Molecular marker associated with 120 kg weight day age of pig and application of molecular marker
Through whole-genome sequencing and association analysis, molecular markers associated with pig 120kg weight day age were screened out, primer pairs and kits were developed for detection, which solved the problems of inefficiency and insufficient accuracy of existing detection methods, and achieved accurate prediction and improvement of pig growth rate and breeding efficiency.
Patent Information
- Application Number
- CN202510064748.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-05-09
AI Technical Summary
The existing pig 120kg weight day-age detection method has problems of inefficiency and insufficient accuracy, and it is difficult to effectively predict the growth rate and breeding efficiency of pigs.
Through whole-genome sequencing and association analysis, molecular markers associated with pig 120kg body weight day age were screened, specifically at the C/A single nucleotide polymorphism site at the 11266105nt position of pig chromosome 7, and primer pairs and kits were developed for detecting the molecular marker.
Accurate prediction of the pig's weight of 120kg was achieved, especially by breeding AA individuals, which significantly shortened the pig's weight of 120kg, thereby improving the economic benefits of breeding.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of Duroc pigs, and in particular to molecular markers associated with pigs 120 kg in weight and age, and their application. Background Art
[0002] The age at which a pig reaches 120kg in weight refers to the number of days it takes for a pig to grow to a weight of 120kg. It is an important indicator for measuring the growth rate and breeding efficiency of pigs. Different breeds of pigs, feeding and management conditions and other factors will lead to differences in the age at which a pig reaches 120kg in weight. For example, under good feeding and management conditions, some excellent lean pig breeds may reach a weight of 120kg at around 160-180 days of age, while some local breeds or breeds with slower growth rates may need more than 200 days of age to reach the weight.
[0003] Traditional methods for detecting the age of pigs at 120kg include visual weight estimation combined with growth curve method, sampling weight combined with correction method, and body length and chest circumference weight estimation method. The shorter the age of pigs at 120kg, the shorter the time it takes for the pigs to grow to the target weight. Under the condition of relatively stable breeding costs, the pigs can be put on the market faster, the funds can be recovered faster, and the breeding economic benefits are higher. Summary of the invention
[0004] The inventors of the present application used whole genome sequencing and association analysis to screen and obtain molecular markers associated with the age of 120 kg body weight in pigs. The molecular markers were located at the single nucleotide polymorphism site of C / A at the 11266105 nt position of chromosome 7 of pigs. Among them, the age of 120 kg body weight in individuals with AA genotype at this site was significantly lower than that of other individuals, indicating that the A-type nucleotide at this site can serve as an allele that is beneficial to shortening the age of 120 kg body weight in Duroc pigs.
[0005] To this end, the embodiments of the present application disclose at least the following technical solutions:
[0006] In a first aspect, the embodiments disclose molecular markers associated with a pig's age of 120 kg body weight, including a nucleotide sequence formed by a single nucleotide polymorphism of C / A at position 11266105 nt on chromosome 7 of the International Pig Reference Genome version 11.1.
[0007] In a second aspect, the embodiment discloses a primer pair for detecting the molecular marker described in the first aspect, and the nucleotide sequence of the primer pair is shown in SEQ ID NO:1 and SEQ ID NO:2.
[0008] In a third aspect, an embodiment discloses a kit comprising the primer pair described in the second aspect and reagents required for PCR amplification.
[0009] In a fourth aspect, an embodiment discloses a method for detecting the molecular markers described in the first aspect, the method comprising the following steps: extracting the genomic DNA of the pig to be tested; performing PCR amplification on the genomic DNA of the pig to be tested to obtain a PCR amplification product; sequencing the PCR amplification product to obtain a sequencing result; based on the sequencing result, interpreting the polymorphism of the 11266105nt site on chromosome 7 of the International Porcine Reference Genome 11.1 version.
[0010] In a fifth aspect, the embodiments disclose molecular markers associated with the age of pigs at 120 kg body weight, including nucleotide sequences shown as SEQ ID NO:3 and SEQ ID NO:4.
[0011] In a sixth aspect, an embodiment discloses a method for breeding a group of short-lived pigs weighing 120 kg, the method comprising the following steps: detecting the genotype of the 11266105nt site of chromosome 7 of the International Pig Reference Genome 11.1 version, and selecting AA type individuals at the 11266105nt site as breeding pigs.
[0012] In a seventh aspect, an embodiment discloses a method for breeding a group of short-lived pigs weighing 120 kg, comprising: if a nucleotide sequence as shown in SEQ ID NO: 3 is detected in the pig genomic DNA, the corresponding homozygous genotype individual is used as a breeding pig.
[0013] In an eighth aspect, the embodiment discloses the use of a SNP site or primer pair associated with Duroc pigs 120 kg in screening a short 120 kg weight pig population, wherein the SNP site is the 11266105 nt site of chromosome 7 of the International Porcine Genome Version 11.1 reference sequence, and has a C / A polymorphism. The nucleotide sequences of the primer pairs are shown in SEQ ID NO: 1 and SEQ ID NO: 2. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 The electrophoresis detection diagram of the pig individual genomic DNA amplification product provided in the embodiment is 2% agarose gel. M represents the 1000bp Marker molecular weight standard, and the other lanes are the DNA products amplified from the SNP mutation sites C and A Duroc pig genomic DNA, and the target fragment size is 404bp. Among them, the 1000bp Marker molecular weight bands are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp from top to bottom.
[0015] Figure 2 The sequencing peak diagram of the pig individual genomic DNA amplification product provided in the example is compared with the sequence of the relevant gene fragments of the pig in GenBank. Figure 2Figure A shows the sequence alignment result of the pig chromosome 7 at position 11266105nt, Figure 2 B is the sequence alignment result of A at the 11266105 nt position of pig chromosome 7. DETAILED DESCRIPTION
[0016] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.
[0017] The age of pigs at 120kg is affected by many factors, including genes, environment, and management. Therefore, in order to better predict the age of pigs at 120kg, it is necessary to consider multiple factors, not just the role of a single molecular marker. Secondly, due to the large number of pig breeds and the large genetic differences between different breed groups, it is necessary to conduct systematic research in multiple different breeds to find more reliable and effective molecular markers.
[0018] The present application uses genome-wide association analysis (GWAS) to detect the polymorphism of genetic markers in multiple individuals across the genome, obtain genotypes, and then perform statistical analysis of genotypes and phenotypes at the population level, and screen out the SNP sites that are most likely to affect the 120kg weight-day-age trait of pigs based on significance tests. The present application discovered a single nucleotide polymorphism site with C / A at the 11266105nt position of chromosome 7 of the international pig genome version 11.1 reference sequence (GCA_000003025.6), wherein the 120kg weight-day-age of individuals with the AA genotype at this site is significantly lower than that of other individuals, indicating that the A-type nucleotide at this site can be used as an allele that is beneficial to obtaining the 120kg weight-day-age of short Duroc.
[0019] Based on this, the embodiment discloses molecular markers associated with a pig with a body weight of 120 kg, including a nucleotide sequence formed by a single nucleotide polymorphism of C / A at position 11266105 nt of chromosome 7 of the International Pig Reference Genome 11.1.
[0020] The embodiment discloses a primer pair for detecting the molecular marker, and the nucleotide sequence of the primer pair is shown in SEQ ID NO:1 and SEQ ID NO:2.
[0021] The embodiment also discloses a kit, comprising the primer pair and reagents required for PCR amplification.
[0022] The embodiment discloses a method for detecting the molecular markers, which comprises the following steps: extracting the genomic DNA of the pig to be tested; performing PCR amplification on the genomic DNA of the pig to be tested to obtain a PCR amplification product; sequencing the PCR amplification product to obtain a sequencing result; and based on the sequencing result, interpreting the polymorphism of the 11266105nt site of chromosome 7 of the International Pig Reference Genome 11.1 version.
[0023] The embodiment discloses a method for breeding a group of short-lived pigs weighing 120 kg, the method comprising the following steps: detecting the genotype of the 11266105nt site of chromosome 7 of the International Pig Reference Genome 11.1 version, and selecting AA-type individuals at the 11266105nt site as breeding pigs.
[0024] The embodiment discloses a method for breeding a group of short-120 kg body weight day-old pigs, comprising: if a nucleotide sequence as shown in SEQ ID NO: 3 is detected in the pig genomic DNA, the corresponding homozygous genotype individuals are used as breeding pigs.
[0025] The embodiment discloses the use of a SNP site or primer pair associated with Duroc pigs 120 kg in weight age in screening a short 120 kg weight age pig population, wherein the SNP site is the 11266105 nt site of chromosome 7 of the international porcine genome version 11.1 reference sequence, and has a C / A polymorphism. The nucleotide sequence of the primer pair is shown in SEQ ID NO: 1 and SEQ ID NO: 2.
[0026] 1. Test sample collection
[0027] The experimental pigs were 464 purebred Duroc pigs from a pig farm in Hubei. The pigs were allowed to eat and drink freely, and the entire feeding method and feeding conditions were kept consistent, which was a conventional method.
[0028] 2. Extraction and Detection of Pig Genomic DNA
[0029] In this experiment, the genomic DNA kit (TIANamp Genomic DNA Kit) produced by Beijing Tiangen Biochemical Technology Co., Ltd. was used to extract pig genomic DNA from pig ear tissue. The specific operation steps are as follows:
[0030] 1) Use alcohol-disinfected ophthalmic surgical scissors to cut the pig ear sample into a paste, add 200 μL buffer GA (provided in the kit), and shake until thoroughly suspended.
[0031] 2) Add 20 μL of proteinase K solution (provided in the kit), mix well, and digest overnight in a 56°C water bath.
[0032] 3) Add 200 μL of buffer GB (provided with the kit), mix thoroughly by inversion, and place at 70°C for 10 minutes. When the solution becomes clear, centrifuge briefly to remove water droplets on the inner wall of the tube cap.
[0033] 4) Add 200 μL of anhydrous ethanol and vortex thoroughly for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.
[0034] 5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12000 rpm for 30 seconds, pour out the waste liquid, and put the adsorption column CB3 back into the collection tube.
[0035] 6) Add 500 μL of buffer GD (provided in the kit) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube.
[0036] 7) Add 600 μL of rinsing solution PW (provided in the kit) to the adsorption column CB3, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube.
[0037] 8) Repeat step 7.
[0038] 9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 min, discard the waste liquid, and place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.
[0039] 10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μL of elution buffer TE to the middle part of the adsorption membrane, let it stand at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the solution into the centrifuge tube.
[0040] 11) Take 2 μL of the DNA solution obtained in the previous step and 1 μL of the loading buffer solution, mix them, load them on a 1.2% agarose gel, and perform electrophoresis at 120V for about 20 minutes. Observe the electrophoresis results under ultraviolet light and take pictures to determine the integrity of the DNA. The extracted DNA was quality tested using a NanoDrop 2000 nucleic acid protein analyzer (Thermo Fisher Scientific, USA). The ratio of A260 / A280 was between 1.7-2.1, and the ratio of A260 / A230 was between 1.8-2.2, which was considered qualified. The concentration of qualified DNA was measured, and then the concentration was uniformly diluted to 200 ng / μL and stored in a refrigerator at -20°C. Unqualified DNA samples need to be re-extracted.
[0041] 3. PCR Testing
[0042] The porcine genomic DNA obtained according to the above steps was amplified by PCR using the primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2. The PCR reaction system contained 1 μL porcine genomic DNA, 0.5 μL DNA shown in SEQ ID NO: 2, 0.5 μL DNA shown in SEQ ID NO: 3, and 10 μL PCR Mix reagent (2×M5TaqHiFiPCRMix, Mei5bio) in 12 μL. The PCR reaction steps included 94°C, 3 min; 30 cycles (94°C, 25 s; 60°C, 25 s; 72°C, 10 s); 72°C, 5 min; 4°C, ∞.
[0043] Figure 1 The figure shows the electrophoresis detection of the PCR product obtained by PCR amplification of a pig genomic DNA in the above steps in a 2% agarose gel. In the figure, the size of the target fragment amplified by the PCR product is about 404 bp.
[0044] The 404 bp were sequenced to obtain the nucleotide sequences shown in SEQ ID NOs: 3 and 4. Figure 2 The results of sequence alignment between the sequence near the SNP site in the nucleotide sequence shown in SEQ ID NO:3 and the related gene fragments of pigs in GenBank, and the results of sequence alignment between the sequence near the SNP site in the nucleotide sequence shown in SEQ ID NO:4 and the related gene fragments of pigs in GenBank are shown.
[0045] Statistical Analysis
[0046] The general linear model of SAS software was used to conduct association analysis between genotype and phenotype at the age of 120 kg body weight. The model is as follows:
[0047] Y ijnk =u i +G j +S n +D k +e jnk
[0048] Where: Y ijnk It is the trait of pigs reaching 120kg body weight at the age of 120kg; G j represents the genotype fixed effect of the jth SNP;
[0049] S n represents the fixed effect of gender; D k represents the fixed effect of birth year and season; e jnk is the residual.
[0050] Table 1 shows the relationship between the genotype of the SNP loci provided in the embodiment and the 120kg body weight age phenotype. In Table 1, the 120kg body weight age of the AA genotype, CA genotype and CC genotype are statistically analyzed, and the significance level of the 120kg body weight age between these genotypes is compared. Capital letters indicate extremely significant differences P<0.01.
[0051] Table 1
[0052]
[0053] As shown in Table 1, the age at 120kg for individuals with AA genotype is significantly lower than that for individuals with CA genotype and CC genotype, and A is an allele that is beneficial to the age at 120kg for Duroc pigs. This shows that breeding AA and CA type individuals in Duroc pigs can help shorten the age at 120kg, thereby improving the economic benefits of the pig farming industry.
[0054] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.
Claims
1. Molecular markers associated with pigs 120 kg in age, including a nucleotide sequence formed by a single nucleotide polymorphism of C / A at position 11266105 on chromosome 7 of the International Pig Reference Genome 11.
1.
2. A primer pair for detecting the molecular marker according to claim 1, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO:
2.
3. A kit comprising the primer pair according to claim 2 and reagents required for PCR amplification.
4. A method for detecting the molecular marker according to claim 1, comprising: Extracting genomic DNA from the pig to be tested; Performing PCR amplification on the genomic DNA of the pig to be tested to obtain a PCR amplification product; Sequencing the PCR amplification product to obtain a sequencing result; Based on the sequencing results, the polymorphism of the 11266105nt site on chromosome 7 of the International Pig Reference Genome 11.1 version was determined.
5. Molecular markers associated with pigs at 120 kg body weight, comprising the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO:
4.
6. A method for breeding a group of pigs with a body weight of less than 120 kg, comprising: The genotype of the 11266105nt site on chromosome 7 of the International Pig Reference Genome 11.1 version was detected, and the AA type individuals at the 11266105nt site were selected as breeding pigs.
7. A method for breeding a group of pigs with a body weight of less than 120 kg, comprising: If the nucleotide sequence shown in SEQ ID NO: 3 is detected in the pig genomic DNA, the corresponding homozygous genotype individual is used as a breeding pig.
8. The use of a SNP site or primer pair associated with Duroc pigs weighing 120 kg per day in screening a short 120 kg per day pig population, wherein the SNP site is the 11266105 nt site of chromosome 7 of the International Swine Genome Version 11.1 reference sequence, and has a C / A polymorphism, and the nucleotide sequence of the primer pair is shown in SEQ ID NO: 1 and SEQ ID NO: 2.