Product of SNP (Single Nucleotide Polymorphism) molecular marker related to sow litter size character detection and application thereof

Abstract

The invention relates to a product of an SNP (Single Nucleotide Polymorphism) molecular marker related to sow litter size character detection and application of the product. The SNP molecular marker comprises Cgt, Cgt, Cgt, Cgt, Cgt, Cgt, Cgt, Cgt, Cgt, Cgt, Cgt, Cgt, Cgt, Cgt, Cgt, Cgt, Cgt, Cgt The nucleotide sequence of the gene is shown as SEQ ID No.1, and the nucleotide sequence of the gene is shown as SEQ ID No.1. 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.2, and the nucleotide sequence of the downstream primer is as shown in SEQ ID No.3. The SNP molecular marker provided by the invention can be used for breeding, breeding and evaluating multi-variety sows with high reproductive performance.

Description

Technical Field

[0001] This invention relates to a SNP molecular marker technology, specifically to products and applications for detecting SNP molecular markers related to sow litter size traits. Background Technology

[0002] The reproductive performance of sows (such as total number born (TNB), number born live (NBA), and number born healthy (NBH)) is a key indicator affecting the economic benefits of the pig farming industry.

[0003] Single nucleotide polymorphisms (SNPs) are densely distributed throughout the genome (approximately one SNP every 300 bp in the pig genome), comprehensively covering coding regions, regulatory regions (such as promoters and enhancers), and non-coding functional regions, avoiding the "blind spots" of traditional QTL mapping. Even if an SNP associated with a target trait does not directly cause phenotypic variation, it may indirectly mark key genes (such as ESR1 and VEGFA) through a strong LD relationship with functional mutations (such as missense mutations), improving the efficiency of candidate gene screening. Therefore, SNP molecular markers are widely used in livestock and poultry molecular breeding, greatly accelerating the speed of livestock and poultry genetic improvement. Continuously developing and expanding livestock and poultry genetic marker resource banks is of great significance for sustainable genetic improvement.

[0004] Existing technologies also include some reports on SNP molecular markers related to sow reproductive performance. For example, CN114736975A reports an SNP molecular marker associated with sow litter size, which is an A>G base mutation located at position 7047076 on chromosome 2 of the international swine genome Sscrofa version 11.1. CN119307626A reports a SNP molecular marker associated with pig litter size, located at position 34977540 bp on chromosome 2 of the international swine reference genome Sscrofa version 11.1, with a nucleotide variation site of either G or A. Existing technologies primarily target common Landrace and Large White breeds; the molecular markers have limited effectiveness in identifying reproductive traits in sows of other breeds. Summary of the Invention

[0005] The first technical problem to be solved by this invention is to provide a product for detecting SNP molecular markers related to the litter size trait in sows.

[0006] The second technical problem to be solved by the present invention is to provide a product for detecting SNP molecular markers related to sow litter size traits and its application in the breeding of sow litter size traits.

[0007] The third technical problem to be solved by the present invention is to provide a product for detecting SNP molecular markers related to the litter size trait of sows and its application in breeding high-yielding sow breeds.

[0008] The fourth technical problem to be solved by this invention is to provide a product for detecting SNP molecular markers related to sow litter size traits and its application in improving sow litter size traits.

[0009] The fifth technical problem to be solved by this invention is to provide an application of a product for detecting SNP molecular markers related to sow litter size traits in evaluating sow litter performance.

[0010] The technical solution adopted by this invention to solve its first technical problem is a product for detecting SNP molecular markers related to the sow litter size trait, wherein the SNP molecular markers include:

[0011] The C>T base mutation located at nucleotide site 76367457 in Sscrofa version 11.1 of the international pig genome has the nucleotide sequence shown in SEQ ID No. 1;

[0012] 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. 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 3.

[0013] 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 sow litter size traits in the breeding of sow litter size traits.

[0014] Furthermore, the sow litter size trait includes one or a combination of traits such as total number of piglets, live number of piglets, or healthy number of piglets.

[0015] Furthermore, the method of application includes the following steps:

[0016] 1) Detect the SNP molecular markers in replacement breeding sows;

[0017] 2) Individuals with the CC or CT genotype at the 76367457 nucleic acid site detected in step 1) are selected as breeding sows; individuals with the CC genotype at the 76367457 nucleic acid site can effectively increase the number of piglets born to sows.

[0018] 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 litter size trait of sows in the breeding of high-yielding sow breeds.

[0019] Furthermore, the method of application includes the following steps:

[0020] 1) Detect the SNP molecular markers in replacement breeding sows;

[0021] 2) Select individuals with the CC or CT genotype at the 76367457 nucleic acid site detected in step 1) as breeding sows, and mate these breeding sows;

[0022] 3) Test the SNP molecular markers of the sows born from mating in step 2), retain individuals with the CC or CT genotype at the 76367457 nucleic acid site, and cull individuals with the TT genotype at the 76367457 nucleic acid site. Breed these individuals to develop a high-fertility breeding sow breed with a high number of piglets.

[0023] 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 sow litter size traits in improving the litter size trait of sow populations.

[0024] Furthermore, the method of application includes the following steps:

[0025] 1) Detect the SNP molecular markers in replacement breeding sows;

[0026] 2) Select individuals with the CC or CT genotype at the 76367457 nucleic acid site detected in step 1) as breeding sows, and mate these breeding sows;

[0027] 3) Test the SNP molecular markers of the sows born from mating in step 2), retain individuals with the CC or CT genotype at the 76367457 nucleotide locus, and cull individuals with the TT genotype at the 76367457 nucleotide locus. Breed these sows to increase the frequency of the dominant allele C at the 76367457 nucleotide locus generation by generation, thereby improving the litter size trait of the sow population and increasing the litter size of the sow population.

[0028] The technical solution adopted by this invention to solve its fifth technical problem is the application of products that detect SNP molecular markers related to the litter size trait in sows in evaluating sow reproductive performance. The application involves identifying the genotype of the SNP molecular marker in the sow to be evaluated. When the allele at the 76367457 nucleotide site is CC or CT, the sow to be evaluated has high reproductive performance; when the allele at the 76367457 nucleotide site is TT, the sow to be evaluated has low reproductive performance.

[0029] In this application, 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 typically obtains its genotype through sequencing.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] This invention identifies molecular genetic markers associated with sow reproductive function, facilitating targeted improvements in sow reproductive performance. Utilizing these molecular genetic markers, the reproductive performance of breeding sows can be efficiently assessed, improving the accuracy and convenience of testing. By employing these markers and optimizing breeding strategies through genotype selection, the birth quality and quantity of piglets can be improved, providing technical support for core breeding farms and contributing to more efficient and precise genetic improvement. Through the discovery and application of molecular genetic markers, this invention provides a novel method for improving the reproductive performance of sows in multiple breeds, contributing to enhanced overall economic efficiency and scientific rigor in the pig farming industry. Attached Figure Description

[0032] Figure 1 Manhattan plot of genome-wide association analysis for live piglet number in sows.

[0033] Figure 2 QQ plot of genome-wide association analysis for live piglet number in sows.

[0034] Figure 3 Manhattan plot of genome-wide association analysis for the number of healthy piglets born to sows.

[0035] Figure 4 QQ plot of genome-wide association analysis for the number of healthy piglets born to sows.

[0036] Figure 5 Manhattan plot for genome-wide association analysis of total litter size in sows.

[0037] Figure 6 QQ plot for genome-wide association analysis of total litter size in sows. Detailed Implementation

[0038] Many specific details of the invention are set forth in the following description to enable those skilled in the art to 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.

[0039] 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.

[0040] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The reagents used in this embodiment are all commercially available products.

[0041] Example 1: Screening of SNP molecular markers related to sow litter size

[0042] (1) Data collection and sample preparation for sow reproductive performance

[0043] Ear samples were collected from 839 Duroc, Large White, and Landrace breeding sows from the Changde Breeding Farm of Xinwufeng Company after removing outliers (exceeding 3 standard deviations) from the sow breeding records. Approximately 0.5g of ear sample was collected using ear clipping forceps. After each sample was collected, the forceps were disinfected by wiping with alcohol swabs 3-4 times. The collected ear samples were placed in 2mL centrifuge tubes containing 75% ethanol, labeled with the sow's number, and stored at -20℃.

[0044] (2) Main experimental instruments and reagents

[0045] Experimental instruments included a benchtop refrigerated centrifuge, a -80°C ultra-low temperature freezer, a constant temperature mixer, a DNA dry bath microheater, and a Nanodrop ultra-micro spectrophotometer (all manufactured by Eppendorf GmbH, Germany), and a vortex mixer (manufactured by Shanghai Jingke Industrial Co., Ltd.). Experimental reagents included DEPC buffer, chloroform, proteinase K, anhydrous ethanol, DNA extraction phenol reagent, and DNA lysis buffer, all provided by Changsha Spectrobio Biotechnology Co., Ltd. and Beijing Solarbio Technology Co., Ltd.

[0046] (3) DNA extraction and purity testing

[0047] Approximately 0.5g of ear-like material was chopped into a paste, added to lysis buffer and proteinase K, and heated at 55°C for 1 hour for overnight digestion. DNA was extracted and purified using saturated phenol and chloroform, and the DNA was separated by centrifugation and dissolved in the aqueous phase. DNA was precipitated with anhydrous ethanol, followed by washing the precipitate with 70% ethanol. Finally, the DNA stock solution was dissolved in TE buffer and stored at -20°C.

[0048] (4) Genomic DNA concentration determination and agarose gel electrophoresis detection

[0049] The concentration and purity of DNA samples were determined using a Nanodrop 2000 spectrophotometer, and the OD260 / 280 values ​​were used for purity estimation. A 1% agarose gel was prepared, and DNA sample quality was assessed by electrophoresis to ensure clear bands without any dragging.

[0050] (5) SNP chip detection and genotyping

[0051] Genotyping was performed on 839 individuals using the Compson Microsystems No. 1 chip, yielding 57,466 SNP loci. This chip technology is based on GBTS (Guangzhou-based Genetic Testing Technology), which uses probe capture technology to detect multiple SNPs within a single amplicon.

[0052] (6) Quality control and filling of genotype data

[0053] PLINK software was used to perform quality control on the genotype data, removing unqualified individuals and SNP loci. Beagle 5.4 software was used to self-fill the missing genotypes.

[0054] (7) Principal Component Analysis

[0055] The principal components of the target population were calculated using Plink software, and the PCA data of the SNPs were plotted in three dimensions using the scatterplot3d function in R to eliminate the population stratification effect.

[0056] (8) Genome-wide association study (GWAS)

[0057] Genome-wide association analysis (GWAS) was performed on different reproductive traits using an MLM model based on the rMVP package in R, considering both genetic factors and non-genetic factors such as puberty and prenatal backfat. Significance thresholds were determined using the Bonferroni method, and linkage disequilibrium was analyzed using LDBlockShow v1.40 and PopLDdecay software. For SNPs that showed significant association after GWAS analysis, gene annotation and candidate gene mining were performed using the BioMart tool on the Ensembl website and the NCBI database. The results are shown in Table 1.

[0058] Table 1. Analysis of differences in litter size among different C>T genotypes in Large White pigs using molecular markers.

[0059]

[0060] Note: *** indicates P < 0.001; ** indicates P < 0.01.

[0061] Example 2: Detection of SNP molecular markers related to sow litter size

[0062] This example demonstrates the design of upstream and downstream primers for detecting the C / T nucleotide site at 76367457 bp on chromosome 2, the SNP site obtained in Example 1.

[0063] 1. Primer design for the target gene sequence

[0064] The target gene is a partial sequence on chromosome 2 containing 76,367,457 bp, and its nucleotide sequence is as follows:

[0065]

[0066] Based on the target gene sequence, optimized amplification primers were designed using primer design software and synthesized by Beijing Qingke Biotechnology Co., Ltd. The primer pair sequences are as follows:

[0067] Upstream primer: 5'-GCAGGACACTGGAGCTGGGGCTCTGGGCTC-3' (e.g., SEQ ID No. 2)

[0068] Downstream primer: 5'-CATAGCCAGGGCGCCAAGGGTCACCCGCAG-3' (e.g., SEQ ID No. 3)

[0069] 2. Large White pig genome extraction

[0070] 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.

[0071] 3. PCR amplification of SNP loci associated with litter size in Large White pigs

[0072] Using the extracted DNA as a template, PCR amplification was performed using the primers described above.

[0073] 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.

[0074] Amplification procedure:

[0075] Pre-denaturation at 95℃ for 15 minutes;

[0076] 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.

[0077] After amplification, the temperature was maintained at 4°C to preserve the product and terminate the reaction.

[0078] 4. Sequencing and analysis of amplified products

[0079] 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 genotype of the target SNP locus related to litter size was CC, CT, or TT.

[0080] Example 3: Application of the molecular genetic markers of this application in sow breeding

[0081] This embodiment provides a method for applying the molecular genetic markers of this application to sow breeding or assisted breeding, the specific steps of which are as follows:

[0082] 1) Extract genomic DNA from the sow and detect the deoxynucleotide at position 76367457 on chromosome 2 to determine whether the deoxynucleotide at this position is C or T;

[0083] 2) Based on the genotype of the locus (CC, TC or TT), select the genotype that is suitable for the breeding needs for the next step of selection and / or breeding.

[0084] Specifically, if the goal is to breed sows with high litter sizes, TT-type Large White sows are culled, while CC-type sows are retained to increase the frequency of allele C through successive generations. Studies have shown that CC-type sows have a higher total litter size than TT-type sows, with each CC-type sow producing 1.58 more piglets than a TT-type sow. Assuming there are 10,000 fertile sows, each litter producing 15,800 piglets, and these piglets are fattened and slaughtered at a weight of 300 catties, with an estimated 85% dressing percentage, this could provide an additional 2,749.2 tons of pork. Therefore, the molecular genetic markers proposed in this application have promising application prospects and industrial value in sow breeding.

[0085] Example 4: A method for genetic improvement of sows with high litter size.

[0086] This embodiment provides a method for genetic improvement of Large White pigs with a high litter size trait. The specific steps are as follows:

[0087] 1) SNP molecular markers related to litter size were detected in replacement breeding pigs, genomic DNA was extracted, and the primers and methods described in Example 2 above were used for detection;

[0088] 2) Select individuals with the genotype CC or TC as breeding pigs, and mate the selected boars and sows;

[0089] 3) Perform SNP molecular marker testing on piglets born after mating, retain individuals with genotype CC or TC, and cull individuals with genotype TT, thereby cultivating a high-fertility pig breed with high reproductive performance.

[0090] Furthermore, steps (2) and (3) can be repeated based on step (3) to select breeding pigs with genotype CC or TC generation by generation, eliminate individuals with TT genotype, and gradually increase the frequency of the C allele at this SNP locus in the pig population, thereby increasing the number of piglets born in the offspring.

[0091] 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 sow litter size traits, characterized in that, The SNP molecular markers are: The C>T base mutation located at nucleotide site 76367457 on chromosome 2 of the international pig genome Sscrofa version 11.1, with its nucleotide sequence shown in SEQ ID No. 1; 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. 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID No.

3.

2. The application of the product described in claim 1 for detecting SNP molecular markers related to sow litter size traits in the breeding of sows with litter size traits.

3. The application according to claim 2, characterized in that, The sow litter size trait includes one or a combination of traits such as total number of piglets, live number of piglets, or healthy number of piglets.

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) Selecting individuals with the CC or CT genotype at the 76367457 nucleic acid site detected in step 1) can effectively increase the number of piglets born to sows.

5. The application of the product described in claim 1 for detecting SNP molecular markers related to sow litter size traits in the breeding of high-yielding sow breeds.

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 CC or CT genotype at the 76367457 nucleic acid site detected 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 CC or CT genotype at the 76367457 nucleic acid site, and cull individuals with the TT genotype at the 76367457 nucleic acid site. Breed these individuals to develop a high-fertility breeding sow breed with a high number of piglets.

7. The application of the product for detecting SNP molecular markers related to sow litter size traits as described in claim 1 in improving sow litter size traits.

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 CC or CT genotype at the 76367457 nucleic acid site detected 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 CC or CT genotype at the 76367457 nucleotide locus, and cull individuals with the TT genotype at the 76367457 nucleotide locus. Breed these sows to increase the frequency of the dominant allele C at the 76367457 nucleotide locus generation by generation, thereby improving the litter size trait of the sow population and increasing the litter size of the sow population.

9. The application of the product for detecting SNP molecular markers related to sow litter size traits as described in claim 1 in evaluating sow farrowing performance, characterized in that... The application identifies the genotype of the SNP molecular marker in the sow to be evaluated. When the allele at the 76367457 nucleotide site is CC or CT, the sow to be evaluated has high litter size; when the allele at the 76367457 nucleotide site is TT, the sow to be evaluated has low litter size.

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