CN117025787A - SNP locus related to fine wool sheep net wool rate and application thereof - Google Patents
SNP locus related to fine wool sheep net wool rate and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular genetics, and particularly relates to an SNP locus related to the fine wool sheep wool cleaning rate and application thereof. The SNP molecular marker is located on the 60455332 nucleotide site A/G mutation on the No. 5 chromosome of the International sheep reference genome oar_v4.0 version. The invention also provides application of the reagent for detecting the SNP locus in detecting the wool purifying rate character of the fine wool sheep or in molecular marker assisted breeding of the fine wool sheep, and has great application value in fine wool sheep variety screening, variety identification or molecular marker assisted breeding; the method is used for early breeding selection, shortens the generation interval, accelerates the breeding process, saves a great deal of breeding cost and has wide market prospect.
Description
Technical Field
The invention belongs to the technical field of molecular genetics, and particularly relates to an SNP locus related to the fine wool sheep wool cleaning rate and application thereof.
Background
Wool is a layer of textile-value fiber which is covered on the surface of sheep body and is a derivative of skin. Wool is the main worsted raw material in the wool spinning industry and is mainly used for processing products such as clothing fabrics and the like. In wool production and breeding practice, wool traits are important economic traits, and mainly comprise a plurality of indexes such as wool yield, average fiber diameter, breaking strength, wool cleaning rate and the like. The net wool rate is the weight percentage of the pure wool fiber in the original wool sample weight under the condition of the definite moisture regain, and is the most important economic index of wool. Factors affecting the wool yield are numerous, such as variety, environment, nutrient level, age, sex, etc., which are the most dominant factors.
Fine wool sheep is known for its production of quality wool, which is a natural fiber, an important agricultural product for processing clothing and textiles. The quality of wool is determined by, among other things, fiber diameter, fiber length, net hair rate, and percentage of oil perspiration color. The wool character of the fine wool sheep is always the key point of artificial breeding. In many wool traits, the net wool rate is an important basis for calculating weight and price of wool transactions in the field of wool circulation, and the net wool rate is based on the net wool rate when determining and counting wool production of flocks and individuals in the sheep industry production. Therefore, the net wool rate of the fine wool sheep affects the economic value of wool.
The invention discovers a SNP molecular marker affecting the wool-cleaning rate property of the fine wool sheep, which is positioned at the base of the 60455332 locus on chromosome 5 of the international sheep genome oar_v4.0 version, and the mutation 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 individuals with fine wool of the AA genotype is significantly higher than that of individuals with AG genotype (P < 0.05). The invention can be used for selecting and retaining in early breeding stage by detecting SNP locus related to the fine wool yield character, can increase the genetic progress of the fine wool yield, has the advantage of simple operation, improves the precision of variety selection, reduces the breeding time of fine wool yield excellent character, reduces the breeding cost and increases core competition.
Disclosure of Invention
The invention provides an SNP molecular marker affecting the wool purifying rate character of a fine wool sheep, and the wool purifying rate genotyping of the fine wool sheep is realized by detecting the base type of the SNP molecular marker, wherein when the base of the SNP molecular marker is A, the genotype is AA; when the SNP molecular marker base is G, the genotype is AG; the net wool rate of the fine wool sheep of the genotype AA is obviously higher than that of the genotype AG; the genetic progress of the fine wool sheep wool-cleaning rate can be increased by optimizing the dominant allele of the SNP molecular marker, the method has the advantages of simplicity in operation, accuracy in variety screening is improved, breeding time of fine wool sheep wool-cleaning rate excellent characters is shortened, breeding cost is reduced, and core competition is increased. The method specifically comprises the following steps:
in a first aspect, the invention provides an application of a reagent for detecting SNP molecular markers related to the wool-cleaning rate property of the fine wool sheep, wherein the SNP molecular markers are positioned at the base of the 60455332 site on chromosome 5 of the international sheep genome oar_v4.0 version; the mutant base is A or G.
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 the fine wool sheep of genotype AA is significantly greater than that of genotype AG.
Preferably, the reagents comprise a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.
Preferably, the primer pair has the sequence:
F：5’-TTAGTCCTTCACTTCCAATT-3’；
R：5’-AGTAAGAAGCAAGACACATT-3’。
preferably, the method for detecting the net wool rate of the fine wool sheep comprises the following steps:
(1) Extracting the blood genome DNA of the fine wool sheep as a template DNA;
(2) Carrying out PCR amplification on the genome DNA of the blood of the fine wool sheep to be detected obtained in the step (1) by using a primer pair to obtain a PCR amplification product;
(3) Purifying the PCR amplification product obtained in the step (2), and carrying out genotyping detection, wherein 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 the fine wool sheep of genotype AA is significantly greater than that of genotype AG.
Preferably, the PCR amplification system is 25 μl: gold medal Mix 22. Mu.L, 1. Mu.L each of the upstream and downstream primers, and 1. Mu.L of template DNA.
Preferably, the PCR amplification procedure: 98 ℃ for 2min;98 ℃ for 10s,60 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
In a second aspect, the invention provides an application of a reagent for detecting SNP molecular markers related to the fine wool sheep net hair rate character in early breeding of the fine wool sheep net hair rate character, wherein the SNP molecular markers are positioned at the base of the 60455332 site on chromosome 5 of the international sheep genome oar_v4.0 version 5; the mutant base is A or G.
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 the fine wool sheep of genotype AA is significantly greater than that of genotype AG.
Preferably, the reagents comprise a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.
Preferably, the primer pair has the sequence:
F：5’-TTAGTCCTTCACTTCCAATT-3’；
R：5’-AGTAAGAAGCAAGACACATT-3’。
preferably, the method for realizing the fine wool sheep net wool rate character breeding comprises the following steps:
(1) Extracting the blood genome DNA of the fine wool sheep as a template DNA;
(2) Carrying out PCR amplification on the genome DNA of the blood of the fine wool sheep to be detected obtained in the step (1) by using a primer pair to obtain a PCR amplification product;
(3) Purifying the PCR amplification product obtained in the step (2), and carrying out genotyping detection, wherein 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 the fine wool sheep of the genotype AA is obviously larger than that of the genotype AG; and selecting the fine wool sheep individuals with the genotype AA to carry out fine wool sheep net wool rate character early breeding.
Preferably, the PCR amplification system is 25 μl: gold medal Mix 22. Mu.L, 1. Mu.L each of the upstream and downstream primers, and 1. Mu.L of template DNA.
Preferably, the PCR amplification procedure: 98 ℃ for 2min;98 ℃ for 10s,60 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
The beneficial effects of the invention are as follows: (1) the invention provides an SNP molecular marker affecting the fuzzing rate of fine wool sheep, wherein the SNP molecular marker is positioned at the base of the 60455332 locus 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 the fine wool sheep individuals with the AA genotype is significantly higher than that of the fine wool sheep individuals with the AG genotype (P < 0.05); (2) the method has the advantages that through detecting SNP loci related to the fine wool sheep net wool rate, the fine wool sheep net wool rate can be judged, a basis is provided for fine wool sheep molecular marker assisted breeding, the fine wool sheep early selection can be enhanced, the seed selection accuracy is improved, the cultivation period is shortened, and the cultivation process is accelerated; (3) through the specific primer pair, a high-efficiency and accurate molecular marker assisted breeding technology can be established, the genetic progress of the fine wool sheep net wool rate can be increased through optimizing the dominant allele of the SNP molecular marker, the method has the advantages of simplicity in operation, the precision of variety screening is improved, the breeding time of fine wool sheep net wool rate excellent characters is shortened, the breeding cost is reduced, the core competition is increased, and the method has potential application value in large-scale molecular accurate breeding of fine wool sheep.
Drawings
FIG. 1PCR amplification results;
FIG. 2 genotyping results obtained after purification and sequencing of the PCR products.
Detailed Description
The following describes the technical scheme of the present invention in detail by referring to examples. It should be noted that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
All the experimental methods of the experiments in the following examples are conventional methods unless otherwise specified.
The experimental conditions for all experiments in the examples described below are conventional, unless otherwise specified, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 2001), or as recommended by the manufacturer's instructions.
The SNP is short for single nucleotide polymorphism, and refers to DNA sequence polymorphism caused by single nucleotide variation at genome level.
EXAMPLE 1 correlation between different genotypes and the net wool Rate of Fine wool sheep
1. Sample collection
And (3) collecting 383 fine wool sheep blood samples with production records, taking 5ml of blood from each sheep vein, placing the blood into a blood taking tube added with EDTA-K2 anticoagulant, fully and uniformly mixing, placing the blood into a sampling box containing an ice bag for temporary storage, transporting the blood back to a laboratory, and placing the blood into a refrigerator at the temperature of minus 20 ℃ for freezing and preserving the blood for DNA extraction. The test sample comes from a sheep breeding technology popularization station in Gansu province.
2. Main reagent and instrument
EDTA-K2 vacuum blood collection tubes were purchased from Jiangsu Yuli medical instruments Co., ltd; blood genome extraction kit was purchased from tiangen biochemical technology (beijing) limited; nanoDrop2000 spectrophotometer us Thermo Fisher Scientific company; DL2000 Marker, agarose, nucleic acid dye were all purchased from Beijing Soy Bao technology Co., ltd; gold medal Mix (green) was purchased from beijing engine biotechnology limited; the electrophoresis apparatus is purchased from Beijing Liuyi instrument factory; PCR instrument was purchased from BioRad corporation.
3. Method of
3.1 extraction of genomic DNA from blood
Extraction of DNA from blood samples collected using a blood genome extraction kit from Tiangen Biochemical technology (Beijing) Co., ltdTaking, placing the extracted DNA under ultraviolet spectrophotometer to detect its concentration and purity, and concentrating>20ng/μL、OD 260 /OD 280 The experimental requirements are met when the temperature is between 1.7 and 1.9, and the mixture is stored at the temperature of minus 20 ℃ for standby.
3.2 primer design
A pair of specific primers was designed by using Primer premier5.0 software with reference to the international sheep genome oar_v4.0 version No. 5 chromosome gene sequence (GenBank accession number: NC_019462.2, SNP marker at position 60455332).
Primer sequence:
F：5’-TTAGTCCTTCACTTCCAATT-3’(SEQ ID NO.1)；
R：5’-AGTAAGAAGCAAGACACATT-3’(SEQ ID NO.2)；
the length of the amplified fragment is 156bp, and the primer is synthesized by Beijing qing biological technology Co.
3.3PCR amplification and sequencing
PCR amplification System 25. Mu.L: gold medal Mix (green) 22. Mu.L, 1. Mu.L each for the upstream and downstream primers, and 1. Mu.L for the template.
PCR amplification procedure: 98 ℃ for 2min;94 ℃ for 10s,56 ℃ for 10s and 72 ℃ for 10s, and 30 cycles are carried out; extending at 72℃for 1min.
And detecting the PCR product by using 1.5% agarose gel electrophoresis, sequencing the PCR product by using a direct sequencing method after the PCR product is qualified by using the agarose gel electrophoresis, wherein the amplified nucleotide sequences are shown as SEQ ID NO.3 and SEQ ID NO. 4, and the SNP marker is positioned at 89 positions of the nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO. 4.
Sequencing was done by Beijing Optimu Biotechnology Co. Comparing the sequencing results of the PCR products by using biological analysis software, and analyzing a sequencing peak diagram to finish typing.
3.4 statistical analysis
And counting the number of individuals with different genotypes at each site according to the genotyping result. The gene frequency, genotype frequency, effective allele factor (Ne), site heterozygosity (He) and Hardy-Weinberg equilibrium test of the SNP locus are calculated by using Popgen32 software, and the polymorphism information content is calculated by using PIC calculation software. The correlation of different genotypes of the fine wool sheep and the net wool rate is analyzed by adopting a general linear model in IBM SPSS Statistics software, and the result is expressed as an average value +/-standard error.
4. Results
4.1PCR amplification and sequencing results
The SNP locus amplification product of the chromosome 5 of the fine wool sheep is detected by using 1.5% agarose gel (see figure 1), the stripe is clear, the band is free, the specificity is good, the size of the PCR product fragment is 156bp and accords with the expected size, and the next experiment can be carried out.
The peak diagram and the sequence obtained after the PCR product is purified and sequenced are shown in figure 2, wherein the SNP locus generates A/G mutation, and the genotypes of AA and AG exist.
4.2 statistical analysis results
The genotype and allele frequency of the SNP locus of chromosome 5 of the fine-wool sheep were analyzed from the perspective of population genetics. As can be seen from Table 1, the AA genotype frequency was highest at the SNP site, and was the dominant genotype, and the A allele frequency was 95.0%, which was expressed as the dominant allele. The SNP site was shown to be in Hardy-Weinberg equilibrium (P > 0.05) by X2 fitness test (Table 1). The expected heterozygosity of the locus is 0.1, the polymorphism information content (Polymorphism Information Content, PIC for short) is 0.09, and the locus belongs to low-degree polymorphism.
TABLE 1 polymorphism of chromosome 5 SNP locus 60455332 of fine wool sheep
4.3 correlation analysis of different genotypes and Net wool Rate of Fine wool sheep
The correlation of the different genotypes of the fine wool sheep and the net hair rate is analyzed by adopting a general linear model in IBM SPSS Statistics software, and the net hair rate of the fine wool sheep with the AA genotype is obviously higher than that of an AG genotype individual (P < 0.05). The net wool rate of the fine wool sheep can be judged by detecting the base of the SNP locus of chromosome 5 of the fine wool sheep, and the result is shown in Table 2.
TABLE 2 correlation analysis between different genotypes and net wool Rate of Fine wool sheep
Note that: the same row of data is marked with different lowercase letters to indicate significant differences (p < 0.05).
In summary, the SNP molecular marker is positioned at 60455332 bases of chromosome 5 of the international sheep reference genome oar_v4.0 version; the mutation type is A/G, two genotypes exist, and when 60455332 bases on the No. 5 chromosome are A, the genotypes are AA; when 60455332 bases on chromosome 5 are G, the genotype is AG; through correlation analysis of different genotypes and the net hair rate, the net hair rate of the fine wool sheep of the AG genotype is found to be significantly smaller than that of an AA genotype individual, and P is found to be less than 0.05.
The result shows that the fuzzing rate can be judged by detecting the base of the 60455332 nucleotide site on the chromosome 5 of the fuzzing sheep, and a basis is provided for molecular marker assisted breeding of the fuzzing rate. The method is used for the early breeding selection of the fine wool sheep, can realize individual selection of the net wool rate character which is difficult to measure in early stage, shortens the generation interval, accelerates the breeding process, saves a great deal of breeding cost, and provides technical support for the identification, the seed conservation, the genetic improvement and the like of the subsequent fine wool sheep.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. The application of the reagent for detecting the SNP molecular marker related to the wool-cleaning rate character of the fine wool sheep is characterized in that the SNP molecular marker is positioned at the base of the 60455332 locus on the chromosome 5 of the International sheep genome oar_v4.0 version; the mutant base is A or G.
2. The application of the reagent for detecting the SNP molecular marker related to the wool-cleaning rate character of the fine wool sheep in the early breeding of the wool-cleaning rate character of the fine wool sheep is characterized in that the SNP molecular marker is positioned at the base of the 60455332 locus on the chromosome 5 of the international sheep genome oar_v4.0 version; the mutant base is A or G.
3. The use according to claim 1 or 2, wherein 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 the fine wool sheep of genotype AA is significantly greater than that of genotype AG.
4. The use according to claim 3, wherein the reagent comprises a primer pair for amplifying a nucleotide sequence containing the SNP molecular marker.
5. The use of claim 4, wherein the primer pair has the sequence:
F：5’-TTAGTCCTTCACTTCCAATT-3’；
R：5’-AGTAAGAAGCAAGACACATT-3’。
6. the use according to claim 5, wherein the method for detecting the net wool rate of a fine wool sheep comprises:
(1) Extracting the blood genome DNA of the fine wool sheep as a template DNA;
(2) Carrying out PCR amplification on the genome DNA of the blood of the fine wool sheep to be detected obtained in the step (1) by using a primer pair to obtain a PCR amplification product;
(3) Purifying the PCR amplification product obtained in the step (2), and carrying out genotyping detection, wherein 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 the fine wool sheep of genotype AA is significantly greater than that of genotype AG.
7. The use according to claim 5, wherein the method for achieving early breeding of the net wool rate trait of fine wool sheep comprises:
(1) Extracting the blood genome DNA of the fine wool sheep as a template DNA;
(2) Carrying out PCR amplification on the genome DNA of the blood of the fine wool sheep to be detected obtained in the step (1) by using a primer pair to obtain a PCR amplification product;
(3) Purifying the PCR amplification product obtained in the step (2), and carrying out genotyping detection, wherein 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 the fine wool sheep with the genotype AA is obviously larger than that of the genotype AG, and the fine wool sheep individual with the genotype AA is selected for early breeding of the net wool rate character of the fine wool sheep.
8. The use according to claim 6 or 7, wherein the PCR amplification system is 25 μl: gold medal Mix 22. Mu.L, 1. Mu.L each of the upstream and downstream primers, and 1. Mu.L of template DNA.
9. The use according to claim 6 or 7, wherein the PCR amplification procedure: 98 ℃ for 2min;98 ℃ for 10s,60 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
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