A SNP locus associated with the net wool rate of fine wool sheep and its application
By detecting the base types of specific SNP sites in the genome of fine wool sheep and identifying and selecting high-brown wool genotypes, the problem of difficulty in improving the net wool wool in the prior art is solved, and the effect of significantly improving the net wool and simplifying the breeding process is achieved.
Patent Information
- Application Number
- CN202311046511.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-18
- Publication Date
- 2025-03-14
- Estimated Expiration
- 2043-08-18
AI Technical Summary
The prior art is difficult to effectively increase the net wool ratio of fine wool, affecting the economic value of wool.
By detecting the base type of SNP molecular marker located at the 60455332 site of chromosome 5, Oar_v4.0 version of the International Sheep Genome, individuals with the AA genotype, are identified, and the AA genotype is preferred to improve net hair rate.
The net wool ratio of fine wool has been significantly improved, the breeding process has been simplified, the accuracy of variety selection has been improved, the breeding time has been shortened, and the cost has been reduced.
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Figure CN117025787B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular genetics, and in particular relates to a SNP site related to the net wool rate of fine wool sheep and an application thereof. Background Art
[0002] Wool refers to a layer of fiber with textile value covering the surface of the sheep's body. It is a derivative of the skin. Wool is the main raw material for fine spinning in the wool textile industry and is mainly used to process clothing fabrics and other products. In wool production and breeding practices, wool traits are important economic traits, mainly including wool production, average fiber diameter, breaking strength, net wool rate and other indicators. Among them, the net wool rate refers to the percentage of the weight of pure wool fiber under the conditions of the specified regain rate to the weight of the original wool sample, which is the most important economic indicator of wool. There are many factors that affect the net wool rate, such as breed, environment, nutritional level, age, gender and other factors can affect the net wool rate of wool, among which breed and environmental factors are the most important.
[0003] Fine-wool sheep are famous for producing high-quality wool. As a natural fiber, wool is an important agricultural product used in the processing of clothing and textiles. Among them, the quality of wool is determined by fiber diameter, fiber length, net wool rate, and oil and sweat color percentage. The wool traits of fine-wool sheep have always been the focus of artificial breeding. Among the many wool traits, the net wool rate is an important basis for calculating the weight and price of wool transactions in the field of wool circulation, and the net wool volume is used as the basis for measuring and counting the wool production of sheep flocks and individuals in sheep farming production. Therefore, the net wool rate of fine-wool sheep affects the economic value of wool.
[0004] The present invention has discovered a SNP molecular marker that affects the fine wool rate trait of fine wool sheep, which is located at the base of the 60455332 position on chromosome 5 of the international sheep genome Oar_v4.0 version, and the mutant base is A or G; when the SNP molecular marker base is A, the genotype is AA; when the SNP molecular marker base is G, the genotype is AG; the fine wool rate of fine wool sheep individuals with AA genotype is significantly higher than that of individuals with AG genotype (P<0.05). The present invention can be used for early breeding selection by detecting SNP sites related to the fine wool rate trait of fine wool sheep, can increase the genetic progress of the fine wool rate of fine wool sheep, has the advantages of simple operation, improves the accuracy of variety selection, reduces the breeding time of fine wool sheep with excellent fine wool rate traits, reduces breeding costs, and increases core competitiveness. Summary of the invention
[0005] The present invention provides a SNP molecular marker that affects the fine wool rate trait of fine wool sheep, and realizes the genotyping of the fine wool rate of fine wool sheep by detecting the base type of the SNP molecular marker. When the base of the SNP molecular marker is A, the genotype is AA; when the base of the SNP molecular marker is G, the genotype is AG; the fine wool rate of the genotype AA fine wool sheep is significantly greater than that of the genotype AG; by optimizing the dominant allele of the SNP molecular marker, the genetic progress of the fine wool rate of fine wool sheep can be increased, which has the advantages of simple operation, improves the accuracy of variety selection, reduces the breeding time of fine wool sheep with excellent traits of fine wool rate, reduces breeding costs, and increases core competitiveness. Specifically including the following contents:
[0006] In the first aspect, the present invention provides an application of a reagent for detecting a SNP molecular marker associated with the clean wool rate trait of fine wool sheep in detecting the clean wool rate of fine wool sheep, wherein the SNP molecular marker is located at the base at position 60455332 on chromosome 5 of the international sheep genome Oar_v4.0 version; the mutant base is A or G.
[0007] Preferably, when the SNP molecular marker base is A, the genotype is AA; when the SNP molecular marker base is G, the genotype is AG; the net wool rate of fine wool sheep with the genotype AA is significantly greater than that of the genotype AG.
[0008] Preferably, the reagents include a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.
[0009] Preferably, the sequence of the primer pair is:
[0010] F: 5'-TTAGTCCTTCACTTCCAATT-3';
[0011] R: 5'-AGTAAGAAGCAAGACACATT-3'.
[0012] Preferably, the method for detecting the net wool rate of fine wool sheep comprises:
[0013] (1) Extracting genomic DNA from the blood of fine-wool sheep as template DNA;
[0014] (2) using a primer pair to perform PCR amplification on the genomic DNA of the fine-wool sheep blood to be tested obtained in step (1) to obtain a PCR amplification product;
[0015] (3) Purifying the PCR amplification product obtained in step (2) and performing genotyping detection. When the SNP molecular marker base is A, the genotype is AA; when the SNP molecular marker base is G, the genotype is AG; wherein the net wool rate of fine wool sheep with genotype AA is significantly greater than that of genotype AG.
[0016] Preferably, the PCR amplification system is 25 μL: 22 μL of Gold Mix, 1 μL of upstream and downstream primers, and 1 μL of template DNA.
[0017] Preferably, the PCR amplification program is: 98°C for 2 min; 98°C for 10 s, 60°C for 10 s, 72°C for 10 s, for a total of 40 cycles; and extension at 72°C for 2 min.
[0018] In the second aspect, the present invention provides an application of a reagent for detecting SNP molecular markers related to the clean wool yield trait of fine wool sheep in early breeding of the clean wool yield trait of fine wool sheep, wherein the SNP molecular marker is located at the base at position 60455332 on chromosome 5 of the international sheep genome Oar_v4.0 version; the mutant base is A or G.
[0019] Preferably, when the SNP molecular marker base is A, the genotype is AA; when the SNP molecular marker base is G, the genotype is AG; the net wool rate of fine wool sheep with the genotype AA is significantly greater than that of the genotype AG.
[0020] Preferably, the reagents include a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.
[0021] Preferably, the sequence of the primer pair is:
[0022] F: 5'-TTAGTCCTTCACTTCCAATT-3';
[0023] R: 5'-AGTAAGAAGCAAGACACATT-3'.
[0024] Preferably, the method for achieving fine wool sheep clean wool rate trait breeding comprises:
[0025] (1) Extracting genomic DNA from the blood of fine-wool sheep as template DNA;
[0026] (2) using a primer pair to perform PCR amplification on the genomic DNA of the fine-wool sheep blood to be tested obtained in step (1) to obtain a PCR amplification product;
[0027] (3) Purifying the PCR amplification product obtained in step (2) and performing genotyping detection; when the SNP molecular marker base is A, the genotype is AA; when the SNP molecular marker base is G, the genotype is AG; wherein the fine wool rate of fine wool sheep with genotype AA is significantly greater than that of genotype AG; and selecting fine wool sheep individuals with genotype AA for early breeding of fine wool sheep for the fine wool rate trait.
[0028] Preferably, the PCR amplification system is 25 μL: 22 μL of Gold Mix, 1 μL of upstream and downstream primers, and 1 μL of template DNA.
[0029] Preferably, the PCR amplification program is: 98°C for 2 min; 98°C for 10 s, 60°C for 10 s, 72°C for 10 s, for a total of 40 cycles; and extension at 72°C for 2 min.
[0030] The beneficial effects of the present invention are as follows: ① The present invention provides a SNP molecular marker affecting the net wool rate of fine-wool sheep, wherein the SNP molecular marker is located at the base of the 60455332 position on chromosome 5 of the international sheep genome Oar_v4.0 version, and the mutant base is A or G; when the SNP molecular marker base is A, the genotype is AA, and when the SNP molecular marker base is G, the genotype is AG; the net wool rate of fine-wool sheep individuals with AA genotype is significantly higher than that of individuals with AG genotype (P<0.05); ② By detecting the SNP sites related to the net wool rate of fine-wool sheep, it is possible to judge the net wool rate of fine-wool sheep. The net wool rate of sheep provides a basis for molecular marker-assisted breeding of fine-wool sheep, which can strengthen the early selection of fine-wool sheep, improve the accuracy of selection, shorten the breeding cycle, and accelerate the breeding process; ③ Through the specific primer pairs, an efficient and accurate molecular marker-assisted breeding technology can be established. By optimizing the dominant alleles of the SNP molecular marker, the genetic progress of the net wool rate of fine-wool sheep can be increased. It has the advantages of simple operation, improves the accuracy of variety selection, reduces the breeding time of fine-wool sheep's fine wool rate excellent traits, reduces breeding costs, increases core competitiveness, and has potential application value in large-scale molecular precision breeding of fine-wool sheep. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 PCR amplification results;
[0032] Figure 2 The genotyping results were obtained after the PCR products were purified and sequenced. DETAILED DESCRIPTION
[0033] The technical scheme of the present invention is described in detail below in conjunction with embodiments. It should be noted that the following embodiments are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.
[0034] Unless otherwise specified, all experimental methods in the following examples are conventional methods.
[0035] Unless otherwise specified, the experimental conditions of all experiments in the following examples are conventional conditions, such as Sambrook et al. Molecular cloning laboratory manual (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or the conditions recommended by the manufacturer's instructions.
[0036] The SNP described in the present invention is the abbreviation of single nucleotide polymorphism, which refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level.
[0037] Example 1 Correlation between different genotypes of fine wool sheep and net wool rate
[0038] 1. Sample Collection
[0039] Blood samples were collected from 383 fine-wool sheep with production records. 5 ml of blood was collected from each sheep's vein and placed in a blood collection tube with EDTA-K2 anticoagulant. After being fully mixed, it was temporarily stored in a sampling box containing ice packs. After being transported back to the laboratory, it was frozen in a -20℃ refrigerator for DNA extraction. The test samples came from the Gansu Sheep Breeding Technology Promotion Station.
[0040] 2. Main reagents and instruments
[0041] EDTA-K2 vacuum blood collection tubes were purchased from Jiangsu Yuli Medical Instrument Co., Ltd.; blood genome extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; NanoDrop2000 spectrophotometer was purchased from Thermo Fisher Scientific, USA; DL2000 Marker, agarose, and nucleic acid dye were all purchased from Beijing Solebow Technology Co., Ltd.; Gold Mix (green) was purchased from Beijing Qingke Biotechnology Co., Ltd.; electrophoresis instrument was purchased from Beijing Liuyi Instrument Factory; and PCR instrument was purchased from BioRad.
[0042] 3. Methods
[0043] 3.1 Extraction of genomic DNA from blood
[0044] The blood genome extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract DNA from the collected blood samples. The extracted DNA was placed under an ultraviolet spectrophotometer to detect its concentration and purity. The concentration was >20ng / μL and OD 260 / OD 280 A value between 1.7 and 1.9 meets the experimental requirements and can be stored at -20°C for future use.
[0045] 3.2 Primer design
[0046] Referring to the gene sequence of chromosome 5 of the international sheep genome Oar_v4.0 version (GenBank accession number: NC_019462.2, SNP marker located at position 60455332), a pair of specific primers were designed using Primer Premier5.0 software.
[0047] Primer sequences:
[0048] F: 5'-TTAGTCCTTCACTTCCAATT-3' (SEQ ID NO. 1);
[0049] R: 5'-AGTAAGAAGCAAGACACATT-3' (SEQ ID NO. 2);
[0050] The length of the amplified fragment was 156 bp, and the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.
[0051] 3.3 PCR amplification and sequencing
[0052] PCR amplification system 25 μL: Gold Mix (green) 22 μL, upstream and downstream primers 1 μL each, template 1 μL.
[0053] PCR amplification program: 98°C for 2 min; 94°C for 10 s, 56°C for 10 s, 72°C for 10 s, for a total of 30 cycles; extension at 72°C for 1 min.
[0054] The PCR product was detected by 1.5% agarose gel electrophoresis. After the PCR product was qualified by agarose gel electrophoresis, it was sequenced by direct sequencing. The amplified nucleotide sequences are shown in SEQ ID NOs. 3 and 4. The SNP marker is located at position 89 of the nucleotide sequences shown in SEQ ID NOs. 3 and 4.
[0055] Sequencing was completed by Beijing Qingke Biotechnology Co., Ltd. Bioanalysis software was used to compare the sequencing results of PCR products, analyze the sequencing peak graph, and complete the typing.
[0056] 3.4 Statistical analysis
[0057] According to the genotyping results, the number of individuals with different genotypes at each site was counted. The gene frequency, genotype frequency, effective allele number (Ne), site heterozygosity (He), and Hardy-Weinberg equilibrium test of the SNP site were calculated using Popgen32 software, and the polymorphic information content was calculated using PIC calculation software. The general linear model in IBM SPSS Statistics 22 software was used to analyze the association between different genotypes of fine-wool sheep and net wool rate, and the results were expressed as "mean ± standard error".
[0058] 4. Results
[0059] 4.1 PCR amplification and sequencing results
[0060] The amplified product of the SNP site on chromosome 5 of fine-wool sheep was detected by 1.5% agarose gel (see Figure 1 ), the bands were clear without any other bands, the specificity was good, and the size of the PCR product fragment was 156 bp, which was in line with the expected size, so the next step of the experiment could be carried out.
[0061] The peak diagram and sequence obtained after the PCR product was purified and sequenced are shown in Figure 2 It can be seen that the SNP site undergoes an A / G mutation, and there are two genotypes, AA and AG.
[0062] 4.2 Statistical analysis results
[0063] The genotype and allele frequencies of the SNP locus of chromosome 5 of fine-wool sheep were analyzed from the perspective of population genetics. As shown in Table 1, at the SNP locus, the AA genotype frequency is the highest, which is the dominant genotype, and the A allele frequency is 95.0%, which is a dominant allele. The χ2 fitness test shows that the SNP locus is in Hardy-Weinberg equilibrium (P>0.05) (Table 1). The expected heterozygosity of the locus is 0.1, and the polymorphism information content (PIC) is 0.09, which is a low degree of polymorphism.
[0064] Table 1 Polymorphism of SNP locus 60455332 on chromosome 5 of fine-wool sheep
[0065]
[0066] 4.3 Association analysis between different genotypes of fine wool sheep and net wool rate
[0067] The general linear model in IBM SPSS Statistics 22 software was used to analyze the association between different genotypes of fine-wool sheep and the clean wool rate. The clean wool rate of fine-wool sheep with AA genotype was significantly greater than that of individuals with AG genotype (P<0.05). The clean wool rate of fine-wool sheep can be determined by detecting the bases of the SNP site of chromosome 5 of fine-wool sheep. The results are shown in Table 2.
[0068] Table 2 Correlation analysis between different genotypes of fine wool sheep and net wool rate
[0069]
[0070] Note: Data in the same row marked with different lowercase letters indicate significant differences (p < 0.05).
[0071] In summary, the SNP molecular marker described in the present invention is located at the 60455332nd base on chromosome 5 of the international sheep reference genome Oar_v4.0 version; the variation type is A / G, and there are two genotypes. When the 60455332nd base on chromosome 5 is A, the genotype is AA; when the 60455332nd base on chromosome 5 is G, the genotype is AG; through the association analysis of different genotypes and net wool rate, it was found that the net wool rate of fine-wool sheep with AG genotype was significantly lower than that of AA genotype individuals, P<0.05.
[0072] The above results show that by detecting the bases at the 60455332nd nucleotide site on chromosome 5 of fine-wool sheep, the fine-wool sheep's net wool rate can be determined, providing a basis for molecular marker-assisted breeding of fine-wool sheep's net wool rate. For early breeding selection of fine-wool sheep, it can achieve individual selection of the net wool rate trait that is easy to measure in the early stage, shorten the generation interval, accelerate the breeding process, save a lot of breeding costs, and provide technical support for the subsequent identification, seed conservation, and genetic improvement of fine-wool sheep.
[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. Use of a reagent for detecting SNP molecular markers associated with the fine wool rate trait in detecting the fine wool rate of fine wool sheep, characterized in that: The SNP molecular marker is located at the base at position 60455332 on chromosome 5 of the international sheep genome Oar_v4.0 version; the mutant base is A or G; when the SNP molecular marker base is A, the genotype is AA; when the SNP molecular marker base is G, the genotype is AG; the net wool rate of fine wool sheep with genotype AA is significantly greater than that of genotype AG; the reagent includes a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.
2. The use of a reagent for detecting SNP molecular markers associated with the fine wool rate trait in early breeding of the fine wool rate trait of fine wool sheep, characterized in that: The SNP molecular marker is located at the base at position 60455332 on chromosome 5 of the international sheep genome Oar_v4.0 version; the mutant base is A or G; when the SNP molecular marker base is A, the genotype is AA; when the SNP molecular marker base is G, the genotype is AG; the net wool rate of fine wool sheep with genotype AA is significantly greater than that of genotype AG; the reagent includes a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.
3. The use according to claim 1 or 2, characterized in that: The sequences of the primer pairs are: F: 5'-TTAGTCCTTCACTTCCAATT-3'; R: 5'-AGTAAGAAGCAAGACACATT-3'.
4. The use according to claim 3, characterized in that Methods for detecting the net wool rate of fine wool sheep include: (1) Extracting genomic DNA from the blood of fine-wool sheep as template DNA; (2) using a primer pair to perform PCR amplification on the genomic DNA of the fine-wool sheep blood to be tested obtained in step (1) to obtain a PCR amplification product; (3) Purifying the PCR amplification product obtained in step (2) and performing genotyping detection. When the SNP molecular marker base is A, the genotype is AA; when the SNP molecular marker base is G, the genotype is AG; wherein the net wool rate of fine wool sheep with genotype AA is significantly greater than that of genotype AG.
5. The use according to claim 3, characterized in that Methods for achieving early breeding of fine wool sheep for wool yield traits include: (1) Extracting genomic DNA from the blood of fine-wool sheep as template DNA; (2) using a primer pair to perform PCR amplification on the genomic DNA of the fine-wool sheep blood to be tested obtained in step (1) to obtain a PCR amplification product; (3) Purifying the PCR amplification product obtained in step (2) and performing genotyping detection; when the SNP molecular marker base is A, the genotype is AA; when the SNP molecular marker base is G, the genotype is AG; wherein the fine wool rate of the fine wool sheep with the genotype AA is significantly greater than that of the genotype AG, and the fine wool sheep individuals with the genotype AA are selected for early breeding of the fine wool sheep clean wool rate trait.
6. The use according to claim 4 or 5, characterized in that The PCR amplification system 25 μL: premix 22 μL, upstream and downstream primers 1 μL each, template DNA 1 μL.
7. The use according to claim 4 or 5, characterized in that The PCR amplification program was as follows: 98°C for 2 min; 98°C for 10 s, 60°C for 10 s, and 72°C for 10 s, for a total of 40 cycles; and extension at 72°C for 2 min.