CN117721221A - Cloning and application of SNP molecular marker related to Tibetan sheep immune traits - Google Patents
Cloning and application of SNP molecular marker related to Tibetan sheep immune traits 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
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
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- Y02P60/87—Re-use of by-products of food processing for fodder production
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Abstract
The invention belongs to the technical field of molecular biology detection, and particularly relates to cloning and application of SNP molecular markers related to Tibetan sheep immune traits. The SNP molecular marker related to Tibetan sheep immune traits is obtained through screening and positioned at 6966825 bases on chromosome 14 of sheep reference genome oar_v4.0 version, and the mutant bases are C or T. The invention provides a new SNP molecular marker resource for auxiliary selection of Tibetan sheep immune character markers for non-diagnosis purposes. Provides a basis for breeding Tibetan sheep with high immunity.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to cloning and application of SNP molecular markers related to Tibetan sheep immune traits.
Background
The Tibetan sheep is a main livestock variety of the Qinghai-Tibet plateau in China, and is also an important component of grassland animal husbandry in the alpine pasture area of the Qinghai-Tibet plateau. 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 an important carrier for Tibetan Buddhism and Tibetan language inheritance and development. 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, and the problems are one of key factors influencing sustainable development of Tibetan sheep industry.
With the rapid development of DNA molecular marker technology, a foundation is laid for people to study the genetic mechanism of Tibetan sheep disease resistance on a molecular level, and the 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. In the prior art, the auxiliary selection of the molecular markers related to the disease resistance of the Tibetan sheep immunity is not more, so that the SNP molecular markers related to the Tibetan sheep immunity are provided, and are particularly important to be used in auxiliary breeding of the Tibetan sheep molecular markers.
Disclosure of Invention
Accordingly, the present invention aims to provide a cloning and application of SNP molecular markers related to Tibetan sheep immune traits.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an SNP molecular marker related to Tibetan sheep immune traits, which is positioned at 6966825 bases on chromosome 14 of sheep reference genome oar_v4.0 version 14, and the mutant bases are C or T.
Preferably, the Tibetan sheep with the mutated base C has genotype CC or CT;
the genotype of Tibetan sheep with mutant base as T is TT;
the content of immunoglobulin in Tibetan sheep individuals with genotype TT is higher than that in Tibetan sheep individuals with genotype CC or CT.
Preferably, the immunoglobulin comprises one or more of immunoglobulin A, immunoglobulin G and immunoglobulin M.
The invention also provides application of the SNP molecular marker in preparing a product for detecting Tibetan sheep immunity.
The invention also provides application of the SNP molecular marker in preparing Tibetan sheep assisted breeding products.
The invention also provides a primer pair for amplifying the SNP molecular marker gene fragment, and the sequence of the primer pair is shown as SEQ ID NO. 1-2.
The invention also provides application of the primer pair in preparation of a product for detecting Tibetan sheep immunity.
The invention also provides application of the primer pair in preparing a Tibetan sheep assisted breeding product.
The invention also provides a kit for detecting Tibetan sheep immunity, which comprises a reagent for detecting the SNP molecular marker or the primer pair.
The invention also provides a kit for auxiliary breeding of Tibetan sheep, which comprises a reagent for detecting the SNP molecular marker or the primer pair.
The invention provides cloning and application of SNP molecular markers related to Tibetan sheep immune traits, and the invention discovers that SNP loci related to Tibetan sheep immune are positioned at 6966825 bases on chromosome 14 of international sheep reference genome oar_v4.0 version, the variation type is C/T, 3 genotypes exist, and when 6966825 bases on chromosome 14 are C, the genotypes are CC or CT; when 6966825 bases on chromosome 14 are T, the genotype is TT; through correlation analysis of different genotypes and contents of immunoglobulin A, immunoglobulin G and immunoglobulin M, it is found that the immunoglobulin A, immunoglobulin G and immunoglobulin M of Tibetan sheep individuals with TT genotypes are significantly higher than those of individuals with CC and CT genotypes (p < 0.05), and the immunoglobulins of the individuals with CC and CT genotypes do not show significant differences (p > 0.05). The content of immunoglobulin A, immunoglobulin G and immunoglobulin M of Tibetan sheep individuals can be judged by detecting the base of 6966825 nucleotide locus on Tibetan sheep chromosome 14, and the invention provides a novel SNP molecular marker resource for auxiliary selection of Tibetan sheep immune character markers for non-diagnostic purposes. Provides a basis for breeding Tibetan sheep with high immunity.
Drawings
FIG. 1 is an agarose gel electrophoresis;
FIG. 2 is a sequence peak diagram after sequencing of PCR amplification products.
Detailed Description
The invention provides an SNP molecular marker related to Tibetan sheep immune traits, which is positioned at 6966825 bases on chromosome 14 of sheep reference genome oar_v4.0 version 14, and the mutant bases are C or T.
In the present invention, the genotype of Tibetan sheep with mutant base C is CC or CT;
the genotype of Tibetan sheep with mutant base as T is TT;
the content of immunoglobulin in Tibetan sheep individuals with genotype TT is higher than that in Tibetan sheep individuals with genotype CC or CT.
In the present invention, the immunoglobulin includes one or more of immunoglobulin a, immunoglobulin G and immunoglobulin M.
The invention also provides application of the SNP molecular marker in preparing a product for detecting Tibetan sheep immunity.
The invention also provides application of the SNP molecular marker in preparing Tibetan sheep assisted breeding products.
The invention also provides a primer pair for amplifying the SNP molecular marker gene fragment, and the sequence of the primer pair is shown as SEQ ID NO. 1-2.
SEQ ID NO.1 is an upstream primer, the sequence of which is:
5'-CCCCAATTTAATACAGCATCG-3'；
SEQ ID NO.2 is a downstream primer, the sequence of which is:
5'-TGAATTCACGGATGGCATT-3'。
in the invention, the amplification system for amplifying the SNP molecular marker gene fragments comprises the following steps: gold medal Mix (green) 22. Mu.L, 1. Mu.L each of the upstream and downstream primers, and 1. Mu.L of genomic DNA.
The amplification procedure when amplifying the SNP molecular marker gene fragment comprises the following steps: 98 ℃ for 2min;98 ℃ for 10s,58 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
The length of the amplified gene fragment is 460 bp, and the SNP molecular marker is positioned at the 153 th position of the gene fragment.
The invention also provides application of the primer pair in preparation of a product for detecting Tibetan sheep immunity.
The invention also provides application of the primer pair in preparing a Tibetan sheep assisted breeding product.
The invention also provides a kit for detecting Tibetan sheep immunity, which comprises a reagent for detecting the SNP molecular marker or the primer pair.
The invention also provides a kit for auxiliary breeding of Tibetan sheep, which comprises a reagent for detecting the SNP molecular marker or the primer pair.
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.
Example 1
1 sample collection
The sample is from Tibetan sheep group under natural grazing condition, wherein 66 parts of Gannan Tibetan autonomous state in Gansu province, 44 parts of Jade Tree Tibetan autonomous state in Qinghai province, 28 parts of Tibet autonomous region day-karst city, collecting 138 hollow Tibetan sheep blood samples 5mL in a clean procoagulant vacuum blood collection tube, standing for 30min, centrifuging at 3500r/min for 15min, sucking supernatant in a clean PE tube, sealing and storing in a low-temperature refrigerator at-20deg.C; 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 extracting genome DNA.
2 main reagents and instruments
The blood collection tube added with EDTA-K2 anticoagulant is purchased from Jiangsu Yuli medical instruments Co., ltd; the blood genome DNA extraction kit is purchased from Tiangen biochemical technology (Beijing) limited company; nanoDrop2000 spectrophotometer us Thermo Fisher Scientific company; DL2000Marker, 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. Immunoglobulin A (IgA) (E027-1-1), immunoglobulin G (IgG) (E026-1-1), and immunoglobulin M (IgM) (E025-1-1) detection kits were purchased from Nanjing's institute of biological engineering.
3 method
3.1IgA, 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, adding 7 mu L of distilled water, standard solution and sample to be detected into a blank tube, a standard tube and a measuring tube respectively, supplementing R1 solution to 900 mu L, incubating for 5min at 37 ℃, and recording the reading number as A1 at the wavelength of 340 nm; then 180. Mu.L of R2 solution is added into each tube, and the tubes are incubated for 5min at 37 ℃, and the reading number at the wavelength of 340nm is recorded as A2; 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 chromosome 14 gene sequence (GenBank accession number: NC-019471.2) of International sheep genome Oar_v4.0, a pair of specific primers is designed by using Prmerpmier 5.0 software, and the nucleotide of the primer pair is shown as SEQ ID NO. 1-2. SEQ ID NO.1 is an upstream primer, the sequence of which is: 5'-CCCCAATTTAATACAGCATCG-3'; SEQ ID NO.2 is a downstream primer, the sequence of which is: 5'-TGAATTCACGGATGGCATT-3'.
The length of the amplified fragment is 464bp, and the primer is synthesized by Beijing qing biological science and technology 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,58 ℃ 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 agarose gel electrophoresis detection results are shown in FIG. 1. FIG. 1 shows that the length of the PCR amplified sequence is 464bp, and the nucleotide sequence 153 (at 6966825 bases on chromosome 14 of the genome oar_v4.0 version) of the amplified product is subjected to C/T mutation by sequencing. The amplified product has clear band, no impurity band and good specificity. The SNP marker locus of Tibetan sheep was initially identified and designated as g6966825C > T SNP. The sequence obtained by PCR amplification is shown as SEQ ID NO.3, and 153 position of the sequence is C mutation. The fragment size of the PCR amplified product is in accordance with the expected size, and the next experiment can be performed.
SEQ ID NO.3：
CCCCAATTTAATACAGCATCGTTTTTTCACAGGTCCCCATATTTCCTCCCCCACAGGAAATTTCCCACTGTCATGATTTGTCCAGAAATACTCTCTGCTGCCACAGGACTGAGTTTCTTCTGACGTGCTGGGTCCCTCAAACCTTCTTTCCTCGGCTGCCCATCAACACTCAAAATCCTTTCCTCATGGAAGAAGTTGAGCTCTTTCACCCTCAAAAATCTAGGCTTTGCCTGTAAGTTGGCAAAGAAAAAAAAAAAAAGCAAGACAGAGGCTCACCTTTCCCAACCACACAGCCCCATCGAAGTATTTTATCCATCAAAGAGCCCTTCATCTCTAATACTCATGCTGTTCTTTCCCTAAATGACAGAAACTTGACACTCGTTTTTTTCACTAAAGTGCATGTTTTTCTCTCTGCCTCATGATCCTATAAAGCTATATCAAGGCAAATGCCATCCGTGAATTCA。
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 polymorphic information content was calculated using the Popgen32 software to calculate g6966825C > T gene frequency, genotype frequency, effective allele (Ne), site heterozygosity (He), hardy-Weinberg equilibrium test, and the PIC (polymorphism information content, PIC for short). The correlation of the 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
5.1 PCR amplification and sequencing results
The amplification product of the SNP locus of the Tibetan sheep chromosome 14 g6966825C > T (see figure 1) is detected by using 1.5% agarose gel, the band is clear, the band is free from impurity bands, the specificity is good, the size of the PCR product fragment is 464bp, the 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 can be seen from FIG. 2, the g6966825C > T SNP site has C-T mutation, and three genotypes of CC, CT and TT exist.
5.2 statistical analysis results
Genotype and allele frequency of the Tibetan sheep chromosome 14 g6966825C > T 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 g6966825C > T 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 Tibetan sheep chromosome 14 g6966825C > T SNP site polymorphism
Table 1 shows that the CC genotype frequency was highest at the g6966825C > T SNP site, and was the dominant genotype, and the C allele frequency was 94.2%, representing the dominant allele. 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 0.109, PIC is 0.103, PIC is less than 0.25, and the locus belongs to low-level polymorphism.
5.3 correlation analysis of different genotypes with 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 TT genotype is obviously higher than that of Tibetan sheep individuals with CC and CT genotypes (P < 0.05), the immunoglobulin between CC and CT genotype individuals does not show obvious difference (P > 0.05), which indicates that the base of Tibetan sheep chromosome 14 g6966825C > T SNP locus is obviously related to Tibetan sheep IgA, igG, igM (P < 0.05), and the Tibetan sheep strain is the Tibetan sheep IgA, igG, igM related SNP marker. The results are shown in Table 2.
TABLE 2 relationship between different genotypes and immunoglobulins A, G and M
Note that: the same row of data is marked with different lower case letters to indicate that the difference is significant (P < 0.05).
As shown in Table 2, the IgA, igG, igM content of Tibetan sheep individuals with TT genotype is significantly higher than those of individuals with CC and CT genotypes (P < 0.05), and IgA, igG, igM does not show significant difference between individuals with CC and CT genotypes (P > 0.05), which indicates that the base of the Tibetan sheep chromosome 14 g6966825C > T SNP locus is significantly related to Tibetan sheep IgA, igG, igM (P < 0.05), which is a Tibetan sheep IgA, igG, igM related SNP marker.
The SNP molecular marker is positioned at 6966825 bases on chromosome 14 of the international sheep reference genome oar_v4.0 version; the mutation type is C/T, the mutation type is named as g6966825C > T, three genotypes exist, and when 6966825 th base on the 14 th chromosome is C, the genotype is CC or CT; when 6966825 bases on the chromosome 14 are T, the genotype is TT; by correlation analysis of different genotypes and IgA, igG, igM content, igA, igG, igM of Tibetan sheep individuals with the TT genotype was found to be significantly higher than those of CC and CT genotypes (p < 0.05), and IgA, igG, igM did not show significant differences (p > 0.05) between CC and CT genotypes. The IgA, igG, igM content of Tibetan sheep individuals can be judged by detecting the base of 6966825 nucleotide sites on the Tibetan sheep chromosome 14, and the invention provides a novel SNP molecular marker resource for auxiliary selection of Tibetan sheep immune trait markers for non-diagnostic 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 (10)
1. A SNP molecular marker associated with Tibetan sheep immune trait, wherein the SNP molecular marker is located at 6966825 base on chromosome 14 of sheep reference genome oar_v4.0 version 14, and the mutant base is C or T.
2. The SNP molecular marker of claim 1, wherein the genotype of Tibetan sheep with mutated base C is CC or CT;
the genotype of Tibetan sheep with mutant base as T is TT;
the content of immunoglobulin in Tibetan sheep individuals with genotype TT is higher than that in Tibetan sheep individuals with genotype CC or CT.
3. The SNP molecular marker of claim 2, wherein the immunoglobulin comprises one or more of immunoglobulin a, immunoglobulin G and immunoglobulin M.
4. Use of the SNP molecular marker of any one of claims 1-3 in the preparation of a product for detecting Tibetan sheep immunity.
5. Use of the SNP molecular marker of any one of claims 1-3 for preparing a Tibetan sheep assisted breeding product.
6. A primer pair for amplifying the SNP molecular marker gene fragment according to any one of claims 1 to 3, wherein the sequence of the primer pair is shown as SEQ ID NO.1 to 2.
7. Use of the primer pair of claim 6 in the preparation of a product for detecting Tibetan sheep immunity.
8. Use of the primer pair of claim 6 in the preparation of a Tibetan sheep assisted breeding product.
9. A kit for detecting the immunity of Tibetan sheep, comprising a reagent for detecting the SNP molecular markers described in any one of claims 1 to 3 or the primer pair described in claim 6.
10. A kit for auxiliary breeding of Tibetan sheep, comprising a reagent for detecting the SNP molecular markers described in any one of claims 1 to 3 or the primer pair described in claim 6.
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| CN118166117A (en) * | 2024-04-11 | 2024-06-11 | 中国农业科学院兰州畜牧与兽药研究所 | Molecular marker for early identification of Tibetan sheep immune traits and application thereof |
| CN118166119A (en) * | 2024-04-11 | 2024-06-11 | 中国农业科学院兰州畜牧与兽药研究所 | Tibetan sheep immune trait identification SNP molecular marker and application thereof |
| CN118166118A (en) * | 2024-04-11 | 2024-06-11 | 中国农业科学院兰州畜牧与兽药研究所 | ANKRD50 gene as molecular marker of Tibetan sheep immune trait and application thereof |
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| CN118166119A (en) * | 2024-04-11 | 2024-06-11 | 中国农业科学院兰州畜牧与兽药研究所 | Tibetan sheep immune trait identification SNP molecular marker and application thereof |
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