CN117987569B - Molecular marker related to immune traits and used as auxiliary selection of Tibetan sheep marker and application thereof - Google Patents
Molecular marker related to immune traits and used as auxiliary selection of Tibetan sheep marker and application thereof Download PDFInfo
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention provides a molecular marker related to immune traits and used as auxiliary selection of Tibetan sheep markers and application thereof, belonging to the technical field of molecular biological detection. The invention provides an SNP locus related to Tibetan sheep immune traits, which is positioned at 7098151 th base of chromosome 22 of the international sheep genome oar_v4.0 version. According to the scheme of the invention, compared with the traditional detection method, the method for detecting the Tibetan sheep immune trait by utilizing the SNP locus and the molecular marker based on the locus has the advantages of high accuracy, high detection speed, low cost, easy judgment of the result and the like, and provides a new SNP molecular marker resource for auxiliary selection of the Tibetan sheep immune trait marker for non-diagnosis purposes.
Description
The invention claims the priority of Chinese patent with the application number 2024101064983 and the application date 2024, 01 and 25, and the name of 'a molecular marker related to immune characteristics and application thereof as auxiliary selection of Tibetan sheep marker'.
Technical Field
The invention relates to the technical field of molecular biological detection, in particular to a molecular marker which is used as auxiliary selection of Tibetan sheep markers and is related to immune traits and application thereof.
Background
The Tibetan sheep is one of the main domestic animal varieties of the Qinghai-Tibet plateau in China, is also an important component part of grassland animal husbandry in the high-cold pasture area of the Qinghai-Tibet plateau, is a precious livestock genetic resource for producing high-quality carpet wool and organic green meat foods, and has extremely strong adaptability to severe natural environments such as high altitude, low air pressure, strong ultraviolet rays, hypoxia, cold season nutritional stress and the like, and is formed under specific natural conditions and by careful breeding of herders. The Tibetan sheep has the characteristics of large population quantity, wide distribution, unique biological characteristics, good meat performance, excellent carpet wool quality and the like, provides production and living data such as meat, wool, fuel and the like for Tibetan people, is a symbol of wealth, and is a very precious gene treasury in inheritance.
At present, the problems of insufficient breeding, small group size, difficulty in meeting market demands and the like still exist in production traits such as Tibetan sheep fertility, disease resistance and the like in China, and the construction of a breeding technology system is still relatively lagged, so that the problems are one of key factors influencing sustainable development of Tibetan sheep industry in China. Therefore, there is an urgent need to obtain a Tibetan sheep breeding method, so as to develop and establish a modern biological breeding technology system and accelerate the genetic improvement and new variety cultivation progress of Tibetan sheep.
Disclosure of Invention
The invention aims to provide a molecular marker related to immune characteristics, which is used as auxiliary selection of Tibetan sheep markers, and application thereof, so as to solve the problems of insufficient Tibetan sheep breeding and weak disease resistance in the prior art.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides an SNP locus related to Tibetan sheep immune traits, which is positioned at 7098151 th base of chromosome 22 of the international sheep genome oar_v4.0 version;
The variation type of the SNP locus is G/A, and when the base of the SNP locus is G, the genotype is GG or GA; when the base of the SNP locus is A, the genotype is AA.
Preferably, igA, igG, igM of the Tibetan sheep individual is significantly higher than genotype GG when genotype is AA, igA, igG, igM of the Tibetan sheep individual is significantly higher than genotype GG when genotype is GA, and IgA and IgG of the Tibetan sheep individual are significantly higher than genotype GA when genotype is AA.
The invention provides application of the SNP locus in preparation of products related to Tibetan sheep immune traits.
The invention provides an SNP molecular marker related to Tibetan sheep immune traits, wherein the nucleic acid sequence of the SNP molecular marker is shown as SEQ ID No. 1;
the SNP molecular marker comprises the SNP locus, and the SNP locus is positioned at the 262 th position of the nucleotide sequence shown in SEQ ID No. 1.
The invention provides application of the SNP molecular marker in preparation of products related to Tibetan sheep immune traits.
The invention also provides a detection method related to Tibetan sheep immune traits, which comprises the following steps:
(1) Extracting Tibetan sheep genome DNA;
(2) Carrying out PCR amplification on the genome DNA obtained in the step (1) by utilizing a specific primer pair to obtain a PCR amplification product;
(3) Genotyping the PCR amplification product obtained in the step (2), wherein when the base of the SNP locus is G, the genotype is GG or GA; when the base of the SNP locus is A, the genotype is AA.
Preferably, the PCR amplification system comprises 25. Mu.L of gold plate Mix (green) 22. Mu.L, 1. Mu.L of each of the upstream and downstream primers, and 1. Mu.L of genomic DNA.
Preferably, the PCR amplification procedure is 2min at 98 ℃;98 ℃ for 10s,56 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
Preferably, the nucleotide sequence of the specific primer pair is shown as SEQ ID No. 2-3.
The invention also provides application of the detection method in preparing Tibetan sheep assisted breeding products.
The invention has the following technical effects and advantages:
The invention provides a molecular marker related to immune traits and an application thereof, wherein the molecular marker is used as auxiliary selection of Tibetan sheep markers. According to the scheme of the invention, compared with the traditional detection method, the method for detecting the Tibetan sheep immune trait by utilizing the SNP locus and the molecular marker based on the locus has the advantages of high accuracy, high detection speed, low cost, easy judgment of the result and the like, and provides a new SNP molecular marker resource for auxiliary selection of the Tibetan sheep immune trait marker for non-diagnosis purposes.
Drawings
FIG. 1 is a gel electrophoresis diagram of PCR amplification products;
FIG. 2 is a peak diagram and a sequence diagram of PCR amplification products.
Detailed Description
The invention provides an SNP locus related to Tibetan sheep immune traits, which is positioned at 7098151 th base of chromosome 22 of the international sheep genome oar_v4.0 version;
The variation type of the SNP locus is G/A, and when the base of the SNP locus is G, the genotype is GG or GA; when the base of the SNP locus is A, the genotype is AA.
In the invention, igA, igG, igM of Tibetan sheep individuals is obviously higher than genotype GG when the genotype is AA, igA, igG, igM of Tibetan sheep individuals is obviously higher than genotype GG when the genotype is GA, and IgA and IgG of Tibetan sheep individuals are obviously higher than genotype GA when the genotype is AA.
The invention provides application of the SNP locus in preparation of products related to Tibetan sheep immune traits.
The invention provides an SNP molecular marker related to Tibetan sheep immune traits, wherein the nucleic acid sequence of the SNP molecular marker is shown as SEQ ID No. 1;
The sequence of the SEQ ID No.1 is ：ACTTAAAGAGAATGAGATAATGCCATTTGCTGAAACACAGATGAACCTAGAGGCTGTCATATCGAGTGGAATAAGTCAGACAGAGGAGAAGAAATATCAACTAACATCCCTTACACATTCAATCTAAAAAGAAATGACAGAAATGAAGTTACAAAACAGAAACAGACTCACAGTCTTAAAGAGCAAACTTCTGGTTGCTGGGTAGGAAAAATGAGGGGAAGGCATAGTTAAGGAGTTTGGGATGGACATGTACACACTGCTGTATTTCAAGGAGATGACCAACAAAGGTATACTGTATGGCACATGAAACTCTGGTCAATGTTATGTGGCAGCCTGGAAGGGAGGGGAGTTTGAGGGGAGAACACATACATGTATTTGTATGGCTGAGTCCCTTTGCTGTTCACCTGAAATTATTACAACATTGTTAATCAGTTATACCTTGGTAAAAAATGAATTTTTTTTATGTGCTTATTTAAGCAACTGTGGTCATTTCAAGAGATATTCTGCTTGATATTTTTTAGGAACCACAGGAATATTTTCTTGAGGC.
The SNP molecular marker comprises the SNP locus, and the SNP locus is positioned at the 262 th position of the nucleotide sequence shown in SEQ ID No. 1.
The invention provides application of the SNP molecular marker in preparation of products related to Tibetan sheep immune traits.
The invention also provides a detection method related to Tibetan sheep immune traits, which comprises the following steps:
(1) Extracting Tibetan sheep genome DNA;
(2) Carrying out PCR amplification on the genome DNA obtained in the step (1) by utilizing a specific primer pair to obtain a PCR amplification product;
(3) Genotyping the PCR amplification product obtained in the step (2), wherein when the base of the SNP locus is G, the genotype is GG or GA; when the base of the SNP locus is A, the genotype is AA.
In the invention, the PCR amplification system comprises 22 mu L of gold plate Mix (green), 1 mu L of each of the upstream and downstream primers and 1 mu L of genome DNA in 25 mu L.
In the invention, the PCR amplification procedure is 98℃for 2min;98 ℃ for 10s,56 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
In the invention, the nucleotide sequence of the specific primer pair is shown in SEQ ID No. 2-3:
F：5'-ACTTAAAGAGAATGAGATAATGCC-3'(SEQ ID No.2)；
R：5'-GCCTCAAGAAAATATTCCTGT-3'(SEQ ID No.3)。
the invention also provides application of the detection method in preparing Tibetan sheep assisted breeding products.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Examples
1. Sample collection
The sample is from Tibetan sheep group under natural grazing condition, wherein, gansu Gannan Tibetan autonomous state 40 parts, qinghai Gansu Jade Gangzhou autonomous state 45 parts, tibet autonomous region day-click market 43 parts, 128 fasting Tibetan sheep blood samples 5mL are collected in a clean procoagulant vacuum blood collection tube, kept stand for 30min, centrifuged for 15min at 3500r/min, the supernatant is sucked in a clean PE tube, and the sealed is put in a low-temperature refrigerator at-20 ℃; and collecting 5mL of blood sample in a blood collection tube added with EDTA-K2 anticoagulant, quickly and uniformly mixing the blood sample after the blood sample is collected, temporarily storing the blood sample in a sampling box containing an ice bag, and carrying the blood sample back to a laboratory for freezing and preserving the blood sample in a refrigerator at the temperature of minus 20 ℃ for DNA extraction.
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 Thermo FISHER SCIENTIFIC, inc;
DL2000 Marker, agarose, nucleic acid dye were all purchased from Beijing Soy Bao technology Co., ltd;
Gold medal Mix (green), purchased from beijing qingke biotechnology limited;
The electrophoresis apparatus is purchased from Beijing Liuyi instrument factory;
PCR instrument was purchased from BioRad corporation;
IgA, igG, igM the detection kit is purchased from Nanjing institute of biological engineering, igA detection kit is E027-1-1, igG detection kit is E026-1-1, igM detection kit is E025-1-1.
3. Detection method
3.1 Extraction of genomic DNA from blood
Extracting genome DNA from blood sample by blood genome extracting reagent kit, detecting concentration and purity of extracted DNA in spectrophotometer, screening out sample with concentration greater than 20 ng/. Mu. L, OD260/OD280 between 1.7-1.9, and storing at-20 deg.c for further use.
3.2 Primer design
A pair of specific primers was designed using PRIMERPREMIER 5.0.0 software using genomic DNA as a template.
Specific primer sequences:
F：5'-ACTTAAAGAGAATGAGATAATGCC-3'(SEQ ID No.2)；
R：5'-GCCTCAAGAAAATATTCCTGT-3'(SEQ ID No.3)。
the amplified fragment length is 547bp, and the primer is synthesized by Beijing qing biological science and technology Co.
3.3 PCR amplification, sequencing and typing
PCR amplification System 25. Mu.L: gold medal Mix (green) 22. Mu.L, 1. Mu.L each of the upstream and downstream primers, and 1. Mu.L of genomic DNA.
PCR amplification procedure: 98 ℃ for 2min;98 ℃ for 10s,56 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
The PCR product was detected by agarose gel electrophoresis of 1.5%, and after passing the detection, the PCR product was sequenced by Beijing qingke biosciences, inc.
And comparing the sequencing results of the PCR products by using biological analysis software MEGA 6.0, and analyzing a sequencing peak diagram to finish typing.
3.4IgA, igG, igM concentration detection
(1) Standard curves were established using standards.
(2) Distilled water, standard solution and a sample to be detected are respectively added into a blank tube, a standard tube and a measuring tube for 7 mu L respectively, then R1 solution is supplemented to 900 mu L, incubation is carried out for 5min at 37 ℃, the blank tube, the standard tube and the measuring tube are put into a spectrophotometer, the wavelength is adjusted to 340nm, and the reading number is recorded as A1; taking out the blank tube, standard tube and measuring tube, adding 180 μl of R2 solution into each test tube, incubating at 37deg.C for 5min, placing the blank tube, standard tube and measuring tube into spectrophotometer, adjusting wavelength to 340nm, and counting reading number as A2.
(3) Calculating DeltaA=A2-A1, and taking DeltaA into a standard curve equation to calculate the concentration of the sample IgA, igG, igM.
4. Statistical analysis
And counting the number of individuals with different genotypes at each site according to the genotyping result. The genetic frequency, genotype frequency, effective allele (Ne), site heterozygosity (He) and Hardy-Weinberg balance test of SNP sites are calculated by Popgen software, and the polymorphism information content is calculated by PIC (polymorphism information content, PIC for short) calculation software. The correlation of Tibetan sheep different genotypes with IgA, igG, igM was analyzed using a general linear model in IBM SPSS STATISTICS software, and the results were expressed as "mean ± standard error".
5. Results
5.1PCR amplification, sequencing and typing
The amplified product of the SNP locus of the No. 22 Tibetan sheep chromosome is detected by using 1.5% agarose gel, the result is shown in figure 1, the band is clear, the band is free from impurity, the specificity is good, the fragment size of the PCR product is 547bp, and the fragment size is the same as the size of the SNP molecular marker, thereby being in line with expectations.
The peak pattern and sequence obtained after the PCR product is purified and sequenced are shown in FIG. 2. As is clear from FIG. 2, the SNP site has G/A mutation, and three genotypes GG, GA and AA exist.
5.2 Statistical analysis results
Genotype and allele frequency of the Tibetan sheep chromosome 22 SNP site were analyzed from a population genetics perspective. As can be seen from Table 1, GG genotypes were most frequently seen at SNP sites, being dominant genotypes, and G alleles were 92.2% frequently seen as dominant alleles. The χ2 fitness test showed that the SNP site was significantly deviated from Hardy-Weinberg equilibrium (P < 0.05). The expected heterozygosity of the locus is 0.144, PIC is 0.134, PIC is less than 0.25, and the locus belongs to low-level polymorphism.
TABLE 1 Tibetan sheep chromosome 22 SNP site polymorphism
5.3 Correlation analysis of different genotypes with IgA, igG, igM
The correlation of different Tibetan sheep genotypes and IgA, igG, igM content is analyzed by adopting a general linear model in IBM SPSS STATISTICS software, and the result shows that the IgA and IgG of Tibetan sheep individuals with AA genotypes are obviously higher than those of Tibetan sheep individuals with GG and GA genotypes (p < 0.05), and the IgA and IgG of Tibetan sheep individuals with GA genotypes are obviously higher than those of Tibetan sheep individuals with GG genotypes (p < 0.05); the IgM of Tibetan sheep individuals with the GG genotype is significantly lower than that of Tibetan sheep individuals with the GA and AA genotypes (p < 0.05), and the IgM of the Tibetan sheep individuals with the GA and AA genotypes does not show a significant difference (p > 0.05), which indicates that the base of the Tibetan sheep No. 22 chromosome SNP locus is significantly related to Tibetan sheep IgA, igG, igM and can be a Tibetan sheep IgA, igG, igM related SNP molecular marker. The results are shown in Table 2.
TABLE 2 correlation analysis between different genotypes and IgA, igG, igM
In summary, the SNP locus is positioned at 7098151 th base on chromosome 22 of the international sheep reference genome oar_v4.0 version; the variation type is G/A, three genotypes exist, and when 7098151 th base on the 22 nd chromosome is G, the genotype is GG or GA; when 7098151 bases on the 22 nd chromosome are A, the genotype is AA; through correlation analysis of different genotypes and IgA, igG, igM content, the IgA and IgG of Tibetan sheep individuals with the AA genotype are found to be significantly higher than those of Tibetan sheep individuals with GG and GA genotypes (p < 0.05), and the IgA and IgG of Tibetan sheep individuals with the GA genotype are found to be significantly higher than those of Tibetan sheep individuals with the GG genotype (p < 0.05); the IgM of Tibetan sheep individuals of GG genotype was significantly lower than that of GA and AA genotype individuals (p < 0.05), and the IgM did not show significant differences between GA and AA genotype individuals (p > 0.05). By detecting the base at nucleotide position 7098151 on chromosome 22 of Tibetan sheep, the IgA, igG, igM content of Tibetan sheep individual can be judged.
From the above examples, the present invention provides a molecular marker related to immune traits as a marker-assisted selection of Tibetan sheep and its application. According to the scheme of the invention, compared with the traditional detection method, the method for detecting the Tibetan sheep immune trait by utilizing the SNP locus and the molecular marker based on the locus has the advantages of high accuracy, high detection speed, low cost, easy judgment of the result and the like, and provides a new SNP molecular marker resource for auxiliary selection of the Tibetan sheep immune trait marker for non-diagnosis purposes.
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 (2)
1. The application of a reagent for detecting SNP molecular markers related to Tibetan sheep immunity in preparing a product for detecting Tibetan sheep immunity is characterized in that the SNP locus is positioned at 7098151 th base of an international sheep genome oar_v4.0 version 22 chromosome;
The variation type of the SNP locus is G/A, and when the base of the SNP locus is G, the genotype is GG or GA; when the base of the SNP locus is A, the genotype is AA;
When the genotype is AA, igA, igG, igM of Tibetan sheep individuals is obviously higher than genotype GG, when the genotype is GA, igA, igG, igM of Tibetan sheep individuals is obviously higher than genotype GG, and when the genotype is AA, igA and IgG of Tibetan sheep individuals are obviously higher than genotype GA.
2. The application of a reagent for detecting SNP molecular markers related to Tibetan sheep immune traits in preparing a Tibetan sheep assisted breeding product with high immunity is characterized in that the SNP locus is positioned at 7098151 th base of international sheep genome oar_v4.0 version 22 chromosome;
The variation type of the SNP locus is G/A, and when the base of the SNP locus is G, the genotype is GG or GA; when the base of the SNP locus is A, the genotype is AA;
When the genotype is AA, igA, igG, igM of Tibetan sheep individuals is obviously higher than genotype GG, when the genotype is GA, igA, igG, igM of Tibetan sheep individuals is obviously higher than genotype GG, and when the genotype is AA, igA and IgG of Tibetan sheep individuals are obviously higher than genotype GA.
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| CN202410106498.3A CN117737260A (en) | 2024-01-25 | 2024-01-25 | Molecular marker related to immune traits and used as auxiliary selection of Tibetan sheep marker and application thereof |
| CN2024101064983 | 2024-01-25 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114214428A (en) * | 2021-12-16 | 2022-03-22 | 中国农业科学院兰州畜牧与兽药研究所 | SNP molecular marker influencing mohair shearing amount of alpine merino and application thereof |
| CN114752680A (en) * | 2022-04-14 | 2022-07-15 | 中国农业科学院兰州畜牧与兽药研究所 | SNP marker influencing diameter of alpine merino sheep wool fiber and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114214428A (en) * | 2021-12-16 | 2022-03-22 | 中国农业科学院兰州畜牧与兽药研究所 | SNP molecular marker influencing mohair shearing amount of alpine merino and application thereof |
| CN114752680A (en) * | 2022-04-14 | 2022-07-15 | 中国农业科学院兰州畜牧与兽药研究所 | SNP marker influencing diameter of alpine merino sheep wool fiber and application thereof |
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| CN117737260A (en) | 2024-03-22 |
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