CN117778594A - SNP molecular marker related to Tibetan sheep immune traits, detection method and application thereof - Google Patents
SNP molecular marker related to Tibetan sheep immune traits, detection method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention relates to the technical field of molecular markers, and provides a SNP molecular marker related to Tibetan sheep immune traits, a detection method and application thereof, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1. Compared with the traditional detection method, the SNP molecular marker polymorphism detection method has the advantages of high accuracy, high detection speed, low cost, easy judgment of results and the like. SNP locus detection is used for carrying out Tibetan sheep immune trait evaluation, and an early Tibetan sheep disease resistance selection method is established so as to ensure Tibetan sheep organism health and contribute to increasing economic benefit.
Description
Technical Field
The invention relates to the technical field of molecular markers, in particular to a SNP molecular marker related to Tibetan sheep immune traits, a detection method and application thereof.
Background
The Tibetan sheep is one of livestock varieties of major authorities in Qinghai-Tibet plateau in China, and is also an important component of grassland animal husbandry in high and cold pastures of the Qinghai-Tibet plateau. The Tibetan sheep has the characteristics of large population quantity, wide distribution, unique biological characteristics, good meat performance, good carpet wool quality and the like. At present, the problems of insufficient breeding, small group size, difficulty in meeting market demands and the like still exist on production traits such as fertility, disease resistance and the like of Tibetan sheep, and the construction of a breeding technology system is still relatively lagged. Therefore, the Tibetan sheep immunity is improved, and the Tibetan sheep industry development is promoted.
Single nucleotide polymorphisms (Single nucleotide polymorphism, SNPs) are the most basic variant of a genomic DNA sequence, caused by a single base mutation in the DNA sequence that replaces one nucleotide with another. The presence of SNPs within a gene or within a regulatory region may have a direct effect on gene function. Therefore, these SNP variants can be used as biomarkers for locating genes associated with certain traits, and are widely used in marker-assisted breeding, linkage analysis and biodiversity studies. The rapid development of DNA molecular marker technology lays a foundation for people to study the genetic mechanism of Tibetan sheep disease resistance on molecular level, and screening to obtain corresponding molecular markers is convenient for more efficient screening assistance for Tibetan sheep disease resistance breeding, which is a technical problem to be solved urgently in the field. Therefore, development and establishment of a modern biological breeding technology system are urgently needed to accelerate the genetic improvement and new variety cultivation of Tibetan sheep.
Disclosure of Invention
The invention aims to provide an SNP molecular marker related to Tibetan sheep immune traits, a detection method and application thereof, and the SNP molecular marker is screened to obtain corresponding molecular markers so as to facilitate more efficient screening assistance for Tibetan sheep disease-resistant breeding.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a molecular marker related to Tibetan sheep immune traits, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1.
Preferably, the molecular marker is located on the 96216397 th base of chromosome 6 of sheep genome oar_v4.0 version GenBank accession No. nc_ 019463.2.
Preferably, the molecular marker comprises an SNP locus, the SNP locus is positioned at the 176 th position of the molecular marker, and the mutation base of the SNP locus is C or A.
Preferably, when the molecular marker base is C, the genotype is CC or CA; when the molecular marker base is A, the genotype is AA.
The invention also provides application of the molecular marker in preparing products for in-vitro detection of Tibetan sheep immune traits or disease-resistant breeding screening of Tibetan sheep.
The invention also provides a specific primer group of the molecular marker, which comprises an upstream primer with a sequence shown as SEQ ID NO.2 and a downstream primer with a sequence shown as SEQ ID NO. 3.
The invention also provides application of the specific primer group in-vitro detection of Tibetan sheep immune traits or in Tibetan sheep disease-resistant breeding screening.
The invention also provides a method for in vitro detection of Tibetan sheep immune traits or screening Tibetan sheep disease-resistant breeding, which comprises the following steps:
(1) Extracting Tibetan sheep blood genome DNA;
(2) Taking Tibetan sheep blood genome DNA as a template, and adopting the specific primer group to carry out PCR amplification to obtain a PCR amplification product;
(3) Purifying the PCR amplification product, and carrying out genotyping detection, wherein when the SNP molecular marker base is C, the genotype is CC or CA; when the SNP molecular marker base is A, the genotype is AA.
Preferably, the PCR amplification system in step (2) is: gold medal Mix (green) 22. Mu.L, upstream primer 1. Mu.L, downstream primer 1. Mu.L, genomic DNA 1. Mu.L.
Preferably, the PCR amplification procedure in step (2) is: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 60℃for 10s, extension at 72℃for 10s for 40 cycles; final extension at 72℃for 2min.
The invention provides a SNP molecular marker related to Tibetan sheep immune traits, a detection method and application thereof, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1. Compared with the traditional detection method, the SNP molecular marker polymorphism detection method has the advantages of high accuracy, high detection speed, low cost, easy judgment of results and the like. SNP locus detection is used for carrying out Tibetan sheep immune trait evaluation, and an early Tibetan sheep disease resistance selection method is established so as to ensure Tibetan sheep organism health and contribute to increasing economic benefit.
Drawings
FIG. 1 shows the amplification product of the SNP site of Tibetan sheep chromosome 6 g 96216397C > A;
FIG. 2 shows the peak pattern and sequence obtained after purification and sequencing of PCR products.
Detailed Description
The invention provides a molecular marker related to Tibetan sheep immune traits, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1;
the sequence of SEQ ID NO.1 is: CCATTCTCTTGTTTGCCTCTATTTCTT TGCATTGATCACTGAGGAAGGCTTTCTTATTTCTTCTTGCTATTCTTTGGAACTCTGCATTCAGATGCTTATATTCTTGCCTGGAGAATTCCTTAAATAGAAGAGCCTGGTGGGCTACAGTCCATGGGGTCCCAGAGTCGGACAGAACAGAGCAACAAACACTTTAACACACACTTAATTTTTAACTTTCTAATTTACATTGGAGTATAGTTGATTAACAATATTGTGATAGCTTCAAGTGGACAGAAAAGTGATTCAGTTATATGTATACATGTATCTATTCTTTTTCAAATTATTCTCCCATGTATGTTGTTATATAATACCTAGCAGAGTTCCCTGTGCTATGCAGCAGGTCTTTTTTGATTCTTCATTTTAAATATAGCAGTGTGTACATGCTGCATTGGTTTAGACGATACCAGAAAACTGAGCACTCTTCTCACAGATTAAGTCTGTACTCTGCAAAAATGCAGAATACTACAGACCACCTAAACATTCAATTGCCTTCCATTGTATTCTAACCTTGGTGTGCTATGAAAGTATG.
In the present invention, the molecular marker is located on 96216397 th base of chromosome 6 of sheep genome oar_v4.0 version GenBank accession No. nc_ 019463.2.
In the invention, the molecular marker comprises an SNP locus, the SNP locus is positioned at the 176 th position of the molecular marker, and a mutation base of the SNP locus is C or A.
In the invention, when the molecular marker base is C, the genotype is CC or CA; when the molecular marker base is A, the genotype is AA.
The invention also provides application of the molecular marker in preparing products for in-vitro detection of Tibetan sheep immune traits or disease-resistant breeding screening of Tibetan sheep.
The invention also provides a specific primer group of the molecular marker, which comprises an upstream primer with a sequence shown as SEQ ID NO.2 and a downstream primer with a sequence shown as SEQ ID NO. 3.
The invention also provides application of the specific primer group in-vitro detection of Tibetan sheep immune traits or in Tibetan sheep disease-resistant breeding screening.
The invention also provides a method for in vitro detection of Tibetan sheep immune traits or screening Tibetan sheep disease-resistant breeding, which comprises the following steps:
(1) Extracting Tibetan sheep blood genome DNA;
(2) Taking Tibetan sheep blood genome DNA as a template, and adopting the specific primer group to carry out PCR amplification to obtain a PCR amplification product;
(3) Purifying the PCR amplification product, and carrying out genotyping detection, wherein when the SNP molecular marker base is C, the genotype is CC or CA; when the SNP molecular marker base is A, the genotype is AA.
In the present invention, the PCR amplification system in step (2) is preferably: gold medal Mix (green) 22. Mu.L, upstream primer 1. Mu.L, downstream primer 1. Mu.L, genomic DNA 1. Mu.L.
In the present invention, the PCR amplification procedure in step (2) is preferably: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 60℃for 10s, extension at 72℃for 10s for 40 cycles; final extension at 72℃for 2min.
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 67 parts of Gannan Tibetan autonomous state in Gansu province, 44 parts of Jade Tree in Qinghai province, 45 parts of Tibetan autonomous region day click, 5mL of 156 fasting Tibetan sheep blood sample is 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, sealed and stored 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 reagents and instruments
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. IgA (E027-1-1), igG (E026-1-1), igM (E025-1-1) detection kits were purchased from Nanjing's institute of biological engineering.
3 method
3.1 immunoglobulin IgA, igG, igM detection
The detection kit is used for measurement by a spectrophotometry method according to the Nanjing institute of biological engineering IgA, igG, igM. Firstly, establishing a standard curve by using a standard substance; secondly, distilled water, standard solution and 7uL of a sample to be detected are respectively added into a blank tube, a standard tube and a measuring tube, R1 solution is supplemented to 900uL, incubation is carried out for 5min at 37 ℃, and the reading number at the wavelength of 340nm is recorded as A1; then adding 180uL of R2 solution into each tube, incubating for 5min at 37 ℃, and recording the reading number as A2 at the wavelength of 340 nm; finally, calculating DeltaA=A2-A1, and taking DeltaA into a standard curve equation to calculate the concentration of the sample IgA, igG, igM.
3.2 extraction of genomic DNA from blood
Extracting genome DNA from blood sample by adopting a blood genome extraction kit of Tiangen biochemical technology (Beijing) limited company, and placing the extracted DNA under an ultraviolet spectrophotometer to detect the concentration and purity, wherein the concentration is more than 20 ng/mu L, OD260/OD280 and is between 1.7 and 1.9, thus meeting the experimental requirement, and storing at-20 ℃ for standby.
3.3 primer design
Referring to the international sheep genome oar_v4.0 version 6 chromosomal gene sequence (GenBank accession number: NC_ 019463.2), a pair of specific primers was designed using primepremier 5.0 software, containing g 96216397C > A SNP site.
Primer sequence:
F：5'–CCATTCTCTTGTTTGCCTC–3'(SEQ ID NO.2)；
R：5'–CATACTTTCATAGCACACCA–3'(SEQ ID NO.3)。
the amplified fragment length is 568bp, and the primer is synthesized by Beijing qingke biotechnology Co.
3.4PCR amplification and sequencing
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,60 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
And detecting the PCR product by using 1.5% agarose gel electrophoresis, and after the PCR product is qualified by using the agarose gel electrophoresis detection, sequencing by using a direct sequencing method, and completing sequencing by Beijing qingke biotechnology Co. The amplified nucleotide sequence is shown as SEQ ID NO.1, and the SNP marker is located at position 176 of the nucleotide sequence shown as SEQ ID NO. 1.
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.
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 g 962163973C > A are calculated by using Popgen32 software, and the polymorphism information content is calculated by using PIC (polymorphism information content, PIC for short) calculation software. The correlation of Tibetan sheep different genotypes with immunoglobulin IgA, igG, igM was analyzed using a general linear model in IBM SPSS Statistics software and the results are expressed as "mean ± standard error".
5 results
5.1 PCR amplification and sequencing results
The amplified product of the SNP locus of the Tibetan sheep chromosome 6 g 962163977C > A is detected by using 1.5% agarose gel (see figure 1), the stripe is clear, the band is free, the specificity is good, the fragment size of the PCR product is 568bp, the fragment size accords with the expected size, and the next experiment can be carried out.
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 C-A mutation occurred at the SNP site g 96216397C > A, and there were three genotypes of CC, CA and AA.
2.2 statistical analysis results
Genotype and allele frequency of the Tibetan sheep chromosome 6 g 96216397c > a SNP site were analyzed from a population genetics perspective. As can be seen from Table 1, the CC genotype was most frequently found at the g 96216397C > A SNP site, and the C allele frequency was 94.2% for the dominant genotype, which was expressed as the dominant allele. The χ2 fitness test showed that the SNP site was significantly deviated from Hardy-Weinberg equilibrium (P < 0.05) (Table 1). The expected heterozygosity of the locus is 0.109, PIC is 0.103, PIC is less than 0.25, and the locus belongs to low-level polymorphism.
TABLE 1 SNPs of Tibetan sheep chromosome 6 g 96216397C > A
2.3 correlation analysis of different genotypes with immunoglobulin IgA, igG, igM
The correlation of Tibetan sheep different genotypes and the content of immunoglobulin IgA, igG, igM is analyzed by adopting a general linear model in IBM SPSS Statistics software, and the result shows that the immunoglobulin IgA, igG, igM of Tibetan sheep individuals with AA genotypes is obviously higher than that of Tibetan sheep individuals with CC and CA genotypes (P < 0.05), the immunoglobulin IgA, igG, igM of Tibetan sheep individuals with CA genotypes is obviously higher than that of Tibetan sheep individuals with CC genotypes (P < 0.05), which indicates that the Tibetan sheep No. 6 chromosome g 96216397C > A SNP locus is obviously related to Tibetan sheep IgA, igG, igM (P < 0.05), and is the SNP marker related to Tibetan sheep IgA, igG, igM. The results are shown in Table 2.
TABLE 2 correlation analysis between different genotypes and immunoglobulins IgA, igG, igM
Note that: the same row of data is marked with different lower case letters to indicate that the difference is significant (P < 0.05).
In summary, the SNP molecular marker is positioned at 96216397 bases on chromosome 6 of the international sheep reference genome oar_v4.0 version 6; the mutation type is C/A, named g 96216397C > A, three genotypes exist, and when 96216397 bases on the 6 th chromosome are C, the genotypes are CC or CA; when 96216397 bases on the chromosome 6 are A, the genotype is AA; through correlation analysis of different genotypes and immunoglobulin IgA, igG, igM content, the immunoglobulin IgA, igG, igM of Tibetan sheep individuals with the AA genotype is found to be significantly higher than that of Tibetan sheep individuals with the CC and CA genotypes (p < 0.05), and the immunoglobulin IgA, igG, igM of Tibetan sheep individuals with the CA genotype is found to be significantly higher than that of Tibetan sheep individuals with the CC genotype (p < 0.05). The content of immunoglobulin IgA, igG, igM in Tibetan sheep individuals can be judged by detecting the base of 96216397 nucleotide sites on Tibetan sheep chromosome 6, and the invention provides a novel SNP molecular marker resource for auxiliary selection of Tibetan sheep immune trait markers for non-diagnostic purposes.
As can be seen from the above examples, the invention provides a SNP molecular marker related to Tibetan sheep immune traits, a detection method and application thereof, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1. Compared with the traditional detection method, the SNP molecular marker polymorphism detection method has the advantages of high accuracy, high detection speed, low cost, easy judgment of results and the like. SNP locus detection is used for carrying out Tibetan sheep immune trait evaluation, and an early Tibetan sheep disease resistance selection method is established so as to ensure Tibetan sheep organism health and contribute to increasing economic benefit.
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 (10)
1. A molecular marker related to Tibetan sheep immune traits is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1.
2. The molecular marker of claim 1, wherein the molecular marker is located on the 96216397 th base of chromosome 6 of GenBank accession No. NC 019463.2, version oar_v4.0 of the sheep genome.
3. The molecular marker according to claim 2, wherein the molecular marker comprises a SNP site located at position 176 of the molecular marker, and the SNP site mutation base is C or a.
4. The molecular marker according to claim 3, wherein when the molecular marker base is C, the genotype is CC or CA; when the molecular marker base is A, the genotype is AA.
5. Use of the molecular marker according to any one of claims 1 to 4 in the preparation of a product for in vitro detection of immune traits in Tibetan sheep or screening of disease resistant breeding in Tibetan sheep.
6. A specific primer group for detecting the molecular marker as set forth in any one of claims 1 to 4, wherein the primer group contains an upstream primer having a sequence shown as SEQ ID NO.2 and a downstream primer having a sequence shown as SEQ ID NO. 3.
7. The use of the specific primer set as defined in claim 6 in Tibetan sheep immune trait in vitro detection or Tibetan sheep disease-resistant breeding screening.
8. The in vitro detection of Tibetan sheep immune traits or the screening method of Tibetan sheep disease-resistant breeding is characterized by comprising the following steps:
(1) Extracting Tibetan sheep blood genome DNA;
(2) Performing PCR amplification by using Tibetan sheep blood genome DNA as a template and adopting the specific primer set of claim 6 to obtain a PCR amplification product;
(3) Purifying the PCR amplification product, and carrying out genotyping detection, wherein when the SNP molecular marker base is C, the genotype is CC or CA; when the SNP molecular marker base is A, the genotype is AA.
9. The method of claim 8, wherein the PCR amplification system of step (2) is: gold medal Mix (green) 22. Mu.L, upstream primer 1. Mu.L, downstream primer 1. Mu.L, genomic DNA 1. Mu.L.
10. The method of claim 9, wherein the PCR amplification procedure in step (2) is: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 60℃for 10s, extension at 72℃for 10s for 40 cycles; final extension at 72℃for 2min.
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