SNP molecular marker of cyp7a1 gene related to milk production traits of dairy cows and application thereof

Abstract

The application discloses application of a SNP molecular marker of a gene CYP7A1 related to milk production traits of a dairy cow. One technical solution to be protected by the application is application of a substance for detecting polymorphism or genotypes of three SNPs, namely SNP1, SNP2 and SNP3, in identification or auxiliary identification of milk production traits of the dairy cow. Experiments prove that the related molecular marker of the SNP1, the SNP2 and / or the SNP3, and corresponding haplotypes and genotypes can be used for early prediction and screening of the milk production traits of the dairy cow, and help to breed a dairy cow breed with high milk production traits.

Description

Technical Field

[0001] This invention relates to a gene in the field of molecular biology that is associated with milk production traits in dairy cows. CYP7A1 SNP molecular markers and their applications. Background Technology

[0002] Cholesterol 7α-hydroxylase CYP7A1 Cholesterol 7α-monooxygenase (C7A1) is a member of the liver-specific microsomal cytochrome P450 enzyme family. Located on bovine chromosome 14, it consists of 6 exons and has a full-length gene of 10,337 bp. The protein encoded by this gene belongs to the cytochrome P450 (CYP) family and is involved in the synthesis of bile acids and bile salts, cytochrome P450 oxidation, statin pathway, and lipid metabolism through the peroxisome proliferator-activated receptor α (PPARα) signaling pathway. CYP7A1 It is the rate-limiting enzyme in cholesterol catabolism and bile acid biosynthesis, playing a role in converting cholesterol into 7α-hydroxylated cholesterol. Cholesterol is then transferred into high-density lipoprotein particles, primarily through the rate-limiting enzyme. CYP7A1 It returns to the liver and is converted into bile acids. In mammals, CYP7A1 It is expressed only in hepatocytes, and cholesterol synthesis is upregulated in early lactation compared to late pregnancy. CYP7A1 Cholesterol can induce the activity of its substrates. Liver X receptors (LXRα) are substances that cholesterol regulates... CYP7A1 The main substance at that time. LXRα is a cholesterol-activated nuclear receptor that can be activated by specific cholesterol oxidation derivatives. LXRα binds to retinoid X receptors (RXRα) to form a heterodimer RXRα / LXRα to exert its function. CYP7A1 It is a key target gene of LXRα.

[0003] Studies have found that fatty acids can affect cholesterol homeostasis in the body. Specifically, adding cholesterol to polyunsaturated fatty acids (PUFAs) increases CYP7α1 expression, while adding cholesterol to monounsaturated fatty acids (MUFAs) and saturated fatty acids decreases CYP7α1 expression. The intermediate bridge between fatty acid and cholesterol metabolism may be the peroxisome proliferator-activated receptor (PPAR), through which fatty acids can regulate... CYP7A1 PPARs are members of the steroid hormone receptor superfamily and can be activated by fatty acids and their metabolites, as well as exogenous peroxisom proliferators (pp). They regulate the gene expression of certain enzymes involved in lipid metabolism.

[0004] Polymerase chain reaction (PCR) is a rapid nucleic acid amplification technique that mimics the natural DNA replication process in vitro. Its most significant characteristic is its ability to dramatically increase minute amounts of DNA. Invented in 1983 by Mullis et al. in the United States, PCR utilizes the fact that DNA denatures into single strands at high temperatures (Celsius). At low temperatures, primers bind to the single strands according to the principle of complementary base pairing. The temperature is then adjusted to the optimal reaction temperature for DNA polymerase, which synthesizes complementary strands along the phosphate-to-pentose (5'-3') direction. Currently, this technology has become one of the most commonly used and important molecular biology techniques. PCR products can be sequenced after agarose gel electrophoresis for gene polymorphism identification; the detection method is simple and easy to perform. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to identify or assist in the identification of milk production traits in dairy cows or how to conduct dairy cow breeding.

[0006] To solve the above-mentioned technical problems, the present invention first provides an application, wherein the application is any one of the following P1-P5: P1 refers to the application of substances that detect the polymorphisms or genotypes of the three SNPs, SNP1, SNP2, and SNP3, in the identification or auxiliary identification of milk production traits in dairy cows. P2 refers to the application of substances for detecting haplotypes in identifying or assisting in the identification of milk production traits in dairy cows. The haplotype is a polymorphic combination of the three SNPs, SNP1, SNP2, and SNP3, on a single chromosome of a dairy cow. The P3 refers to the application of substances that detect the polymorphism or genotype of SNP1 in the identification or auxiliary identification of milk production traits in dairy cows. The P4 refers to the application of substances that detect the polymorphism or genotype of the SNP2 in the identification or auxiliary identification of milk production traits in dairy cows. The P5 refers to the application of substances that detect the polymorphism or genotype of the SNP3 in the identification or auxiliary identification of milk production traits in dairy cows. SNP1 is a SNP in the bovine genome, located at nucleotide 249 of SEQ ID No. 1 in the sequence listing, and is either C or T; SNP2 is a SNP in the bovine genome, located at nucleotide 1462 of SEQ ID No. 1 in the sequence listing, and is either C or A; SNP3 is a SNP in the bovine genome, located at nucleotide 11400 of SEQ ID No. 1 in the sequence listing, and is either C or T.

[0007] The three single nucleotide polymorphism sites mentioned above, SNP1, SNP2, and SNP3, are located on chromosome 14 of the bovine genome. CYP7A1 gene In the middle, the said CYP7A1 gene Located at positions 24664833-24675169 on chromosome 14 of dairy cows, it is associated with the milk production trait in dairy cows, and its nucleotide sequence is the DNA molecule shown in SEQ ID No. 1 of the sequence listing.

[0008] To address the aforementioned technical problems, the present invention also provides an application, wherein the application is any one of Q1-Q5: Q1 refers to the application of substances that detect the polymorphisms or genotypes of the three SNPs, SNP1, SNP2, and SNP3, in the preparation of products for identification or auxiliary identification of dairy cow milk production traits. Q2 refers to the application of substances for detecting haplotypes in the preparation of products for identifying or assisting in the identification of dairy cow milk production traits; the haplotype is a polymorphic combination of the three SNPs SNP1, SNP2 and SNP3 on a single chromosome of a dairy cow. Q3 refers to the application of substances that detect the polymorphism or genotype of SNP1 in the preparation of products for identifying or assisting in the identification of dairy cow milk production traits. Q4 refers to the application of substances that detect the polymorphism or genotype of SNP2 in the preparation of products for identifying or assisting in the identification of dairy cow milk production traits. Q5 refers to the application of substances that detect the polymorphism or genotype of SNP3 in the preparation of products for identifying or assisting in the identification of dairy cow milk production traits. SNP1 is a SNP in the bovine genome, located at nucleotide 249 of SEQ ID No. 1 in the sequence listing, and is either C or T; SNP2 is a SNP in the bovine genome, located at nucleotide 1462 of SEQ ID No. 1 in the sequence listing, and is either C or A; SNP3 is a SNP in the bovine genome, located at nucleotide 11400 of SEQ ID No. 1 in the sequence listing, and is either C or T.

[0009] To address the aforementioned technical problems, the present invention also provides an application, wherein the application is any one of E1-E5: E1 refers to the application of substances that detect the polymorphisms or genotypes of the three SNPs, SNP1, SNP2, and SNP3, in dairy cattle breeding or the preparation of dairy cattle breeding products. The E2 refers to the application of substances for detecting haplotypes in dairy cow breeding or the preparation of dairy cow breeding products; the haplotype is a polymorphic combination of the three SNPs, SNP1, SNP2 and SNP3, on a single chromosome of a dairy cow. The E3 refers to the application of substances used to detect the polymorphism or genotype of the SNP1 in dairy cattle breeding or the preparation of dairy cattle breeding products. The E4 refers to the application of substances used to detect the polymorphism or genotype of the SNP2 in dairy cattle breeding or the preparation of dairy cattle breeding products. The E5 refers to the application of substances used to detect the polymorphism or genotype of the SNP3 in dairy cattle breeding or the preparation of dairy cattle breeding products. SNP1 is a SNP in the bovine genome, located at nucleotide 249 of SEQ ID No. 1 in the sequence listing, and is either C or T; SNP2 is a SNP in the bovine genome, located at nucleotide 1462 of SEQ ID No. 1 in the sequence listing, and is either C or A; SNP3 is a SNP in the bovine genome, located at nucleotide 11400 of SEQ ID No. 1 in the sequence listing, and is either C or T.

[0010] The dairy cow's milk production traits can be any one or a combination of milk yield, milk fat content, milk fat percentage, milk protein content, and milk protein percentage.

[0011] The genotype (i.e., allele) of SNP1 can be genotype CC, genotype TT, or genotype CT. Genotype CC is homozygous for SNP1 being C, genotype TT is homozygous for SNP1 being T, and genotype CT is heterozygous for SNP1 being both C and T. The genotype (i.e., allele) of SNP2 can be genotype CC, genotype AA, or genotype CA. Genotype CC is homozygous for SNP2 being C, genotype AA is homozygous for SNP2 being A, and genotype CA is heterozygous for SNP2 being both C and A. The genotype (i.e., allele) of SNP3 can be genotype CC, genotype TT, or genotype CT. Genotype CC is homozygous for SNP1 being C, genotype TT is homozygous for SNP1 being T, and genotype CT is heterozygous for SNP1 being both C and T.

[0012] The haplotypes can specifically be haplotypes H1, H2, and H3; haplotype H1 (TAC) is where SNP1 is T, SNP2 is A, and SNP3 is C; haplotype H2 (TCT) is where SNP1 is T, SNP2 is C, and SNP3 is T; and haplotype H3 (CCT) is where SNP1 is TC, SNP2 is C, and SNP3 is T.

[0013] The genotypes of SNPs 1, 2, and 3 can be TTAACC, TTCACT, and / or TTCCTT. Genotype TTAACC is a combination of three SNPs with genotypes TT for SNP1, AA for SNP2, and CC for SNP3; genotype TTCACT is a combination of three SNPs with genotypes TT for SNP1, CA for SNP2, and CT for SNP3; genotype TTCCTT is a combination of three SNPs with genotypes TT for SNP1, CC for SNP2, and TT for SNP3. Cows with the genotype TTAACC for SNPs 1, 2, and 3 have higher or potential higher milk production traits than cows with the genotypes TTCACT or TTCCTT for SNPs 1, 2, and 3. Cows with the genotype TTAACC for SNPs 1, 2, and 3 have higher or potentially higher milk yield and / or milk fat content than cows with the genotype TTCACT for SNPs 1, 2, and 3, and cows with the genotype TTCCTT for SNPs 1, 2, and 3. Cows with the genotype TTAACC for SNPs 1, 2, and 3 have higher or potentially higher milk protein content than cows with the genotype TTCCTT for SNPs 1, 2, and 3.

[0014] To solve the above-mentioned technical problems, the present invention also provides a product containing the above-mentioned substance for detecting the polymorphism or genotype of SNP1, SNP2 and SNP3 in the bovine genome, or the above-mentioned substance for detecting haplotypes, or the above-mentioned product for detecting the polymorphism or genotype of SNP1 in the bovine genome, or the above-mentioned product for detecting the polymorphism or genotype of SNP2 in the bovine genome, or the above-mentioned product for detecting the polymorphism or genotype of SNP3 in the bovine genome, and may be any one of the following G1)-G3): G1) Products that detect single nucleotide polymorphisms or genotypes related to milk production traits in dairy cows; G2) Products used to identify or assist in identifying milk production traits in dairy cows; G3) is a product used for dairy cow breeding.

[0015] To address the aforementioned technical problems, the present invention also provides a method for identifying or assisting in the identification of milk production traits in dairy cows, wherein the method is method A or method B.

[0016] Method A is a method for identifying or assisting in the identification of milk production traits in dairy cows, including detecting the genotypes of three SNPs (SNP1, SNP2, and SNP3) in the dairy cow to be tested, and identifying or assisting in the identification of milk production traits based on the genotypes of the three SNPs in the dairy cow to be tested: the milk production trait of dairy cows with the genotype TTAACC of the three SNPs is higher or a candidate higher than that of dairy cows with the genotypes TTCACT or TTCCTT of the three SNPs; genotype TTAACC is a combination genotype of three SNPs with genotypes TT for SNP1, AA for SNP2, and CC for SNP3; genotype TTCACT is a combination genotype of three SNPs with genotypes TT for SNP1, CA for SNP2, and CT for SNP3; genotype TTCCTT is a combination genotype of three SNPs with genotypes TT for SNP1, CC for SNP2, and TT for SNP3.

[0017] Method B is a method for identifying or assisting in the identification of milk production traits in dairy cows, including detecting the haplotypes in the dairy cows to be tested, and identifying or assisting in the identification of milk production traits in dairy cows based on the haplotypes of the dairy cows to be tested: the milk production trait of the homozygous genotype corresponding to haplotype H1 is higher or candidate higher than that of the homozygous genotype corresponding to haplotype H2; the haplotype H1 (TAC) is T for SNP1, A for SNP2 and C for SNP3; the haplotype H2 (TCT) is T for SNP1, C for SNP2 and T for SNP3.

[0018] To address the aforementioned technical problems, the present invention also provides a method for identifying or assisting in the identification of milk production traits in dairy cows, comprising detecting the genotype of the aforementioned SNP1 in the dairy cow to be tested, and identifying or assisting in the identification of milk production traits in the dairy cow based on the genotype of the SNP1 in the dairy cow to be tested: dairy cows with the SNP1 genotype CC have higher or candidate higher milk production traits than dairy cows with the genotype TT or CT; CC is a homozygous type of SNP1 with C; TT is a homozygous type of SNP1 with T; and CT is a heterozygous type of SNP1 with both C and T.

[0019] To address the aforementioned technical problems, this invention also provides a method for identifying or assisting in the identification of milk production traits in dairy cows, comprising detecting the genotype of the aforementioned SNP2 in the dairy cow to be tested, and identifying or assisting in the identification of milk production traits in the dairy cow based on the genotype of the SNP2 in the dairy cow to be tested: dairy cows with the genotype AA for SNP2 have higher or candidate higher milk production traits than dairy cows with the genotype CC or CA; AA is a homozygous type of SNP2 being A; CC is a homozygous type of SNP3 being C; and CA is a heterozygous type of SNP2 being C and A.

[0020] To address the aforementioned technical problems, this invention also provides a method for identifying or assisting in the identification of milk production traits in dairy cows, comprising detecting the genotype of the aforementioned SNP3 in the dairy cow to be tested, and identifying or assisting in the identification of milk production traits in the dairy cow based on the genotype of the SNP3 in the dairy cow to be tested: dairy cows with the SNP3 genotype CC have higher or candidate higher milk production traits than dairy cows with the genotype TT or CT; CC is a homozygous type of SNP3 C; TT is a homozygous type of SNP3 T; and CT is a heterozygous type of SNP3 C and T.

[0021] The application of the above-mentioned methods for identifying or assisting in the identification of milk production traits in dairy cows in dairy cow breeding is also within the scope of protection of this invention.

[0022] This invention also provides a method for breeding dairy cows, comprising: detecting the polymorphism or genotype of three SNPs, SNP1, SNP2, and SNP3, in the dairy cow genome, or detecting the type of haplotypes described above in the dairy cow genome, and selecting dairy cows with the genotype combination TTAACC as parents for breeding; or selecting dairy cows with haplotype H1 (TAC) as parents for breeding; or selecting homozygous dairy cows with SNP1 locus C as parents for breeding; or selecting homozygous dairy cows with SNP2 locus A as parents for breeding; or selecting homozygous dairy cows with SNP3 locus C as parents for breeding.

[0023] The dairy cattle breeding mentioned above aims to develop dairy cattle breeds with high milk production traits.

[0024] The milk production traits mentioned above can specifically refer to milk yield, milk fat content, milk fat percentage, milk protein content, and milk protein percentage.

[0025] In the above applications and methods, the substances used to detect the polymorphisms or genotypes of SNPs SNP1, SNP2, and SNP3, or the substances used to detect haplotypes, or the substances used to detect the polymorphisms or genotypes of SNP1, SNP2, and SNP3, can be used to determine the nucleotide types of SNP1, SNP2, and / or SNP3 sites in the above-mentioned dairy cow genome through at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP microarrays. The SNP microarrays include microarrays based on nucleic acid hybridization reactions, microarrays based on single-base extension reactions, microarrays based on allele-specific primer extension reactions, microarrays based on one-step reactions, microarrays based on primer ligation reactions, microarrays based on restriction endonuclease reactions, microarrays based on protein-DNA binding reactions, and microarrays based on fluorescent molecule-DNA binding reactions.

[0026] In the above applications or methods, the substance used to detect the polymorphism or genotype of the three SNPs SNP1, SNP2, and SNP3, or the substance used to detect haplotypes, or the substance used to detect the polymorphism or genotype of SNP3, may be D1), D2), or D3): D1) Contains PCR primers that amplify bovine genomic DNA fragments including the SNP1, SNP2 and / or SNP3 sites; D2) PCR reagents containing the PCR primers described in D1); D3) A kit containing the PCR primers described in D1) or the PCR reagents described in D2).

[0027] The PCR primers mentioned above are 1F / 1R, 3F / 3R, and / or 11F / 11R: 1F / 1R, a primer set consisting of single-stranded DNA represented by positions 4-23 of SEQ ID No. 1 in the sequence listing and single-stranded DNA that is reverse complementary to positions 598-617 of SEQ ID No. 1; 3F / 3R, a primer set consisting of single-stranded DNA represented by positions 1011-1030 of SEQ ID No. 1 in the sequence listing and single-stranded DNA that is reverse complementary to positions 1702-1721 of SEQ ID No. 1; 11F / 11R is a primer set consisting of single-stranded DNA from positions 11191-11209 of SEQ ID No. 1 in the sequence listing and single-stranded DNA that is reverse complementary to positions 11623-11642 of SEQ ID No. 1.

[0028] In the above applications and methods, the PCR primers may or may not be labeled with a marker. The marker refers to any atom or molecule that can be used to provide detectable results and can be linked to nucleic acids. Markers include, but are not limited to, dyes; radioactive markers, such as... 32 P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties whose emission spectra can be inhibited or shifted by fluorescence resonance energy transfer (FRET). The label can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The label can be a charged moieties (positive or negative) or, optionally, charge-neutral. The label can include nucleic acid or protein sequences or combinations thereof, provided that the sequence containing the label is detectable. In some embodiments, nucleic acids are detected directly without labeling (e.g., direct sequence reading).

[0029] In the above applications and methods, the product may be a reagent, kit, or system. The system may include a combination of reagents or kits, instruments, and analysis software, such as a product consisting of PCR primers, PARMS master mix reagents, an ELISA reader, and online SNP decoder software (http: / / www.snpway.com / snpdecoder01 / ), or a combination of PCR primers, PARMS master mix reagents, online SNP decoder software, and a real-time PCR instrument. The product may include the substances described above for detecting polymorphisms or genotypes of SNP1, SNP2, and / or SNP3 sites in the bovine genome.

[0030] In embodiments of the present invention, by analyzing dairy cow-related groups CYP7A1 gene Genetic variation analysis revealed three SNPs: SNP1, SNP2, and SNP3, which are located in genes related to milk production traits in the dairy cow genome. CYP7A1 gene The SNPs are located at positions 249, 1462, and 11400 of SEQ ID No. 1. In the embodiments of this invention, the dominant allele for SNP1 is C, the dominant allele for SNP2 is A, and the dominant allele for SNP3 is C, indicating that SNP1, SNP2, and / or SNP3 can be used for marker-assisted selection breeding of dairy cows and the selection of dairy cow breeds with high milk production traits. These three SNP combinations contain three haplotypes: haplotype H1 (TAC), haplotype H2 (TCT), and haplotype H3 (CCT). Experiments have shown that dairy cows with homozygous genotype H1 (TAC) have significantly higher milk production traits than those with homozygous genotypes of other haplotypes. The H1 (TAC) molecular marker can be used for early prediction and screening of dairy cow milk production traits, and also for marker-assisted selection breeding of dairy cows and the selection of dairy cow breeds with high milk production traits. In addition, in the embodiments of the present invention, the milk production trait of dairy cows with the genotype TTAACC for the three SNPs SNP1, SNP2, and SNP3 is higher or a candidate higher than that of dairy cows with the genotypes TTCACT or TTCCTT for the three SNPs SNP1, SNP2, and SNP3. Attached Figure Description

[0031] Figure 1 The mutation locations are g.24676224G>A, g.24675708G>T, and g.24665770A>G.

[0032] Figure 2For the chain disequilibrium estimation of SNP1 (g.24676224G>A), SNP2 (g.24675708G>T) and SNP3 (g.24665770A>G). Detailed Implementation

[0033] 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.

[0034] 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.

[0035] The Chinese Holstein cows in the following examples came from the Hebei Provincial Livestock Breeding Station.

[0036] The data in the examples are all from lactation period 1. Lactation period 1 refers to the lactation period after the first delivery.

[0037] In this example, the milk yield refers to the milk yield of an individual cow over 305 days, which is the total milk production from the first day of calving to the 305th day. When the actual milking days are less than 305 days, the actual milk yield is used as the 305-day milk yield; when the actual milking days exceed 305 days, the milk yield after the 306th day is not included. Milk yield is obtained from monthly DHI (Dairy Herd Improvement) measurements. The 305-day milk yield for the lactation period can be calculated by plotting a milk yield-lactation curve using more than three DHI data points within the same lactation period.

[0038] In this example, the milk fat content refers to the milk fat content over 305 days, which is calculated as: milk fat percentage × milk production over 305 days. The milk fat percentage is obtained from monthly DHI (Dairy Herd Improvement) measurements. The average milk fat percentage for the lactation period can be calculated by plotting a milk fat percentage lactation curve using more than three DHI data points within the same lactation period.

[0039] The milk protein content in this example refers to the milk protein content over 305 days. 305-day milk protein content = milk protein percentage × milk production over 305 days. The milk protein percentage is obtained from monthly DHI (Dairy Herd Improvement) measurements. The average milk protein percentage for the lactation period can be calculated by plotting a lactation curve using milk protein percentage data from at least three DHI measurements within the same lactation period.

[0040] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0041] Example 1: Discovery of Molecular Markers I. Relevant Basic Research The inventor's research group used liver tissue from three Chinese Holstein cattle at different lactation stages (dry period, early lactation, and peak lactation) as experimental material. They employed next-generation sequencing technology for transcriptome sequencing (RNA-sequencing, RNA-seq) and small RNA sequencing (small RNA-seq). They found that compared to the dry period, CYP7A1 Its expression was significantly upregulated during the early and peak stages of lactation. P <0.01), can promote increased bile acid synthesis, thereby improving lipid absorption for milk synthesis.

[0042] II. Gene Polymorphism Detection 1. A total of 111 Chinese Holstein cows from Hebei Province were selected as the experimental population for gene polymorphism detection. These 111 cows were randomly divided into 5 groups (one group of 23 cows and the other 4 groups of 22 cows each). Genomic DNA was extracted from blood samples. The concentration of the DNA was accurately determined using a nucleic acid quality analyzer. All DNA samples were diluted to a concentration of 50 ng / μL and mixed in equal amounts to form 5 DNA pools, which were then used as templates for PCR amplification.

[0043] 2. According to cattle CYP7A1 The gene sequence (Ensembl ID is ENSBTAG00000005287, as shown in SEQ ID No. 1) was used to design 16 pairs of primers as shown in Table 1.

[0044] Table 1 CYP7A1 Gene PCR amplification primer sequence information

[0045] 3. Using the pooled DNA obtained in step 1 as a template, PCR amplification was performed using each primer pair to obtain the PCR amplification products. The PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.

[0046] Table 2 PCR reaction system

[0047] Table 3 PCR reaction conditions

[0048] 4. Sequencing of the PCR amplification products. Results showed that the cow herd... CYP7A1The gene contains two SNP markers (SNP1 and SNP2) in the flanking sequence 2000 bp upstream, and one SNP marker (SNP3) in the 3' UTR. The three SNP markers are shown in Table 4, and the mutation locations are as follows: Figure 1 As shown.

[0049] Table 4 CYP7A1 Three SNPs discovered in genes

[0050] SNP1, corresponding to g.24676224G>A, was obtained by sequencing analysis of the product amplified by PCR using primers 1F and 1R (as shown in positions 4-617 of SEQ ID No. 1). Its nucleotides are C or T, corresponding to position 249 from the 5' end of SEQ ID No. 1 in the sequence listing. SNP2, corresponding to g.24675708G>T, was obtained by sequencing analysis of the product amplified by PCR using primers 3F and 3R (as shown in positions 1011-1721 of SEQ ID No. 1). Its nucleotides are C or A, corresponding to position 1462 from the 5' end of SEQ ID No. 1 in the sequence listing. SNP3, corresponding to g.24665770A>G, was obtained by sequencing analysis of the product from PCR amplification using primers 11F and 11R (the PCR amplification product is shown as positions 11191-11642 of SEQ ID No. 1). Its nucleotides are T or C, corresponding to position 11400 from the 5' end of SEQ ID No. 1 in the sequence listing. In the sequence listing, Y in SEQ ID No. 1 represents T or C, and M represents C or A.

[0051] g.24676224G>A, g.24675708G>T, and g.24665770A>G are the names of SNP1, SNP2, and SNP3, respectively. SNP naming generally follows the pattern observed when the SNP was first discovered. DNA has a double-stranded structure and follows the complementary base pairing principle. SEQ ID No. 1 in this invention is the reverse complementary strand of the same double-stranded DNA as the sequence at which SNP1, SNP2, and SNP3 were discovered. Therefore, in this invention, the polymorphic form of g.24676224G>A is C or T, the polymorphic form of g.24675708G>T is C or A, and the polymorphic form of g.24665770A>G is T or C.

[0052] III. Correlation Analysis (a) Obtaining the test group The test population consisted of 1,123 Chinese Holstein cows.

[0053] (ii) Genotyping Each individual in the test population was genotyped separately.

[0054] I. Genotyping based on g.24676224G>A.

[0055] 1. Take blood from the test individual and extract genomic DNA.

[0056] 2. Using genomic DNA as a template, PCR amplification was performed using primer pairs consisting of 1F (as shown in positions 4-23 of SEQ ID No. 1) and 1R (reverse complementary to positions 598-617 of SEQ ID No. 1). The PCR amplification products were then recovered and sequenced.

[0057] The reaction system for PCR amplification is shown in Table 5. The reaction conditions for PCR amplification are shown in Table 6.

[0058] The PCR amplification products of each test individual were 614bp, with the 246th position being g.24676224G>A, which is SNP1 (corresponding to the 249th position from the 5' end of SEQ ID No. 1 in the sequence listing).

[0059] Table 5

[0060] Table 6

[0061] II. Genotyping based on g.24675708G>T.

[0062] 1. Take blood from the test individual and extract genomic DNA.

[0063] 2. Using genomic DNA as a template, PCR amplification was performed using primer pairs consisting of 3F (as shown in positions 1011-1030 of SEQ ID No. 1) and 3R (reverse complementary to positions 1702-1721 of SEQ ID No. 1). The PCR amplification products were then recovered and sequenced.

[0064] The reaction system for PCR amplification is shown in Table 7. The reaction conditions for PCR amplification are shown in Table 8.

[0065] The PCR amplification products of each test individual were 711bp, with the 452nd position being g.24675708G>T, which is SNP2 (corresponding to the 1462nd position from the 5' end of SEQ ID No. 1 in the sequence listing).

[0066] Table 7

[0067] Table 8

[0068] III. Genotyping based on g.24665770A>G.

[0069] 1. Take blood from the test individual and extract genomic DNA.

[0070] 2. Using genomic DNA as a template, PCR amplification was performed using primer pairs consisting of 11F (as shown in positions 11191-11209 of SEQ ID No. 1) and 11R (reverse complementary to positions 11623-11642 of SEQ ID No. 1). The PCR amplification products were then recovered and sequenced.

[0071] The reaction system for PCR amplification is shown in Table 9. The reaction conditions for PCR amplification are shown in Table 10.

[0072] The PCR amplification products of each test individual were 452bp, with the 210th position being g.24665770A>G, which is SNP3 (corresponding to the 11400th position from the 5' end of SEQ ID No. 1 in the sequence listing).

[0073] Table 9

[0074] Table 10

[0075] (III) Testing milk production characteristics Each cow in the test group underwent individual testing for milk production traits.

[0076] Milk production traits include the following five indicators: milk yield, milk fat content, milk fat percentage, milk protein content, and milk protein percentage.

[0077] Each individual's record includes, in order: cow individual number, father number, mother number, grandfather number, grandmother number, maternal grandfather number, maternal grandmother number, date of birth, lactation period, calving date, milk yield, milk fat content, and milk protein content.

[0078] (iv) Association analysis model between a single SNP site and a trait SNP1 site (i.e.) CYP7A1 Gene g.24676224G>A), SNP2 site (i.e. CYP7A1 Gene g.24675708G>T) and SNP3 locus (i.e. CYP7A1 The genotypes and milk production phenotypes of the gene g.24665770A>G are shown in Tables 11, 12 and 13.

[0079] Table 11. Phenotypes of some milk production traits and genotypes of three SNP loci in 1123 Chinese Holstein cows.

[0080] Table 12. Descriptive statistics of five milk production traits in a herd of 1,123 Chinese Holstein cows.

[0081] Table 13 Allele frequencies and genotype frequencies at three SNP loci in the CYP7A1 gene

[0082] The results showed that there were three genotypes at the SNP1 locus (referred to as SNP1 genotypes): CC, TT, or CT. Genotype CC is homozygous for SNP1 with C, genotype TT is homozygous for SNP1 with T, and genotype CT is heterozygous for SNP1 with both C and T. There were three genotypes at the SNP2 locus (referred to as SNP2 genotypes): CC, AA, or CA. Genotype CC is homozygous for SNP2 with C, genotype AA is homozygous for SNP2 with A, and genotype CA is heterozygous for SNP2 with both C and A. There were three genotypes at the SNP3 locus (referred to as SNP3 genotypes): CC, TT, or CT. Genotype CC is homozygous for SNP1 with C, genotype TT is homozygous for SNP1 with T, and genotype CT is heterozygous for SNP1 with both C and T.

[0083] Association analysis was performed on five indicators of milk production traits and genotypes using the MIXED procedure in SAS 9.2 software. An animal model was used for the association analysis; the specific model is as follows:

[0084] Wherein, Y: observed values ​​of milk production traits (milk yield, milk fat content, milk fat percentage, milk protein content or milk protein percentage); μ: population mean; hys: field-year-season effect; b: regression coefficient of covariate M; M: calving age effect; G: genotype effect; a: individual random additive genetic effect; e: random residual effect.

[0085] SNP1 locus in a population of 1123 heads (i.e. CYP7A1 The gene g.24676224G>A was successfully genotyped in 1120 pigs, and the results of the association analysis with milk production traits are shown in Table 14.

[0086] Table 14 CYP7A1Association analysis between gene g.24676224G>A and milk production trait (least square mean ± standard error)

[0087] Note: * P <0.05 indicates a significant difference; ** P <0.01 indicates a highly significant difference. a,b Different superscripts in the same column of data indicate significant differences. A,B Different superscripts in the same column of data indicate highly significant differences.

[0088] As shown in Table 14, SNP1 (g.24676224G>A) was significantly associated with milk yield, milk protein content, and milk protein percentage (P=0.0236-0.0037). For the traits of milk yield, milk protein content, and milk protein percentage, the dominant allele was C. The milk production of CC genotype cows was higher than that of TT genotype cows. There was no significant difference in milk production between CC genotype cows and CT genotype cows, and there was no significant difference in milk production between TT genotype cows and CT genotype cows.

[0089] The milk protein content of CC genotype cows was higher than that of TT genotype cows, and the milk protein content of CT genotype cows was higher than that of TT genotype cows. There was no significant difference in milk protein content between CT genotype cows and CC genotype cows.

[0090] The milk protein content of CC genotype cows was higher than that of CT genotype cows. There was no significant difference in milk protein content between CC genotype cows and TT genotype cows, and there was no significant difference in milk protein content between TT genotype cows and CT genotype cows.

[0091] SNP2 locus in a population of 1123 heads (i.e. CYP7A1 The gene g.24675708G>T was successfully genotyped in 1118 animals, and the results of the association analysis with milk production traits are shown in Table 15.

[0092] Table 15 CYP7A1 Association analysis between gene g.24675708G>T and milk production trait (least square mean ± standard error)

[0093] Note: * P <0.05 indicates a significant difference; ** P <0.01 indicates a highly significant difference. a,b Different superscripts in the same column of data indicate significant differences. A,B Different superscripts in the same column of data indicate highly significant differences.

[0094] As shown in Table 15, SNP2 (g.24675708G>T) was significantly associated with milk yield, milk fat content, milk protein content, and milk protein percentage (P=0.0202~0.0005). For the traits of milk yield, milk fat content, milk protein content, and milk protein percentage, the dominant allele was A. Milk production in AA genotype cows was higher than that in CC genotype cows. There was no significant difference in milk production between CA genotype cows and AA genotype cows, and between CA genotype cows and CC genotype cows.

[0095] The milk fat percentage of CC genotype cows was higher than that of AA genotype cows. There was no significant difference in milk fat percentage between CC genotype cows and CA genotype cows, and there was no significant difference in milk fat percentage between AA genotype cows and CA genotype cows.

[0096] The milk protein content of AA genotype cows was higher than that of CC genotype cows, and the milk protein content of CA genotype cows was higher than that of CC genotype cows. There was no significant difference in milk protein content between CA genotype cows and AA genotype cows.

[0097] The milk protein content of CC genotype cows was higher than that of AA genotype cows. The milk protein content of CA genotype cows was not significantly different from that of CC genotype cows, and the milk protein content of CA genotype cows was not significantly different from that of AA genotype cows.

[0098] SNP3 locus in a population of 1123 heads (i.e. CYP7A1 The gene g.24665770A>G was successfully genotyped in 1116 animals, and the results of the association analysis with milk production traits are shown in Table 16.

[0099] Table 16 CYP7A1 Association analysis between gene g.24665770A>G and milk production trait (least square mean ± standard error)

[0100] Note: * P <0.05 indicates a significant difference; ** P <0.01 indicates a highly significant difference. a,b Different superscripts in the same column of data indicate significant differences. A,B Different superscripts in the same column of data indicate highly significant differences.

[0101] As shown in Table 16, SNP3 (g.24665770A>G) was significantly associated with milk yield, milk fat content, milk fat percentage and milk protein content (P=0.0018~<0.0001). For the traits of milk yield, milk fat content, milk fat percentage and milk protein content, the dominant allele was C.

[0102] Cows with the CT genotype produce more milk than cows with the TT genotype.

[0103] Cows with the CC genotype had higher milk fat content, milk fat percentage, and milk protein content than cows with the TT genotype. Cows with the CT genotype had higher milk fat content, milk fat percentage, and milk protein content than cows with the TT genotype. Cows with the CC genotype had no significant differences in milk fat content, milk fat percentage, and milk protein content compared to cows with the CT genotype.

[0104] (V) Analysis of Genetic Effects The significance of SNP additive effects, dominant effects, and substitution effects was tested using SAS 9.2 software.

[0105] The basic calculation formula is as follows: a = (AA - BB) / 2, d = AB - (AA + BB) / 2, α = a + d(qp); a is the additive effect, d is the dominant effect, and α is the allele substitution effect; AA, AB, and BB are the least squares mean values ​​of the milk production trait for the corresponding genotypes; p is the frequency of allele A, and q is the frequency of allele B.

[0106] The results of tests for additive effects, dominant effects, and allele substitution effects are shown in Table 17.

[0107] Table 17 CYP7A1 Results of tests for additive, dominant, and substitution effects of gene alleles.

[0108] Note: * P <0.05 indicates a significant difference; ** P <0.01 indicates a highly significant difference.

[0109] The results showed that the additive, dominant, and allele substitution effects of SNP1 (g.24676224G>A) on milk yield, milk protein content, and milk protein percentage were significant or highly significant. Specifically, each T allele replacing a C allele resulted in a decrease of 81.622 kg in milk yield (P<0.01), a decrease of 1.898 kg in milk protein content (P<0.01), and an increase of 0.009% in milk protein percentage (P<0.05). SNP2 (g.24675708G>T) showed significant additive and allele substitution effects on milk yield, milk fat percentage, milk protein mass, and milk protein percentage. Specifically, each C allele replacing an A allele resulted in a 90.947 kg decrease in milk yield (P < 0.01), a 0.039% increase in milk fat percentage (P < 0.01), a 2.216 kg decrease in milk protein mass (P < 0.01), and a 0.011% increase in milk protein percentage (P < 0.01). SNP3 (g.24665770A>G) also showed significant additive and allele substitution effects on milk protein mass and milk protein percentage. Specifically, each T allele replacing a C allele resulted in a 3.057 kg decrease in milk protein mass (P < 0.05) and a 0.023% decrease in milk protein percentage (P < 0.05).

[0110] (vi) Haplotype analysis Haplotypes were constructed using Haploview 4.2 software, haplotype frequencies and linkage analysis were performed, and association analysis with traits was conducted. The model was constructed using the MIXED procedure in SAS 9.2 software, as follows:

[0111] Y: Observed values ​​of milk production traits (milk yield, milk fat content, milk fat percentage, milk protein content, and milk protein percentage); μ: Population mean; hys: Field-year-season effect; b: Regression coefficient of covariate M; M: Calving age effect; G: Haplotype combination effect; a: Individual random additive genetic effect; e: Random residual effect.

[0112] The haplotype analysis results are shown in Table 18.

[0113] Table 18 CYP7A1 Haplotypes consisting of 3 SNPs in a gene

[0114] In the aforementioned research group of 1123 heads CYP7A1Three SNPs (SNP1, SNP2, and SNP3) constitute a haplotype block, forming three haplotype combinations: haplotype H1, haplotype H2, and haplotype H3. Haplotype H1 (TAC) is a combination on a chromosome where SNP1 is T, SNP2 is A, and SNP3 is C. Haplotype H2 (TCT) is a combination on a chromosome where SNP1 is T, SNP2 is C, and SNP3 is T. Haplotype H3 (CCT) is a combination on a chromosome where SNP1 is C, SNP2 is C, and SNP3 is T. The frequencies of haplotype H1, H2, and H3 are 49%, 28.3%, and 22.1%, respectively. Linkage disequilibrium estimation of these three SNPs indicates that they are in a fully linked state (rlinked). 2 =1), such as Figure 2 As shown.

[0115] Haplotypes represent the linkage between two SNPs on the same chromosome. The cattle are diploid, and the haplotype combinations of the two chromosomes in the 1123 tested individuals were H1H1, H1H2, and H2H2, respectively.

[0116] The cow corresponding to H1H1 has the genotype TTAACC, which is a combination of three SNPs: SNP1 genotype TT, SNP2 genotype AA, and SNP3 genotype CC. The cow corresponding to H1H2 has the genotype TTCACT, which is a combination of three SNPs: SNP1 genotype TT, SNP2 genotype CA, and SNP3 genotype CT. The cow corresponding to H2H2 has the genotype TTCCTT, which is a combination of three SNPs: SNP1 genotype TT, SNP2 genotype CC, and SNP3 genotype TT.

[0117] CYP7A1 The results of the association analysis between haplotype combinations and milk production traits are shown in Table 19 (the haplotype combination H3H3 with a frequency of less than 0.05 was removed, and although the frequency of H2H3 was >0.05, the genotype CTCCTT of the H2H3 combination did not appear in this population, so it was also removed). CYP7A1 The haplotype combinations of the three SNPs of the gene were significantly associated with milk yield, milk fat content, milk fat percentage, milk protein content, and milk protein percentage (P=0.0088<0.0001), with haplotype H1 being the dominant haplotype for all five milk production traits.

[0118] Table 19 CYP7A1 Association analysis results between haplotype combinations and milk production traits (least square mean ± standard error)

[0119] Cows with the haplotype H1H1 (i.e., genotype TTAACC) produced more milk than cows with the haplotype H1H2 (i.e., genotype TTCACT) and the haplotype H2H2 (i.e., genotype TTCCTT).

[0120] Cows with the haplotype H1H1 (i.e., genotype TTAACC) had higher milk fat content than cows with the haplotype H1H2 (i.e., genotype TTCACT) and the haplotype H2H2 (i.e., genotype TTCCTT).

[0121] Cows with the haplotype H2H2 (i.e., genotype TTCCTT) had a higher milk fat percentage than cows with the haplotype H1H2 (i.e., genotype TTCACT) and the haplotype H1H1 (i.e., genotype TTAACC).

[0122] Cows with haplotype H1H1 (i.e., genotype TTAACC) had higher milk protein content than cows with haplotype H2H2 (i.e., genotype TTCCTT), and cows with haplotype H1H2 (i.e., genotype TTCACT) had higher milk protein content than cows with haplotype H2H2 (i.e., genotype TTCCTT).

[0123] Compared to single-marker analysis, haplotype analysis often expresses more information, and changes in some site interactions within a gene can have a significant impact on phenotypic traits. Therefore, haplotype analysis is significantly superior to conventional single-marker analysis in complex trait association analysis.

[0124] The molecular markers disclosed in this invention can be used to assist in the identification of dairy cow populations with excellent milk production traits (305-day milk yield, milk fat content, milk fat percentage, milk protein content, and milk protein percentage). They have the following advantages: they are simple, rapid, sensitive, and provide reliable, stable, and accurate results, making them suitable for large-scale laboratory testing.

[0125] 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.

Claims

1. Use, characterized in that: The application is any one of the following P1-P4: P1 refers to the application of substances for detecting haplotypes in identifying or assisting in the identification of milk yield, milk fat content, milk fat percentage, milk protein content and / or milk protein percentage in dairy cows. The haplotype is a polymorphic combination of three SNPs, SNP1, SNP2 and SNP3, on a single chromosome of a dairy cow. The P2 refers to the application of substances that detect the polymorphism or genotype of SNP1 in identifying or assisting in the identification of milk yield, milk protein content, and / or milk protein percentage in dairy cows. The P3 refers to the application of substances that detect the polymorphism or genotype of the SNP2 in identifying or assisting in the identification of milk yield, milk fat percentage, milk protein content and / or milk protein percentage in dairy cows. The P4 refers to the application of substances that detect the polymorphism or genotype of the SNP3 in identifying or assisting in the identification of milk yield, milk fat content, milk fat percentage, and / or milk protein content in dairy cows. SNP1 is a SNP in the bovine genome, located at nucleotide 249 of SEQ ID No. 1 in the sequence listing, and is either C or T; SNP2 is a SNP in the bovine genome, located at nucleotide 1462 of SEQ ID No. 1 in the sequence listing, and is either C or A; SNP3 is a SNP in the bovine genome, located at nucleotide 11400 of SEQ ID No. 1 in the sequence listing, and is either C or T. The dairy cows mentioned are Holstein cattle; The milk production is the individual's milk production over 305 days, obtained from monthly DHI measurements, and calculated by plotting a milk production lactation curve using more than 3 DHI data within the same lactation period. The milk fat content mentioned is the milk fat content of an individual over 305 days, where 305-day milk fat content = milk fat percentage × milk production over 305 days; The milk fat percentage was obtained from monthly DHI measurements and calculated by plotting a milk fat percentage lactation curve using more than three DHI data points within the same lactation period. The milk protein content mentioned is the individual's milk protein content over 305 days, where 305-day milk protein content = milk protein percentage × milk production over 305 days; The milk protein percentage is obtained from monthly DHI measurements and is calculated by plotting a milk protein percentage lactation curve using more than three DHI data points within the same lactation period. The lactation period refers to the lactation period following the first childbirth.

2. Application, characterized in that: The application is any one of Q1-Q4: Q1 refers to the application of substances for detecting haplotypes in the preparation of products for identifying or assisting in the identification of milk yield, milk fat content, milk fat percentage, milk protein content, and / or milk protein percentage in dairy cows; the haplotype is a polymorphic combination of three SNPs, SNP1, SNP2, and SNP3, on a single chromosome of a dairy cow. Q2 refers to the application of substances that detect the polymorphism or genotype of SNP1 in the preparation of products for identifying or assisting in the identification of milk yield, milk protein content, and / or milk protein percentage in dairy cows. Q3 refers to the application of substances that detect the polymorphism or genotype of SNP2 in the preparation of products for identifying or assisting in the identification of milk yield, milk fat percentage, milk protein content and / or milk protein percentage in dairy cows; Q4 refers to the application of substances that detect the polymorphism or genotype of SNP3 in the preparation of products for identifying or assisting in the identification of milk yield, milk fat content, milk fat percentage and / or milk protein content in dairy cows; SNP1 is a SNP in the bovine genome, located at nucleotide 249 of SEQ ID No. 1 in the sequence listing, and is either C or T; SNP2 is a SNP in the bovine genome, located at nucleotide 1462 of SEQ ID No. 1 in the sequence listing, and is either C or A; SNP3 is a SNP in the bovine genome, located at nucleotide 11400 of SEQ ID No. 1 in the sequence listing, and is either C or T. The dairy cows mentioned are Holstein cattle; The milk production is the individual's milk production over 305 days, obtained from monthly DHI measurements, and calculated by plotting a milk production lactation curve using more than 3 DHI data within the same lactation period. The milk fat content mentioned is the milk fat content of an individual over 305 days, where 305-day milk fat content = milk fat percentage × milk production over 305 days; The milk fat percentage was obtained from monthly DHI measurements and calculated by plotting a milk fat percentage lactation curve using more than three DHI data points within the same lactation period. The milk protein content mentioned is the individual's milk protein content over 305 days, where 305-day milk protein content = milk protein percentage × milk production over 305 days; The milk protein percentage is obtained from monthly DHI measurements and is calculated by plotting a milk protein percentage lactation curve using more than three DHI data points within the same lactation period. The lactation period refers to the lactation period following the first childbirth.

3. Application, characterized in that: The application is any one of E1-E4: E1 refers to the application of substances that detect haplotypes in dairy cow breeding or the preparation of dairy cow breeding products; the haplotype is a polymorphic combination of three SNPs, SNP1, SNP2, and SNP3, on a single chromosome of a dairy cow; the breeding objective is to select dairy cows with high milk yield, milk fat content, milk fat percentage, milk protein content, and / or milk protein percentage. The E2 refers to the application of substances that detect the polymorphism or genotype of the SNP1 in dairy cattle breeding or the preparation of dairy cattle breeding products; the goal of the breeding is to select dairy cattle with high milk yield, milk protein content, and / or milk protein percentage. The E3 refers to the application of substances that detect the polymorphism or genotype of the SNP2 in dairy cow breeding or the preparation of dairy cow breeding products; the goal of the breeding is to select dairy cows with high milk yield, milk fat percentage, milk protein content and / or milk protein percentage. The E4 refers to the application of substances used to detect the polymorphism or genotype of the SNP3 in dairy cattle breeding or the preparation of dairy cattle breeding products; the goal of the breeding is to select dairy cattle with high milk yield, high milk fat content, high milk fat percentage, and / or high milk protein content. SNP1 is a SNP in the bovine genome, located at nucleotide 249 of SEQ ID No. 1 in the sequence listing, and is either C or T; SNP2 is a SNP in the bovine genome, located at nucleotide 1462 of SEQ ID No. 1 in the sequence listing, and is either C or A; SNP3 is a SNP in the bovine genome, located at nucleotide 11400 of SEQ ID No. 1 in the sequence listing, and is either C or T. The dairy cows mentioned are Holstein cattle; The milk production is the individual's milk production over 305 days, obtained from monthly DHI measurements, and calculated by plotting a milk production lactation curve using more than 3 DHI data within the same lactation period. The milk fat content mentioned is the milk fat content of an individual over 305 days, where 305-day milk fat content = milk fat percentage × milk production over 305 days; The milk fat percentage was obtained from monthly DHI measurements and calculated by plotting a milk fat percentage lactation curve using more than three DHI data points within the same lactation period. The milk protein content mentioned is the individual's milk protein content over 305 days, where 305-day milk protein content = milk protein percentage × milk production over 305 days; The milk protein percentage is obtained from monthly DHI measurements and is calculated by plotting a milk protein percentage lactation curve using more than three DHI data points within the same lactation period. The lactation period refers to the lactation period following the first childbirth.

4. A method for identifying or assisting in the identification of milk production traits in dairy cows, wherein the method is method A or method B; Method A is a method for identifying or assisting in the identification of milk production traits in dairy cows, comprising detecting the genotypes of three SNPs (SNP1, SNP2, and SNP3) in the dairy cow to be tested, and identifying or assisting in the identification of milk yield, milk fat content, and / or milk protein content based on the genotypes of the three SNPs in the dairy cow to be tested: The milk yield, milk fat content, and / or milk protein content of dairy cows with the genotype TTAACC of the three SNPs are higher than or candidate to be higher than those of dairy cows with the genotype TTCAC. Cows with genotype T or TTCCTT; genotype TTAACC is a combination of three SNPs with genotypes TT for SNP1, AA for SNP2, and CC for SNP3; genotype TTCACT is a combination of three SNPs with genotypes TT for SNP1, CA for SNP2, and CT for SNP3; genotype TTCCTT is a combination of three SNPs with genotypes TT for SNP1, CC for SNP2, and TT for SNP3. Method B is a method for identifying or assisting in the identification of milk production traits in dairy cows, comprising detecting the haplotypes described in claim 1 in the dairy cows to be tested, and identifying or assisting in the identification of milk yield, milk fat content, and / or milk protein content based on the haplotypes of the dairy cows to be tested: the milk yield, milk fat content, and / or milk protein content of the homozygous genotype corresponding to haplotype H1 is higher than or candidate higher than that of the homozygous genotype corresponding to haplotype H2; haplotype H1 is defined as SNP1 being T, SNP2 being A, and SNP3 being C; haplotype H2 is defined as SNP1 being T, SNP2 being C, and SNP3 being T; The dairy cows mentioned are Holstein cattle; The milk production is the individual's milk production over 305 days, obtained from monthly DHI measurements, and calculated by plotting a milk production lactation curve using more than 3 DHI data within the same lactation period. The milk fat content mentioned is the milk fat content of an individual over 305 days, where 305-day milk fat content = milk fat percentage × milk production over 305 days; The milk fat percentage was obtained from monthly DHI measurements and calculated by plotting a milk fat percentage lactation curve using more than three DHI data points within the same lactation period. The milk protein content mentioned is the individual's milk protein content over 305 days, where 305-day milk protein content = milk protein percentage × milk production over 305 days; The milk protein percentage is obtained from monthly DHI measurements and is calculated by plotting a milk protein percentage lactation curve using more than three DHI data points within the same lactation period. The lactation period refers to the lactation period following the first childbirth.

5. A method for identifying or assisting in the identification of milk production traits in dairy cows, characterized in that: The method includes detecting the genotype of SNP1, SNP2, or SNP3 as described in claim 1 in the dairy cow to be tested, and identifying or assisting in the identification of milk production traits in the dairy cow based on the genotype of SNP1, SNP2, or SNP3 of the dairy cow to be tested: Cows with the SNP1 genotype CC have higher or candidate higher milk yield and / or milk protein content than cows with the genotype TT or CT; CC is a homozygous SNP1 genotype of C; TT is a homozygous SNP1 genotype of T; CT is a heterozygous SNP1 genotype of both C and T. Cows with the SNP2 genotype AA have higher or candidate higher milk yield and / or milk protein content than cows with the genotype CC or CA; AA is a homozygous type with SNP2 of A; CC is a homozygous type with SNP2 of C; and CA is a heterozygous type with SNP2 of both C and A. Cows with the SNP3 genotype CC have higher or candidate higher milk yield, milk fat content, and / or milk protein content than cows with the genotype TT or CT; CC is a homozygous SNP3 type C; TT is a homozygous SNP3 type T; and CT is a heterozygous SNP3 type C and T. The dairy cows mentioned are Holstein cattle; The milk production is the individual's milk production over 305 days, obtained from monthly DHI measurements, and calculated by plotting a milk production lactation curve using more than 3 DHI data within the same lactation period. The milk fat content mentioned is the milk fat content of an individual over 305 days, where 305-day milk fat content = milk fat percentage × milk production over 305 days; The milk fat percentage was obtained from monthly DHI measurements and calculated by plotting a milk fat percentage lactation curve using more than three DHI data points within the same lactation period. The milk protein content mentioned is the individual's milk protein content over 305 days, where 305-day milk protein content = milk protein percentage × milk production over 305 days; The milk protein percentage is obtained from monthly DHI measurements and is calculated by plotting a milk protein percentage lactation curve using more than three DHI data points within the same lactation period. The lactation period refers to the lactation period following the first childbirth.

6. The application of the method according to claim 4 or 5 in dairy cow breeding, characterized in that: The goal of the breeding program is to select dairy cows with high milk yield, high milk fat content, and / or high milk protein content. The dairy cows mentioned are Holstein cattle; The milk production is the individual's milk production over 305 days, obtained from monthly DHI measurements, and calculated by plotting a milk production lactation curve using more than 3 DHI data within the same lactation period. The milk fat content mentioned is the milk fat content of an individual over 305 days, where 305-day milk fat content = milk fat percentage × milk production over 305 days; The milk fat percentage was obtained from monthly DHI measurements and calculated by plotting a milk fat percentage lactation curve using more than three DHI data points within the same lactation period. The milk protein content mentioned is the individual's milk protein content over 305 days, where 305-day milk protein content = milk protein percentage × milk production over 305 days; The milk protein percentage is obtained from monthly DHI measurements and is calculated by plotting a milk protein percentage lactation curve using more than three DHI data points within the same lactation period. The lactation period refers to the lactation period following the first childbirth.

7. The application according to any one of claims 1-3 or 6, or the method according to any one of claims 4-5, characterized in that: The substance used to detect haplotypes, or to detect SNP3 polymorphisms or genotypes, is as follows: D1) or D2). D1) Contains PCR primers that amplify bovine genomic DNA fragments including the SNP1, SNP2, and SNP3 sites; D2) A kit containing the PCR primers described in D1).

8. The application or method according to claim 7, characterized in that: The PCR primers were 1F / 1R, 3F / 3R, and 11F / 11R: 1F / 1R, a primer set consisting of single-stranded DNA represented by positions 4-23 of SEQ ID No. 1 in the sequence listing and single-stranded DNA that is reverse complementary to positions 598-617 of SEQ ID No. 1; 3F / 3R, a primer set consisting of single-stranded DNA represented by positions 1011-1030 of SEQ ID No. 1 in the sequence listing and single-stranded DNA that is reverse complementary to positions 1702-1721 of SEQ ID No. 1; 11F / 11R is a primer set consisting of single-stranded DNA from positions 11191-11209 of SEQ ID No. 1 in the sequence listing and single-stranded DNA that is reverse complementary to positions 11623-11642 of SEQ ID No. 1.

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