A SNP molecular marker for gene APOA5 related to milk production traits in dairy cows and its application

Abstract

The present invention discloses the use of SNP molecular markers for the gene APOA5, which is associated with milk production in dairy cows. One technical solution protected by the present invention is the use of a substance that detects the polymorphisms or genotypes of three SNPs, SNP1, SNP2, and SNP3, for identifying or assisting in the identification of milk production traits in dairy cows. Experiments have demonstrated that molecular markers associated with SNP1 and / or SNP2 and / or SNP3, as well as their corresponding haplotypes and genotypes, can be used for early prediction and screening of milk production traits in dairy cows, facilitating the selection and breeding of high-milk-producing dairy cows.

Description

Technical Field

[0001] The present invention relates to a SNP molecular marker of a gene APOA5 related to milk production traits of dairy cows and application thereof in the field of molecular biotechnology. Background Art

[0002] The apolipoprotein A5 (APOA5) gene, a member of the apolipoprotein family, is located on bovine chromosome 15. It encodes a protein involved in plasma triglyceride transport. Studies have shown that plasma triglyceride concentrations in mice overexpressing APOA5 are significantly lower than in control mice, while plasma triglyceride concentrations in APOA5 knockout mice are significantly higher than in normal control mice, indicating that APOA5 is a key determinant of plasma triglyceride levels. The APOA5 gene, encoding an apolipoprotein, plays a crucial role in regulating plasma triglyceride levels and is also involved in the peroxisome proliferator-activated receptor α (PPARα) signaling pathway, chylomicron-mediated lipid transport, and lipoprotein metabolism. During the periparturient period, as dairy cows prepare for lactation, the APOA5 gene is involved in lipoprotein metabolism and is therefore highly expressed in the liver. APOA5 participates in fatty acid and lipoprotein metabolism in the liver, thereby influencing lipid synthesis and milk production traits.

[0003] Invented in 1983 by Mullis et al. in the United States, the polymerase chain reaction (PCR) is a rapid nucleic acid amplification technique that simulates the natural DNA replication process in vitro. Its greatest feature is its ability to significantly increase trace amounts of DNA. PCR utilizes the fact that DNA denatures into single strands at high temperatures in vitro. At low temperatures, primers pair with the single strands based on complementary bases. 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 technique has become one of the most commonly used and important molecular biology techniques. PCR products can be sequenced after agarose gel electrophoresis to identify genetic polymorphisms, making the detection method simple and easy. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to identify or assist in identifying the milk production traits of dairy cows or how to breed dairy cows.

[0005] In order to solve the above technical problems, the present invention first provides an application, which is P1, P2 or P3;

[0006] The P1 is an application of a substance for detecting the polymorphism or genotype of three SNPs, SNP1, SNP2, and SNP3, in identifying or assisting in identifying the milk production trait of a dairy cow, wherein SNP1 is a SNP in the dairy cow genome, which is the 2773rd nucleotide of SEQ ID No. 1 in the sequence listing, and is A or G; SNP2 is a SNP in the dairy cow genome, which is the 2071st nucleotide of SEQ ID No. 1 in the sequence listing, and is G or A; and SNP3 is a SNP in the dairy cow genome, which is the 857th nucleotide of SEQ ID No. 1 in the sequence listing, and is T or C;

[0007] The P2 is an application of a substance for detecting haplotypes in identifying or assisting in identifying the milk production traits of dairy cows, wherein the haplotype is a polymorphic combination of the three SNPs SNP1, SNP2 and SNP3 on a chromosome of a dairy cow;

[0008] The P3 is an application of a substance for detecting the polymorphism or genotype of any SNP site among the SNP1, the SNP2 and the SNP3 in identifying or assisting in identifying the milk production traits of dairy cows.

[0009] In order to solve the above technical problems, the present invention further provides an application, wherein the application is Q1, Q2 or Q3;

[0010] Said Q1 is the use of a substance for detecting the polymorphism or genotype of three SNPs, SNP1, SNP2 and SNP3, in preparing a product for identifying or assisting in identifying the milk production trait of a dairy cow; said SNP1 is a SNP in the dairy cow genome, which is the 2773rd nucleotide of SEQ ID No.1 in the sequence listing, which is A or G; said SNP2 is a SNP in the dairy cow genome, which is the 2071st nucleotide of SEQ ID No.1 in the sequence listing, which is G or A; said SNP3 is a SNP in the dairy cow genome, which is the 857th nucleotide of SEQ ID No.1 in the sequence listing, which is T or C;

[0011] Said Q2 is the use of a substance for detecting haplotypes in the preparation of a product for identifying or assisting in identifying the milk production traits of dairy cows; said haplotype is a polymorphic combination of the three SNPs, SNP1, SNP2 and SNP3, on a chromosome of a dairy cow;

[0012] The Q3 is the use of a substance for detecting the polymorphism or genotype of any SNP site among the SNP1, the SNP2 and the SNP3 in the preparation of a product for identifying or assisting in identifying the milk production traits of dairy cows.

[0013] In order to solve the above technical problems, the present invention further provides an application, wherein the application is E1, E2 or E3;

[0014] The E1 is a use of a substance for detecting the polymorphism or genotype of three SNPs, SNP1, SNP2, and SNP3, in dairy cow breeding or preparing dairy cow breeding products; the SNP1 is a SNP in the dairy cow genome, which is the 2773rd nucleotide of SEQ ID No. 1 in the sequence listing, which is A or G; the SNP2 is a SNP in the dairy cow genome, which is the 2071st nucleotide of SEQ ID No. 1 in the sequence listing, which is G or A; the SNP3 is a SNP in the dairy cow genome, which is the 857th nucleotide of SEQ ID No. 1 in the sequence listing, which is T or C;

[0015] The E2 is a substance for detecting haplotypes in dairy cattle breeding or the preparation of dairy cattle breeding products; the haplotype is a polymorphic combination of the three SNPs SNP1, SNP2 and SNP3 on a chromosome of a dairy cow;

[0016] The E3 is the use of a substance for detecting the polymorphism or genotype of any SNP site among the SNP1, the SNP2 and the SNP3 in dairy cow breeding or the preparation of dairy cow breeding products.

[0017] The milk production traits of dairy cows may be any one of milk yield, milk fat content, milk fat rate, milk protein content and milk protein rate and / or a combination of multiple ones.

[0018] The genotype (i.e., allele) of the SNP1 may be genotype AA, genotype GG or genotype GA, genotype AA is the homozygous type of SNP1 being A, genotype GG is the homozygous type of SNP1 being G, and genotype GA is the heterozygous type of SNP1 being A and G; the genotype (i.e., allele) of the SNP2 may be genotype AA, genotype GG or genotype GA, genotype AA is the homozygous type of SNP2 being A, genotype GG is the homozygous type of SNP1 being G, and genotype GA is the heterozygous type of SNP2 being A and G; the genotype (i.e., allele) of the SNP3 may be genotype CC, genotype TT or genotype TC, genotype CC is the homozygous type of SNP3 being C, genotype TT is the homozygous type of SNP3 being T, and genotype TC is the heterozygous type of SNP3 being T and C.

[0019] To solve the above technical problems, the present invention also provides a product, which contains the above-mentioned substance for detecting the polymorphism or genotype of the three SNPs SNP1, SNP2 and SNP3 in the dairy cow genome, or contains the above-mentioned substance for detecting haplotypes, or contains the above-mentioned product for detecting the polymorphism or genotype of any SNP site among the SNP1, SNP2 and SNP3 in the dairy cow genome, and can be any one of the following G1)-G3):

[0020] G1) Products for detecting single nucleotide polymorphisms or genotypes related to milk production traits in dairy cows;

[0021] G2) Products that identify or assist in identifying the milk production traits of dairy cows;

[0022] G3) Products for dairy cattle breeding.

[0023] The above-mentioned milk production traits may be milk yield and / or milk fat content and / or milk protein content.

[0024] In order to solve the above technical problems, the present invention also provides a method for identifying or assisting in identifying the milk production traits of dairy cows, which is method A or method B;

[0025] The method A is a method for identifying or assisting in identifying the milk production traits of dairy cows, comprising detecting the genotypes of the three SNPs SNP1, SNP2 and SNP3 in claim 1 in the dairy cow to be tested, and identifying or assisting in identifying the milk production traits of the dairy cow according to the genotypes of the three SNPs in the dairy cow to be tested: the milk production and / or milk fat content and / or milk protein content of the dairy cow whose genotype of the three SNPs is genotype GGAACC is higher than or candidate higher than that of the dairy cow whose genotype of the three SNPs is genotype GAGATC and genotype AAGGTT, and the milk production of the dairy cow whose genotype of the three SNPs is genotype GAGATC and genotype AAGGTT is higher than or candidate higher than that of the dairy cow whose genotype of the three SNPs is genotype GAGATC and genotype AAGGTT. The amount and / or milk fat amount and / or milk protein amount have no difference or the candidate is no difference; the genotype GGAACC is a three-SNP combination genotype of the SNP1 genotype GG, the SNP2 genotype AA and the SNP3 genotype CC, the genotype GAGATC is a three-SNP combination genotype of the SNP1 genotype GA, the SNP2 genotype GA and the SNP3 genotype TC, the genotype AAGGTT is a three-SNP combination genotype of the SNP1 genotype AA, the SNP2 genotype GG and the SNP3 genotype TT;

[0026] The method B is a method for identifying or assisting in identifying the milk production traits of dairy cows, comprising detecting the haplotype described in claim 1 in the dairy cow to be tested, and identifying or assisting in identifying the milk production traits of the dairy cow according to the haplotype of the dairy cow to be tested: the milk production and / or milk fat content and / or milk protein content of the homozygous genotype dairy cow corresponding to haplotype H1 is higher or candidate higher than that of the homozygous genotype dairy cow corresponding to haplotype H2, the haplotype H1 is a haplotype in which the SNP1 is G, the SNP2 is A, and the SNP3 is C, and the haplotype H2 is a haplotype in which the SNP1 is A, the SNP2 is G, and the SNP3 is T.

[0027] In order to solve the above technical problems, the present invention also provides a method for identifying or assisting in identifying the milk production traits of dairy cows, detecting the genotype of any one of the SNP sites of SNP1, SNP2 and SNP3 in claim 1 in the dairy cow to be tested, and identifying or assisting in identifying the milk production traits of the dairy cow based on the genotype of the SNP of the dairy cow to be tested:

[0028] The milk production of cows with the GG genotype of SNP1 is higher or potentially higher than that of cows with the AA or GA genotypes, and there is no significant difference in milk production between cows with the AA genotype and cows with the GA genotype; GG is a homozygous type of SNP1 being G; AA is a homozygous type of SNP1 being A; AG is a heterozygous type of SNP1 being A and G;

[0029] The milk production and / or milk protein content of dairy cows with the genotype of SNP2 being AA is higher or is higher than that of dairy cows with the genotype of GG or GA, and there is no significant difference in milk production between cows with the GG genotype and cows with the GA genotype; GG is a homozygous type of SNP2 being G; AA is a homozygous type of SNP2 being A; AG is a heterozygous type of SNP2 being A and G;

[0030] The milk production and / or milk protein content of cows with the CC genotype of SNP3 is higher or potentially higher than that of cows with the TT or TC genotype, and there is no significant difference in milk production between cows with the TT genotype and cows with the TC genotype; the TT is a homozygous type with SNP3 being T; the CC is a homozygous type with SNP3 being C; and the TC is a heterozygous type with SNP3 being T and C.

[0031] The present invention also provides a method for breeding dairy cows, comprising selecting dairy cows whose SNP1 genotype is GG and / or whose SNP2 genotype is AA and / or whose SNP3 genotype is CC as parents for breeding; the SNP1 is a SNP in the dairy cow genome, which is the 2773rd nucleotide of SEQ ID No.1 in the sequence list, which is G or A, and the GG is a homozygous type of the SNP1 being G; the SNP2 is a SNP in the dairy cow genome, which is the 2071st nucleotide of SEQ ID No.1 in the sequence list, which is G or A, and the AA is a homozygous type of the SNP2 being A; the SNP3 is a SNP in the dairy cow genome, which is the 857th nucleotide of SEQ ID No.1 in the sequence list, which is T or C, and the CC is a homozygous type of the SNP3 being C.

[0032] The dairy cow breeding mentioned above is to cultivate dairy cow breeds with high milk composition.

[0033] The milk components mentioned above may specifically be milk yield, milk fat content, milk fat rate, milk protein content and / or milk protein rate.

[0034] In the above applications and methods, the substance for detecting the polymorphism or genotype of the three SNPs SNP1, SNP2 and SNP3, or the substance for detecting the haplotype, or the substance for detecting the polymorphism or genotype of any SNP site among the SNP1, SNP2 and SNP3, can be used to determine the nucleotide type of the SNP1 and / or SNP2 and / or SNP3 sites in the above dairy cow genome by at least one of the following methods: DNA sequencing, restriction enzyme fragment length polymorphism, single-stranded conformation polymorphism, denaturing high-performance liquid chromatography and SNP chip. Among them, the SNP chip includes a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction endonuclease reaction, a chip based on protein DNA binding reaction, and a chip based on fluorescent molecule DNA binding reaction.

[0035] In the above application or method, the substance for detecting the polymorphism or genotype of the three SNPs SNP1, SNP2 and SNP3, or the substance for detecting the haplotype, or the substance for detecting the polymorphism or genotype of any SNP site among the SNP1, the SNP2 and the SNP3, may be as follows (D1), D2) or D3):

[0036] D1) containing PCR primers for amplifying a dairy cow genomic DNA fragment including the SNP1, SNP2 and / or SNP3 sites;

[0037] D2) a PCR reagent containing the PCR primers described in D1);

[0038] D3) A kit containing the PCR primers described in D1) or the PCR reagents described in D2).

[0039] The above PCR primers are Y1, Y2 and / or Y3:

[0040] Y1, a primer set consisting of the single-stranded DNA shown at positions 2463-2482 of SEQ ID No. 1 in the sequence listing, and the single-stranded DNA reversely complementary to positions 2842-2861 of SEQ ID No. 1;

[0041] Y2, a primer set consisting of the single-stranded DNA represented by positions 1937-1955 of SEQ ID No. 1 in the sequence listing, and the single-stranded DNA reversely complementary to positions 2417-2437 of SEQ ID No. 1;

[0042] Y3. A primer set consisting of the single-stranded DNA represented by positions 729 to 751 of SEQ ID No. 1 in the sequence listing, and the single-stranded DNA reversely complementary to positions 1151 to 1170 of SEQ ID No. 1.

[0043] In the above applications and methods, the PCR primers may or may not be labeled with a label. The label refers to any atom or molecule that can be used to provide a detectable effect and can be attached to a nucleic acid. Labels include but are not limited to dyes; radioactive labels such as 32 P; a binding moiety such as biotin; a hapten such as digoxigenin (DIG); a luminescent, phosphorescent or fluorescent moiety; and a fluorescent dye alone or in combination with a moiety that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The label can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, etc. The label can be a charged moiety (positive or negative) or, alternatively, can be charge neutral. The label can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence comprising the label is detectable. In some embodiments, the nucleic acid is directly detected (e.g., directly reading the sequence) without a label.

[0044] In the above applications and methods, the product may be a reagent, a kit, or a system. The system may include a combination of a reagent or kit, an instrument, and analytical software, such as a product consisting of PCR primers, a PARMS master mix reagent, a microplate reader, and the online software SNP decoder (http: / / www.snpway.com / snpdecoder01 / ), or a combination product consisting of PCR primers, a PARMS master mix reagent, the online software SNP decoder, and a fluorescence quantitative PCR instrument. The product may include the above-mentioned substance for detecting the polymorphism or genotype of the SNP1 and / or SNP2 and / or SNP3 sites in the cow genome.

[0045] In an embodiment of the present invention, genetic variation analysis of the APOA5 gene in a related population of dairy cows revealed three SNPs: SNP1, SNP2, and SNP3, located in the APOA5 gene and its 2000bp upstream flanking sequence, respectively, in the dairy cow genome, which is associated with milk production. These SNPs are located at positions 2773, 2071, and 857 of SEQ ID No. 1. In this embodiment of the present invention, the dominant allele at SNP1 is G, the dominant allele at SNP2 is A, and the dominant allele at SNP3 is C. This indicates that SNP1, SNP2, and / or SNP3 can be used for molecular marker-assisted selection breeding of dairy cows and the selection of high-milk-producing dairy cow breeds. These three SNP combinations exist in three haplotypes: haplotype H1 (GAC) and haplotype H2 (AGT). Experiments have shown that cows homozygous for haplotype H1 (GAC) exhibit significantly higher milk production than cows homozygous for other haplotypes. The haplotype H1 (GAC) molecular marker can be used for early prediction and screening of milk production traits in dairy cows, and can also be used for molecular marker-assisted selection breeding of dairy cows and the selection of high-milk-producing dairy cow breeds. In addition, in the embodiment of the present invention, the milk production traits of dairy cows whose genotypes of the three SNPs SNP1, SNP2 and SNP3 are genotype GGAACC are higher or are candidates for being higher than those of dairy cows whose genotypes of the three SNPs SNP1, SNP2 and SNP3 are genotypes AAGGTT or genotype GAGATC. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Linkage disequilibrium estimates for SNP1 (g.27445825T>C), SNP2 (g.27446527C>T), and SNP3 (g.27447741A>G). DETAILED DESCRIPTION

[0047] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0048] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0049] The Chinese Holstein bulls and cows in the following examples were all from the Beijing Dairy Center.

[0050] The data in the examples are all data of lactation period 1. Lactation period 1 refers to the lactation period after the first parturition.

[0051] The milk yield in the examples refers to the individual 305-day milk yield, which refers to the total milk production from the first day of calving to the 305th day. If the actual number of milking days is less than 305, the actual milk production is used as the 305-day milk yield. If the actual number of milking days exceeds 305, milk production after 306 days is not included. Milk yield is determined by monthly DHI (Dairy Herd Improvement) measurements. The 305-day milk yield for that lactation period can be calculated by plotting a milk yield lactation curve using three or more DHI data from the same lactation period.

[0052] The milk fat content in the examples refers to the 305-day milk fat content, which is calculated as milk fat percentage x 305-day milk production. Milk fat percentage is determined by measuring the monthly Dairy Herd Improvement (DHI). The average milk fat percentage for a lactation period can be calculated by plotting a milk fat percentage lactation curve using three or more DHI data points within the same lactation period.

[0053] The milk protein content in the examples refers to the 305-day milk protein content, which is calculated as milk protein rate x 305-day milk production. The milk protein rate is determined by measuring the monthly Dairy Herd Improvement (DHI). The average milk protein rate for a lactation period can be calculated by plotting a milk protein rate lactation curve using three or more DHI data from the same lactation period.

[0054] Example 1. Discovery of molecular markers

[0055] 1. Related Basic Research

[0056] The inventor's research group used liver tissues from three Chinese Holstein cows at different lactation stages (dry period, early lactation, and peak lactation) as experimental materials and conducted proteomic analysis, finding significant differences in APOA5 expression across different lactation stages (P < 0.05).

[0057] 2. Gene polymorphism detection

[0058] 1. A total of 45 Chinese Holstein bulls in the Beijing area were selected as the test population for genetic polymorphism detection. Genomic DNA was extracted from frozen semen samples of the 45 Chinese Holstein bulls. The DNA concentration was accurately measured using a nucleic acid quality detector. The DNA was diluted to a concentration of 50 ng / μL, and equal amounts of DNA were mixed and used as templates for PCR amplification.

[0059] 2. Based on the bovine APOA5 gene sequence and its upstream and downstream regulatory region sequences (Ensembl ID: ENSBTAG00000019764, as shown in SEQ ID No. 1, wherein the APOA5 gene sequence is positions 2523-4662 of SEQ ID No. 1), 13 pairs of primers as shown in Table 1 were designed.

[0060] Table 1 APOA5 gene PCR amplification primer sequence information

[0061]

[0062]

[0063] 3. Using the pooled DNA obtained in step 1 as a template, perform PCR amplification using each primer pair to obtain a PCR amplification product. The PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.

[0064] Table 2 PCR reaction system

[0065]

[0066] Table 3 PCR reaction conditions

[0067]

[0068] 4. Sequencing of the PCR amplified products revealed that the bull population APOA5 gene contained one SNP marker (designated SNP1) in exon 1 and two SNP markers (designated SNP2 and SNP3) in the upstream 2000 bp flanking sequence. The three SNP markers are listed in Table 4.

[0069] Table 4 Three SNPs found in the APOA5 gene

[0070] Gene location name SNPs physical location Polymorphic Form Exon 1 SNP1 g.27445825T>C Chr15:27445825bp A / G 5' regulatory region SNP2 g.27446527C>T Chr15:27446527bp G / A 5' regulatory region SNP3 g.27447741A>G Chr15:27447741bp T / C

[0071] Among them, SNP1 corresponds to g.27445825T>C, and was obtained by sequencing analysis of the product obtained by PCR amplification using a primer pair consisting of 6F and 6R (the PCR amplification product is shown in positions 2463-2861 of SEQ ID No.1). Its nucleotide is A or G, corresponding to position 2773 of SEQ ID No.1 in the sequence listing, and is represented by R. SNP2 corresponds to g.27446527C>T, and was obtained by sequencing analysis of the product obtained by PCR amplification using a primer pair consisting of 5F and 5R (the PCR amplification product is shown in positions 1937-2437 of SEQ ID No.1). Its nucleotide is G or A, corresponding to position 2071 of SEQ ID No.1 in the sequence listing, and is represented by R. SNP3 corresponding to g.27447741A>G was obtained by sequencing analysis of the product obtained by PCR amplification using a primer pair consisting of 2F and 2R (the PCR amplification product is shown in positions 729-1170 of SEQ ID No.1), and its nucleotide is T or C, corresponding to position 857 of SEQ ID No.1 in the sequence listing, represented by Y.

[0072] g.27445825T>C, g.27446527C>T, and g.27447741A>G are the names of SNP1, SNP2, and SNP3, respectively. SNP naming is generally based on the rules when the SNP was first discovered. DNA has a double-stranded structure and follows the principle of complementary base pairing. SEQ ID No. 1 in the present invention and the sequences when SNP1, SNP2, and SNP3 were discovered are the reverse complementary strands of the same double-stranded DNA. Therefore, in the present invention, the polymorphic form of g.27445825T>C is A or G, the polymorphic form of g.27446527C>T is G or A, and the polymorphic form of g.27447741A>G is T or C.

[0073] 3. Correlation Analysis

[0074] (1) Obtaining the test population

[0075] The experimental group consisted of 944 Chinese Holstein cows.

[0076] (2) Genotyping

[0077] Each individual in the test group was genotyped separately.

[0078] I. Genotyping based on g.27445825T>C.

[0079] 1. Take blood from the test individual and extract genomic DNA.

[0080] 2. Using genomic DNA as a template, PCR amplification was performed using a primer pair consisting of 6F (as shown in positions 2463-2482 of SEQ ID No. 1) and 6R (reverse complementary to positions 2842-2861 of SEQ ID No. 1), and the PCR amplification product was recovered and sequenced.

[0081] The reaction system for PCR amplification is shown in Table 5. The reaction conditions for PCR amplification are shown in Table 6.

[0082] The PCR amplification products of each test individual were all 399 bp, wherein the 311th position was g.27445825T>C, namely SNP1 (corresponding to the 2773rd position from the 5' end of SEQ ID No. 1 in the sequence listing).

[0083] Table 5

[0084]

[0085] Table 6

[0086]

[0087] II. Genotyping based on g.27446527C>T.

[0088] 1. Take blood from the test individual and extract genomic DNA.

[0089] 2. Using genomic DNA as a template, PCR amplification was performed using a primer pair consisting of 5F (as shown in positions 1937-1955 of SEQ ID No. 1) and 5R (reverse complementary to positions 2417-2437 of SEQ ID No. 1), and the PCR amplification product was recovered and sequenced.

[0090] The reaction system for PCR amplification is shown in Table 7. The reaction conditions for PCR amplification are shown in Table 8.

[0091] The PCR amplification products of each test individual were all 501 bp, wherein the 135th position was g.27446527C>T, namely SNP2 (corresponding to the 2071st position from the 5' end of SEQ ID No. 1 in the sequence listing).

[0092] Table 7

[0093]

[0094] Table 8

[0095]

[0096] III Genotyping based on g.27447741A>G.

[0097] 1. Take blood from the test individual and extract genomic DNA.

[0098] 2. Using genomic DNA as a template, PCR amplification was performed using a primer pair consisting of 2F (as shown in positions 729-751 of SEQ ID No. 1) and 2R (reverse complementary to positions 1151-1170 of SEQ ID No. 1), and then the PCR amplification product was recovered and sequenced.

[0099] The reaction system for PCR amplification is shown in Table 9. The reaction conditions for PCR amplification are shown in Table 10.

[0100] The PCR amplification products of each test individual were all 442 bp, wherein the 129th position was g.27447741A>G, namely SNP3 (corresponding to the 857th position from the 5' end of SEQ ID No. 1 in the sequence listing).

[0101] Table 9

[0102]

[0103]

[0104] Table 10

[0105]

[0106] (3) Detection of milk production traits

[0107] Each cow in the test group was tested for milk production traits.

[0108] Milk production traits include the following five indicators: milk yield, milk fat content, milk fat rate, milk protein content and milk protein rate.

[0109] The records of each individual include the cow's individual number, father number, mother number, grandfather number, grandmother number, maternal grandfather number, maternal grandmother number, date of birth, lactation period, calving date, milk production, milk fat content and milk protein content.

[0110] (IV) Association analysis model between single SNP loci and traits

[0111] The genotypes and milk production phenotypes of the SNP1 site (i.e., APOA5 gene g.27445825T>C), SNP2 site (i.e., APOA5 gene g.27446527C>T) and SNP3 site (i.e., APOA5 gene g.27447741A>G) are shown in Tables 11, 12 and 13.

[0112] Table 11 Phenotypes of some milk production traits and genotypes of three SNP loci in 944 Chinese Holstein cows

[0113]

[0114]

[0115]

[0116] Table 12 Descriptive statistics of phenotypic values of five milk production traits in a population of 944 Chinese Holstein cows

[0117] Traits average value Standard deviation Minimum Maximum Coefficient of variation Milk production (kg) 10421.30 1483.48 6057.96 14505.68 0.14 Milk fat content (kg) 352.57 61.68 184.80 537.97 0.17 Milk protein amount (kg) 315.40 48.55 157.73 457.53 0.15 Milk fat content (%) 3.39 0.43 1.95 4.74 0.13 Milk protein rate (%) 3.03 0.20 2.24 3.51 0.07

[0118] Table 13 Allele frequencies and genotype frequencies of the three SNP sites in the APOA5 gene

[0119]

[0120] The results showed that there were three genotypes at the SNP1 site (referred to as SNP1 genotypes), namely AA, GG or AG. Genotype AA is the homozygous type of SNP1 with A, genotype GG is the homozygous type of SNP1 with G, and genotype AG is the heterozygous type of SNP1 with A and G; there were three genotypes at the SNP2 site (referred to as SNP2 genotypes), namely GG, AA or GA. Genotype GG is the homozygous type of SNP2 with G, genotype AA is the homozygous type of SNP2 with A, and genotype GA is the heterozygous type of SNP2 with G and A; there were three genotypes at the SNP3 site (referred to as SNP3 genotypes), namely CC, TT or CT. Genotype CC is the homozygous type of SNP3 with C, genotype TT is the homozygous type of SNP3 with T, and genotype CT is the heterozygous type of SNP3 with C and T.

[0121] The MIXED procedure in SAS 9.2 software was used to conduct association analysis between the five indicators of milk production and genotypes. The association analysis used an animal model, and the specific model is as follows:

[0122] Y=μ+hys+b×M+G+a+e

[0123] Where Y is the observed value of milk production traits (milk yield, milk fat content, milk fat percentage, milk protein content or milk protein percentage); μ is the overall mean; hys is the field year and season effect; b is the regression coefficient of the covariate M; M is the effect of calving age; G is the genotype effect; a is the individual random additive genetic effect; and e is the random residual effect.

[0124] The results of the association analysis between SNP1 site (i.e., APOA5 gene g.27445825T>C) and milk production traits are shown in Table 14.

[0125] Table 14 Association analysis between APOA5 gene g.27445825T>C and milk production traits (least squares mean ± standard error)

[0126] genotype Milk production (kg) Milk fat content (kg) Milk fat content (%) Milk protein amount (kg) Milk protein rate (%) GG(427) <![CDATA[10341±64.1416 a ]]> 341.09±2.8302 3.3297±0.02638 <![CDATA[304.45±2.0605 a ]]> 2.9628±0.01961 GA(420) <![CDATA[10212±62.4416 b ]]> 338.42±2.7621 3.3325±0.02571 <![CDATA[301.32±2.0108 b ]]> 2.9631±0.01918 AA(97) <![CDATA[10134±93.1163 b ]]> 337.23±3.9251 3.3429±0.03772 <![CDATA[300.3±2.8605 ab ]]> 2.9798±0.02591 P-value 0.0116* 0.342 0.9297 0.075 0.7157

[0127] Note: * P<0.05 indicates a significant difference. a,b Data in the same column with different superscripts indicate significant differences.

[0128] As shown in Table 14, SNP1 (g.27445825T>C) is significantly associated with milk yield (P=0.0116). For the milk yield trait, the dominant allele is G:

[0129] The milk production of GG genotype cows was higher than that of AA or GA genotype cows, and there was no significant difference in milk production between AA genotype cows and GA genotype cows.

[0130] The results of the association analysis between SNP2 site (i.e., APOA5 gene g.27446527C>T) and milk production traits are shown in Table 15.

[0131] Table 15 Association analysis between APOA5 gene g.27446527C>T and milk production traits (least squares mean ± standard error)

[0132] genotype Milk production (kg) Milk fat content (kg) Milk fat content (%) Milk protein amount (kg) Milk protein rate (%) GG(101) <![CDATA[10094±91.6949 B ]]> 335.92±3.8691 3.3415±0.03716 <![CDATA[299.25±2.8196 b ]]> 2.9798±0.02558 GA(427) <![CDATA[10212±62.1995 b ]]> 338.1±2.7544 3.331±0.02562 <![CDATA[301.17±2.0052 b ]]> 2.9626±0.01915 AA(416) <![CDATA[10362±64.6715 Aa ]]> 342.04±2.8491 3.3316±0.02659 <![CDATA[305.14±2.0744 a ]]> 2.963±0.01972 P-value 0.0012** 0.0841 0.9506 0.0108* 0.705

[0133] Note: * P<0.05 indicates significant difference; ** P<0.01 indicates that the difference is extremely significant. a,b Data in the same column with different superscripts indicate significant differences; A,B Data in the same column with different superscripts indicate extremely significant differences.

[0134] As shown in Table 15, SNP2 (g.27446527C>T) is extremely significantly associated with milk yield (P=0.0012) and significantly associated with milk protein content (P=0.0108). For milk yield and milk protein content, the dominant allele is A:

[0135] The milk yield of cows with AA genotype was higher than that of cows with GG and GA genotypes. There was no significant difference in milk yield between cows with GG genotype and cows with GA genotype.

[0136] The milk protein content of cows with AA genotype was higher than that of cows with GG and GA genotypes, and there was no significant difference in milk protein content between cows with GG genotype and cows with GA genotype.

[0137] The results of the association analysis between SNP3 site (i.e., APOA5 gene g.27447741A>G) and milk production traits are shown in Table 16.

[0138] Table 16 Association analysis between APOA5 gene g.27447741A>G and milk production traits (least squares mean ± standard error)

[0139] genotype Milk production (kg) Milk fat content (kg) Milk fat content (%) Milk protein amount (kg) Milk protein rate (%) TT(92) <![CDATA[10103±94.7461 B ]]> 334.86±3.9891 3.3316±0.03837 <![CDATA[299.36±2.9072 b ]]> 2.98±0.02629 TC(415) <![CDATA[10206±62.6452 B ]]> 338.49±2.77 3.3348±0.02579 <![CDATA[301.02±2.0167 b ]]> 2.962±0.01922 CC(437) <![CDATA[10352±63.9228 A ]]> 341.48±2.8212 3.3299±0.02629 <![CDATA[304.93±2.054 a ]]> 2.9642±0.01954 P-value 0.0025** 0.1214 0.9732 0.0146* 0.7157

[0140] Note: * P<0.05 indicates significant difference; ** P<0.01 indicates that the difference is extremely significant. a,b Data in the same column with different superscripts indicate significant differences; A,B Data in the same column with different superscripts indicate extremely significant differences.

[0141] As shown in Table 16, SNP3 (g.27447741A>G) was extremely significantly associated with milk yield (P=0.0025) and significantly associated with milk protein content (P=0.0146). For the milk yield and milk protein content traits, the dominant allele was C.

[0142] The milk yield of CC genotype cows was higher than that of TT and TC genotype cows, and there was no significant difference in milk yield between TT genotype cows and TC genotype cows.

[0143] The milk protein content of CC genotype cows was higher than that of TT and TC genotype cows, and there was no significant difference in milk protein content between TT genotype cows and TC genotype cows.

[0144] (V) Analysis of genetic effects

[0145] SAS 9.2 software was used to perform significance tests of SNP additive effect, dominant effect and substitution effect.

[0146] The basic calculation formula is as follows:

[0147] a=(AA-BB) / 2, d=AB-(AA+BB) / 2, α=a+d(qp); a is the additive effect, d is the dominant effect, α is the allele substitution effect; AA, AB, BB are the least square means of the milk production traits of the corresponding genotypes; p is the frequency of allele A, and q is the frequency of allele B.

[0148] The results of the tests for additive effect, dominance effect and allele substitution effect are shown in Table 17.

[0149] Table 17 Test results of additive effect, dominant effect and substitution effect of APOA5 gene alleles

[0150]

[0151] Note: * P<0.05 indicates significant difference; ** P<0.01 indicates that the difference is extremely significant.

[0152] The results show that SNP1 (g.27445825T>C) has a significant additive effect on milk yield; SNP2 (g.27446527C>T) has extremely significant and significant additive effects on milk yield and milk fat percentage, respectively, and the allele substitution effect on milk yield is significant; SNP3 (g.27447741A>G) has extremely significant and significant additive effects on milk yield and milk fat percentage, respectively, and the allele substitution effect on milk yield is significant.

[0153] (6) Haplotype analysis

[0154] Haploview 4.2 software was used to construct haplotypes, estimate haplotype frequencies and linkage analysis, and conduct association analysis with traits. Using the MIXED procedure in SAS 9.2 software, the model was as follows:

[0155] Y=μ+hys+b×M+G+a+e

[0156] Y: observed values of milk production traits (milk yield, milk fat content, milk fat percentage, milk protein content and milk protein percentage); μ: overall mean; hys: field year and 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.

[0157] The results of haplotype analysis are shown in Table 18.

[0158] Table 18 Haplotypes composed of three SNPs in the APOA5 gene

[0159] Haplotype type Haplotype combination frequency H1 GAC 0.667 H2 AGT 0.317

[0160] In the above-mentioned study population of 944 individuals, the three SNPs (SNP1, SNP2, and SNP3) of the APOA5 gene together constitute a haplotype block and form two haplotype combinations, namely haplotype H1 and haplotype H2. Haplotype H1 (GAC) is a combination of SNP1 being G, SNP2 being A, and SNP3 being C on a chromosome. Haplotype H2 (AGT) is a combination of SNP1 being A, SNP2 being G, and SNP3 being T on a chromosome. The frequency of haplotype H1 is 66.7%, and the frequency of haplotype H2 is 31.7%. Linkage disequilibrium estimation was performed on these three SNPs, and the results showed that these three SNPs are in a completely linked state (r 2 =1), such as Figure 1 shown.

[0161] The haplotype represents the linkage relationship between the two SNPs on the same chromosome. Cattle are diploid, and the haplotype combinations of the two chromosomes of individuals in the population of 944 tested were H1H1, H1H2, and H2H2, respectively.

[0162] The genotype of the cow corresponding to H1H1 is GGAACC, which is a combination of three SNPs: SNP1 genotype GG, SNP2 genotype AA, and SNP3 genotype CC. The genotype of the cow corresponding to H1H2 is GAGATC, which is a combination of three SNPs: SNP1 genotype GA, SNP2 genotype GA, and SNP3 genotype TC. The genotype of the cow corresponding to H2H2 is AAGGTT, which is a combination of three SNPs: SNP1 genotype AA, SNP2 genotype GG, and SNP3 genotype TT.

[0163] The results of association analysis between APOA5 gene haplotype combinations and milk production traits are shown in Table 19. The haplotype combinations of the three SNPs in the APOA5 gene were significantly associated with milk yield, milk fat content, and milk protein content (P = 0.0259-0.0011), among which haplotype H1 was the dominant haplotype for all five milk production traits.

[0164] Table 19 Association analysis results between APOA5 gene haplotype combinations and milk production traits (least squares mean ± standard error)

[0165]

[0166]

[0167] Note: * P<0.05 indicates significant difference; ** P<0.01 indicates that the difference is extremely significant. a,b Data in the same column with different superscripts indicate significant differences; A,B Data in the same column with different superscripts indicate extremely significant differences.

[0168] The milk yield of cows with the haplotype combination H1H1 (ie, genotype GGAACC) is higher than that of cows with the haplotype combination H1H2 (ie, genotype GAGATC) and cows with the haplotype combination H2H2 (ie, genotype AAGGTT).

[0169] The milk fat content of cows with the haplotype combination H1H1 (ie, genotype GGAACC) is higher than that of cows with the haplotype combination H2H2 (ie, genotype AAGGTT).

[0170] The milk protein content of cows with the haplotype combination H1H1 (i.e., genotype GGAACC) is higher than that of cows with the haplotype combination H2H2 (i.e., genotype AAGGTT) and cows with the haplotype combination H2H2 (i.e., genotype AAGGTT).

[0171] Compared with single marker analysis, haplotype analysis often expresses more information, and changes in the interaction of some sites within the gene will have a great impact on phenotypic traits. Therefore, haplotype analysis is significantly superior to conventional single marker analysis in complex trait association analysis.

[0172] The molecular markers disclosed in the present invention can be used to assist in identifying dairy cow groups with excellent milk production traits (305-day milk production, milk fat content, milk fat percentage, milk protein content and milk protein percentage). They have the following advantages: simplicity, speed, sensitivity, reliable, stable and accurate results, and are suitable for the needs of large-scale laboratory group testing.

[0173] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

[0174] Sequence Listing

[0175] SEQ ID No.1

[0176]

Claims

1. Application, characterized by: The application is P1, P2, P3, P4 or P5; The P1 is an application of a substance for detecting the polymorphism or genotype of three SNPs, SNP1, SNP2, and SNP3, in identifying or assisting in identifying the milk production trait of a dairy cow, wherein SNP1 is a SNP in the dairy cow genome, which is the 2773rd nucleotide of SEQ ID No.1 in the sequence listing, and is A or G; SNP2 is a SNP in the dairy cow genome, which is the 2071st nucleotide of SEQ ID No.1 in the sequence listing, and is G or A; SNP3 is a SNP in the dairy cow genome, which is the 857th nucleotide of SEQ ID No.1 in the sequence listing, and is T or C; and the milk production trait is any one and / or a combination of multiple of milk yield, milk fat content, and milk protein content; The P2 is an application of a substance for detecting haplotypes in identifying or assisting in identifying the milk production traits of dairy cows, wherein the haplotype is a polymorphic combination of the three SNPs SNP1, SNP2, and SNP3 on a chromosome of the dairy cow; the milk production trait is any one and / or a combination of multiple of milk yield, milk fat content, and milk protein content; The P3 is a use of a substance for detecting the polymorphism or genotype of the SNP1 in identifying or assisting in identifying the milk production trait of dairy cows; the milk production trait is milk yield; The P4 is an application of a substance for detecting the polymorphism or genotype of the SNP2 site in identifying or assisting in identifying the milk production traits of dairy cows; The milk production trait is milk yield and / or milk protein content; The P5 is an application of a substance for detecting the polymorphism or genotype of the SNP3 site in identifying or assisting in identifying the milk production traits of dairy cows; The milk production trait is milk yield and / or milk protein content; The dairy cows are Holstein cows.

2. Application, characterized by: The application is Q1, Q2, Q3, Q4 or Q5; Said Q1 is the use of a substance for detecting the polymorphism or genotype of three SNPs, SNP1, SNP2 and SNP3, in preparing a product for identifying or assisting in identifying the milk production trait of a dairy cow; said SNP1 is a SNP in the dairy cow genome, which is the 2773rd nucleotide of SEQ ID No.1 in the sequence listing, which is A or G; said SNP2 is a SNP in the dairy cow genome, which is the 2071st nucleotide of SEQ ID No.1 in the sequence listing, which is G or A; said SNP3 is a SNP in the dairy cow genome, which is the 857th nucleotide of SEQ ID No.1 in the sequence listing, which is T or C; said milk production trait is any one and / or a combination of multiple of milk yield, milk fat content and milk protein content; The Q2 is an application of a substance for detecting haplotypes in the preparation of a product for identifying or assisting in identifying the milk production traits of dairy cows; the haplotype is a polymorphic combination of three SNPs, namely, SNP1, SNP2, and SNP3, on a chromosome of a dairy cow; and the milk production trait is any one and / or a combination of multiple of milk yield, milk fat content, and milk protein content; Q3 is the use of a substance for detecting the polymorphism or genotype of the SNP1 site in the preparation of a product for identifying or assisting in identifying the milk production trait of a dairy cow; the milk production trait is milk yield; Said Q4 is the use of a substance for detecting the polymorphism or genotype of said SNP2 site in the preparation and identification or auxiliary identification of the milk production trait of dairy cows; The milk production trait is milk yield and / or milk protein content; Said Q5 is the use of a substance for detecting the polymorphism or genotype of said SNP3 site in the preparation and identification or auxiliary identification of the milk production trait of dairy cows; The milk production trait is milk yield and / or milk protein content; The dairy cows are Holstein cows.

3. Application, characterized by: The application is E1, E2, E3, E4 or E5; The E1 is an application of a substance for detecting the polymorphism or genotype of three SNPs, SNP1, SNP2, and SNP3, in dairy cow breeding or the preparation of dairy cow breeding products; the SNP1 is a SNP in the dairy cow genome, which is the 2773rd nucleotide of SEQ ID No.1 in the sequence listing, and is A or G; the SNP2 is a SNP in the dairy cow genome, which is the 2071st nucleotide of SEQ ID No.1 in the sequence listing, and is G or A; the SNP3 is a SNP in the dairy cow genome, which is the 857th nucleotide of SEQ ID No.1 in the sequence listing, and is T or C; the breeding is the selection of dairy cow breeds with high milk production traits; the milk production traits are any one and / or a combination of multiple of milk yield, milk fat content, and milk protein content; The E2 is the use of a substance 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 chromosome of a dairy cow; the breeding is the selection of dairy cow breeds with high milk production traits; the milk production traits are any one and / or a combination of multiple of milk production, milk fat content and milk protein content; the E3 is the use of a substance for detecting the polymorphism or genotype of the SNP1 in dairy cow breeding or the preparation of dairy cow breeding products; the breeding is the selection of dairy cow breeds with high milk production traits; the milk production trait is milk production; The E4 is the use of a substance for detecting the polymorphism or genotype of the SNP2 in dairy cow breeding or the preparation of dairy cow breeding products; the breeding is the selection of dairy cow breeds with high milk production traits; the milk production traits are milk yield and / or milk protein content; The E5 is a use of a substance for detecting the polymorphism or genotype of the SNP3 in dairy cow breeding or the preparation of dairy cow breeding products; the breeding is the selection of dairy cow breeds with high milk production traits; the milk production traits are milk yield and / or milk protein content; The dairy cows are Holstein cows.

4. A method for identifying or assisting in identifying the milk production traits of dairy cows, the method being method A or method B; The method A is a method for identifying or assisting in identifying the milk production traits of dairy cows, comprising detecting the genotypes of the three SNPs SNP1, SNP2 and SNP3 in claim 1 in the dairy cow to be tested, and identifying or assisting in identifying the milk production traits of the dairy cow according to the genotypes of the three SNPs in the dairy cow to be tested: the milk production and / or milk fat content and / or milk protein content of the dairy cow whose genotype of the three SNPs is genotype GGAACC is higher or candidate is higher than that of the dairy cow whose genotype of the three SNPs is genotype GAGATC or genotype AAGGTT, and the milk production and / or milk fat content and / or milk protein content of the dairy cow whose genotype of the three SNPs is genotype GAGATC and genotype AAGGTT is not different or candidate is not different; the genotype GGAACC is the three SNP combination genotype of the genotype of SNP1 is GG, the genotype of SNP2 is AA and the genotype of SNP3 is CC, the genotype GAGATC is the genotype of SNP1 is GA and the genotype of SNP2 is GA. The genotype of the three SNPs is GA and the genotype of the SNP3 is TC, the genotype AAGGTT is the genotype of the three SNPs in which the genotype of the SNP1 is AA, the genotype of the SNP2 is GG and the genotype of the SNP3 is TT; the genotype of the SNP1 is GG and the homozygous type of the SNP1 is G, the genotype of the SNP1 is AA and the homozygous type of the SNP1 is A, the genotype of the SNP1 is GA and the genotype of the SNP1 is A and The genotype of the SNP2 is AA, which is the homozygous type of the SNP2 A; the genotype of the SNP2 is GG, which is the homozygous type of the SNP2 G; the genotype of the SNP2 is GA, which is the heterozygous type of the SNP2 A and G; the genotype of the SNP3 is CC, which is the homozygous type of the SNP3 C; the genotype of the SNP3 is TT, which is the homozygous type of the SNP3 T; the genotype of the SNP3 is TC, which is the heterozygous type of the SNP3 T and C; The method B is a method for identifying or assisting in identifying the milk production traits of dairy cows, comprising detecting the haplotype described in claim 1 in a dairy cow to be tested, and identifying or assisting in identifying the milk production traits of the dairy cow according to the haplotype of the dairy cow to be tested: the milk production and / or milk fat content and / or milk protein content of the homozygous genotype dairy cow corresponding to haplotype H1 is higher or candidate higher than that of the homozygous genotype dairy cow corresponding to haplotype H2, the haplotype H1 is a haplotype in which the SNP1 is G, the SNP2 is A, and the SNP3 is C, and the haplotype H2 is a haplotype in which the SNP1 is A, the SNP2 is G, and the SNP3 is T; The dairy cows are Holstein cows.

5. A method for identifying or assisting in identifying the milk production traits of dairy cows, characterized in that: The method comprises detecting the genotype of the SNP1 of claim 1 in the tested dairy cow, and identifying or assisting in identifying the milk production trait of the dairy cow according to the genotype of the SNP in the tested dairy cow: The milk production of cows with the GG genotype of SNP1 is higher or potentially higher than that of cows with the AA or GA genotypes, and there is no significant difference in milk production between cows with the AA genotype and cows with the GA genotype; GG is a homozygous type of SNP1 being G; AA is a homozygous type of SNP1 being A; GA is a heterozygous type of SNP1 being A and G; The dairy cows are Holstein cows.

6. A method for identifying or assisting in identifying the milk production traits of dairy cows, characterized in that: The method comprises detecting the genotype of the SNP2 of claim 1 in the tested dairy cow, and identifying or assisting in identifying the milk production trait of the dairy cow based on the genotype of the SNP in the tested dairy cow: The milk production and / or milk protein content of cows with the genotype of SNP2 being AA is higher or is higher than that of cows with the genotype of GG or GA, and there is no significant difference in milk production between cows with the GG genotype and cows with the GA genotype; GG is a homozygous type of SNP2 being G; AA is a homozygous type of SNP2 being A; GA is a heterozygous type of SNP2 being A and G; The dairy cows are Holstein cows.

7. A method for identifying or assisting in identifying the milk production traits of dairy cows, characterized in that: The method comprises detecting the genotype of the SNP3 of claim 1 in the tested dairy cow, and identifying or assisting in identifying the milk production trait of the dairy cow based on the genotype of the SNP in the tested dairy cow: The milk production and / or milk protein content of dairy cows with the CC genotype of the SNP3 is higher or potentially higher than that of dairy cows with the TT or TC genotypes, and there is no significant difference in milk production between cows with the TT genotype and cows with the TC genotype; the TT is a homozygous type with SNP3 being T; the CC is a homozygous type with SNP3 being C; and the TC is a heterozygous type with SNP3 being T and C. The dairy cows are Holstein cows.

8. Application of the method of claim 4 in dairy cow breeding; the dairy cows are Holstein cows; the breeding is the selection of dairy cow breeds with high milk production traits; the milk production traits are any one and / or a combination of multiple of milk yield, milk fat content and milk protein content.

9. Application of the method of claim 5 in dairy cow breeding; the dairy cows are Holstein cows; the breeding is the selection of dairy cow breeds with high milk production traits; the milk production trait is milk yield.

10. Application of the method according to claim 6 or 7 in dairy cow breeding; the dairy cow is Holstein cattle; the breeding is the selection of dairy cow breeds with high milk production traits; the milk production traits are milk yield and / or milk protein content.

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