SNP molecular markers associated with mastitis resistance in dairy cows and their application

Abstract

The invention discloses a SNP molecular marker associated with the mastitis resistance of dairy cows and its application, and belongs to the field of dairy cow breeding markers. The nucleotide sequence of the SNP molecular marker associated with the mastitis resistance of dairy cows provided by the invention is shown in SEQ ID No.1, and the polymorphic site is located at the 129th position of the sequence shown in SEQ ID No.1, and the polymorphism is C or T. The molecular marker can accurately predict the mastitis resistance characteristics of dairy cows under different genetic backgrounds, and can be used to identify dairy cows with high mastitis resistance traits, thereby improving the efficiency of molecular design breeding. And according to the identification results of the above-mentioned SNP sites, breeders can select favorable genotype individuals for seed retention, thereby improving the mastitis resistance traits of dairy cow groups, improving the mastitis resistance of dairy cow groups, reducing treatment costs, and improving the economic benefits of pastures.

Description

Technical Field

[0001] The invention belongs to the field of dairy cow breeding markers, and in particular relates to a SNP molecular marker associated with dairy cow mastitis resistance and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Mastitis is one of the most common and costly diseases in dairy cow production, which reduces milk production and quality, leads to increased farm treatment costs, and has a negative impact on animal welfare. The incidence, severity, and outcome of mastitis are affected by multiple factors, including pathogen type, host immunity, and environment. On the host side, mastitis resistance, cow age, and lactation stage affect mastitis risk. Improving dairy cows' resistance to mastitis from a genetic perspective through molecular breeding is one of the fundamental ways to reduce the incidence of mastitis.

[0004] Early studies reported QTLs (quantitative trait locus) that affect clinical mastitis in cattle, however, these studies could not identify specific candidate gene variants. In recent years, with the increase of high-density SNP chip data, the chances of identifying pathogenic mutations are getting higher and higher. Identifying causal variants or assigning QTLs to one or several genes through association mapping may help identify genes that control mastitis resistance, such as NPFFR2 gene, TRAPPC gene, ARHGAP39 gene, LY6K gene, LY6D gene, LYNX1 gene, etc. However, mastitis resistance in dairy cows is a complex trait controlled by multiple genes, and it is still necessary to further explore related gene loci.

[0005] According to the annotation of the cattle reference genome UMD3.1 version, the SH3PXD2B gene is located in the 4010453-4094221bp region of chromosome 20 of the cattle reference genome (UMD3.1: GK000020.2), located on the antisense strand. The gene name of its Ensembl version is ENSBTAG00000009019.5. The gene encodes one transcript, the transcript name is SH3PXD2B-201, the transcript ID is ENSBTAT00000011874.5, the transcript has 13 exons, of which 13 are coding exons, the transcript length is 2667nt, and the encoded protein length is 888 amino acid residues. The gene encodes an adaptor protein characterized by a PX domain and four Src homology domains. The encoded protein is required for podosome formation and is involved in cell adhesion and migration in many cell types. A switch protein involved in invadopodia and podosome formation and extracellular matrix degradation. Binds matrix metalloproteinases (ADAMs), NADPH oxidases (NOXs), and inositol phosphates. As an organizer protein, it allows NOX1- or NOX3-dependent reactive oxygen species (ROS) generation and localization of ROS. Studies have shown that the SH3PXD2B gene plays a role in early mitotic clonal expansion of adipocyte differentiation, but its effect on mastitis resistance in dairy cows has not been reported. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the present invention provides SNP molecular markers associated with cow mastitis resistance and applications thereof.

[0007] In order to achieve the technical purpose, the present invention designs and screens molecular marker-assisted breeding related to cow mastitis resistance, thereby reducing the incidence of cow mastitis, reducing treatment costs, and improving cow profitability. The present invention uses the SH3PXD2B gene as a candidate gene for cow mastitis resistance to study the correlation between its mutation site and the cow mastitis resistance trait.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention provides a SNP molecular marker associated with cow mastitis resistance, the molecular marker is from the cow SH3PXD2B gene, its nucleotide sequence is shown in SEQ ID No.1, the polymorphic site is located at the 129th position of the sequence shown in SEQ ID No.1, and the polymorphism is C or T.

[0010] The polymorphic site of the SNP molecular marker described above is located at 4011529 bp of the bovine chromosome 20 sequence (the bovine reference genome version is UMD3.1, NCBI's Assembly accession GCA_000003055.3, and the chromosome number is GK000020.2).

[0011] The above-mentioned dairy cow SH3PXD2B gene is located in Chromosome 20:4011529 of the cattle reference genome UMD3.1, and its name is rs133650873. The polymorphic site of the SNP molecular marker is located at the 1654th nt of the transcript, and the corresponding codon is GGC or AGC, encoding glycine (G) or serine (S), which is a missense mutation, and the corresponding amino acid encoded is located at position 552 of the entire protein (a total of 888 amino acid residues).

[0012] The present invention provides a cow mastitis resistance trait-related SH3PXD2B gene by a PCR method, and identifies a SNP site on the 12th exon of the gene by a Bovine SNP50 chip. In a Holstein cattle population, an association analysis is performed on the genotype of a SNP on the 12th exon of the SH3PXD2B gene and the breeding value of the mastitis resistance trait genome of the cattle in the population, and the analysis result shows that the SNP molecular marker is significantly correlated with the mastitis resistance of the cow. The above-mentioned SNP molecular marker can accurately predict the mastitis resistance characteristics of the cow under different genetic backgrounds, can be used to identify the cows with high mastitis resistance traits, and improve the efficiency of molecular design breeding. And according to the identification result of the above-mentioned SNP site, breeders can select favorable genotype individuals for seed retention, thereby improving the mastitis resistance trait of the cow population, improving the mastitis resistance of the cow population, reducing treatment costs, and improving the economic benefits of pastures.

[0013] The SNP identification method used in the present invention is as follows: genotype identification was performed in the dairy cow population using the Bovine SNP50 chip, and a SNP (chr20:g.4011529C>T) was found on the 12th exon of the SH3PXD2B gene ( Figure 1 ); through the association analysis of dairy cow population genotype and dairy cow mastitis resistance trait genome estimated breeding value, it was proved that the SNP site was significantly associated with dairy cow mastitis resistance trait.

[0014] Based on the above research results, the SNP molecular marker on the 12th exon of the SH3PXD2B gene can be used as a marker to identify the high mastitis resistance trait of dairy cows and assist breeding, and can be used to screen parent individuals with higher mastitis resistance, effectively increase the service life of cows, save treatment costs, reduce breeding costs, and increase pasture income. In addition, the relevant detection reagents of the above molecular markers can also be developed into products related to the breeding of Chinese Holstein cows.

[0015] SEQ ID NO: 1:

[0016] .

[0017] In some embodiments of the present invention, the SNP molecular marker is obtained by amplifying the cow genome as a template using primers having sequences as shown in SEQ ID No. 2-3.

[0018] SEQ ID NO.2: 5'-TTTGGAGATAGGTCGCAGGC-3';

[0019] SEQ ID NO. 3: 5'-TGGCCCTGCCTCCATTCTTA-3'.

[0020] In some embodiments of the present invention, when the polymorphic site of the SNP molecular marker is C, it corresponds to high mastitis resistance.

[0021] The second aspect of the present invention provides a set of primer pairs for detecting mastitis resistance in dairy cows, wherein the primer pairs are used to detect the SNP molecular markers described in the first aspect.

[0022] According to the location of the SNP molecular marker in the genome and its upstream and downstream sequences provided above, technicians with the skill in the art can develop various types of primers for amplifying the SNP molecular marker.

[0023] The above primers may be any type of primers that can be used to detect the genotype of SNP molecular markers.

[0024] Preferably, the primer pair comprises primers shown in SEQ ID No.2-3, wherein SEQ ID No.2 is an upstream primer and SEQ ID No.3 is a downstream primer.

[0025] The primers described above can achieve efficient amplification and genotyping for the above-mentioned SNP molecular markers.

[0026] The third aspect of the present invention provides a kit for detecting mastitis resistance in dairy cows, the kit comprising the primer pair described above.

[0027] The above-mentioned kit may also include a sampling tool, reagents for PCR expansion, including but not limited to DNA polymerase, PCR reaction buffer solution, probe, dNTP, Mg 2+ , water, etc.

[0028] The above reagents can be packaged separately or provided as a premix after mixing.

[0029] The fourth aspect of the present invention provides the use of the above-mentioned SNP molecular marker or its detection reagent in any one of the following 1)-4);

[0030] 1) Application in detecting or assisting in detecting resistance to mastitis in dairy cows;

[0031] 2) Application in screening or identifying superior strains of dairy cows with resistance to mastitis;

[0032] 3) Application in molecular marker-assisted breeding for mastitis resistance in dairy cows;

[0033] 4) Application in improving the germplasm resources for mastitis resistance in dairy cows.

[0034] The detection reagent is the primer pair or the kit described above.

[0035] Preferably, in the above application, the nucleotide sequence of the SNP molecular marker is as shown in SEQ ID No.1, the polymorphic site is located at position 129 of the sequence shown in SEQ ID No.1, and the polymorphism is C or T.

[0036] Specifically, the SNP molecular marker is obtained by amplifying the cow genome as a template using primers having sequences as shown in SEQ ID No. 2-3.

[0037] When the polymorphic site of the above-mentioned SNP molecular marker is C, it corresponds to high mastitis resistance.

[0038] Preferably, the primer pair comprises primers whose sequences are shown as SEQ ID No.2-3, wherein SEQ ID No.2 is an upstream primer and SEQ ID No.3 is a downstream primer.

[0039] A fifth aspect of the present invention provides a method for screening or identifying a superior strain of cow mastitis resistance, comprising the following steps:

[0040] (1) Extracting genomic DNA from the cow to be tested;

[0041] (2) using the genomic DNA of the cow to be tested as a template and the primer pair described above to amplify the exon region of the cow SH3PXD2B gene by PCR reaction;

[0042] (3) Detect the PCR amplification product. If the base at 129 bp in the amplification product sequence is C, the tested dairy cow belongs to a dominant dairy cow strain with high resistance to mastitis.

[0043] Preferably, the cow to be tested is one of Chinese Holstein cow, Sanhe cow, Xinjiang brown cow or grassland red cow, and more preferably Chinese Holstein cow.

[0044] Preferably, the primer pair comprises primers with sequences as described in SEQ ID No.2-3, wherein SEQ ID No.2 is an upstream primer and SEQ ID No.3 is a downstream primer.

[0045] A sixth aspect of the present invention provides a method for assisted breeding of dairy cows, comprising the following steps:

[0046] The dairy cow individuals with the superior genotype obtained by the method described in the fifth aspect are used as parents for breeding.

[0047] A seventh aspect of the present invention provides a method for identifying a dairy cow with high mastitis resistance, comprising the following steps:

[0048] Detecting the genotype of a SNP molecular marker associated with cow mastitis resistance; the polymorphic site of the SNP molecular marker is located at 4011529bp of the chromosome 20 sequence of the UMD3.1 reference genome, and the polymorphism is C or T;

[0049] The polymorphic site of the SNP molecular marker is C, corresponding to high mastitis resistance.

[0050] Preferably, the nucleotide sequence of the SNP molecular marker is as shown in SEQ ID No.1, the polymorphic site is located at position 129 of the sequence shown in SEQ ID No.1, and the polymorphism is C or T.

[0051] Specifically, the SNP molecular marker is obtained by amplifying the cow genome as a template using primers having sequences as shown in SEQ ID No. 2-3.

[0052] In the above method, the genotype of the SNP molecular marker is preferably detected by the following method:

[0053] The genome of the cow to be tested is used as a DNA template, and PCR amplification is performed using primers with sequences as shown in SEQ ID No. 2-3, and the genotype of the PCR amplification product is detected.

[0054] The beneficial effects of the present invention are:

[0055] The present invention discloses for the first time a SNP molecular marker associated with the resistance to mastitis in dairy cows, which is from the SH3PXD2B gene of dairy cows, whose nucleotide sequence is shown in SEQ ID No.1, and the polymorphic site is located at the 129th position of the sequence shown in SEQ ID No.1, and the polymorphism is C or T, which can improve the resistance to mastitis in dairy cows, can be used as a marker for identifying the high resistance to mastitis in dairy cows and assisting breeding, can be used to screen parental individuals with higher resistance to mastitis, effectively improve the service life of cows, save treatment costs, reduce breeding costs, and increase pasture income. In addition, the relevant detection reagents of the above molecular markers can also be developed into products related to the breeding of Chinese Holstein dairy cows.

[0056] The present invention utilizes the information of a SNP site on the 12th exon of the SH3PXD2B gene of the cattle, adopts molecular biology-related technology to identify the genotype of the dairy cow at the site, and realizes the early selection of the genotype individuals with high mastitis resistance. The present invention identifies a SNP site, detects the genotype of the SH3PXD2B gene of the individual dairy cow at the site by molecular biology-related technology, and selects favorable genotype individuals for seed preservation through association analysis with the estimated breeding value of the dairy cow mastitis resistance trait, which can increase the frequency of the SH3PXD2B gene high mastitis resistance genotype in the dairy cow population, thereby reducing the incidence of mastitis in the dairy cow population, increasing the service life of the dairy cow population, reducing breeding costs, and increasing the economic benefits of the pasture, providing a new method for improving the mastitis resistance of dairy cows. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0058] Figure 1 This is the Sanger sequencing peak diagram of the chr20:g.4011529C>T mutation site on the 12th exon sequence of the SH3PXD2B gene. DETAILED DESCRIPTION

[0059] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0060] Unless otherwise specified, the test methods used in the following examples are conventional methods.

[0061] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0062] Example 1

[0063] The 12th exon of the SH3PXD2B gene affecting the resistance to mastitis in dairy cows and its molecular marker and application are described in detail below. The specific scheme is:

[0064] 1. Screening of SH3PXD2B gene as a candidate gene for mastitis resistance in dairy cows

[0065] (1) Blood or hair follicle samples were collected from 1,069 Chinese Holstein cows in four large-scale farms

[0066] Chinese Holstein cattle samples were collected from four ranches across the country. The locations of the ranches and the number of samples collected from each ranch are shown in Table 1.

[0067] Table 1. Location of farms where samples were collected and number of samples collected

[0068] Ranch location Number of samples Ranch location Number of samples Shandong Dongying 268 Guangdong Huamei 196 Xinjiang Shawan 200 Tongliao, Inner Mongolia 399

[0069] (2) The genotypes of the collected samples were determined using the Illumina BovineSNP50 chip. The genotype determination was performed by Neogen Biotech (Shanghai) Co., Ltd., and the genotypes of a total of 47,843 SNP marker loci were obtained.

[0070] (3) The genomic breeding value for mastitis resistance of each individual was estimated based on the genotype. The genomic breeding value estimation was performed by Neogen Biotech (Shanghai) Co., Ltd.

[0071] (4) Based on genotype data, GEMMA software was used to conduct genome-wide association analysis on the genomic breeding value of mastitis resistance. The analysis model used a mixed linear model, with pasture and cattle age as covariates. The FDR method was used for multiple test correction. The results of the association analysis showed that the most significant SNP site was located on the 12th exon of the SH3PXD2B gene, with a significant P value of 1.172e-21.

[0072] 2. Identification of the dominant genotype at exon 12 of the SH3PXD2B gene

[0073] Among the 1,069 Chinese Holstein cattle collected, 288 individuals with the CC homozygous genotype were identified, with an average estimated breeding value of 0.77 for the mastitis resistance genome; 488 individuals with the CT genotype, with an average estimated breeding value of 0.33 for the mastitis resistance genome; and 286 individuals with the TT genotype, with an average estimated breeding value of -0.0056 for the mastitis resistance genome. In genomic genetic evaluation, the higher the estimated breeding value of the mastitis resistance genome, the better, so the CC genotype is the dominant genotype.

[0074] 3. Amplification of the Partial Sequence of the Bovine SH3PXD2B Gene

[0075] (1) Blood collection from the cattle tail vein

[0076] Cows with high mastitis resistance and cows with low mastitis resistance were selected as test materials, and blood was collected from the tail veins of the cows.

[0077] (2) Genomic DNA extraction

[0078] Take 500μL of whole blood, add 500μL of STE lysis buffer, add 50μL of 10% SDS, 5μL of proteinase K (20mg / mL) in sequence, and lyse at 56℃ for about 3h until the lysate is clear. Add the same volume of saturated phenol (250μL), chloroform / isoamyl alcohol (24:1) (250μL), shake gently for 20min, and centrifuge at 12000rpm for 10min. Take the supernatant and repeat the above steps until there is no protein layer between the aqueous phase and the organic phase. Take the supernatant, add the same volume of chloroform / isoamyl alcohol, shake gently for 20min, and centrifuge at 12000rpm for 10min. Take the supernatant, add 1 / 10 volume of 3M NaAc (pH5.2) and 2 times the volume of cold anhydrous ethanol, shake well, let stand at -20℃ for 20min, and centrifuge at 12500rpm for 20min. Precipitate the nucleic acid at the bottom of the tube. Discard the supernatant and wash the precipitate with 70% ethanol. Collect the precipitate and dry it in air until the ethanol is completely evaporated. Add 20 μL TE (containing RNase A) to dissolve the DNA, let it stand at 37°C for about 30 minutes, and then store it at 4°C. Detect the DNA sample by 1% agarose gel electrophoresis, and detect the concentration and purity by UV spectrophotometer.

[0079] (3) Primer design

[0080] According to the gene sequence of cattle SH3PXD2B gene, a pair of specific primers were designed.

[0081] The forward primer is SH3PXD2B-5F (SEQ ID NO.2):

[0082] 5′-TTTGGAGATAGGTCGCAGGC-3′;

[0083] The reverse primer is SH3PXD2B-3R (SEQ ID NO.3):

[0084] 5′-TGGCCCTGCCTCCATTCTTA-3′;

[0085] (4) Multiple enzyme chain reaction

[0086] This primer was used for PCR amplification, and the reaction system was as follows: 10×Buffer 1μL, 2.5mM dNTP 0.8μL, 2.5mM MgCl2, 0.6μL, forward primer (10μM) 0.1μL, reverse primer (10μM) 0.5μL, Taq enzyme (5U / μL) 0.1μL, template 0.5μL, LCGreen saturated fluorescent dye 0.7μL, and H2O was added to make up to 10μL.

[0087] The amplification reaction was completed on an Applied Biosystem PCR system under the following reaction conditions: 95°C for 5 min; 95°C for 30 s, 59°C for 30 s, 72°C for 1 min; 35 cycles; 72°C for 5 min.

[0088] The genotypes of PCR products were determined by Sanger sequencing.

[0089] By selecting individuals from the core group of dairy cows and using the above-mentioned molecular biology-related techniques to detect the genotype of the chr20:g.4011529C>T site of the SH3PXD2B gene, and selecting individuals with a favorable genotype for breeding, the mastitis resistance trait of the dairy cow group can be improved, the incidence of mastitis in the dairy cow group can be reduced, the breeding costs can be reduced, the breeding income can be increased, and the foundation can be laid for breeding a new breed of excellent dairy cows with high mastitis resistance.

[0090] Example 2

[0091] The Sanger sequencing method was used to detect the genotype and allele frequency distribution of a SNP site in the 12th exon of the SH3PXD2B gene in the Chinese Holstein cattle population. The results are shown in Table 2, which includes Chinese Holstein cattle in multiple pastures. The test results showed that there were three genotypes in the Chinese Holstein dairy cow population; in all the populations tested, the CC gene frequency was 62.6%, the CT genotype frequency was 33.5%, the TT genotype frequency was 3.9%, and the CC genotype was the dominant genotype (see Table 2).

[0092] Table 2 Distribution of bovine SH3PXD2B gene in Chinese Holstein cattle population

[0093]

[0094] Table 2 Description: The above group materials are all Chinese Holstein cattle groups, among which: Group 1 is a Chinese Holstein cattle group from a ranch in Gansu, Group 2 is a Chinese Holstein cattle group from a ranch in Hebei, and Group 3 is a Chinese Holstein cattle group from a ranch in Shandong.

[0095] In order to determine whether the bovine SH3PXD2B genotype is associated with bovine mastitis resistance, 971 Chinese Holstein cows were selected for association analysis of SH3PXD2B genotype and genomic breeding value of mastitis resistance. The average genomic breeding value of CC genotype individuals was 0.4097, the average genomic breeding value of CT genotype individuals was 0.067, and the average genomic breeding value of TT genotype individuals was -0.1601. PLINK v1.90 software was used to analyze the correlation between different genotypes and the mastitis resistance trait in dairy cows, and the results showed that the genotype and genomic breeding value were significantly correlated (P value was 1.974e-05).

[0096] In summary, the present invention identifies a SNP site, detects the genotype of the SH3PXD2B gene of individual dairy cows at this site through molecular biology related technologies, and selects favorable genotype individuals for breeding through association analysis with the genomic estimated breeding value of the cow's mastitis resistance trait, which can increase the frequency of the dominant genotype of mastitis resistance of the SH3PXD2B gene in the dairy cow population, thereby improving the mastitis resistance of the dairy cow population, reducing the incidence of mastitis in the dairy cow population, reducing the cost of mastitis treatment, reducing breeding costs, and improving breeding benefits, providing a new method for genetic improvement of mastitis resistance in dairy cows.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A SNP molecular marker associated with cow mastitis resistance, or a set of primer pairs for detecting the SNP molecular marker, or a kit for detecting the SNP molecular marker, used in any one of the following 1)-4); 1) Application in the preparation of products for testing or assisting in the testing of mastitis resistance in Chinese Holstein cows; 2) Application in screening or identifying superior strains of Chinese Holstein cows with resistance to mastitis; 3) Application of molecular marker-assisted breeding for mastitis resistance in Holstein cows in China; 4) Application in improving germplasm resources for mastitis resistance in Chinese Holstein cows; The molecular marker is from the cow SH3PXD2B gene, and its nucleotide sequence is shown in SEQ ID No.

1. The polymorphic site is located at the 129th position of the sequence shown in SEQ ID No.1, and the polymorphism is C or T.

2. The use according to claim 1, characterized in that: The polymorphic site of the SNP molecular marker is C, corresponding to high mastitis resistance.

3. The use according to claim 1, characterized in that The primer pair comprises primers shown in SEQ ID No.2-3, wherein SEQ ID No.2 is an upstream primer and SEQ ID No.3 is a downstream primer.

4. The use according to claim 1, characterized in that The kit comprises the primer pair according to claim 3.

5. A method for screening or identifying a superior strain of dairy cows with resistance to mastitis, characterized in that: The following steps are involved: (1) Extracting genomic DNA from Chinese Holstein cows to be tested; (2) using the genomic DNA of the Chinese Holstein cow to be tested as a template, and using the primer pair described in claim 3, amplifying the exon region of the cow SH3PXD2B gene by PCR reaction; (3) Detect the PCR amplification product. If the base at 129 bp in the amplification product sequence is C, the Chinese Holstein cow to be tested belongs to the dominant strain of Chinese Holstein cow with high resistance to mastitis.

6. A method for assisted breeding of dairy cows, characterized in that: The following steps are involved: The Chinese Holstein dairy cow individuals with the superior genotype obtained by the method according to claim 5 are used as parents for breeding.

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