Molecular marker related to immune traits of male semi-fine wool sheep as well as detection primer and application of molecular marker

Abstract

The invention provides a molecular marker related to immune traits of elephant male semi-fine wool sheep and a detection primer and application thereof, and belongs to the technical field of molecular assisted breeding. The nucleotide sequence of the molecular marker is shown as SEQ ID NO.1; the molecular marker comprises an SNP site, the SNP site is located at the 117th site of the molecular marker, the mutation basic group of the SNP site is A or G, and the immune globulin content of an individual male semi-fine wool sheep with the genotype of AG or GG at the SNP site is remarkably higher than that of an individual with the genotype of AA. The content of immune globulins IgA, IgG and IgM of an individual elephant male semi-fine-wool sheep can be judged according to the genotype of the molecular marker, the immune traits of the elephant male semi-fine-wool sheep can be rapidly and accurately evaluated, the high-immunity elephant male semi-fine-wool sheep can be screened, the molecular marker has important significance on early-stage auxiliary breeding of the elephant male semi-fine-wool sheep, the breeding period is remarkably shortened, and the breeding cost is reduced. The breeding efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular assisted breeding, and particularly relates to a molecular marker related to the immune traits of Xiongxiong semi-fine wool sheep, its detection primers and applications. Background Art

[0002] In the process of the development of modern animal husbandry, breeding livestock breeds with excellent immune traits is of great significance for improving breeding efficiency, ensuring animal health and reducing the cost of epidemic prevention and control. As a characteristic breed adapted to the local plateau environment through long-term breeding, Xiongxiong semi-fine wool sheep occupies an important position in plateau animal husbandry. However, the traditional phenotypic selection breeding method has certain limitations in improving its immune performance and is difficult to meet the growing demand for efficient and precise breeding.

[0003] With the rapid development of molecular biology technology, the SNP (single nucleotide polymorphism) molecular marker technology has emerged, opening up a new way for livestock genetic breeding research. SNP molecular markers have significant advantages such as wide distribution, rich quantity, high genetic stability and easy automated detection, and can reveal genetic differences between animal individuals at the molecular level. In the field of sheep breeding, through the precise screening and identification of SNP molecular markers closely related to immune traits, the genetic mechanism of sheep immune ability can be deeply analyzed. For example, several SNP loci related to the immunoglobulin content of Tibetan sheep have been successfully discovered, and by detecting these loci, the contents of immunoglobulin A, immunoglobulin G and immunoglobulin M in Tibetan sheep individuals can be effectively judged, providing key molecular marker resources for marker-assisted selection of Tibetan sheep immune traits.

[0004] For Xiongxiong semi-fine wool sheep, deeply exploring SNP molecular markers related to its immune traits and applying them to assisted breeding work has great potential value. On the one hand, this helps to accurately screen out breeding sheep with excellent immune traits at an early stage, significantly improve the selection efficiency and accuracy, and accelerate the genetic improvement process of the excellent immune characteristics of Xiongxiong semi-fine wool sheep. On the other hand, by using SNP molecular marker-assisted breeding technology, the limitation of traditional breeding relying only on phenotypic selection can be broken, accurately locate individuals carrying dominant immune genes, avoid selection deviation caused by environmental factors, and thus breed new strains of Xiongxiong semi-fine wool sheep with stronger immunity and better adaptability, further promoting the sustainable and healthy development of plateau animal husbandry. However, at present, the research on SNP molecular markers related to the immune traits of Xiongxiong semi-fine wool sheep is still in its infancy, and the lack of relevant molecular marker resources seriously restricts the breeding progress of this breed in improving immune performance. There is an urgent need to carry out systematic and in-depth research work to fill the gap in this field. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a molecular marker related to the immune traits of Xiongxiong semi-fine wool sheep, as well as its detection primers and applications. By detecting the genotype of the molecular marker related to the immune traits in Xiongxiong semi-fine wool sheep, it is possible to quickly and accurately evaluate the immune traits of Xiongxiong semi-fine wool sheep, screen Xiongxiong semi-fine wool sheep with high immunity, which is of great significance for the early assisted breeding of Xiongxiong semi-fine wool sheep. Screening can be achieved at the young age of sheep, significantly shortening the breeding cycle and improving the breeding efficiency.

[0006] The present invention provides a molecular marker related to the immune traits of Xiongxiong semi-fine wool sheep. The nucleotide sequence of the molecular marker is shown as SEQ ID NO.1. The molecular marker includes an SNP site, which is located at the 117th position of the molecular marker. The mutant base of the SNP site is A or G. The immunoglobulin content of Xiongxiong semi-fine wool sheep individuals with the SNP site genotype of AG or GG is significantly higher than that of individuals with the genotype of AA.

[0007] Preferably, the immune traits include the content of immunoglobulin.

[0008] Preferably, the immunoglobulin includes one or more of IgA, IgG, and IgM.

[0009] The present invention provides a primer pair for amplifying the molecular marker, including primer F and primer R. The nucleotide sequence of primer F is shown as SEQ ID NO.2, and the nucleotide sequence of primer R is shown as SEQ ID NO.3.

[0010] The present invention provides the application of the primer pair in the preparation of a reagent for detecting the immune ability of Xiongxiong semi-fine wool sheep.

[0011] The present invention provides the application of the reagent for detecting the molecular marker or the primer pair in the assisted breeding of Xiongxiong semi-fine wool sheep.

[0012] Preferably, it includes the following steps:

[0013] 1) Extract the genomic DNA of the Xiongxiong semi-fine wool sheep to be bred. Using the genomic DNA of the Xiongxiong semi-fine wool sheep as a template, perform PCR amplification with the primer pair to obtain an amplification product.

[0014] 2) Sequence the amplification product to determine the genotype of the SNP site, and select the Xiongxiong semi-fine wool sheep to be bred with the genotype of AG or GG for subsequent breeding.

[0015] Preferably, the amplification system of the PCR amplification is 25 μL, including 22 μL of PCR enzyme, 1 μL of primer F, 1 μL of primer R, and 1 μL of template DNA.

[0016] Preferably, the amplification program for PCR amplification is as follows: 98°C for 2 min; 98°C for 10 s, 59°C for 10 s, 72°C for 10 s, for a total of 40 cycles; 72°C for extension for 2 min.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a molecular marker related to the immune traits of Xiongxiong semi-fine wool sheep. The nucleotide sequence of the molecular marker is shown as SEQ ID NO.1; the molecular marker includes an SNP locus, the SNP locus is located at the 117th position of the molecular marker, the mutated base of the SNP locus is G, and the genotype is AG or GG. Through the correlation analysis of different genotypes of Xiongxiong semi-fine wool sheep samples with the contents of immunoglobulin IgA, IgG, and IgM, the present invention determines that the levels of immunoglobulin IgA, IgG, and IgM in individuals with AG and GG genotypes are significantly higher than those in individuals with AA genotype. The present invention provides a new SNP molecular marker resource for marker-assisted selection of the immune traits of Xiongxiong semi-fine wool sheep for non-diagnostic purposes.

[0019] By detecting the genotype of the molecular marker related to the immune traits in Xiongxiong semi-fine wool sheep, the present invention can quickly and accurately evaluate the immune traits of Xiongxiong semi-fine wool sheep, which is of great significance for the early assisted breeding of Xiongxiong semi-fine wool sheep. Screening can be achieved at the young age of sheep, significantly shortening the breeding cycle and improving the breeding efficiency. Description of the Drawings

[0020] Figure 1 It is an agarose gel electrophoresis diagram of PCR amplification products; where M is a 1000bp Marker, and 1-3 are the electrophoresis bands of samples corresponding to the three genotypes of AA, AG, and GG;

[0021] Figure 2 It is a sequencing peak diagram of three genotypes of the SNP locus. Detailed Embodiments

[0022] The present invention provides a molecular marker related to the immune traits of Xiongxiong semi-fine wool sheep. The nucleotide sequence of the molecular marker is shown as SEQ ID NO.1; the molecular marker is 329bp in total, includes an SNP locus, the SNP locus is located at the 117th position of the molecular marker (i.e., the underlined and bolded locus below), the mutated base of the SNP locus is G, and the genotype is AG or GG; specifically as follows:

[0023]

[0024] In the present invention, the immunoglobulin content of Zhangzhung semi-fine wool sheep individuals with the SNP locus genotype of AG or GG is significantly higher than that of individuals with the genotype of AA. The immunoglobulin preferably includes one or several of IgA, IgG, and IgM.

[0025] The present invention provides a primer pair for amplifying the molecular marker, including primer F and primer R; the nucleotide sequence of primer F is as shown in SEQ ID NO.2, and the nucleotide sequence of primer R is as shown in SEQ ID NO.3; specifically as follows:

[0026] Primer F (SEQ ID NO.2): 5'-AATCTTAAAGCCTCAGACTCC-3';

[0027] Primer R (SEQ ID NO.3): 5'-TGAAATCAAGGCTCTAGACCA-3'.

[0028] The present invention also provides the application of the primer pair in the preparation of a reagent for detecting the immune ability of Zhangzhung semi-fine wool sheep.

[0029] The present invention also provides the application of the reagent for detecting the molecular marker or the primer pair in the assisted breeding of Zhangzhung semi-fine wool sheep.

[0030] The application preferably includes the following steps:

[0031] 1) Extract the genomic DNA of the Zhangzhung semi-fine wool sheep to be bred. Using the genomic DNA of the Zhangzhung semi-fine wool sheep as a template, perform PCR amplification with the primer pair to obtain an amplification product;

[0032] 2) Sequence the amplification product to determine the genotype of the SNP locus, and select the Zhangzhung semi-fine wool sheep to be bred with the genotype of AG or GG for subsequent breeding.

[0033] In the present invention, first extract the genomic DNA of the Zhangzhung semi-fine wool sheep to be bred. Preferably, extract the genomic DNA of a blood sample. The present invention has no special limitation on the method for extracting the genomic DNA, and a conventional method for extracting genomic DNA in the art can be used. In the specific implementation process of the present invention, preferably use the blood genomic DNA extraction kit of TransGen Biotech Co., Ltd. The concentration of the genomic DNA is preferably >20 ng / μL, and the OD260 / OD280 is preferably between 1.7 and 1.9. The genomic DNA is preferably stored at -20°C.

[0034] After obtaining the genomic DNA, using the genomic DNA of the Zhangzhung semi-fine wool sheep as a template, PCR amplification is carried out with the primer pairs described above to obtain an amplification product. In the present invention, the amplification system for the PCR amplification is calculated based on 25 μL, and preferably includes 22 μL of PCR enzyme, 1 μL of primer F, 1 μL of primer R; 1 μL of template DNA; the amplification procedure for the PCR amplification is preferably as follows: 98 °C for 2 min; 98 °C for 10 s, 59 °C for 10 s, 72 °C for 10 s, for a total of 40 cycles; 72 °C for extension for 2 min.

[0035] After the present invention obtains the amplification product, the amplification product is sequenced to determine the genotype of the SNP locus, and the Zhangzhung semi-fine wool sheep to be bred with the genotype of AG or GG is selected for subsequent breeding. In the present invention, the amplification product is preferably purified before sequencing. The present invention has no special limitation on the purification method, and a conventional purification method in the art can be used. In the present invention, the sequencing is preferably carried out by the direct sequencing method, and the sequencing is preferably entrusted to Beijing Qingke Biotechnology Co., Ltd. to complete.

[0036] After the sequencing of the present invention is completed, the genotype of the SNP locus is determined. Preferably, the sequencing results of the PCR products are compared using the biological analysis software MEGA6.0, the sequencing peak map is analyzed, typing is completed, and the genotype of the SNP locus is determined; then the Zhangzhung semi-fine wool sheep to be bred with the genotype of AG or GG is selected for subsequent breeding.

[0037] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0038] Example 1

[0039] 1 Sample collection

[0040] In the Zhangzhung semi-fine wool sheep breeding farm in Ngari Prefecture, Tibet Autonomous Region, 118 adult Zhangzhung semi-fine wool sheep under natural grazing conditions were randomly selected. 5 mL of blood samples were collected on an empty stomach in a coagulation-promoting vacuum blood collection tube, left to stand for 30 min, then centrifuged at 3500 r / min for 15 min, and the supernatant was aspirated into a clean PE tube, sealed and stored in a -20 °C low-temperature refrigerator for determination of immune indexes; another 5 mL of blood samples were collected in a blood collection tube added with EDTA-K2 anticoagulant, and after the blood samples were collected, they were quickly mixed evenly, placed in a sampling box containing ice packs for temporary storage, and stored frozen in a -20 °C refrigerator after being transported back to the laboratory for DNA extraction.

[0041] 2 Main reagents and instruments

[0042] EDTA-K2 vacuum blood collection tubes were purchased from Jiangsu Yuli Medical Instrument Co., Ltd.; blood genome extraction kits, DL1000 Marker, agarose, nucleic acid dyes, and PCR enzymes were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; NanoDrop 2000 spectrophotometer was purchased from Thermo Fisher Scientific, USA; electrophoresis instrument was purchased from Beijing Liuyi Instrument Factory; PCR instrument was purchased from BioRad. IgA, IgG, and IgM detection kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0043] 3 Methods

[0044] 3.1 Immunoglobulin IgA, IgG, and IgM detection

[0045] Serum was tested using IgA, IgG, and IgM test kits. First, a standard curve was established using standard products; second, distilled water, standard solution, and 7 μL of the sample to be tested were added to the blank tube, standard tube, and test tube, respectively, and R1 solution was added to 900 μL, incubated at 37°C for 5 minutes, and the reading at a wavelength of 340 nm was recorded as A1; then 180 μL of R1 solution was added to each tube, incubated at 37°C for 5 minutes, and the reading at a wavelength of 340 nm was recorded as A2; finally, △A was calculated as A2-A1, and △A was substituted into the standard curve equation to calculate the sample IgA, IgG, and IgM concentrations.

[0046] 3.2 Extraction of genomic DNA from blood

[0047] The blood genome extraction kit of Beijing Quanshijin Biotechnology Co., Ltd. was used to extract genomic DNA from the blood samples. The extracted DNA was placed under an ultraviolet spectrophotometer to detect the concentration and purity. The concentration >20ng / μL and OD260 / OD280 between 1.7-1.9 met the experimental needs and was stored at -20℃ for future use.

[0048] 3.3 Primer design

[0049] According to the chromosome 1 gene sequence (GenBank accession number: NC_056054.1) of the Rambouillet reference genome (ARS-UI_Ramb_v3.0, GCF_016772045.2), a pair of specific primers including the g31667355A>G SNP site were designed using Oligo 7 software.

[0050] Primer sequences:

[0051] F: 5'-AATCTTAAAGCCTCAGACTCC-3';

[0052] R: 5'-TGAAATCAAGGCTCTAGACCA-3'.

[0053] The amplified fragment length was 329 bp. Beijing Tsingke Biotechnology Co., Ltd. was commissioned to sequence the amplified fragment, and the primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0054] 3.4 PCR Amplification and Sequencing

[0055] The PCR amplification system was 25 μL: 22 μL of PCR enzyme, 1 μL of primer F, 1 μL of primer R, and 1 μL of template DNA.

[0056] The PCR amplification program was: 98°C for 2 min; 98°C for 10 s, 59°C for 10 s, 72°C for 10 s, for a total of 40 cycles; 72°C for extension for 2 min.

[0057] The PCR products were detected by 1.5% agarose gel electrophoresis. After the PCR products passed the agarose gel electrophoresis detection, direct sequencing was used for sequencing, which was completed by Sangon Biotech (Shanghai) Co., Ltd. The amplified nucleotide sequence was as shown in SEQ ID No.1, and the SNP marker was located at position 117 of the nucleotide sequence shown in SEQ ID No.1.

[0058] The sequencing results of the PCR products were aligned using the biological analysis software MEGA 6.0, and the sequencing peak maps were analyzed to complete the genotyping.

[0059] 4 Statistical Analysis

[0060] According to the gene typing results, the number of individuals with different genotypes at each locus was counted. The Popgen32 software was used to calculate the gene frequency, genotype frequency, effective allele number (Ne), locus heterozygosity (He), and Hardy-Weinberg equilibrium test of g31667355A>G, and the PIC (polymorphism information content) calculation software was used to calculate the polymorphism information content. The general linear model in IBM SPSS Statistics 22 software was used to analyze the correlation between different genotypes of Xiong semi-fine wool sheep and immunoglobulins IgA, IgG, and IgM, and the results were expressed as "mean ± standard error".

[0061] 5 Results

[0062] 5.1 PCR Amplification and Sequencing Results

[0063] The amplified products of the g31667355A>G SNP locus on chromosome 1 of Xiong semi-fine wool sheep were detected by 1.5% agarose gel (see Figure 1) The bands were clear without background bands, indicating good specificity. The size of the PCR product fragment was 329 bp, which was consistent with the expected size, and the next experiment could be carried out.

[0064] The peak map and sequence obtained after purification and sequencing of the PCR product are shown in Figure 2 . It can be seen from Figure 2 that there was an A-G mutation at the g31667355A>G SNP locus, and there were three genotypes: AA, AG, and GG.

[0065] 5.2 Statistical analysis results

[0066] The genotypes and allele frequencies of the g31667355A>G SNP locus on chromosome 1 of Xiangxiong semi-fine wool sheep were analyzed from the perspective of population genetics. As shown in Table 1, at the g31667355A>G SNP locus, the frequency of the GG genotype was the highest, which was the dominant genotype, and the frequency of the G allele was 92%, showing a dominant allele. The χ2 goodness-of-fit test showed that the SNP locus significantly deviated from the Hardy-Weinberg equilibrium state (P<0.05) (Table 1). The expected heterozygosity of this locus was 0.155, and the PIC was 0.143. PIC<0.25, indicating low polymorphism.

[0067] Table 1 Polymorphism of the g31667355A>G SNP locus on chromosome 1 of Xiangxiong semi-fine wool sheep

[0068]

[0069]

[0070] 5.3 Association analysis between different genotypes and immunoglobulins IgA, IgG, and IgM

[0071] The general linear model in IBM SPSS Statistics 22 software was used to analyze the association between different genotypes of Xiangxiong semi-fine wool sheep and the contents of immunoglobulins IgA, IgG, and IgM. The results showed that the contents of immunoglobulins IgA, IgG, and IgM in Xiangxiong semi-fine wool sheep individuals with the AA genotype were significantly lower than those in individuals with the AG and GG genotypes (p<0.05), and there was no significant difference between individuals with the AG and GG genotypes (p>0.05). This indicated that the base at the g31667355A>G SNP locus on chromosome 1 of Xiangxiong semi-fine wool sheep was significantly correlated with IgA, IgG, and IgM in Xiangxiong semi-fine wool sheep (p<0.05), and it was an SNP marker related to IgA, IgG, and IgM in Xiangxiong semi-fine wool sheep. The results are shown in Table 2.

[0072] Table 2 Correlation analysis between different genotypes and immunoglobulins IgA, IgG, and IgM

[0073]

[0074] Note: Different lowercase letters in the same row of data indicate significant differences (p < 0.05).

[0075] In summary, the SNP molecular marker of the present invention is located at the 31,667,355th base on chromosome 1 of the Rambouillet reference genome (ARS-UI_Ramb_v3.0, GCF_016772045.2); the mutation type is A / G, named g31667355A>G, and there are three genotypes. When the 31,667,355th base on chromosome 1 is A, the genotype is AA or AG; when the 31,667,355th base on chromosome 1 is G, the genotype is GG; through the association analysis of different genotypes with the contents of immunoglobulins IgA, IgG, and IgM, it is found that the contents of immunoglobulins IgA, IgG, and IgM in the individuals of the Xiangxiong semi-fine wool sheep with the AA genotype are significantly lower than those of the individuals with the AG and GG genotypes (p < 0.05), and there is no significant difference between the individuals with the AG and GG genotypes (p > 0.05). By detecting the base at the 31,667,355th nucleotide site on chromosome 1 of the Xiangxiong semi-fine wool sheep, the contents of immunoglobulins IgA, IgG, and IgM in the individuals of the Xiangxiong semi-fine wool sheep can be judged. The present invention provides a new SNP molecular marker resource for marker-assisted selection of the immune traits of the Xiangxiong semi-fine wool sheep for non-diagnostic purposes.

[0076] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A molecular marker related to the immune traits of Zhangzhung semi-fine wool sheep, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1; the molecular marker includes an SNP site, the SNP site is located at the 117th position of the molecular marker, the mutated base of the SNP site is A or G, and the immunoglobulin content of the Zhangzhung semi-fine wool sheep individuals with the SNP site genotype of AG or GG is significantly higher than that of the individuals with the genotype of AA.

2. The molecular marker according to claim 1, characterized in that The immunoglobulin includes one or more of IgA, IgG, and IgM.

3. A primer pair for amplifying the molecular marker according to claim 1, characterized in that, It includes primer F and primer R; the nucleotide sequence of primer F is shown in SEQ ID NO.2, and the nucleotide sequence of primer R is shown in SEQ ID NO.

3.

4. Use of the primer pair according to claim 3 in the preparation of a reagent for detecting the immune traits of Zhangzhung semi-fine wool sheep.

5. Use of the reagent for detecting the molecular marker according to claim 1 or the primer pair according to claim 3 in the assisted breeding of Zhangzhung semi-fine wool sheep.

6. The application according to claim 5, wherein It includes the following steps: 1) Extract the genomic DNA of the Zhangzhung semi-fine wool sheep to be bred. Using the genomic DNA of the Zhangzhung semi-fine wool sheep as a template, perform PCR amplification with the primer pair according to claim 3 to obtain an amplification product. 2) Sequence the amplification product to determine the genotype of the SNP site, and select the Zhangzhung semi-fine wool sheep to be bred with the genotype of AG or GG for subsequent breeding.

7. The application according to claim 6, wherein The amplification system of the PCR amplification is 25 μL in total, including 22 μL of PCR enzyme, 1 μL of primer F, 1 μL of primer R, and 1 μL of template DNA.

8. The application according to claim 7, characterized in that, The amplification program of the PCR amplification is as follows: 98°C for 2 min; 98°C for 10 s, 59°C for 10 s, 72°C for 10 s, a total of 40 cycles; 72°C for extension for 2 min.

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