A method for detecting insertion / deletion markers of the CTLA-4 gene in cashmere goats and its application

Abstract

The present invention discloses a method for detecting insertion / deletion markers of the CTLA-4 gene in cashmere goats and its application. Using the whole genome of the cashmere goat to be detected as a template and the designed primer pairs P1 and P2 as amplification primers, a partial fragment of the CTLA-4 gene in cashmere goats is amplified by PCR, and then the genotypes of the insertion / deletion polymorphic sites at positions NC_030809.1:rs655778058,g.43841952_43841968 and NC_030809.1:rs656541224,g.43843568_43843579 of the CTLA-4 gene in cashmere goats are identified by agarose gel electrophoresis and sequencing technology. The relevant analysis results show that there is a significant correlation between different genotypes of the two InDel sites of the CTLA-4 gene in cashmere goats and the Brucella resistance of Shaanbei white cashmere goats, which can be used as an effective DNA marker for Brucella resistance breeding of Shaanbei white cashmere goats. According to the detected genotypes of the insertion / deletion of the InDel sites of the CTLA-4 gene in cashmere goats, the present invention can be used as an effective DNA marker for the prevention and control of Brucella in cashmere goats and Brucella resistance genetic breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of livestock breeding, and relates to a method for detecting an insertion / deletion marker of a CTLA-4 gene in cashmere goats and its application. Background Art

[0002] The goat breeding industry is one of the important pillars for the rapid development of China's animal husbandry. With the improvement of the national economic level, the society's demand for cashmere, mutton, cashmere and wool has increased accordingly. However, brucellosis caused by Brucella seriously hinders the development of the goat breeding industry. Brucellosis is a highly contagious and extremely harmful chronic zoonosis. After humans or goats are infected with brucellosis, it can have a great impact on the reproductive system. At present, detecting positive livestock for brucellosis and immediately culling, burning and harmlessly treating them are effective means for brucellosis prevention and control. In China, goats, as the main source of infection for brucellosis, can be infected through the digestive tract, respiratory tract, reproductive tract, and damaged skin and mucous membranes. However, there is currently no selection of brucellosis-resistant goats using traditional breeding methods.

[0003] With the rapid development of molecular biology and bioinformatics, using molecular marker-assisted selection (MAS) technology to screen and identify genes related to livestock economic traits, growth traits and disease resistance has become an important way to improve livestock production performance. The screening and identification of MAS technology is a molecular breeding method based on genetic mutations of genes, which mainly includes genetic mutation methods such as SNPs, INDELs and CNVs.

[0004] InDel (Insertion / Deletion) molecular markers refer to insertion or deletion markers with different numbers of bases at the same locus in the genomes of the same species or related species. The inserted or deleted fragments range from 1 bp to 50 bp. The generation of insertions or deletions is mainly related to the base types of genomic sequences and the number of DNA replications or slippages. Because simple agarose gel electrophoresis can be used to genotype InDel, combined with high-throughput sequencing technology, the development cost of InDel markers is significantly reduced, and they are widely used in the breeding of various crops and livestock and poultry. As a new type of molecular marker, InDel molecular markers have a lower abundance than SNP markers, but can exclude single-base nonsense mutations of SNP markers. Based on the characteristics of high accuracy and stable variation of InDel markers, they are widely used in fields such as constructing genetic maps of animal and plant populations, molecular assisted breeding and medical diagnosis.

[0005] Currently, there have been reports on InDel research in livestock: Wang et al. found that a 27-bp InDel mutation in the intron region of the DNAH1 gene could significantly affect the litter size of Shaanbei white cashmere goats (Wang et al., 2022); Zhou et al. reported that a 19-bp InDel mutation in the PLAG1 gene of Yunling cattle was significantly correlated with body height, rump height, and chest girth (Zhou et al., 2019); Cui et al. analyzed the correlation of InDel mutation sites in the intron of the KDM5B gene in Landrace pigs and found that a 35-bp InDel mutation could extremely significantly affect the testis weight and the short axis length of the testis of 40-day-old Landrace pigs (Cui et al., 2018). The above studies all indicate that InDel mutations have research value and reference significance for molecular-assisted breeding of livestock.

[0006] Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) belongs to the members of the immunoglobulin superfamily, is located on chromosome 2q33 encoding immunoregulatory molecules, is an important structure for signal transmission between cells of the immune system, and participates in regulating cell surface molecules of T cells. In recent years, CTLA-4 has been widely studied in viral infectious diseases and the induction of cellular immunity in the body. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for detecting insertion / deletion markers of the CTLA-4 gene in cashmere goats and its application, which helps to conduct early selection of Brucella-resistant sheep flocks through screening and combining molecular marker-assisted breeding, accelerate the establishment of Brucella-resistant sheep flocks and carry out rapid propagation.

[0008] To achieve the above purpose, the present invention adopts the following technical solutions:

[0009] A method for detecting insertion / deletion polymorphisms of the CTLA-4 gene in cashmere goats, comprising the following operations:

[0010] Using the whole genome of the cashmere goat to be detected as a template, PCR amplify the fragment containing the insertion / deletion polymorphisms of two mutation sites in the 5' regulatory region of the CTLA-4 gene, perform agarose gel electrophoresis on the PCR amplification product, and identify the genotypes of the insertion / deletion polymorphisms of the mutation sites according to the electrophoresis results;

[0011] The fragment amplified by the primer pair P1 contains a 12-bp insertion / deletion polymorphism site at positions NC_030809.1:rs655778058,g.43841952_43841968 of the CTLA-4 gene in cashmere goats;

[0012] The fragment amplified by primer pair P2 contains the 12 bp insertion / deletion polymorphism site at positions 43843568_43843579 of the Cashmere goat CTLA-4 gene NC_030809.1:rs656541224;

[0013] The primer pair P1 is as follows:

[0014] Forward primer: 5’-ATTCCATCACCCACAG-3’

[0015] Reverse primer: 5’-ATTACCCAGTTATCCTTC-3’

[0016] The primer pair P2 is as follows:

[0017] Forward primer: 5’-CACAGCCATACCCATTC-3’

[0018] Reverse primer: 5’-GCACCATAAGGAGCAA-3’.

[0019] The PCR reaction program for primer pair P1 is as follows:

[0020] Pre-denaturation at 95℃ for 5 min, denaturation at 94℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 30 s, for a total of 38 cycles; extension at 72℃ for 10 min, and preservation at 4℃;

[0021] The PCR reaction program for primer pair P2 is: pre-denaturation at 95℃ for 5 min, denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s, for a total of 38 cycles;

[0022] Extension at 72℃ for 10 min, and preservation at 4℃. All electrophoresis was performed using a 3% agarose gel by mass concentration.

[0023] For the insertion / deletion polymorphism site at positions 43841952_43841968, the insertion / insertion genotype II shows a single band of 285 bp, the insertion / deletion genotype ID shows two bands of 285 bp and 273 bp, and the deletion / deletion genotype DD shows a single band of 273 bp;

[0024] For the insertion / deletion polymorphism site at positions 43843568_43843579, the insertion / insertion genotype II shows a single band of 291 bp, the insertion / deletion genotype ID shows two bands of 291 bp and 279 bp, and the deletion / deletion genotype DD shows a single band of 279 bp.

[0025] A kit for detecting the insertion / deletion polymorphism of the CTLA-4 gene in cashmere goats. The kit includes primer pairs and a PCR reaction solution for PCR amplification of the insertion / deletion polymorphic sites at positions NC_030809.1:rs655778058, g.43841952_43841968 and NC_030809.1:rs656541224, g.43843568_43843579 of the CTLA-4 gene in cashmere goats; the primer pairs include primer pair P1 and primer pair P2;

[0026] The primer pair P1 is as follows:

[0027] Forward primer: 5’-ATTCCATCACCCACAG-3’

[0028] Reverse primer: 5’-ATTACCCAGTTATCCTTC-3’

[0029] The primer pair P2 is as follows:

[0030] Forward primer: 5’-CACAGCCATACCCATTC-3’

[0031] Reverse primer: 5’-GCACCATAAGGAGCAA-3’

[0032] The application of the insertion / deletion at positions NC_030809.1:rs655778058, g.43841952_43841968 and NC_030809.1:rs656541224, g.43843568_43843579 of the CTLA-4 gene as a molecular marker in the assistant selection breeding of cashmere goats resistant to Brucellosis.

[0033] For the genotype at positions 43841952_43841968, the insertion / insertion genotype is selected as the Brucellosis resistance genotype of cashmere goats;

[0034] For the genotype at positions 43843568_43843579, the insertion / insertion genotype is selected as the Brucellosis resistance genotype of cashmere goats.

[0035] For the insertion / deletion polymorphic site at positions 43841952_43841968, the insertion / insertion genotype shows a single band of 285 bp in nucleic acid electrophoresis;

[0036] For the insertion / deletion polymorphic site at positions 43843568_43843579, the insertion / insertion genotype shows a single band of 291 bp in nucleic acid electrophoresis.

[0037] Compared with the prior art, the present invention has the following beneficial technical effects:

[0038] The present invention designs primers according to the 5' regulatory region sequence of the CTLA-4 gene of cashmere goats. Using the genomic DNA of cashmere goats as a template, through PCR amplification, agarose gel electrophoresis, and direct sequencing techniques, it can accurately, quickly, and at low cost detect the insertion / deletion polymorphisms at positions NC_030809.1:rs655778058,g.43841952_43841968 and NC_030809.1:rs656541224,g.43843568_43843579 of the CTLA-4 gene of cashmere goats. The present invention uses PCR amplification technology to detect the insertion / deletion polymorphisms of the CTLA-4 gene of cashmere goats and analyze the genotype and allele frequencies. For the first time, it is found that there is a significant correlation between the CTLA-4 gene and the brucellosis resistance of northern Shaanxi white cashmere goats, which can be used as an effective DNA marker for the genetic breeding of northern Shaanxi white cashmere goats against brucellosis.

[0039] The risk levels of the insertion / deletion and deletion / deletion genotypes of the insertion / deletion polymorphic locus at positions 43841952_43841968 for contracting brucellosis are 1.337 times and 1.501 times that of the insertion / insertion genotype respectively. The insertion / insertion genotype may be the brucellosis resistance genotype of cashmere goats;

[0040] The risk levels of the insertion / deletion and deletion / deletion genotypes of the insertion / deletion polymorphic locus at positions 43843568_43843579 for contracting brucellosis are 1.512 times and 1.186 times that of the insertion / insertion genotype respectively. Its insertion / insertion genotype may be the brucellosis resistance genotype of cashmere goats;

[0041] Positions 43841952_43841968 and 43843568_43843579 can be used as important candidate molecular marker sites for the breeding of cashmere goats against brucellosis. The present invention screens and combines molecular marker-assisted selection (MAS) technology to detect DNA candidate marker sites closely related to brucellosis susceptibility or resistance, and conducts correlation analysis based on gene polymorphisms and brucellosis susceptibility or resistance, so as to early select brucellosis-resistant flocks, accelerate the establishment of brucellosis-resistant flocks and carry out rapid expansion. Description of the Drawings

[0042] Figure 1 The result of 3% agarose gel electrophoresis of the product amplified by primer pair P1 for the CTLA-4 gene of cashmere goats; where M represents Marker I.

[0043] Figure 2This is the sequencing map of the PCR amplification product of the CTLA-4 gene. The part marked by the black square represents a 12-bp insertion sequence: NC_030809.1:rs655778058,g.43841952_43841968. The expected insertion sequence is a 17-bp insertion sequence: CAGGAATCTCCTGCACT, and the actual sequencing sequence is a 12-bp insertion sequence: TGTGAATTTTCC.

[0044] Figure 3 This is the result of 3% agarose gel electrophoresis of the product amplified by primer pair P2 for the CTLA-4 gene of cashmere goats. Among them, M represents Marker I.

[0045] Figure 4 This is the sequencing map of the PCR amplification product of the CTLA-4 gene. The part marked by the black square represents a 12-bp insertion sequence: NC_030809.1:rs656541224,g.43843568_43843579. The expected insertion sequence is the same as the actual sequencing sequence, which is a 12-bp insertion sequence CCACACAAAATG.

[0046] Figure 5 This is the linkage disequilibrium analysis of the InDel mutation sites at positions 43841952_43841968 and 43843568_43843579 of the CTLA-4 gene. Among them, D’ represents the deviation of the observed haplotype frequency from the expected frequency in the equilibrium state, and r 2 represents the statistical correlation between two loci. Specific embodiments

[0047] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0048] The polymorphisms of multiple SNP sites of the CTLA-4 gene are related to the susceptibility of several autoimmune diseases such as human systemic lupus erythematosus. At the same time, the expression of immune checkpoint molecules such as CTLA-4 shows differences during the process of avian virus infection, suggesting that CTLA-4 may play an important role in the virus pathogenesis mechanism and the induction of cellular immunity in the body.

[0049] As an important genetic marker, InDel is a small fragment of DNA with an insertion or deletion of 5 - 50 bp in length in the genome. However, there is currently no report on the research related to the polymorphism of the InDel locus of the CTLA-4 gene in cashmere goats and its susceptibility or resistance to brucellosis.

[0050] Referring to the base sequences of the goat CTLA-4 gene published in the NCBI database and the Ensembl database (https: / / asia.ensembl.org / index.html), the InDel mutation sites at positions 43841952_43841968 and 43843568_43843579 were selected as candidate genetic variation sites for research.

[0051] The present invention uses the PCR technique to detect the possible insertion / deletion polymorphisms generated by the mutations at positions 43841952_43841968 and 43843568_43843579 of the CTLA-4 gene (NC_030809.1) in cashmere goats, and conducts a correlation analysis with the Brucella resistance of cashmere goats to verify that it can be used as a molecular marker for assistant selection in the breeding of cashmere goats resistant to Brucella.

[0052] 1. Experimental drugs and reagents

[0053] Biochemical and biological reagents: ① Taq DNA polymerase (purchased from Fermantas, i.e., MBI company); ② Proteinase K (purchased from Huamei Engineering Biology Company); ③ Marker I (purchased from Tiangen Biochemical (Beijing) Co., Ltd.); ④ Rose Bengal plate agglutination antigen, standard negative serum, and standard positive serum were all purchased from Harbin Pharmaceutical Group Biological Vaccines Co., Ltd.

[0054] Common reagents: Common reagents were purchased from Huamei Engineering Biology Company and were imported and repackaged products: glucose, citric acid, sodium citrate, Tris, EDTA, NaCl, NaOH, Kcl, Na2HPO4, KH2PO4, Tris-saturated phenol, isopentyl alcohol, chloroform, absolute ethanol, sodium acetate, sodium dodecyl sulfate (SDS), ethidium bromide (EB), bromophenol blue, dimethylbenzene cyan FF, sucrose, boric acid, acetic acid, agarose, etc.

[0055] Solutions and buffers: All solutions and buffers were prepared with deionized ultrapure water. The autoclaving conditions were 15 bf / in (1.034×105 Pa) and 25 min. The reagent preparation methods all refer to "Molecular Cloning: A Laboratory Manual" edited by Sambrook et al.

[0056] Solutions for extracting tissue sample DNA

[0057] ① Common solutions for extracting genomic DNA;

[0058] ② 2 mol / L NaCl: 11.688 g was dissolved in water, and the volume was fixed to 100 mL and then autoclaved at high temperature;

[0059] ③ Tissue DNA extraction solution (100 mL): 1 mL of 1 mol / L Tris-HCl (pH 8.0), 20 mL of 0.5 mol / L EDTA (pH 8.0), 5 mL of 2 mol / L NaCl, and made up to 100 mL.

[0060] Solutions for agarose gel electrophoresis analysis

[0061] ① 0.5 X TBE buffer: Take 50 mL of 10 X TBE and make up to 1000 mL;

[0062] ② Loading buffer: Containing 0.25% bromophenol blue and 0.25% dimethylbenzene cyan FF, and the solvent is 40.0% (w / v) sucrose aqueous solution.

[0063] 2. Design primers for InDel loci of the CTLA-4 gene in cashmere goats

[0064] Retrieve the sequence of the goat CTLA-4 gene on NCBI, and use Primer 5.0 to design primers that can amplify the DNA fragment of the candidate InDel locus of the CTLA-4 gene. Among them, the primer pair that can amplify the InDel locus in the region of positions 43841952_43841968 in the 5' regulatory region of the cashmere goat CTLA-4 gene is P1, and the primer pair that can amplify the InDel locus in the region of positions 43843568_43843579 in the 5' regulatory region of the cashmere goat CTLA-4 gene is P2. The primer sequences are shown in Table 1:

[0065]

[0066] 3. PCR amplification of the 5' regulatory region fragment of the CTLA-4 gene in the tested cashmere goats

[0067] Collection of cashmere goat ear tissue samples

[0068] The experimental animals were selected from the northern Shaanxi white cashmere goats bred by Shaanxi Haoli Cashmere Goat Science and Technology Development Co., Ltd. in Yulin City, Shaanxi Province, with a total of 810 samples. The sampling time was April 2019. Use ear cutting pliers to cut about soybean-sized ear tissue samples of the test goats, place them in 70% ethanol for preservation, and store them in a -80°C refrigerator after bringing them back to the laboratory in an ice box.

[0069] Extraction of genomic DNA from tissue samples

[0070] Refer to "Molecular Cloning: A Laboratory Manual" (2002) edited by Sambrook et al. and the following references: Lan Xianyong. Genetic Analysis of Important Functional Genes in Goats and Their Relationship with Economic Traits [D]. Doctoral Dissertation of Northwest A&F University, Yangling, Shaanxi, 2007.

[0071] 3.3 Detection of DNA by Agarose Gel Electrophoresis

[0072] Refer to "Molecular Cloning: A Laboratory Manual" (2002) edited by Sambrook et al.

[0073] 3.4 Purification of DNA

[0074] Refer to "Molecular Cloning: A Laboratory Manual" (2002) edited by Sambrook et al.

[0075] 3.5 Detection of DNA by Spectrophotometry

[0076] Use a UV spectrophotometer to measure the OD values of the extracted DNA sample at 260 nm and 280 nm. Calculate the DNA content and the ratio of OD 260 / OD 280 . If the ratio of OD 260 / OD 280 is less than 1.6, it indicates that the sample contains more protein or phenol and should be purified; if the ratio is greater than 1.8, RNA removal should be considered for purification.

[0077] DNA concentration (ng / μL) = 50 × OD 260 value × dilution factor

[0078] After DNA detection is completed, take a certain amount and dilute it to 20 ng / μL, store it at -20 °C in the refrigerator for later use, and store the remaining sample at -80 °C in the refrigerator.

[0079] 4. Detection of Brucella by Rose Bengal Plate Agglutination Test

[0080] 4.1 Collection of Cashmere Goat Serum

[0081] Use a disposable blood collection syringe to collect 2 mL of jugular vein blood from the experimental goats. After standing for 2 hours, the serum is separated and used for serological detection of Brucella.

[0082] 4.2 Serological Detection of Brucella in Cashmere Goats

[0083] Use the RBPT method to conduct serological detection of Brucella in the separated serum. Take 20 μL of the serum to be detected and 20 μL of the Rose Bengal plate agglutination antigen, gently pipette and mix them evenly on the Rose Bengal plate, let it stand at room temperature for 5 min, observe the changes in the serum, and use the negative serum as a control. Standard positive serum shows different degrees of sandy particles or flocculent agglutination, and standard negative serum shows no change. Use this as a standard to judge the serum to be detected. To ensure the accuracy of the results, retest the positive serum. If it is still positive, it is determined as Brucella infection.

[0084] 5. PCR Amplification

[0085] The PCR reaction uses the mixed loading method. According to the quantity of each component required for each reaction system and the number of PCR reactions required for one-time reaction, the total amount of each reaction component is calculated, mixed into a 2 mL centrifuge tube, thoroughly pipetted and mixed, then centrifuged instantaneously, and then successively aliquoted into 0.2 mL PCR tubes. Then, template DNA is added successively, and after instantaneous centrifugation, PCR amplification is carried out.

[0086] The PCR reaction system includes 6.5 μL of 2×Taq SuperMix (including Taq DNA polymerase, dNTPs and optimized reaction buffer, with a concentration of 2X), 0.5 μL of each of the upstream and downstream primers (each of the upstream and downstream primers is 10 pmol / μL), 0.7 μL of genomic DNA (with a concentration of 20 ng / μL), and 4.8 μL of deionized water; a total of 13 μL volume of PCR amplification system.

[0087] PCR reaction procedure

[0088] The PCR reaction procedure for primer pair P1 is: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, for a total of 38 cycles; extension at 72°C for 10 min, and preservation at 4°C. The PCR reaction procedure for primer pair P2 is: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, for a total of 38 cycles; extension at 72°C for 10 min, and preservation at 4°C.

[0089] Agarose gel electrophoresis detection and analysis of PCR products

[0090] Prepare a 3% agarose gel, stain it with ethidium bromide (EB) nucleic acid dye, load 6 μL of sample, and electrophorese at 110 V for 1 - 2 hours;

[0091] After the DNA fragments of different molecular weights are clearly separated, image them on the Invitrogen iBright gel imaging system;

[0092] Analyze genotype typing according to the results of agarose gel electrophoresis;

[0093] Using primer pair P1 for PCR amplification of cashmere goats, a fragment containing the 5' regulatory region of the cashmere goat CTLA-4 gene at positions NC_030809.1:rs655778058,g.43841952_43841968 can be amplified, as shown in the electrophoresis detection map of the amplified fragment ( Figure 1), where II is the insertion / insertion genotype, showing a single band of 285 bp; ID is the insertion / deletion genotype, showing two bands of 285 bp and 273 bp; DD is the deletion / deletion genotype, showing a single band of 273 bp; after sequencing and identification of the amplified fragment (the sequencing results are as Figure 2 shown), the expected sequencing sequence is an insertion / deletion of 17 bp CAGGAATCTCCTGCACT, and the actual sequencing sequence is an insertion / deletion sequence of 12 bp TGTGAATTTTCC.

[0094] Using primer pair P2 to perform PCR amplification on cashmere goats, a fragment containing the 5'-regulatory region of the cashmere goat CTLA-4 gene at positions NC_030809.1:rs656541224,g.43843568_43843579 can be amplified. The electrophoretic detection map of the amplified fragment is shown ( Figure 3 ), where II is the insertion / insertion genotype, showing a single band of 291 bp; ID is the insertion / deletion genotype, showing two bands of 291 bp and 279 bp; DD is the deletion / deletion genotype, showing a single band of 279 bp; after sequencing and identification of the amplified fragment (the sequencing results are as Figure 4 shown), the expected sequencing sequence is the same as the actual sequencing sequence, which is an insertion / deletion sequence of 12 bp CCACACAAAATG.

[0095] 6. Statistical analysis of the InDel site frequencies of the cashmere goat CTLA-4 gene

[0096] Genotype frequency refers to the proportion of the number of a certain genotype in a population to the total number of individuals.

[0097] The genotype frequencies of the insertion / deletion polymorphic sites at positions NC_030809.1:rs655778058, g.43841952_43841968 and NC_030809.1:rs656541224, g.43843568_43843579 in the 5'-regulatory region of the cashmere goat CTLA-4 gene are shown in Tables 2 and 3.

[0098] 7. Correlation analysis of the gene effects of the InDel sites of the cashmere goat CTLA-4 gene

[0099] Genotype data: Genotypes identified by agarose gel electrophoresis and sequencing after PCR amplification; Production data: Positive and negative data of Brucella in Shaanbei white cashmere goats detected by Rose Bengal plate agglutination test.

[0100] The correlation between InDel polymorphic sites of the CTLA-4 gene and Brucella resistance in Shaanbei white cashmere goats was analyzed using SPSS 26.0 software. During the data processing, considering individual effects, gene interactions, and genotype effects, a fixed model was used for correlation analysis. The Logistic regression model was used to calculate the OR value and 95% confidence interval to detect the effects of different factors on Brucella resistance / susceptibility. The statistical analysis model was Y ijk =μ+G i +y k where Y ijk represents whether the goat is diseased, G i represents the genotype effect, and y k represents the age effect.

[0101] The results of the correlation analysis are shown in Tables 4 and 5.

[0102] As can be seen from Table 4, there were significant differences in different genotypes at positions 43841952_43841968 of the CTLA4 gene between the Brucella-positive group and the negative group (P = 0.047). The Brucella susceptibility of Shaanbei white cashmere goats with ID and DD genotypes was significantly higher than that of the II genotype. The OR values were 1.337 (0.968 - 1.846) and 1.501 (0.853 - 2.643) respectively, that is, the probabilities of goats with ID and DD genotypes getting Brucella were 1.337 times and 1.501 times that of the II genotype.

[0103]

[0104] As can be seen from Table 5, there were significant differences in different genotypes at positions 43843568_43843579 of CTLA4 between the Brucella-positive group and the negative group (P = 0.039). The Brucella susceptibility of Shaanbei white cashmere goats with ID and DD genotypes was significantly higher than that of the II genotype. The OR values were 1.512 (1.096 - 2.086) and 1.186 (0.556 - 2.532) respectively, that is, the probabilities of goats with ID and DD genotypes getting Brucella were 1.512 times and 1.186 times that of the II genotype.

[0105]

[0106] Linkage disequilibrium analysis of InDel sites of the CTLA-4 gene in cashmere goats

[0107] The results of the linkage disequilibrium analysis showed that ( Figure 5 ), the D’ value between the InDel sites at positions 43841952_43841968 and 43843568_43843579 of the CTLA-4 gene was 0.108, r 2The value is 0.009, indicating that there is no linkage relationship between the two mutations.

[0108] The present invention uses the PCR amplification method to detect the insertion / deletion polymorphism sites at positions NC_030809.1:rs655778058,g.43841952_43841968 and NC_030809.1:rs656541224,g.43843568_43843579 of the CTLA-4 gene in cashmere goats, and conducts a correlation analysis with the Brucella resistance or susceptibility of Shaanbei white cashmere goats, and finds that it can be used as an effective molecular marker for the prevention and control of goat brucellosis and Brucella-resistant genetic breeding.

[0109] The above-given embodiments are the preferred examples for implementing the present invention, and the present invention is not limited to the above embodiments. Any non-essential addition or replacement made by those skilled in the art according to the technical features of the technical solution of the present invention shall fall within the protection scope of the present invention.

Claims

1. Application of the insertion / deletion at rs656541224 in the CTLA-4 gene NC_030809.1 as a molecular marker in the assisted selection breeding of Shaanbei white cashmere goats against brucellosis; the inserted sequence is CCACACAAAATG.

2. The application according to claim 1, characterized in that: Select the insertion / insertion genotype as the brucellosis resistance genotype of cashmere goats.

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