Large white pig tlr4 gene kasp molecular marker related to lactation intake of sows during lactation and application thereof
Patent Information
- Application Number
- CN202611332274.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-08-31
- Publication Date
- 2026-10-16
AI Technical Summary
哺乳期采食量不足会导致母猪体况过度消耗、断奶至发情间隔延长、下一胎产仔数下降等问题，给养猪生产带来严重的经济损失
（1）首次发现TLR4基因与母猪哺乳期采食量的关联：本发明通过GWAS技术，首次在大白猪群体中发现TLR4基因SNP位点（Chr1_290018621）与哺乳期采食量显著关联（P =4.84×10-5），为母猪采食量的遗传改良提供了全新的分子靶点。
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Figure CN122918218A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of animal molecular breeding technology, and particularly relates to a Large White pig species related to feed intake during lactation. TLR4 KASP molecular markers for genes and their applications. Background Technology
[0002] Feed intake during lactation is a key economic trait affecting sow lactation performance, piglet weaning weight, sow body condition recovery, and subsequent reproductive performance. Insufficient feed intake during lactation can lead to excessive body condition depletion in sows, prolonged weaning-to-estrus intervals, and decreased litter size in the next litter, resulting in significant economic losses for pig production. However, traditional breeding methods for sow feed intake during lactation rely on long-term measurements using automated feeding equipment. These methods suffer from drawbacks such as long measurement cycles, high costs, difficulties in data collection, and susceptibility to interference from environmental and disease factors, making large-scale application in breeding practice difficult.
[0003] With the development of molecular biology techniques, marker-assisted selection (MAS) has provided a new technical approach for animal genetic improvement. Genome-wide association studies (GWAS) can efficiently identify genetic variation sites that are significantly associated with target traits, providing targets for molecular marker development.
[0004] Toll-like receptor 4 TLR4 Lipopolysaccharide (LPS) is a major receptor in the innate immune system that recognizes pathogenic microorganisms and plays an important role in the body's immune defense. Recent studies have found that... TLR4 In addition to its role in immune regulation, the gene also affects the hypothalamic appetite regulation center through the NF-κB and MAPK inflammatory signaling pathways, potentially participating in the regulation of animal feeding behavior. However, no evidence has yet been found of pigs being used in this process. TLR4 Reports on the association of specific gene variants with feed intake in lactating sows and the development of KASP molecular markers.
[0005] Kompetitive allele-specific PCR (KASP) is a high-throughput genotyping technique based on SNPs. It has the advantages of low cost, high accuracy, high throughput, and simple operation, and is suitable for genotyping of large-scale breeding populations.
[0006] This invention addresses the shortcomings of existing technologies. Summary of the Invention
[0007] In view of this, the purpose of this invention is to provide a KASP molecular marker of the TLR4 gene in Large White pigs related to feed intake during lactation and its application. This invention uses GWAS technology to screen for the KASP marker in a Large White sow lactation population. TLR4 The study identified a significant associated locus of the gene and developed the first KASP molecular marker targeting this locus, providing an efficient molecular detection tool for early screening and genetic improvement of high-feed-intake sows.
[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a Large White pig related to feed intake during lactation. TLR4 The KASP gene molecular marker is located at 290,018,621 bp on chromosome 1 and has G / A polymorphism, where G is the high feed intake associated allele and A is the low feed intake associated allele. The TLR4 The nucleotide sequence of the gene is shown in SEQ ID NO: 4.
[0009] The present invention also provides an amplification of the Large White pig described above. TLR4 A primer set for the KASP gene molecular marker, comprising forward primer F1, forward primer F2, and reverse primer R; The nucleic acid sequence of the forward primer F1 is shown in SEQ ID NO: 1; The nucleic acid sequence of the forward primer F2 is shown in SEQ ID NO: 2; The nucleic acid sequence of the reverse primer R is shown in SEQ ID NO: 3.
[0010] Preferably, the 5′ end of the forward primer F1 carries a FAM fluorescent reporter group sequence, and the 5′ end of the forward primer F2 carries a VIC fluorescent reporter group sequence.
[0011] The present invention also provides a method for detecting the aforementioned Large White pig. TLR4 A kit for KASP gene molecular markers, including the primer set described above.
[0012] Preferably, the kit further includes any one or more of 2× KASP Master Mix, sterile deionized water, 384-well PCR plate, or optical-grade sealing film.
[0013] This invention also provides a method for identifying the TLR4 gene genotype in Large White pigs, the method comprising the following steps: (1) Extract genomic DNA from the Large White pig samples to be tested; (2) Using the genomic DNA extracted in step (1) as a template, perform KASP-PCR amplification using the primer set described in claim 2 or 3; (3) Detect the fluorescence signal of the KASP-PCR amplification product and determine the genotype based on the fluorescence signal type; The criteria for determining genotype are as follows: when the test sample shows only FAM fluorescence signal, it is determined to be a low-feed-intake-associated homozygote AA; when the test sample shows only VIC fluorescence signal, it is determined to be a high-feed-intake-associated homozygote GG; when the test sample shows both FAM and VIC fluorescence signals, it is determined to be a heterozygote AG.
[0014] Preferably, the KASP-PCR amplification reaction system comprises: 1.25 μL template DNA, 2.5 μL 2× KASPMaster Mix, and 2.5 μL primer set as described in claim 2 or 3.
[0015] Preferably, the reaction program for KASP-PCR amplification is as follows: 95℃ pre-denaturation / activation for 10 min; 95℃ denaturation for 20 s, 61℃→55℃ cyclic cooling annealing extension, decreasing by 0.6℃ per cycle for 60 s, for a total of 10 cycles; 95℃ denaturation for 20 s, 55℃ annealing extension for 60 s, for a total of 27 cycles; signal reading at 25℃ for 30 s.
[0016] The present invention also provides the aforementioned white pig TLR4 The application of the gene KASP molecular marker, the primer set, and the kit, wherein the application includes: (1) Genotyping of the Large White pig TLR4 gene; (2) Early screening and breeding of sows with high feed intake during lactation; (3) Genetic improvement of feed intake traits in lactating sows; (4) Identification and genetic assessment of Large White pig germplasm resources.
[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) First discovery TLR4 The association between genes and feed intake during lactation in sows: This invention, using GWAS technology, is the first to discover this association in a Large White pig population. TLR4 The gene SNP locus (Chr1_290018621) was significantly associated with feed intake during lactation (P = 4.84 × 10⁻⁶). -5 This provides a novel molecular target for the genetic improvement of sow feed intake.
[0018] (2) High accuracy of KASP tagging: This invention targets TLR4KASP primers were designed for gene-specific SNP sites. After validation with 250 independent samples, the genotyping success rate reached 100%. The three genotypes (G:G, A:G, A:A) showed clear clustering and were completely distinguishable on the fluorescence scatter plot.
[0019] (3) Fast detection speed and high throughput: The entire process from DNA extraction to result interpretation can be completed in 3-4 hours, and it is suitable for high-throughput detection in 96-well or 384-well plates.
[0020] (4) Low cost: The cost of single sample detection of this invention can be controlled at RMB 0.5-1, which is more than 95% lower than the cost of traditional sequencing methods, making it suitable for large-scale promotion and application in breeding farms.
[0021] (5) Early screening: This invention can complete genotyping by taking ear tissue samples at the time of piglet birth, enabling early screening of individuals with high feed intake potential and significantly shortening the generation interval of breeding.
[0022] (6) Simple operation: This invention does not require complex operation steps such as gel electrophoresis and enzyme digestion. Only a PCR instrument and a fluorescence plate reader are needed to interpret the results, which has low technical requirements for operators. Attached Figure Description
[0023] Figure 1 It's a big white pig. TLR4 Scatter plot of KASP genotyping results for gene SNP locus (Chr1_290018621) (the horizontal axis is the FAM fluorescence signal intensity (corresponding to the A allele), and the vertical axis is the VIC fluorescence signal intensity (corresponding to the G allele)). Detailed Implementation
[0024] This invention provides a Large White pig related to feed intake during lactation. TLR4 The KASP gene molecular marker is located at 290,018,621 bp on chromosome 1 and has G / A polymorphism, where G is the high feed intake associated allele and A is the low feed intake associated allele. The TLR4 The nucleotide sequence of the gene is shown in SEQ ID NO: 4, specifically TCTCCTAAGTGTTGGGATTCAGAACCTATTCATCCAAGAGTCGTATTCCT[A / G]GGCTCTGAAATATTTCTCAAACTTTCTAGAAAATTTGGTAACTATTTCA.
[0025] The present invention also provides an amplification of the Large White pig described above. TLR4A primer set for the KASP gene molecular marker, comprising forward primer F1, forward primer F2, and reverse primer R; The nucleic acid sequence of the forward primer F1 is shown in SEQ ID NO: 1, specifically GAAGGTGACCAAGTTCATGCTCTATTCATCCAAGAGTCGTATTCCTA; The nucleic acid sequence of the forward primer F2 is shown in SEQ ID NO: 2, specifically GAAGGTCGGAGTCAACGGATTCTATTCATCCAAGAGTCGTATTCCTG; The nucleic acid sequence of the reverse primer R is shown in SEQ ID NO: 3, specifically CCCACCCTCCACTTGAAATAGTTA.
[0026] In this invention, the 5′ end of the forward primer F1 carries a FAM fluorescent reporter group sequence, and the 5′ end of the forward primer F2 carries a VIC fluorescent reporter group sequence.
[0027] The present invention also provides a method for detecting the aforementioned Large White pig. TLR4 A kit for KASP gene molecular markers, comprising the primer set described above. In this invention, the kit further comprises any one or more of 2× KASP Master Mix, sterile deionized water, a 384-well PCR plate, or an optical-grade sealing film.
[0028] This invention also provides a method for identifying the TLR4 gene genotype in Large White pigs, the method comprising the following steps: (1) Extract genomic DNA from the Large White pig samples to be tested; (2) Using the genomic DNA extracted in step (1) as a template, KASP-PCR amplification was performed using the primer set described above; (3) Detect the fluorescence signal of the KASP-PCR amplification product and determine the genotype based on the fluorescence signal type; The criteria for determining genotype are as follows: when the test sample shows only FAM fluorescence signal, it is determined to be a low-feed-intake-associated homozygote AA; when the test sample shows only VIC fluorescence signal, it is determined to be a high-feed-intake-associated homozygote GG; when the test sample shows both FAM and VIC fluorescence signals, it is determined to be a heterozygote AG.
[0029] In this invention, the KASP-PCR amplification reaction system comprises: 1.25 μL template DNA, 2.5 μL 2×KASP Master Mix, and 2.5 μL of the primer set described above; the KASP-PCR amplification reaction program is as follows: 95℃ pre-denaturation / activation for 10 min; 95℃ denaturation for 20 s, followed by 61℃→55℃ cyclic cooling annealing extension, decreasing the temperature by 0.6℃ for 60 s per cycle, for a total of 10 cycles; 95℃ denaturation for 20 s, followed by 55℃ annealing extension for 60 s, for a total of 27 cycles; and signal reading at 25℃ for 30 s.
[0030] The present invention also provides the aforementioned white pig TLR4 The application of the gene KASP molecular marker, the primer set, and the kit, wherein the application includes: (1) Genotyping of the Large White pig TLR4 gene; (2) Early screening and breeding of sows with high feed intake during lactation; (3) Genetic improvement of feed intake traits in lactating sows; (4) Identification and genetic assessment of Large White pig germplasm resources.
[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0032] Example 1
[0033] TLR4 GWAS screening of gene-significantly associated SNP sites
[0034] 1.1 GWAS Analysis Population
[0035] One hundred Large White sows were selected as the GWAS analysis population. All experimental pigs were raised in a standard breeding farm, and the average daily feed intake (ADFI) of each sow during the 21-day lactation period was recorded.
[0036] Ear tissue samples were collected from experimental pigs, and genomic DNA was extracted using the phenol-chloroform method. Whole-genome genotyping was performed using a porcine whole-genome SNP microarray. High-quality SNP markers were obtained after strict quality control (individual detection rate >95%, SNP detection rate >95%, minimum allele frequency >0.05) for association analysis.
[0037] 1.2 GWAS Association Analysis
[0038] Genome-wide association analysis was performed using a mixed linear model (MLM), with a kinship matrix included as a covariate to control for population stratification.
[0039] GWAS analysis revealed a significant SNP locus on chromosome 1 of Large White pigs associated with feed intake during lactation (SNP number: Chr1_290018621, P = 4.84 × 10⁻⁶). -5 The SNP site is located at... TLR4 Within the gene coding region, the polymorphism is A / G.
[0040] 1.3 Genotype-phenotype association analysis
[0041] To further confirm the association between different alleles at this SNP locus and food intake, this invention performed regression analysis on the genotype and ADFI phenotype of the GWAS population.
[0042] The results showed that the effect size (BETA) of this SNP locus was positive (P = 4.84 × 10⁻⁶). -5 This indicates that the G allele has a positive additive effect on feed intake, meaning that carrying the G allele can increase feed intake during lactation.
[0043] The above results fully demonstrate that G is the allele associated with high feed intake and A is the allele associated with low feed intake.
[0044] Example 2
[0045] TLR4 Design and synthesis of gene KASP primers
[0046] According to the identification in Example 1 TLR4 For the gene SNP site (Chr1_290018621, A / G), flanking sequences of approximately 50-80 bp upstream and downstream of this site (SEQ ID NO:4) were taken, and KASP primers were designed using Primer3 software.
[0047] Two allele-specific forward primers (F1 and F2) and one universal reverse primer (R) were designed. The primer sequences are shown in Table 1.
[0048] Table 1 KASP primer sequence information
[0049] The 3′ end of F1 is A, which perfectly matches the low feed intake associated allele (A); the 3′ end of F2 is G, which perfectly matches the high feed intake associated allele (G). The 5′ ends of F1 and F2 carry FAM and VIC fluorescent tag sequences, respectively.
[0050] The newly synthesized primers were diluted to 10 μM with TE buffer (pH 8.0) and then mixed in a volume ratio of forward genotyping primer F1: forward genotyping primer F2: downstream universal primer R = 1:1:3 for later use.
[0051] Example 3
[0052] Genotyping validation of an independent Large White pig population using KASP markers
[0053] To verify the reliability and stability of the KASP molecular marker of the present invention, a population of 250 independent Large White sows was selected, and KASP genotyping was performed using the primer set designed in Example 2.
[0054] 3.1 DNA extraction and KASP-PCR amplification
[0055] Ear tissue samples were collected from experimental pigs, and genomic DNA was extracted. The DNA samples were diluted to the lowest concentration of 5 ng / μL, and the entire batch of samples was diluted.
[0056] The KASP reaction was performed using a 5 μL system. In a 384-well PCR plate, samples were added as follows: 2.5 μL of 2×KASP Master Mix, 1.25 μL of primer mixture (composed of forward primer F1, forward primer F2, and reverse primer R in a 1:1:3 volume ratio), and then 1.25 μL of diluted DNA sample, for a total of 5 μL. Two technical replicates were performed for each sample, including an NTC (template-free control) and known genotype controls (one each for G:G, A:G, and A:A).
[0057] After adding the samples, seal the 384-well PCR reaction plate with sealing film, shake, and centrifuge to ensure that the reaction system is mixed evenly.
[0058] KASP-PCR amplification was performed on a QuantStudio™ 7 Flex System. The reaction program was as follows: First, pre-denaturation / activation was performed at 95°C for 10 minutes; then, 10 cycles of fall-off PCR were performed, each cycle consisting of 20 seconds of denaturation at 95°C and 60 seconds of annealing extension, with the annealing temperature starting at 61°C and decreasing by 0.6°C each cycle until reaching 55°C; followed by 27 cycles, each cycle consisting of 20 seconds of denaturation at 95°C and 60 seconds of annealing extension at 55°C; finally, signal reading was performed at 25°C for 30 seconds. If the initial genotyping results were unsatisfactory, an optimization cycle of 3 cycles of 20 seconds of denaturation at 95°C and 60 seconds of annealing extension at 55°C could be added after the above program.
[0059] 3.2 Classification Results
[0060] Statistical analysis was performed on the KASP genotyping results of 250 independent Large White sows. The cluster scatter plots of the three genotypes are shown below. Figure 1As shown, the three genotype samples formed three clearly distinguishable independent clusters on the scatter plot, with no ambiguous or failed genotyping samples, and the genotyping success rate reached 100% (250 / 250).
[0061] The distribution of each genotype is shown in Table 2.
[0062] Table 2. Distribution of TLR4 genotypes in an independent Large White sow population (N=250)
[0063] In this population, the frequency of the G allele was as high as 78.2%, the GG genotype accounted for 60.4%, and the AA genotype accounted for only 4.0%. This is highly consistent with the results of the GWAS analysis showing a positive association between the G allele and high feed intake, further verifying the dominant position of the G allele.
[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A Large White pig species associated with feed intake during lactation. TLR4 The KASP gene molecular marker is characterized by, The KASP molecular marker is located at 290,018,621 bp on chromosome 1 and has G / A polymorphism, where G is the high feed intake associated allele and A is the low feed intake associated allele. The TLR4 The nucleotide sequence of the gene is shown in SEQ ID NO:
4.
2. Expanding the Large White pig as described in claim 1 TLR4 The primer set for the KASP gene molecular marker is characterized by, The primer set includes forward primer F1, forward primer F2, and reverse primer R; The nucleic acid sequence of the forward primer F1 is shown in SEQ ID NO: 1; The nucleic acid sequence of the forward primer F2 is shown in SEQ ID NO: 2; The nucleic acid sequence of the reverse primer R is shown in SEQ ID NO:
3.
3. The primer set according to claim 2, characterized in that, The forward primer F1 has a FAM fluorescent reporter group sequence at its 5′ end, and the forward primer F2 has a VIC fluorescent reporter group sequence at its 5′ end.
4. A method for detecting the Large White pig as described in claim 1 TLR4 A kit for KASP gene molecular markers, characterized in that, Includes the primer set as described in claim 2 or 3.
5. The reagent kit according to claim 4, characterized in that, The kit also includes any one or more of the following: 2× KASP MasterMix, sterile deionized water, 384-well PCR plate, or optical-grade sealing film.
6. A method for identifying the TLR4 gene genotype in Large White pigs, characterized in that, The method includes the following steps: (1) Extract genomic DNA from the Large White pig samples to be tested; (2) Using the genomic DNA extracted in step (1) as a template, perform KASP-PCR amplification using the primer set described in claim 2 or 3; (3) Detect the fluorescence signal of the KASP-PCR amplification product and determine the genotype based on the fluorescence signal type; The criteria for determining genotype are as follows: when the test sample shows only FAM fluorescence signal, it is determined to be a low-feed-intake-associated homozygote AA; when the test sample shows only VIC fluorescence signal, it is determined to be a high-feed-intake-associated homozygote GG; when the test sample shows both FAM and VIC fluorescence signals, it is determined to be a heterozygote AG.
7. The method according to claim 6, characterized in that, The KASP-PCR amplification reaction system comprises: 1.25 μL template DNA, 2.5 μL 2× KASP Master Mix, and 2.5 μL primer set as described in claim 2 or 3.
8. The method according to claim 6, characterized in that, The reaction program for KASP-PCR amplification was as follows: 95℃ pre-denaturation / activation for 10 min; 95℃ denaturation for 20 s, followed by 61℃→55℃ cyclic cooling annealing extension, decreasing the temperature by 0.6℃ for 60 s per cycle, for a total of 10 cycles; 95℃ denaturation for 20 s, followed by 55℃ annealing extension for 60 s, for a total of 27 cycles; signal reading at 25℃ for 30 s.
9. The white pig according to claim 1 TLR4 The application of the gene KASP molecular marker, the primer set as described in claim 2 or 3, and the kit as described in claim 4 or 5, is characterized by, The applications include: (1) Genotyping of the Large White pig TLR4 gene; (2) Early screening and breeding of sows with high feed intake during lactation; (3) Genetic improvement of feed intake traits in lactating sows; (4) Identification and genetic assessment of Large White pig germplasm resources.