, and the mean ± 2 was excluded. 1134 birth weight records were retained as abnormal values ​​outside the standard. The average birth weight of the Landrace pig population used in this example was 1.42 ± 0.19 kg, with a minimum of 1.00 kg and a maximum of 2.20 kg.

[0045] 2. Statistical Analysis of Factors Influencing Birth Weight

[0046] Least squares mean (LSM) hypothesis testing and multiple comparison analysis (Tukey's HSD) were used to assess the significance of various influencing factors on birth weight. Fit correction was performed using the R package emmeans. Factors influencing each trait were included in the linear model and subsequent genome-wide association analysis only if they reached a significant level (P ≤ 0.05). In this example, the environmental factors analyzed to have a significant impact on Landrace pig birth weight included farm, year, season, parity, and sex (as shown in Table 1).

[0047] Table 1 Results of least squares means multiple comparisons of factors affecting birth weight traits in Landrace pigs

[0048] Traits / Factors Sessions years season Parity gender Birth weight (Kg) 0.0037** 0.0001*** 7.23E-05*** 0.0015** 0.0001***

[0049] Note: *** indicates P ≤ 0.001; ** indicates 0.001 ≤ P ≤ 0.01; * indicates 0.01 ≤ P ≤ 0.05.

[0050] 3. Sample Collection and DNA Extraction

[0051] Landrace pig ear tissue samples were collected and immediately stored at -80°C until subsequent DNA extraction. Genomic DNA was extracted using a genomic DNA tissue extraction kit strictly following the manufacturer's instructions. DNA purity was determined using a NanoDrop 2000 UV spectrophotometer, ensuring an OD260 / OD280 ratio between 1.8 and 2.0. DNA integrity was assessed by 1% agarose gel electrophoresis to ensure that the DNA quality met the requirements for subsequent microarray analysis.

[0052] 4. Genotyping and Quality Control

[0053] Genotyping was performed using the "SMIC-1" porcine 50K SNP chip, covering approximately 56,000 SNPs across the genome for subsequent genome-wide association studies (GWAS). Following steps such as DNA amplification, fragmentation, resuspension, chip hybridization, chip cleaning, single-base extension, and chip scanning, the results were imported into signal recognition software for visualization, ultimately generating genotyping data for each sample.

[0054] Genotype data were quality controlled using PLINK v1.9, eliminating SNP sites with a detection rate below 95%, SNP sites with a minimum gene frequency below 0.01, and SNP sites without map information. Quality control was also performed on individual samples, eliminating individuals with a genotype detection rate below 95%. Finally, a total of 46,829 high-quality SNP sites and 1,145 samples passed quality control (e.g., Figure 3 shown).

[0055] 5. Genome-wide Association Analysis

[0056] This embodiment adopts the fixed and random model cyclic probability unified method (FarmCPU) to analyze the population principal component analysis results (PCA=3, such as Figure 4 As shown in the figure, significant factors affecting birth weight, including farm number, year, season, parity, and sex, were added as fixed effects into the genome-wide association analysis model to screen SNP sites significantly associated with birth weight in Landrace pigs (such as Figure 1 、 2 Bonferroni multiple test correction was performed on the candidate SNPs, and P < 0.05 / the number of effective SNPs was set as the significance threshold to ensure the reliability of the analysis results.

[0057] After testing, a total of six SNPs were found to be significantly associated with birth weight in Landrace pigs (as shown in Table 2). Based on the porcine reference genome (Sscrofa version 11.1), the MARCHF11 gene was further mapped as a candidate gene. Among them, CNC10160101 is a candidate marker associated with birth weight. This marker is located at 5379864 bp on chromosome 16 and has a mutation from cytosine C to thymine T, represented by g. 5379864 T>C.

[0058] Table 2 SNP markers and candidate genes associated with birth weight in Landrace pigs

[0059] Marker site chromosome Site information (bp) Allele P-value Genes within the locus CNC10031242 3 61739022 A / G 1.29E-07 -- CNC10032309 3 117474270 T / G 7.51E-07 -- CNC10060592 6 28761105 T / A 2.44E-09 -- CNC10082703 8 133752515 C / T 1.60E-07 -- CNC10160101 16 5379864 T / C 7.56E-07 MARCHF11

[0060] 6. Application and Validation of Molecular Markers

[0061] The candidate marker site g. 5379864 T>C was screened and the corresponding genotypes (CC, CT, TT) were compared with the birth weight of Landrace pigs through analysis of variance (ANOVA). Tukey HSD test was further used to compare the significance of the differences between different genotypes (as shown in Table 3). The R package ggplot2 was used to draw violin plots and add significant markers (as shown in Table 3). Figure 5 shown).

[0062] Table 3 Analysis of birth weight differences among Landrace pigs with different genotypes of molecular marker g. 5379864 T>C

[0063] genotype Number of individuals Genotype frequency (%) Birth weight (Kg) Multiple comparison results TT 473 41.31 1.39±0.17 TT vs. CC: ** CT 538 46.99 1.42±0.21 CT vs. CC: ** CC 134 11.70 1.48±0.20 CT vs. CC: *

[0064] Note: ** indicates extremely significant differences in birth weights between different genotypes (P ≤ 0.01); * indicates significant differences in birth weights between different genotypes (P ≤ 0.05).

[0065] Analysis results showed that at the marker locus g. 5379864 T>C, the birth weight of Landrace pigs with the TT genotype was significantly higher (1.48±0.20 kg) than that of the CT genotype (1.42±0.21 kg) and extremely significantly higher than that of the CC genotype (1.39±0.17 kg). Therefore, this molecular marker g. 5379864 T>C could be used for marker-assisted selection of Landrace pig birth weight, with individuals with the TT genotype being selected for further selection or breeding to improve the birth weight trait of Landrace pigs.

[0066] Example 2 Application of SNP Molecular Markers Associated with Landrace Pig Birth Weight in Breeding Landrace Pigs with High / Low Birth Weight

[0067] This example is the design of upstream and downstream primers and amplification results for the detection of the C / T at the SNP site 5379864 bp on chromosome 16 obtained in Example 1.

[0068] 1. Primer design for target gene sequence

[0069] According to the target gene sequence, the optimized amplification primers were designed using primer design software. The primer pair sequences are as follows:

[0070] Upstream primer: 5'-ACAGTCACCTTGGGGTTTCC-3' (as shown in SEQ ID NO: 2)

[0071] Downstream primer: 5'-AGAGGTTTCCCTTCCCCCTT-3' (as shown in SEQ ID NO: 3)

[0072] 2. Landrace Pig Genome Extraction

[0073] Collect ear tissue samples or blood samples from Landrace pigs to be tested, extract genomic DNA, and store at -20℃ after quality and concentration testing.

[0074] 3. PCR amplification of SNP sites associated with birth weight in Landrace pigs

[0075] In the experiment, DNA samples of Landrace pig blood genes were mixed to prepare a mixed pool DNA template, and PCR amplification was performed using the above primers.

[0076] PCR amplification system (total volume 20.0 μL):

[0077] 10.0 μL of 2xTaq PCR Master Mix, 0.5 μL of upstream primer (10 μmol / L), 0.5 μL of downstream primer (10 μmol / L), 1.0 μL of genomic DNA template, and 8.0 μL of ddH2O.

[0078] The PCR amplification procedure is:

[0079] Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, for a total of 30 cycles; extension at 72°C for 5 min.

[0080] The PCR products were detected by 1.5% agarose gel electrophoresis and then subjected to first-generation Sanger sequencing.

[0081] 4. Sequencing and analysis of amplified products

[0082] The PCR product was analyzed by agarose gel electrophoresis, revealing a 113 bp target fragment (shown in SEQ ID NO: 6). The product was sequenced and compared with relevant Landrace pig gene fragments from GenBank (SEQ ID NO: 4 for the TT genotype and SEQ ID NO: 5 for the CC genotype) to determine whether the genotype of the target SNP associated with birth weight in this sequence was CC, CT, or TT.

[0083] Based on the above results, high / low birth weight Landrace pigs were selected. If high birth weight Landrace pigs were selected, Landrace pigs with the TT genotype were selected; if low birth weight Landrace pigs were selected, Landrace pigs with the CC genotype were selected.

[0084] Example 3

[0085] Those skilled in the art can readily refer to other primers designed based on the aforementioned SNP sites of the present invention or probes for identifying the molecular markers to detect the genetic markers, for example, by amplifying the molecular genetic markers by PCR and then cloning and sequencing to obtain the corresponding sequence, or by detecting RFLP polymorphisms. Therefore, the present invention also includes other primers for amplifying the molecular genetic markers or probes for identifying the molecular genetic markers, as well as kits containing the primers or probes.

[0086] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solutions and concepts of the present invention, and all these changes or substitutions should fall within the scope of protection of the claims attached to the present invention.

Claims

1. Application of a pig birth weight-related SNP molecular marker in the genetic breeding of Landrace pig birth weight traits, characterized in that: The site of the SNP molecular marker corresponds to the nucleic acid site at 5379864 bp on chromosome 16 of the pig reference genome Sscrofa11.1 version sequence. The base of the site is C or T and is contained in the intron of the MARCHF11 gene.

2. A method for breeding or assisting in breeding high / low birth weight Landrace pigs using the pig birth weight-related SNP molecular markers according to claim 1, characterized in that: The following steps are involved: The base of the SNP molecular marker site associated with pig birth weight as described in claim 1 at 5379864 bp on chromosome 16 of the pig genome is detected to be C or T, thereby determining whether the genotype of the Landrace pig to be tested is CC, CT or TT, and then selecting Landrace pigs with TT genotype or CC genotype for the next step of breeding and / or breeding according to production needs.

3. The method according to claim 2, characterized in that If you want to breed pigs with high birth weight, choose pigs with TT genotype; if you want to breed pigs with low birth weight, choose pigs with CC genotype.

4. Use of a primer pair in breeding or assisting in breeding high / low birth weight Landrace pigs, characterized in that: The primer pair comprises: an upstream primer F1 having a sequence as shown in SEQ ID NO: 2 and a downstream primer R1 having a sequence as shown in SEQ ID NO:

3. The application method is: using the primer pair to detect whether the base of the pig birth weight-related SNP molecular marker site according to claim 1 at 5379864 bp on chromosome 16 of the pig genome is C or T, thereby determining whether the genotype of the Landrace pig to be tested is CC, CT or TT, and then selecting Landrace pigs with TT genotype or CC genotype for the next step of breeding and / or breeding according to production needs.

5. Use of the SNP molecular marker according to claim 1 in breeding high birth weight Landrace pig strains, characterized in that: The application comprises the following steps: (1) Detecting the SNP molecular marker associated with pig birth weight as described in claim 1 on reserve breeding pigs; (2) Selecting individuals with the TT genotype detected in step (1) as breeding pigs, and mating the breeding boars and sows; (3) The piglets born from mating in step (2) are tested for the SNP molecular marker as described in claim 1, individuals with the genotype of the site being TT are retained, and individuals with the genotype being CC are eliminated, thereby breeding a pig breed with high birth weight.

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