Molecular marker related to immune traits of elephant male semi-fine wool sheep and application of molecular marker

Abstract

The invention provides a molecular marker related to immune traits of elephant male semi-fine wool sheep and application of the molecular marker, and belongs to the technical field of molecular marker application, and 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 363rd site of the molecular marker, the basic group of the SNP site is T or C, and the immune globulin content of a male semi-fine wool sheep individual with the genotype of TC or CC at the SNP site is remarkably higher than that of a male semi-fine wool sheep individual with the genotype of TT. By detecting the genotype of the molecular marker SNP site related to the immune traits in the male semi-fine-wool sheep, the immune traits of the male semi-fine-wool sheep can be rapidly and accurately evaluated, and the molecular marker SNP site has important significance on early-stage auxiliary breeding of the male semi-fine-wool sheep.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular marker application, and in particular relates to a molecular marker related to the immune traits of Xiangxiong semi-fine wool sheep and an application thereof. Background Art

[0002] In the livestock industry, animal immunity is crucial. Good immune performance enables sheep to effectively resist the invasion of various pathogens, reducing the incidence of disease, lowering mortality and morbidity during the breeding process, and thus ensuring the health and stability of the herd. This not only helps improve sheep growth and production performance, ensuring the quality and yield of livestock products such as meat, wool, and milk, but also significantly reduces breeding costs and the dosage of drugs used for disease prevention and control, in line with the development of modern green and sustainable livestock farming. This is especially true for the Xiangxiong semi-fine wool sheep, which live in unique environments such as the plateau. Faced with complex and changing climatic conditions and potential pathogen threats, strong immunity is crucial for their survival and efficient production.

[0003] SNP molecular markers have been widely used in various areas of sheep genetic research. For example, SNP locus analysis of different sheep breeds allows for breed identification and genetic diversity assessment, clarifying the relationships and genetic differences between breeds, and providing a scientific basis for the rational conservation and utilization of sheep germplasm resources. In the study of genes related to economic traits, SNP molecular markers can help locate loci associated with important economic traits such as growth rate, meat production, wool quality, and reproductive performance. By screening these loci and using marker-assisted selection, the selection and breeding of superior breeds can be accelerated, breeding efficiency improved, and breeds more in line with market demand can be cultivated.

[0004] The complex plateau environment of Xiangxiong semi-wool sheep, raised on the plateau, places extremely high demands on their immune performance. Currently, the assessment of immune traits in the breeding and selection of Xiangxiong semi-wool sheep relies primarily on traditional methods, such as observing symptoms, mortality, and morbidity after natural or artificial infection with pathogens, and measuring conventional immune indicators such as serum antibody levels and immune cell counts. While these methods can provide a certain degree of insight into the immune status of sheep, they have significant limitations. Traditional observation methods are highly subjective, easily influenced by environmental factors and observer experience, and are unable to delve into the genetic basis of immune traits. Furthermore, measuring conventional immune indicators often only reflects the current immune response of sheep and cannot predict their future resistance to different pathogens. Furthermore, the testing process is cumbersome, time-consuming, and costly.

[0005] In-depth research on SNP molecular markers associated with the immune traits of Xiangxiong semi-fine wool sheep is crucial for further exploring their excellent immune gene resources, improving their disease resistance in plateau environments, and breeding new Xiangxiong semi-fine wool sheep strains with enhanced immune performance through molecular marker-assisted selection. However, research on SNP molecular markers for the immune traits of Xiangxiong semi-fine wool sheep is still relatively scarce. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a molecular marker related to the immune traits of Xiangxiong semi-fine wool sheep and its application. By detecting the genotype of the molecular marker SNP site related to the immune traits in Xiangxiong semi-fine wool sheep, the immune traits of Xiangxiong semi-fine wool sheep can be quickly and accurately evaluated, which is of great significance for the early assisted breeding of Xiangxiong semi-fine wool sheep.

[0007] The present invention provides a molecular marker associated with the immune traits of Xiangxiong semi-fine wool sheep. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1. The molecular marker includes a SNP site, the SNP site is located at position 363 of the molecular marker, the base of the SNP site is T or C, and the immunoglobulin content of Xiangxiong semi-fine wool sheep individuals with a genotype of TC or CC at the SNP site is significantly higher than that of individuals with a genotype of TT.

[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, comprising a primer F and a primer R; the nucleotide sequence of the primer F is shown in SEQ ID NO.2, and the nucleotide sequence of the primer R is shown in SEQ ID NO.3.

[0010] The present invention provides the use of a reagent for detecting the molecular marker in preparing an in vitro detection reagent for immune traits of Xiangxiong semi-fine wool sheep. The immunoglobulin content of Xiangxiong semi-fine wool sheep individuals with the SNP site genotype of TC or CC is significantly higher than that of individuals with the genotype of TT.

[0011] The present invention provides the use of a reagent for detecting the molecular marker in preparing an auxiliary breeding reagent for a high-immunity Xiangxiong semi-fine wool sheep variety.

[0012] The present invention also provides a method for detecting molecular markers related to immune traits of Xiangxiong semi-fine wool sheep for non-diagnostic purposes, comprising the following steps:

[0013] 1) extracting genomic DNA of the Xiangxiong semi-fine wool sheep to be bred, using the genomic DNA of the Xiangxiong semi-fine wool sheep as a template and performing PCR amplification with the primer pair to obtain an amplified product;

[0014] 2) Sequencing the amplified product to determine the genotype of the SNP site.

[0015] Preferably, the PCR amplification system, in a volume of 25 μL, includes 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 of the PCR amplification is as follows: 98°C for 2 min; 98°C for 10 s, 58°C for 10 s, 72°C for 10 s, for a total of 40 cycles; and extension at 72°C 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 associated with the immune traits of Xiangxiong semi-fine wool sheep. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1. The molecular marker includes a single nucleotide polymorphism (SNP) site located at position 363 of the molecular marker, and the base of the SNP site is T or C. The present invention analyzes the correlation between different genotypes of male semi-fine wool sheep samples and the levels of immunoglobulins IgA, IgG, and IgM, and determines that the immunoglobulin content of Xiangxiong semi-fine wool sheep individuals with TC or CC genotypes is significantly higher than that of individuals with TT genotypes. By detecting the genotype of the molecular marker associated with the immune traits in Xiangxiong semi-fine wool sheep, the present invention can assess the immunoglobulin content of the male semi-fine wool sheep, thereby screening Xiangxiong semi-fine wool sheep with high immunity in the early stages of breeding, thereby improving breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is an agarose gel electrophoresis diagram of the PCR amplification product; M is a 1000 bp marker, and 1 to 3 are the electrophoresis bands of the samples corresponding to the three genotypes of TT, TC, and CC;

[0020] Figure 2 The figure shows the sequencing peaks of three genotypes of SNP sites. DETAILED DESCRIPTION

[0021] The present invention provides a molecular marker associated with the immune traits of Xiangxiong semi-fine wool sheep. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1. The molecular marker includes a SNP site, the SNP site is located at position 363 of the molecular marker (i.e., the site in bold and underlined below), the base of the SNP site is T or C, and the immunoglobulin content of Xiangxiong semi-fine wool sheep individuals with a genotype of TC or CC at the SNP site is significantly higher than that of individuals with a genotype of TT. The immunoglobulin preferably includes one or more of IgA, IgG and IgM.

[0022] SEQ ID NO.1:

[0023]

[0024]

[0025] The present invention provides a primer pair for amplifying the molecular marker, comprising 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; specifically as follows:

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

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

[0028] The present invention provides the use of a reagent for detecting the molecular marker in preparing an in vitro reagent for detecting immune traits in Xiangxiong semi-fine wool sheep. Xiangxiong semi-fine wool sheep individuals with the TC or CC genotype at the SNP locus have significantly higher immunoglobulin levels than those with the TT genotype. The present invention does not specifically limit the type of reagent for detecting the molecular marker, and includes, but is not limited to, amplification primers, sequencing reagents, and genotyping tools.

[0029] The present invention provides the use of a reagent for detecting the molecular marker in preparing a reagent for assisting breeding of a high-immunity Xiangxiong semi-fine wool sheep breed. In the present invention, the genotype of the SNP site of the molecular marker in the Xiangxiong semi-fine wool sheep to be bred is preferably detected, and Xiangxiong semi-fine wool sheep to be bred with a genotype of TC or CC are selected as high-immunity individuals for subsequent breeding.

[0030] The present invention also provides a method for detecting molecular markers related to immune traits of Xiangxiong semi-fine wool sheep for non-diagnostic purposes, comprising the following steps:

[0031] 1) extracting genomic DNA of the Xiangxiong semi-fine wool sheep to be bred, using the genomic DNA of the Xiangxiong semi-fine wool sheep as a template and performing PCR amplification with the primer pair to obtain an amplified product;

[0032] 2) Sequencing the amplified product to determine the genotype of the SNP site.

[0033] In the present invention, genomic DNA of the Xiangxiong semi-fine wool sheep to be bred is first extracted, preferably genomic DNA from a blood sample. The present invention has no special limitation on the method for extracting the genomic DNA, and conventional genomic DNA extraction methods in the art can be used. In the specific implementation of the present invention, a blood genomic extraction kit of Beijing Quanshijin Biotechnology Co., Ltd. is preferably used. The concentration of the genomic DNA is preferably greater than 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 genomic DNA, the present invention uses the genomic DNA of Xiangxiong semi-fine wool sheep as a template and performs PCR amplification using the primer pair to obtain an amplified product. In the present invention, the PCR amplification system preferably includes 22 μL of PCR enzyme, 1 μL of primer F, 1 μL of primer R, and 1 μL of template DNA in 25 μL. The PCR amplification program is preferably as follows: 98°C for 2 minutes; 98°C for 10 seconds, 58°C for 10 seconds, and 72°C for 10 seconds, for a total of 40 cycles; and extension at 72°C for 2 minutes.

[0035] After obtaining the amplified product, the present invention sequences the amplified product to determine the genotype of the SNP site. In the present invention, the amplified product is preferably purified before sequencing. The present invention does not particularly limit the purification method, and conventional purification methods in the art can be used. In the present invention, the sequencing is preferably performed using direct sequencing, and the sequencing is preferably commissioned to Beijing Qingke Biotechnology Co., Ltd.

[0036] After sequencing, the present invention determines the genotype of the SNP site. Preferably, the bioanalysis software MEGA6.0 is used to compare the sequencing results of the PCR products, analyze the sequencing peak graph, complete typing, and determine the genotype of the SNP site.

[0037] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] 1. Sample collection

[0040] At the Xiangxiong semi-wool sheep breeding farm in Ngari Prefecture, Tibet Autonomous Region, 119 adult Xiangxiong semi-wool sheep, grazing under natural conditions, were randomly selected. Fasting blood samples (5 mL) were collected in a coagulant vacuum tube. The tubes were allowed to stand for 30 minutes and then centrifuged at 3500 rpm for 15 minutes. The supernatant was aspirated into a clean PE tube, sealed, and stored in a -20°C freezer for immune marker determination. Another 5 mL blood sample was collected in a tube containing EDTA-K2 anticoagulant. After collection, the blood samples were quickly mixed and placed in a sampling box containing ice packs. After transport to the laboratory, they were frozen at -20°C 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 markers, agarose, nucleic acid dyes, and PCR enzymes were all purchased from Beijing Quanshijin Biotechnology Co., Ltd.; a NanoDrop 2000 spectrophotometer was purchased from Thermo Fisher Scientific; an electrophoresis instrument was purchased from Beijing Liuyi Instrument Factory; and a 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 testing

[0045] Serum was assayed using IgA, IgG, and IgM detection kits from the Nanjing Jiancheng Bioengineering Institute. First, a standard curve was established using standard samples. Then, 7 μL of distilled water, standard solution, and sample to be tested were added to a blank tube, a standard tube, and a test tube, respectively. The volume was filled to 900 μL with R1 solution, incubated at 37°C for 5 minutes, and the reading at a wavelength of 340 nm was recorded as A1. Next, 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, ΔA was calculated as A2-A1, and the IgA, IgG, and IgM concentrations of the samples were calculated by substituting ΔA into the standard curve equation.

[0046] 3.2 Extraction of genomic DNA from blood

[0047] Genomic DNA was extracted from blood samples using a blood genome extraction kit from Beijing Quanshijin Biotechnology Co., Ltd. The extracted DNA was placed under an ultraviolet spectrophotometer to detect concentration and purity. A concentration >20 ng / μL and OD260 / OD280 between 1.7 and 1.9 met the experimental requirements and was stored at -20°C for future use.

[0048] 3.3 Primer design

[0049] Based on the chromosome 2 gene sequence (GenBank accession number: NC_056055.1) of the Rambouillet reference genome (ARS-UI_Ramb_v3.0, GCF_016772045.2), a pair of specific primers containing the g246858305T>C SNP site were designed using Oligo 7 software.

[0050] Primer sequences:

[0051] Primer F (SEQ ID NO. 2): 5′-ACTCAGCCATGATGTCGTCCA-3′;

[0052] Primer R (SEQ ID NO. 3): 5'-TAGGATCCCTCATGCCTCGT-3'.

[0053] The length of the amplified fragment was 847 bp. Beijing Qingke Biotechnology Co., Ltd. was commissioned to sequence the amplified fragment, and the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0054] 3.4 PCR amplification and sequencing

[0055] PCR amplification system 25 μL: PCR enzyme 22 μL, upstream and downstream primers 1 μL each, template DNA 1 μL.

[0056] PCR amplification program: 98°C for 2 min; 98°C for 10 s, 58°C for 10 s, and 72°C for 10 s, for a total of 40 cycles; and extension at 72°C for 2 min.

[0057] The PCR products were detected by 1.5% agarose gel electrophoresis. After the PCR products passed the agarose gel electrophoresis test, they were sequenced by direct sequencing, and the sequencing was completed by Sangon Biotech (Shanghai) Co., Ltd. The amplified nucleotide sequence is shown in SEQ ID No. 1, and the SNP marker is located at position 363 of the nucleotide sequence shown in SEQ ID No. 1.

[0058] The bioanalysis software MEGA6.0 was used to compare the sequencing results of PCR products, analyze the sequencing peak graphs, and complete the typing.

[0059] 4 Statistical analysis

[0060] Based on the genotyping results, the number of individuals with different genotypes at each locus was counted. Popgen32 software was used to calculate the g246858305T>C gene frequency, genotype frequency, effective number of alleles (Ne), locus heterozygosity (He), and Hardy-Weinberg equilibrium test. PIC (polymorphism information content) software was used to calculate the polymorphic information content. General linear models in IBM SPSS Statistics 22 software were used to analyze the associations between different genotypes and immunoglobulin IgA, IgG, and IgM in Xiangxiong semi-fine wool sheep. Results are presented as mean ± standard error.

[0061] 5 Results

[0062] 5.1 PCR amplification and sequencing results

[0063] The amplified product of g246858305T>CSNP site on chromosome 2 of Xiangxiong semi-fine wool sheep was detected by 1.5% agarose gel (see Figure 1 ), the bands were clear without any other bands, the specificity was good, and the PCR product fragment size was 847 bp, which was in line with the expected size, so the next experiment could be carried out.

[0064] The peak diagram and sequence of the PCR product after purification and sequencing are shown in Figure 2 .Depend on Figure 2 It can be seen that the TC mutation occurred at the g246858305T>CSNP site, and there are three genotypes: TT, TC, and CC.

[0065] 5.2 Statistical analysis results

[0066] From a population genetics perspective, the genotype and allele frequencies of the g246858305T>C SNP locus on chromosome 2 in male semi-wool sheep were analyzed. As shown in Table 1, the CC genotype is the most frequent and dominant genotype at the g246858305T>C SNP locus, while the C allele frequency is 87%, indicating a dominant allele. The chi-squared fitness test indicated that the SNP locus significantly deviated from Hardy-Weinberg equilibrium (P < 0.05) (Table 1). The expected heterozygosity and PIC of this locus are 0.233 and 0.206, respectively, with a PIC < 0.25, indicating low polymorphism.

[0067] Table 1 Polymorphism of the g246858305T>C SNP locus on chromosome 2 of Xiangxiong semi-fine wool sheep

[0068]

[0069] 5.3 Association analysis between different genotypes and immunoglobulins IgA, IgG, and IgM

[0070] A general linear model analysis using IBM SPSS Statistics 22 software was used to analyze the association between different genotypes and immunoglobulin IgA, IgG, and IgM levels in Xiangxiong semi-fine wool sheep. The results showed that individuals with the TT genotype had significantly lower levels of immunoglobulins IgA, IgG, and IgM than those with the TC and CC genotypes (p < 0.05). No significant differences were observed between individuals with the TC and CC genotypes (p > 0.05). This suggests that the g246858305T>C SNP on chromosome 2 of Xiangxiong semi-fine wool sheep is significantly associated with IgA, IgG, and IgM levels (p < 0.05), making it a SNP marker associated with IgA, IgG, and IgM levels in Xiangxiong semi-fine wool sheep. The results are shown in Table 2.

[0071] Table 2 Correlation analysis between different genotypes and immunoglobulins IgA, IgG, and IgM

[0072]

[0073]

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

[0075] In summary, the SNP molecular marker described in the present invention is located at the 246858305th base on chromosome 2 of the Rambouillet reference genome (ARS-UI_Ramb_v3.0, GCF_016772045.2); the variation type is T / C, named g246858305T>C, and there are three genotypes. When the 246858305th base on chromosome 2 is T, the genotype is TT or TC; when the 246858305th base on chromosome 2 is C, the genotype is CC; through the association analysis of different genotypes with the content of immunoglobulins IgA, IgG, and IgM, it was found that the immunoglobulins IgA, IgG, and IgM of the Xiangxiong semi-fine wool sheep individuals with the TT genotype were significantly lower than those of the TC and CC genotype individuals (p<0.05), and no significant difference was shown between the TC and CC genotype individuals (p>0.05). By detecting the base at the 246858305th nucleotide position on chromosome 2 of the Xiangxiong semi-fine wool sheep, the immunoglobulin IgA, IgG, and IgM contents of the individual Xiangxiong semi-fine wool sheep can be determined. The present invention provides a new SNP molecular marker resource for marker-assisted selection of immune traits of Xiangxiong semi-fine wool sheep for non-diagnostic purposes.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection 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 363rd position of the molecular marker, the base of the SNP site is T or C, and the immunoglobulin content of the Zhangzhung semi-fine wool sheep individuals with the SNP site genotype of TC or CC is significantly higher than that of the individuals with the genotype of TT.

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 a reagent for detecting the molecular marker according to claim 1 in the preparation of an in vitro detection reagent for the immune traits of Zhangzhong semi-fine wool sheep, characterized in that, The immunoglobulin content of the Zhangzhung semi-fine wool sheep individuals with the SNP site genotype of TC or CC is significantly higher than that of the individuals with the genotype of TT.

5. Use of the reagent for detecting the molecular marker described in claim 1 in the preparation of an auxiliary breeding reagent for the Zhangzhung semi-fine wool sheep breed with high immunity.

6. A method for detecting molecular markers related to the immune traits of Zhangzhung semi-fine wool sheep for non-diagnostic purposes, characterized in that, 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 described in claim 3 to obtain an amplification product. 2) Sequence the amplification product to determine the genotype of the SNP site.

7. The application according to claim 6, characterized in that, The amplification system of the PCR amplification is calculated based on 25 μL, 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, 58 °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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