A primer combination, method and application for identifying Chinese holstein cow mastitis resistance

Abstract

The application discloses a primer combination for identifying Chinese Holstein cow mastitis resistance, a method and application thereof. The method comprises the following steps: detecting genotypes of a to-be-detected Chinese Holstein cow based on SNP1 site (rs135854456), SNP11 site (104010752 of chromosome 5 of a Chinese Holstein cow genome), SNP15 site (rs109421300) and SNP16 site (rs109350371); if the genotype based on the SNP1 site is CC homozygous type, the genotype based on the SNP11 site is CC homozygous type, the genotype based on the SNP15 site is AA homozygous type and the genotype based on the SNP16 site is AA homozygous type, the to-be-detected Chinese Holstein cow has mastitis resistance. The method provided by the application can accurately identify Chinese Holstein cow mastitis resistance and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a primer combination, method, and application for identifying mastitis resistance in Chinese Holstein bovines. Background Technology

[0002] Bovine mastitis, an inflammation of the udder in dairy cows, is the most common and complex disease of the mammary glands. It involves varying degrees of pathological changes in the mammary tissue, leading to an increase in somatic cell count (SCC) in milk, resulting in a significant reduction in milk yield and quality, and has a substantial impact on the global dairy industry. On the one hand, in actual farming, farms typically manage the udder health of their herds by monitoring SCC, using it as an indirect indicator of mastitis (e.g., a healthy cow has an SCC below 100,000 / mL) to control milk quality. However, the standards for using SCC to diagnose mastitis are not standardized, and the results are unstable and inaccurate due to environmental influences. On the other hand, traditional dairy breeding often aims to indirectly improve mastitis resistance by achieving a low somatic cell score (SCS), but this selection method has yielded limited genetic progress. Therefore, under the trend of standardized drug use, large-scale implementation of antibiotic alternatives and antibiotic reduction farming strategies, establishing a standard for identifying mastitis resistance in Chinese Holstein cattle is particularly important for improving dairy cow production performance and increasing the breeding efficiency of mastitis-resistant cattle.

[0003] In recent years, SNPs, as third-generation molecular markers, have received widespread attention due to their advantages such as large number, wide distribution, and genetic stability. Analysis based on the genetic information of Chinese Holstein cattle can uncover more stable and efficient SNP loci. Therefore, it is necessary to explore effective methods for identifying mastitis resistance in Chinese Holstein cattle based on gene polymorphism. Using allele competitive specific PCR, specific primers can be developed to obtain the genotype of the sample at the SNP locus, ultimately identifying mastitis resistance in Chinese Holstein cattle. Summary of the Invention

[0004] The purpose of this invention is to identify mastitis resistance in Chinese Holstein cattle.

[0005] This invention first protects primer combinations. The primer combinations may include primer sets 1 through 4;

[0006] The primer set 1 consists of the forward primer 01F1 shown in SEQ ID NO:1, the forward primer 01F2 shown in SEQ ID NO:2, and the reverse primer 01R shown in SEQ ID NO:3;

[0007] The primer set 2 consists of the forward primer 02F1 shown in SEQ ID NO:4, the forward primer 02F2 shown in SEQ ID NO:5, and the reverse primer 02R shown in SEQ ID NO:6;

[0008] The primer set 3 consists of the forward primer 03F1 shown in SEQ ID NO:7, the forward primer 03F2 shown in SEQ ID NO:8, and the reverse primer 03R shown in SEQ ID NO:9;

[0009] The primer set 4 consists of the forward primer 04F1 shown in SEQ ID NO:10, the forward primer 04F2 shown in SEQ ID NO:11, and the reverse primer 04R shown in SEQ ID NO:12.

[0010] In the above primer combination, forward primers 01F1, 02F1, 03F1, and 04F1 can be FAM fluorescently labeled. Forward primers 01F2, 02F2, 03F2, and 04F2 can be HEX fluorescently labeled.

[0011] The primer combination may specifically consist of primer set 1, primer set 2, primer set 3, and primer set 4.

[0012] The application of any of the primer combinations described above in the preparation of a kit for identifying or assisting in the identification of whether Chinese Holstein cattle have mastitis resistance is also within the scope of protection of this invention.

[0013] The application of any of the primer combinations described above in the preparation of a kit for assisting in the screening of Chinese Holstein cattle with mastitis resistance is also within the scope of protection of this invention.

[0014] This invention also protects the use of any of the primer combinations described above in the auxiliary screening of Chinese Holstein cattle with mastitis resistance; the use is for non-disease diagnosis and treatment.

[0015] This invention also protects the use of any of the primer combinations described above in identifying or assisting in the identification of whether Chinese Holstein cattle have mastitis resistance; the application is for non-disease diagnosis and treatment.

[0016] In any of the above-described applications, the primer combinations described above can be used to detect the genotypes of SNP1 (rs135854456), SNP11 (located at position 104010752 on chromosome 5 of the Chinese Holstein bovine genome), SNP15 (rs109421300), and SNP16 (rs109350371). If the genotype of the Chinese Holstein bovine being tested is CC homozygous based on SNP1 and CC homozygous based on SNP11, the genotype can be determined by the primer combination. If the genotype based on SNP15 is homozygous (AA) and the genotype based on SNP16 is homozygous (AA), then the tested Chinese Holstein cattle are mastitis resistant. If the genotype based on SNP1 is homozygous (TT) and / or the genotype based on SNP11 is homozygous (TT) and / or the genotype based on SNP16 is homozygous (GG) and / or the genotype based on SNP15 is homozygous (GG), then the tested Chinese Holstein cattle are not mastitis resistant (i.e., are susceptible).

[0017] This invention also protects a method for assisting in screening whether a Chinese Holstein cattle to be tested has mastitis resistance, which may include the following steps: detecting the genotype of the Chinese Holstein cattle to be tested based on SNP1, SNP11, SNP15, and SNP16 loci, and then making the following judgment:

[0018] If the Chinese Holstein cattle to be tested have a CC homozygous genotype based on SNP1, a CC homozygous genotype based on SNP11, an AA homozygous genotype based on SNP15, and an AA homozygous genotype based on SNP16, then the Chinese Holstein cattle to be tested have mastitis resistance.

[0019] If the Chinese Holstein cattle to be tested have a TT homozygous genotype based on SNP1 and / or a TT homozygous genotype based on SNP11 and / or a GG homozygous genotype based on SNP16 and / or a GG homozygous genotype based on SNP15, then the Chinese Holstein cattle to be tested do not have mastitis resistance (i.e., they are susceptible).

[0020] The SNP1 site is rs135854456;

[0021] SNP11 is located at position 104010752 on chromosome 5 of the Chinese Holstein cattle genome;

[0022] SNP15 is located at rs109421300;

[0023] SNP16 is located at rs109350371;

[0024] The method is used for the diagnosis and treatment of non-disease conditions.

[0025] In the above method, the steps for detecting the genotype of the Chinese Holstein cattle to be tested based on SNP1, SNP11, SNP15, and SNP16 loci are as follows:

[0026] (1) Using the genomic DNA of the Chinese Holstein cattle to be tested as a template, PCR amplification was performed using the primer sets in any of the primer combinations described above to obtain PCR amplification products;

[0027] (2) After completing step (1), the fluorescence signal of the PCR amplification product is detected by an instrument, and the genotype of the Chinese Holstein cattle to be tested is obtained based on the color of the fluorescence signal at the SNP1, SNP11, SNP15 and SNP16 sites.

[0028] In the above method, the PCR amplification can be performed using the HuaNiu microarray. The specific steps are as follows:

[0029] (a) Take the Huaniu chip and inject the template and primer set into each chip well;

[0030] (b) After completing step (a), inject PCR premixed solution (2×) into the wells of the ThermoScientific chip. TM Then seal the inlet and outlet.

[0031] Each well of the chip contains 1 μL of reaction mixture, including 0.14 μL of mixed primers (12 μM forward primer 1 (primer containing "F1" in its name), 12 μM forward primer 2 (primer containing "F2" in its name), and 30 μM reverse primer (primer containing "R" in its name), and 0.5 μL of PCR premix (2×) (Thermo Scientific). TM ), 0.3 μL template (20 ng / μL) and ddH2O.

[0032] (c) After completing step (b), place the Hua Niu chip in a centrifuge and centrifuge at 4000 rpm for 1 min;

[0033] (d) After completing step (c), place the Hua Niu chip in a heat sealer for heat sealing for 1 second;

[0034] (e) After completing step (d), take the Huaniu chip and perform PCR amplification in each well simultaneously;

[0035] The reaction program was as follows: 95℃ pre-denaturation for 15 min; 95℃ denaturation for 20 s, 61℃-55℃ (using the touch down program, decreasing by 1℃ per cycle) for 1 min, amplification for 10 cycles; 95℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, continued amplification for 26 cycles; 37℃ extension for 60 sec.

[0036] (f) After completing step (e), place the Hua Niu chip in a fluorescence signal scanner to generate a scanned image, and determine the genotype of the Chinese Holstein cattle to be tested based on each SNP locus according to the fluorescence signal color. The specific judgment principles are as follows: If the Chinese Holstein cattle being tested show a red fluorescent signal based on a certain SNP site, then the genotype of the Chinese Holstein cattle based on that SNP site is homozygous, consisting of the 3' terminal first base of the primer that amplifies the SNP site and whose name contains "F1" or its complementary base; if the Chinese Holstein cattle being tested show a green fluorescent signal based on a certain SNP site, then the genotype of the Chinese Holstein cattle based on that SNP site is homozygous, consisting of the 3' terminal first base of the primer that amplifies the SNP site and whose name contains "F2" or its complementary base; if the Chinese Holstein cattle being tested show a yellow fluorescent signal based on a certain SNP site, then the genotype of the Chinese Holstein cattle based on that SNP site is heterozygous, with one base being the 3' terminal first base of the primer that amplifies the SNP site and whose name contains "F1" or its complementary base, and the other base being the 3' terminal first base of the primer that amplifies the SNP site and whose name contains "F2" or its complementary base.

[0037] In the above method, the steps for detecting the genotype of the Chinese Holstein cattle to be tested based on SNP1, SNP11, SNP15, and SNP16 loci are as follows:

[0038] (1) Using the genomic DNA of the Chinese Holstein cattle to be tested as a template, PCR amplification was performed using the primer sets in any of the primer combinations described above to obtain PCR amplification products;

[0039] (2) Take the PCR amplification product obtained in step (1) and sequence it;

[0040] (3) Based on the sequencing results obtained in step (2), obtain the genotypes of the Chinese Holstein cattle to be tested based on SNP1, SNP11, SNP15 and SNP16 sites.

[0041] This invention also protects the application of the genotypes of SNP1, SNP11, SNP15, and SNP16 loci in Chinese Holstein cattle for the identification or auxiliary identification of mastitis resistance; the SNP1 locus is rs135854456; the SNP11 locus is located at position 104010752 on chromosome 5 of the Chinese Holstein cattle genome; the SNP15 locus is rs109421300; and the SNP16 locus is rs109350371; the application is for non-disease diagnosis and treatment.

[0042] Experiments have shown that the method and primer combination provided by this invention can accurately identify mastitis resistance in Chinese Holstein bovines. This invention has significant application value. Attached Figure Description

[0043] Figure 1 The results of SNP1 testing are for Chinese Holstein cattle from Ranch A, Ranch B, and Ranch C.

[0044] Figure 2 The results of SNP11 testing are for Chinese Holstein cattle from Ranch A, Ranch B, and Ranch C.

[0045] Figure 3 The results of SNP15 testing are for Chinese Holstein cattle from Ranch A, Ranch B, and Ranch C.

[0046] Figure 4 The results of SNP16 testing are for Chinese Holstein cattle from Ranch A, Ranch B, and Ranch C.

[0047] Figure 5 The results of SNP1 testing are for Chinese Holstein cattle at Ranch D.

[0048] Figure 6 The results of SNP11 testing are for Chinese Holstein cattle at Ranch D.

[0049] Figure 7 The results of SNP15 testing were obtained for Chinese Holstein cattle at Ranch D.

[0050] Figure 8 The results of SNP16 testing were obtained for Chinese Holstein cattle at Ranch D.

[0051] Figure 9 The results of SNP1 testing are for Chinese Holstein cattle at Ranch E.

[0052] Figure 10 The results of SNP11 testing are for Chinese Holstein cattle at Ranch E.

[0053] Figure 11 The results of SNP15 testing were obtained for Chinese Holstein cattle at Ranch E.

[0054] Figure 12 The results of SNP16 testing were obtained for Chinese Holstein cattle at Ranch E. Detailed Implementation

[0055] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0057] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0058] Huaniu Chip is a product of Beijing Bio-Tech Biotechnology Co., Ltd., with product number G020010.

[0059] Example 1: Obtaining primer combinations for identifying mastitis resistance in Chinese Holstein bovines

[0060] I. Discovery of 4 SNP sites

[0061] Genotyping was performed on 20 SNP loci related to udder health in Chinese Holstein cattle using the IMAP microfluidic SNP chip system. After quality control, combined with phenotypic data of production performance of Chinese Holstein cattle (such as SCC and milk yield at 305 days), mixed linear model analysis was used to screen out SNP loci with two or more months of significant correlation. Finally, four SNP loci that were significantly associated with mastitis resistance in Chinese Holstein cattle were obtained, namely SNP1, SNP11, SNP15 and SNP16.

[0062] Basic information on the four SNP sites is detailed in Table 1.

[0063] Table 1. Basic information of 4 SNP sites

[0064]

[0065] II. Obtaining primer combinations for identifying mastitis resistance in Chinese Holstein bovines

[0066] Based on the four SNP sites identified in step one, a primer combination suitable for identifying mastitis resistance in Chinese Holstein bovines using allele-competitive specific PCR was developed.

[0067] The primer sets consist of four primer groups. The name of each primer group is shown in column 2 of Table 2. Each primer group consists of three primer sequences and is used to amplify one SNP site. The nucleotide sequences of each primer in the four primer groups are shown in column 4 of Table 2.

[0068] Table 2. Nucleotide sequences of four primer sets and their primers

[0069]

[0070]

[0071] Note: FAM indicates FAM fluorescent labeling, and HEX indicates HEX fluorescent labeling.

[0072] Example 2: Validation of the primer combination developed in Example 1 on 837 Chinese Holstein cattle from five ranches in Beijing (Restaurant A, Ranch B, Ranch C, Ranch D, and Ranch E of Shougang Group).

[0073] 1. Obtaining genomic DNA from 837 Chinese Holstein cattle

[0074] Tadal vein blood was collected from 837 Chinese Holstein cattle (895 samples), and genomic DNA was extracted using the CTAB method, resulting in genomic DNA from 837 Chinese Holstein cattle (actually 895 samples, with duplicate sampling).

[0075] The quality and concentration of genomic DNA from Chinese Holstein cattle must meet PCR requirements. The standards are as follows: agarose gel electrophoresis should show a single DNA band without obvious diffusion; the A260 / A280 ratio should be greater than or equal to 1.7 and the A260 / A230 ratio should be greater than 1.8 as detected by a Nanodrop 2000 (Thermo) UV spectrophotometer; the concentration of genomic DNA from Chinese Holstein cattle should be greater than 20 ng / μL, and the sample concentration should be kept as consistent as possible.

[0076] 2. Using genomic DNA from 895 blood samples of Chinese Holstein cattle as templates, PCR amplification was performed using four primer sets to obtain the genotypes of Chinese Holstein cattle based on four SNP loci.

[0077] PCR amplification was performed using the HuaNiu microarray, and the specific steps are as follows:

[0078] (1) Take the Huaniu chip and inject the template and primer set into each chip well;

[0079] (2) After completing step (1), inject PCR premixed solution (2×) into the well of the ThermoScientific chip. TM Then seal the inlet and outlet;

[0080] Each well of the chip contains 1 μL of reaction mixture, including 0.14 μL of mixed primers (12 μM forward primer 1 (primer containing "F1" in its name), 12 μM forward primer 2 (primer containing "F2" in its name) and 30 μM reverse primer (primer containing "R" in its name), 0.5 μL of PCR premix (2×) (Thermo Scientific TM), 0.3 μL of template (20 ng / μL) and ddH2O.

[0081] (3) After completing step (2), place the Hua Niu chip in a centrifuge and centrifuge at 4000 rpm for 1 min;

[0082] (4) After completing step (3), place the Hua Niu chip in a heat sealer for heat sealing for 1 second;

[0083] (5) After completing step (4), take the Hua Niu chip and perform PCR amplification in each well simultaneously;

[0084] The reaction program was as follows: 95℃ pre-denaturation for 15 min; 95℃ denaturation for 20 s, 61℃-55℃ (using the touch down program, decreasing by 1℃ per cycle) for 1 min, amplification for 10 cycles; 95℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, continued amplification for 26 cycles; 37℃ extension for 60 sec.

[0085] (6) After completing step (5), the Hua Niu chip is placed in a fluorescence signal scanner to generate a scan image. The genotype of 837 Chinese Holstein cattle based on each SNP locus is determined according to the fluorescence signal color. The specific judgment principles are as follows: If a Chinese Holstein cattle shows a red fluorescent signal based on a certain SNP site, then the genotype of the Chinese Holstein cattle based on that SNP site is homozygous, meaning "the first base at the 3' end of the primer that amplifies the SNP site and whose name contains 'F1' or its complementary base"; if a Chinese Holstein cattle shows a green fluorescent signal based on a certain SNP site, then the genotype of the Chinese Holstein cattle based on that SNP site is homozygous, meaning "the first base at the 3' end of the primer that amplifies the SNP site and whose name contains 'F2' or its complementary base"; if a Chinese Holstein cattle shows a yellow fluorescent signal based on a certain SNP site, then the genotype of the Chinese Holstein cattle based on that SNP site is heterozygous, with one base being "the first base at the 3' end of the primer that amplifies the SNP site and whose name contains 'F1' or its complementary base," and the other base being "the first base at the 3' end of the primer that amplifies the SNP site and whose name contains 'F2' or its complementary base."

[0086] The SNP1-based detection results for Chinese Holstein cattle from Ranch A, Ranch B, and Ranch C are shown below. Figure 1 .

[0087] The SNP11-based test results for Chinese Holstein cattle from Ranch A, Ranch B, and Ranch C are shown below. Figure 2 .

[0088] The SNP15 test results for Chinese Holstein cattle from Ranch A, Ranch B, and Ranch C are shown below. Figure 3 .

[0089] The SNP16-based test results for Chinese Holstein cattle from Ranch A, Ranch B, and Ranch C are shown below. Figure 4 .

[0090] The SNP1-based test results for Chinese Holstein cattle at Ranch D are shown below. Figure 5 .

[0091] The SNP11 test results for Chinese Holstein cattle at Ranch D are shown below. Figure 6 .

[0092] The SNP15 test results for Chinese Holstein cattle at Ranch D are shown below. Figure 7 .

[0093] The SNP16-based test results for Chinese Holstein cattle at Ranch D are shown below. Figure 8 .

[0094] The SNP1-based test results for Chinese Holstein cattle at E Ranch are shown below. Figure 9 .

[0095] The SNP11 test results for Chinese Holstein cattle from E Ranch are shown below. Figure 10 .

[0096] The SNP15 test results for Chinese Holstein cattle from E Ranch are shown below. Figure 11 .

[0097] The SNP16 test results for Chinese Holstein cattle from E Ranch are shown below. Figure 12 .

[0098] Table 3 shows the genotypic statistics of 837 Chinese Holstein cattle based on each SNP locus. A total of 895 samples were tested. Figures 1-12 The total number of NAs is 895, and after deduplication, it is 837. The genotyping detection rate is obtained by dividing the number of NAs by 837.

[0099] Table 3

[0100]

[0101] The results showed that each primer set could achieve good typing results in Chinese Holstein cattle.

[0102] 3. Mixed linear model analysis

[0103] Based on the mixed linear model as follows, significance analysis was performed using the combined phenotypic data SCC.

[0104] scc~p+fys+stage+x

[0105] Where p represents parity effect, stage represents lactation stage effect, fys represents field, year, and season effect, and x represents SNP effect.

[0106] The analysis results are shown in Table 4. Statistical genetic effects showed that SNP1, SNP11, SNP15, and SNP16 had a significant impact on SCC in two or more months, meeting the screening criteria for SNPs significantly associated with mastitis resistance in Example 1. Among them, individuals with the CC type of SNP1 and SNP11, and individuals with the AA type of SNP15 and SNP16, had lower SCC and higher milk production at 305 days, respectively. Therefore, the above genotypes are considered to be mastitis resistance genotypes at the four SNP loci.

[0107] Table 4

[0108]

[0109]

[0110] Note: * indicates a p-value less than 0.05, indicating a significant difference; ** indicates a p-value less than 0.01, indicating an extremely significant difference.

[0111] Chinese Holstein cattle were classified as resistant based on all genotypes of homozygous CC at SNP1, homozygous CC at SNP11, homozygous AA at SNP16, and homozygous AA at SNP15. Chinese Holstein cattle were classified as susceptible based on any of the following genotypes: homozygous TT at SNP1, homozygous TT at SNP11, homozygous GG at SNP16, and homozygous GG at SNP15. Analysis of variance was performed on SCC and 305M in resistant and susceptible cattle, and the results are shown in Table 5.

[0112] The SCC (strained duct occupancy) of resistant cattle was significantly lower than that of susceptible cattle (P < 0.05), and the 305M (mastitis resistant occupancy) was significantly higher in resistant cattle than in susceptible cattle (P < 0.01). Therefore, it is believed that the SNP combination consisting of SNP1, SNP11, SNP15, and SNP16 can be used to screen for Chinese Holstein cattle with mastitis resistance. In this case, the study population showed 174 resistant cattle and 232 susceptible cattle.

[0113] Table 5

[0114]

[0115] Note: ①Susceptible cattle: cattle with a susceptible genotype among the four SNPs.

[0116] ② The asterisk (*) indicates a p-value less than 0.05, indicating a significant difference.

[0117] The above results demonstrate that the primer combination developed in Example 1 can effectively identify mastitis in Chinese Holstein bovines with high accuracy.

[0118] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. <110> China Agricultural University <120> Primer combination, method and application for identifying mastitis resistance in Chinese Holstein bovines <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 21 <212> DNA <213> Artificial sequence <400> 1 cagtcatgtc cgactctttg c 21 <210> 2 <211> 23 <212> DNA <213> Artificial sequence <400> 2 ctcagtcatg tccgactctt tgt 23 <210> 3 <211> 24 <212> DNA <213> Artificial sequence <400> 3 ggagcttggt aggctacact ccat 24 <210> 4 <211> 23 <212> DNA <213> Artificial sequence <400> 4 gaccatgagg gaagtctttt gag 23 <210> 5 <211> 24 <212> DNA <213> Artificial sequence <400> 5 ggaccatgag ggaagtcttt tgaa 24 <210> 6 <211> 25 <212> DNA <213> Artificial sequence <400> 6 tgccaggggg aatcagaaaa ggatt 25 <210> 7 <211> 18 <212> DNA <213> Artificial sequence <400> 7 gcccctgcgc aggcacat 18 <210> 8 <211> 18 <212> DNA <213> Artificial sequence <400> 8 gcccctgcgc aggcacac 18 <210> 9 <211> 22 <212> DNA <213> Artificial sequence <400> 9 aagctctgag actccccacc ca 22 <210> 10 <211> 18 <212> DNA <213> Artificial sequence <400> 10 gggtccatgg ctgctcct 18 <210> 11 <211> 18 <212> DNA <213> Artificial sequence <400> 11 gggtccatgg ctgctccc 18 <210> 12 <211> 30 <212> DNA <213> Artificial sequence <400> 12 ctaaacactt ccttcaaggt tctaagacta 30

Claims

1. Primer sets used to identify whether Chinese Holstein cattle have mastitis resistance, consisting of primer sets 1-4; The primer set 1 consists of the forward primer 01F1 shown in SEQ ID NO:1, the forward primer 01F2 shown in SEQ ID NO:2, and the reverse primer 01R shown in SEQ ID NO:3; The primer set 2 consists of the forward primer 02F1 shown in SEQ ID NO:4, the forward primer 02F2 shown in SEQ ID NO:5, and the reverse primer 02R shown in SEQ ID NO:6; The primer set 3 consists of the forward primer 03F1 shown in SEQ ID NO:7, the forward primer 03F2 shown in SEQ ID NO:8, and the reverse primer 03R shown in SEQ ID NO:9; The primer set 4 consists of the forward primer 04F1 shown in SEQ ID NO:10, the forward primer 04F2 shown in SEQ ID NO:11, and the reverse primer 04R shown in SEQ ID NO:12; Primer set 1, primer set 2, primer set 3 and primer set 4 are used to detect the genotype of Chinese Holstein cattle based on SNP1, SNP11, SNP15 and SNP16 loci, respectively. The SNP1 site is rs135854456; SNP11 is located at position 104010752 on chromosome 5 of the Chinese Holstein cattle genome; SNP15 is located at rs109421300; The SNP16 locus is rs109350371.

2. The primer combination as described in claim 1, characterized in that: The forward primers 01F1, 02F1, 03F1, and 04F1 are labeled with FAM fluorescence. The forward primers 01F2, 02F2, 03F2, and 04F2 are HEX fluorescently labeled.

3. The use of the primer combination of claim 1 or 2 in the preparation of a kit for identifying or assisting in the identification of whether Chinese Holstein cattle have mastitis resistance.

4. The use of the primer combination of claim 1 or 2 in the preparation of a kit for assisting screening of Chinese Holstein cattle with mastitis resistance.

5. The application as described in claim 4, characterized in that: The kit is used to detect the genotype of Chinese Holstein cattle at SNP1, SNP11, SNP15, and SNP16 sites, and then the following judgments are made: If the Chinese Holstein cattle to be tested have a CC homozygous genotype based on SNP1, a CC homozygous genotype based on SNP11, an AA homozygous genotype based on SNP15, and an AA homozygous genotype based on SNP16, then the Chinese Holstein cattle to be tested have mastitis resistance. If the Chinese Holstein cattle to be tested have a genotype of TT homozygous based on SNP1 and / or a genotype of TT homozygous based on SNP11 and / or a genotype of GG homozygous based on SNP16 and / or a genotype of GG homozygous based on SNP15, then the Chinese Holstein cattle to be tested do not have mastitis resistance. The SNP1 site is rs135854456; SNP11 is located at position 104010752 on chromosome 5 of the Chinese Holstein cattle genome; SNP15 is located at rs109421300; The SNP16 locus is rs109350371.

6. The application as described in claim 5, characterized in that: The steps for detecting the genotype of the Chinese Holstein cattle to be tested based on SNP1, SNP11, SNP15, and SNP16 loci are as follows: (1) Using the genomic DNA of the Chinese Holstein cattle to be tested as a template, PCR amplification was performed using the primer sets in the primer combination described in claim 2 to obtain PCR amplification products; (2) After completing step (1), the fluorescence signal of the PCR amplification product is detected by an instrument, and the genotype of the Chinese Holstein cattle to be tested is obtained based on the color of the fluorescence signal at the SNP1, SNP11, SNP15 and SNP16 sites.

7. The application as described in claim 5, characterized in that: The steps for detecting the genotype of the Chinese Holstein cattle to be tested based on SNP1, SNP11, SNP15, and SNP16 loci are as follows: (1) Using the genomic DNA of the Chinese Holstein cattle to be tested as a template, PCR amplification was performed using the primer sets in the primer combination described in claim 1 to obtain PCR amplification products; (2) Take the PCR amplification product obtained in step (1) and sequence it; (3) Based on the sequencing results obtained in step (2), obtain the genotypes of the Chinese Holstein cattle to be tested based on SNP1, SNP11, SNP15 and SNP16 sites.

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