CN116377087A - SNP molecular marker of gene CYP7A1 related to dairy cow milk production traits and application thereof - Google Patents

Abstract

The invention discloses an application of SNP molecular markers of a gene CYP7A1 related to dairy cow milk production traits. The technical scheme to be protected by the invention is the application of substances for detecting the polymorphism or genotype of three SNP (single nucleotide polymorphism) 1, SNP2 and SNP3 in the identification or auxiliary identification of milk production traits of the dairy cows. Experiments prove that the related molecular markers of SNP1, SNP2 and/or SNP3, and the corresponding haplotypes and genotypes can be used for early prediction and screening of milk production traits of cows, and are beneficial to breeding of cow varieties with high milk production traits.

Description

SNP molecular marker of gene CYP7A1 related to dairy cow milk production traits and application thereof

Technical Field

The invention relates to a SNP molecular marker of a gene CYP7A1 related to milk production traits of dairy cows and application thereof, belonging to the technical field of molecular biology.

Background

Cholesterol 7 alpha-hydroxylase (CYP 7A 1), named cholesterol 7 alpha-monooxygenase or cytochrome P4507A1, is a member of the cytochrome P450 enzyme system belonging to liver-specific microsomes, is located on the 14 th chromosome of cattle, is composed of 6 exons, has the total length of 10337bp, and the protein coded by the gene belongs to the cytochrome P450 (CYP) family, and participates in synthesis of bile acid and bile salt, oxidation of cytochrome P450, statin drug pathway, and the receptor alpha (peroxisome proliferators-activated receptors alpha, PPARalpha) signal pathway is activated through peroxisome proliferator-activated receptor alpha, and the lipid metabolism is involved. CYP7A1 is a rate-limiting enzyme for cholesterol catabolism and bile acid biosynthesis, and serves to convert cholesterol into 7α -hydroxylated cholesterol, which is transferred into high density lipoprotein particles and is returned to the liver for conversion to bile acids primarily by the rate-limiting enzyme CYP7A 1. In mammals, CYP7A1 is expressed only in hepatocytes, and synthesis of cholesterol is up-regulated in the early stages of lactation compared to the late stages of gestation. As a substrate for CYP7A1, cholesterol can induce it. The X receptor (liver X receptors, lxrα) is the major substance of cholesterol in the regulation of CYP7 A1. Lxrα is a cholesterol-activated nuclear receptor that can be activated by specific cholesterol-oxidizing derivatives to act in combination with retinol X receptors (retinoid X receptors, rxrα) as heterodimers rxrα/lxrα. CYP7A1 is a target gene critical to LXRalpha.

Fatty acids have been found to have an effect on cholesterol homeostasis in the body, where polyunsaturated fatty acids (PUFA (polyunsaturated fatty acid, PUFA) cholesterol addition results in increased CYP7Al expression, while monounsaturated fatty acids (monounsaturated fatty acid, MUFA) and saturated fatty acids cholesterol addition results in decreased CYP7Al expression the intermediate bridge of fatty acid and cholesterol metabolism may be peroxisome proliferator-activated receptors (PPAR) with PPAR mediation, which can modulate CYP7a1.PPAR is a member of the steroid hormone receptor superfamily, which can be activated by fatty acids and their metabolites as well as exogenous peroxisome proliferators (peroxisom proliferators, pp), and the gene expression of certain enzymes of lipid metabolism is regulated by them.

The polymerase chain reaction (Polymerase Chain Reaction, PCR) is a nucleic acid rapid amplification technique which simulates the natural DNA replication process in vitro, and is characterized by greatly increasing the trace amount of DNA, and has been invented by Mullis et al in the United states in 1983. PCR is to use DNA to become single chain at high temperature and primer and single chain at low temperature to combine based on the complementary pairing principle, and then regulate the temperature to the optimal reaction temperature of DNA polymerase to synthesize complementary chain along the direction from phosphate to five-carbon sugar (5 '-3'). Currently, this technology has become one of the most common and also important molecular biology techniques. The PCR product can be sequenced after agarose gel electrophoresis to identify the gene polymorphism, and the detection method is simple and easy to implement.

Disclosure of Invention

The invention aims to solve the technical problems of identifying or assisting in identifying milk production traits of cows or breeding cows.

In order to solve the technical problems, the invention firstly provides an application, wherein the application is any one of the following P1-P5:

the P1 is the application of a substance for detecting the polymorphism or genotype of three SNP such as SNP1, SNP2 and SNP3 in the identification or auxiliary identification of the milk production character of the dairy cow;

the P2 is the application of a substance for detecting haplotypes in identification or auxiliary identification of milk production traits of cows, wherein the haplotypes are polymorphism combinations of the SNP1, the SNP2 and the SNP3 on one chromosome of the cows;

the P3 is the application of a substance for detecting the polymorphism or genotype of the SNP1 in the identification or auxiliary identification of the milk production traits of the dairy cows;

the P4 is the application of a substance for detecting the polymorphism or genotype of the SNP2 in the identification or auxiliary identification of the milk production traits of the dairy cows;

the P5 is the application of a substance for detecting the polymorphism or genotype of the SNP3 in the identification or auxiliary identification of the milk production traits of the dairy cows;

SNP1 is one SNP of a dairy cow genome, is 249 th nucleotide of SEQ ID No.1 in a sequence table, and is C or T; SNP2 is one SNP of a dairy cow genome, is 1462 nucleotide of SEQ ID No.1 in a sequence table, and is C or A; SNP3 is one SNP of the genome of the dairy cow, is 11400 nucleotide of SEQ ID No.1 in the sequence table, and is C or T.

The three single nucleotide polymorphism sites of the SNP1, the SNP2 and the SNP3 are positioned in CYP7A1 genes on the 14 th chromosome of the dairy cow genome, the CYP7A1 genes are positioned at the 24664833-24675169 th chromosome of the dairy cow, and the nucleotide sequence of the CYP7A1 genes is a DNA molecule shown by SEQ ID No.1 in a sequence table and is related to the dairy cow milk production property.

In order to solve the technical problems, the invention also provides an application, wherein the application is any one of Q1-Q5:

the Q1 is the application of a substance for detecting the polymorphism or genotype of three SNP such as SNP1, SNP2 and SNP3 in preparing and identifying or assisting in identifying dairy cow milk production character products;

the Q2 is the application of a substance for detecting haplotype in preparing and identifying or assisting in identifying dairy cow milk production character products; the haplotype is a polymorphism combination of three SNP1, SNP2 and SNP3 on one chromosome of the dairy cow;

the Q3 is the application of a substance for detecting the polymorphism or genotype of the SNP1 in preparing and identifying or assisting in identifying dairy cow milk production character products;

the Q4 is the application of a substance for detecting the polymorphism or genotype of the SNP2 in preparing and identifying or assisting in identifying dairy cow milk production character products;

the Q5 is the application of a substance for detecting the polymorphism or genotype of the SNP3 in preparing and identifying or assisting in identifying dairy cow milk production character products;

SNP1 is one SNP of a dairy cow genome, is 249 th nucleotide of SEQ ID No.1 in a sequence table, and is C or T; SNP2 is one SNP of a dairy cow genome, is 1462 nucleotide of SEQ ID No.1 in a sequence table, and is C or A; SNP3 is one SNP of the genome of the dairy cow, is 11400 nucleotide of SEQ ID No.1 in the sequence table, and is C or T.

In order to solve the technical problems, the invention also provides an application, wherein the application is any one of E1-E5:

the E1 is the application of a substance for detecting polymorphism or genotype of three SNP (single nucleotide polymorphism) 1, SNP2 and SNP3 in cow breeding or preparing cow breeding products;

the E2 is the application of a substance for detecting haplotype in cow breeding or preparing cow breeding products; the haplotype is a polymorphism combination of three SNP1, SNP2 and SNP3 on one chromosome of the dairy cow;

the E3 is the application of a substance for detecting the polymorphism or genotype of the SNP1 in cow breeding or preparing cow breeding products;

the E4 is the application of a substance for detecting the polymorphism or genotype of the SNP2 in cow breeding or preparing cow breeding products;

the E5 is the application of a substance for detecting the polymorphism or genotype of the SNP3 in cow breeding or preparing cow breeding products;

SNP1 is one SNP of a dairy cow genome, is 249 th nucleotide of SEQ ID No.1 in a sequence table, and is C or T; SNP2 is one SNP of a dairy cow genome, is 1462 nucleotide of SEQ ID No.1 in a sequence table, and is C or A; SNP3 is one SNP of the genome of the dairy cow, is 11400 nucleotide of SEQ ID No.1 in the sequence table, and is C or T.

The dairy cow milk production trait may be any one or a combination of milk yield, milk fat amount, milk fat rate, milk protein amount, and milk protein rate.

The genotype (i.e., allele) of the SNP1 can be genotype CC, genotype TT or genotype CT, wherein the genotype CC is homozygous for the SNP1 with C, the genotype TT is homozygous for the SNP1 with T, and the genotype CT is heterozygous for the SNP1 with C and T; the genotype (i.e., allele) of the SNP2 can be genotype CC, genotype AA or genotype CA, wherein the genotype CC is homozygous for the SNP2 with C, the genotype AA is homozygous for the SNP2 with A, and the genotype CA is heterozygous for the SNP2 with 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 as C, genotype TT is homozygous for SNP1 as T, genotype CT is heterozygous for SNP1 as C and T.

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

The genotypes of the three SNPs SNP1, SNP2 and SNP3 may be genotype ttaac c, genotype ttact and/or genotype ttact. Genotype ttaac is three SNP combined genotypes with SNP1 genotype TT, SNP2 genotype AA and SNP3 genotype CC; genotype TTCACT is three SNP combined genotypes with SNP1 genotype TT, SNP2 genotype CA and SNP3 genotype CT; genotype TTCCTT is three SNP combined genotypes with SNP1 genotype TT, SNP2 genotype CC and SNP3 genotype TT. Cows with genotype of genotype TTAACC of three SNP1, SNP2 and SNP3 have milk production traits higher or candidates higher than those of genotype TTCACT or genotype TTCCCT of three SNP of SNP1, SNP2 and SNP 3. Milk yield and/or milk fat level of cows with genotype of three SNPs SNP1, SNP2 and SNP3 being genotype ttaac are higher or candidate are higher than those of cows with genotype of three SNPs SNP1, SNP2 and SNP3 being genotype ttact and those of three SNPs SNP1, SNP2 and SNP3 being genotype ttact. Cows with genotypes of three SNPs, SNP1, SNP2 and SNP3, being genotype ttaac have higher milk protein levels or candidates than cows with genotypes of three SNPs, SNP1, SNP2 and SNP3, being genotype ttact.

In order to solve the above technical problems, the present invention also provides a product comprising the above-mentioned substance for detecting polymorphisms or genotypes of three SNPs, i.e., SNP1, SNP2 and SNP3, or comprising the above-mentioned substance for detecting haplotype, or comprising the above-mentioned substance for detecting SNP1 polymorphism or genotype of cow genome, or comprising the above-mentioned substance for detecting SNP2 polymorphism or genotype of cow genome, or comprising the above-mentioned substance for detecting SNP3 polymorphism or genotype of cow genome, and may be any one of the following G1) to G3):

g1 A product for detecting single nucleotide polymorphism or genotype related to milk production traits of the dairy cows;

g2 Identification or auxiliary identification of products of milk production traits of the dairy cows;

g3 A product for breeding dairy cows.

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

The method A is a method for identifying or assisting in identifying milk production traits of cows, and comprises the steps of detecting genotypes of three SNP1, SNP2 and SNP3 in cows to be tested, and identifying or assisting in identifying the milk production traits of the cows according to the genotypes of the three SNP of the cows to be tested: the dairy cows with genotype of the three SNP are the dairy cows with genotype of TTAACC, and the dairy cows with milk production characters higher than or candidates higher than the genotype of the three SNP are the dairy cows with genotype of TTCACT or genotype of TTCCTT; genotype ttaac is three SNP combined genotypes with SNP1 genotype TT, SNP2 genotype AA and SNP3 genotype CC; genotype TTCACT is three SNP combined genotypes with SNP1 genotype TT, SNP2 genotype CA and SNP3 genotype CT; genotype TTCCTT is three SNP combined genotypes with SNP1 genotype TT, SNP2 genotype CC and SNP3 genotype TT.

The method B is a method for identifying or assisting in identifying milk production traits of cows, and comprises the steps of detecting haplotypes in cows to be tested, and identifying or assisting in identifying milk production traits of the cows to be tested according to the haplotypes of the cows to be tested: the milk production character of the homozygous genotype dairy cows corresponding to the haplotype H1 is higher than or the candidate is higher than that of the homozygous genotype dairy cows corresponding to the haplotype H2; the haplotype H1 (TAC) is that the SNP1 is T, the SNP2 is A and the SNP3 is C; the haplotype H2 (TCT) is T for the SNP1, C for the SNP2, and T for the SNP 3.

In order to solve the technical problems, the invention also provides a method for identifying or assisting in identifying milk production traits of cows, which comprises detecting genotypes of the SNP1 of cows to be tested, and identifying or assisting in identifying the milk production traits of the cows according to the genotypes of the SNP1 of the cows to be tested: the milk production character of the dairy cows with the genotype of CC of SNP1 is higher than or the candidate is higher than that of the dairy cows with the genotype of TT or CT; the CC is homozygous with SNP1 as C; the TT is homozygous with SNP1 as T; the CT is heterozygous in which SNP1 is C and T.

In order to solve the technical problems, the invention also provides a method for identifying or assisting in identifying milk production traits of cows, which comprises detecting genotypes of the SNP2 of cows to be tested, and identifying or assisting in identifying the milk production traits of the cows according to the genotypes of the SNP2 of the cows to be tested: the milk producing character of the dairy cows with the genotype of AA of SNP2 is higher or the candidate is higher than that of the dairy cows with the genotype of CC or CA; the AA is homozygous of SNP 2A; the CC is homozygous with SNP3 as C; the CA is heterozygous with SNP2 of C and A.

In order to solve the technical problems, the invention also provides a method for identifying or assisting in identifying milk production traits of cows, which comprises detecting genotypes of the SNP3 of cows to be tested, and identifying or assisting in identifying the milk production traits of the cows according to the genotypes of the SNP3 of the cows to be tested: the milk production character of the dairy cows with the genotype of CC of SNP3 is higher than or the candidate is higher than that of the dairy cows with the genotype of TT or CT; the CC is homozygous with SNP3 as C; the TT is homozygous with SNP3 as T; the CT is heterozygous in which SNP3 is C and T.

The application of the method for identifying or assisting in identifying milk production traits of cows in cow breeding also belongs to the protection scope of the invention.

The invention also provides a method for breeding dairy cows, which comprises the following steps: detecting polymorphism or genotype of three SNP (single nucleotide polymorphism) 1, SNP2 and SNP3 in the genome of the dairy cow, or detecting the type of haplotype in the genome of the dairy cow, and selecting the dairy cow with genotype combined into genotype TTAACC as a parent for breeding; or selecting a milk cow of haplotype H1 (TAC) as a parent for breeding; or selecting homozygous cows with SNP1 locus C as parents for breeding; or selecting homozygous cows with SNP2 locus A as parents for breeding; or selecting homozygous cows with SNP3 locus C as parents for breeding.

The breeding of the dairy cows is to cultivate the dairy cow variety with high milk yield.

The milk-producing traits described above may be, in particular, milk yield, milk fat amount, milk fat percentage, milk protein amount and milk protein percentage.

In the above application and method, the substance for detecting the polymorphism or genotype of three SNPs, SNP1, SNP2 and SNP3, or the substance for detecting haplotype, or the substance for detecting SNP1 polymorphism or genotype, or the substance for detecting SNP2 polymorphism or genotype, or the substance for detecting SNP3 polymorphism or genotype may be a substance for determining the nucleotide type of SNP1, SNP2 and/or SNP3 site in the genome of the above-mentioned dairy cow by at least one of the following methods: DNA sequencing, restriction enzyme fragment length polymorphism, single-stranded conformational polymorphism, denaturing high performance liquid chromatography and SNP chips. The SNP chip comprises 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 method reaction, a chip based on primer connection reaction, a chip based on restriction enzyme reaction, a chip based on protein DNA binding reaction and a chip based on fluorescent molecule DNA binding reaction.

In the above application or method, the substance for detecting polymorphism or genotype of three SNPs, SNP1, SNP2 and SNP3, or the substance for detecting haplotype, or the substance for detecting polymorphism or genotype of SNP3 may be the following D1), D2) or D3):

d1 A PCR primer for amplifying the cow genome DNA fragments including SNP1, SNP2 and/or SNP3 locus;

d2 A PCR reagent comprising D1) the PCR primer;

d3 A kit containing D1) the PCR primer or D2) the PCR reagent.

The PCR primers are 1F/1R, 3F/3R and/or 11F/11R:

1F/1R, a primer group consisting of single-stranded DNA shown in positions 4-23 of SEQ ID No.1 and single-stranded DNA reversely complementary to positions 598-617 of SEQ ID No.1 in the sequence table;

3F/3R, a primer group consisting of single-stranded DNA shown in 1011 th to 1030 th positions of SEQ ID No.1 and single-stranded DNA reversely complementary to 1702 th to 1721 th positions of SEQ ID No.1 in the sequence table;

11F/11R, a primer set consisting of single-stranded DNA shown in SEQ ID No.1 at 11191-11209 of the sequence Listing and single-stranded DNA reversely complementary to SEQ ID No.1 at 11623-11642.

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 that can be attached to a nucleic acid. Markers include, but are not limited to, dyes; radiolabels, e.g. ³² P is as follows; binding moieties such as biotin (biotin); hapten such as Digoxin (DIG); a luminescent, phosphorescent or fluorescent moiety; and fluorescent dyes alone or in combination with a portion of the emission spectrum that can be suppressed or shifted by Fluorescence Resonance Energy Transfer (FRET). The label may provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, or the like. The label may be a charged moiety (positive or negative) or alternatively may be charge neutral. The label may comprise or be a combination of nucleic acid or protein sequences, provided that the sequence comprising the label is detectable. In some embodiments, the nucleic acid is directly detected without a label (e.g., directly reading the sequence).

In the above applications and methods, the product may be a reagent or a kit or a system, which may include a combination of a reagent or a kit, an instrument and analysis software, such as a product consisting of a PCR primer, a PARMS master mix reagent, an enzyme-labeled instrument and an on-line software SNP decoder (http:// www.snpway.com/SNP decoder01 /), a combination of a PCR primer, a PARMS master mix reagent, an on-line software SNP decoder and a fluorescent quantitative PCR instrument. The product may include the above-described substances for detecting polymorphisms or genotypes of SNP1, SNP2 and/or SNP3 sites in the genome of a dairy cow.

In the embodiment of the invention, through genetic variation analysis of CYP7A1 genes in dairy cow related groups, 3 SNP, SNP1, SNP2 and SNP3 are found to be respectively positioned in gene CYP7A1 genes related to milk production traits in dairy cow genomes, namely 249 th, 1462 nd and 11400 th positions of a sequence table SEQ ID No. 1. In the embodiment of the invention, the dominant allele of the SNP1 locus is C, the dominant allele of the SNP2 locus is A, and the dominant allele of the SNP3 locus is C, which indicates that the SNP1 locus, the SNP2 locus and/or the SNP3 locus can be used for auxiliary selection breeding of dairy cow molecular markers and breeding of dairy cow varieties with high milk yield traits. These 3 SNP combinations total three haplotypes: haplotype H1 (TAC), haplotype H2 (TCT), and haplotype H3 (CCT). Experiments prove that the milk production characters of the homozygous genotype dairy cows corresponding to the haplotype H1 (TAC) are obviously higher than those of the homozygous genotype dairy cows corresponding to other haplotypes. The molecular marker of haplotype H1 (TAC) can be used for early prediction and screening of milk production traits of cows, and can also be used for auxiliary selective breeding of the molecular markers of the cows and breeding of cow varieties with high milk production traits. In addition, in the embodiment of the invention, the dairy cows with genotype of three SNP1, SNP2 and SNP3 are dairy cows with genotype of TTAACC, and the dairy cows with genotype of TTCACT or genotype of TTCCCT are higher or candidate higher than those of three SNP1, SNP2 and SNP 3.

Drawings

FIG. 1 shows the mutation positions of g.2467224G > A, g.2467570G > T and g.24665770A > G.

FIG. 2 is an estimate of linkage disequilibrium for SNP1 (g.24676852G > A), SNP2 (g.2467570G > T), and SNP3 (g.24665770A > G).

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The chinese holstein cows in the examples below were from the stock improvement station in the northwest province.

The data in the examples are all data for lactation 1. Lactation 1 refers to the lactation after the first delivery.

The milk yield in the examples is the individual's milk yield on day 305, which refers to the total milk yield from day one to day 305 of calving of the cow. When the actual milking days are less than 305 days, taking the actual milk yield as the milk yield of 305 days; when the actual milking days exceeded 305 days, the milk yield after 306 days was not counted. The milk yield is measured by monthly DHI (DHI, dairy Herd Improvement), and the milk yield in the lactation period of 305 days can be obtained by drawing a milk yield lactation curve according to the DHI data of more than 3 times in the same lactation period.

The milk fat amount in the examples refers to a milk fat amount of 305 days, a milk fat amount of 305 days=milk fat ratio×milk yield of 305 days. The milk fat percentage is determined by monthly DHI (DHI, dairy Herd Improvement), and the average milk fat percentage in the lactation period can be calculated by drawing a milk fat percentage lactation curve according to the DHI data of more than 3 times in the same lactation period.

The milk protein amount in the examples refers to 305 days milk protein amount, 305 days milk protein amount=milk protein rate×305 days milk yield. The milk protein rate is measured by monthly DHI (DHI, dairy Herd Improvement), and the average milk protein rate in the lactation period can be obtained by drawing a milk protein rate lactation curve through more than 3 DHI data in the same lactation period.

Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged.

Example 1 discovery of molecular markers

1. Related basic research

The present inventors have found that the subject group uses liver tissue of 3 different lactation stages (dry period, initial lactation period, peak lactation period) of Holstein cattle in China as test material, and uses the second generation sequencing technology to carry out transcriptome sequencing (RNA-sequencing) and small RNA sequencing (small RNA sequencing, small RNA-seq). It was found that significant upregulation of CYP7A1 expression (P < 0.01) during the initial and peak lactation period compared to the dry period can promote increased bile acid synthesis and thus increased lipid absorption for milk synthesis.

2. Gene polymorphism detection

1. 111 Hebei Hetao Hesi cows are selected as test populations for gene polymorphism detection. The 111 Chinese Holstein cows are randomly divided into 5 groups (one group of 23 heads and the other 4 groups of 22 heads), genomic DNA of a blood sample is extracted, the concentration of the DNA is accurately measured by a nucleic acid quality detector, the DNA is diluted to 50 ng/mu L, and the DNA is mixed into 5 pools of DNA in equal quantity, and the pool of DNA is used as a template for PCR amplification.

2. 16 pairs of primers as shown in Table 1 were designed based on the bovine CYP7A1 gene sequence (Ensembl ID is ENSBTAG00000005287 as shown in SEQ ID No. 1).

TABLE 1 PCR amplification primer sequence information for CYP7A1 Gene

3. And (3) taking the pool DNA obtained in the step (1) as a template, and respectively adopting each primer pair to carry out PCR amplification to obtain PCR amplification products. The PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.

TABLE 2PCR reaction System

TABLE 3 PCR reaction conditions

4. The PCR amplification product was sequenced. As a result, it was found that 2 SNP markers (referred to as SNP1 and SNP2, respectively) were present in the upstream 2000bp flanking sequences of the CYP7A1 gene in the cow population, and 1 SNP (referred to as SNP 3) marker was present in the 3' UTR. The 3 SNP markers are shown in Table 4, and the mutation positions are shown in FIG. 1.

TABLE 4 CYP7A1 Gene found 3 SNPs

Gene position	Name of the name	SNPs	Physical location	Polymorphic forms
					5' regulatory region	SNP1	g.24676224G>A	Chr14:24676224bp	C/T
5' regulatory region	SNP2	g.24675708G>T	Chr14:24675708bp	C/A
					3'UTR	SNP3	g.24665770A>G	Chr14:24665770bp	T/C

Wherein SNP1 corresponds to g.24676852G > A, and is obtained by sequencing a product obtained by PCR amplification by using a primer pair consisting of 1F and 1R (the PCR amplification product is shown as 4 th-617 th site of SEQ ID No. 1), and the nucleotide is C or T, and corresponds to 249 th site of the SEQ ID No.1 from the 5' end in the sequence table. SNP2 corresponds to g.246757088G > T, and is obtained by sequencing a product obtained by PCR amplification by using a primer pair consisting of 3F and 3R (the PCR amplification product is shown as 1011 st-1721 st of SEQ ID No. 1), and the nucleotide is C or A, and corresponds to 1462 nd from the 5' end of SEQ ID No.1 in a sequence table. SNP3 corresponds to g.24665770A > G, is obtained by sequencing a product obtained by PCR amplification by using a primer pair consisting of 11F and 11R (the PCR amplification product is shown as 11191-11642 of SEQ ID No. 1), and the nucleotide is T or C, and corresponds to 11400 th position from the 5' end of SEQ ID No.1 in a sequence table. Y of SEQ ID No.1 in the sequence Listing represents T or C, M represents C or A.

g.246767224G > A, g.2467570G > T and g.24665770A > G are the nomenclature of SNP1, SNP2 and SNP3 respectively, and the nomenclature of SNP is generally based on the rule when the SNP is first discovered. The DNA is of a double-stranded structure and follows the base complementary pairing principle, and the sequence of SEQ ID No.1 and the sequence of the SNP1, the SNP2 and the SNP3 in the invention are the reverse complementary strand of the same double-stranded DNA. Thus, in the present invention the polymorphic form of g.246767224G > A is C or T, the polymorphic form of g.2467570G > T is C or A, and the polymorphic form of g.24665770A > G is T or C.

3. Correlation analysis

(one) obtaining a test population

The test population consisted of 1123 chinese holstein cows.

(II) genotyping

Genotyping was performed separately for each individual in the test population.

I. Genotyping based on g.24676858 g > a.

1. Blood of a subject is taken, and genomic DNA is extracted.

2. The genomic DNA is used as a template, a primer pair consisting of 1F (shown as 4-23 th positions of SEQ ID No. 1) and 1R (reversely complementary to 598-617 th positions of SEQ ID No. 1) is adopted for PCR amplification, and then the PCR amplification product is recovered and sequenced.

The reaction system of PCR amplification is shown in Table 5. The reaction conditions for PCR amplification are shown in Table 6.

The PCR amplified products of each subject were 614bp, where position 246 was g.24676852G > A, namely SNP1 (corresponding to position 249 from the 5' end of SEQ ID No.1 in the sequence Listing).

TABLE 5

TABLE 6

II. Genotyping based on g.246757088 g > T.

1. Blood of a subject is taken, and genomic DNA is extracted.

2. PCR amplification was performed using the genomic DNA as a template, using a primer set composed of 3F (as shown at 1011-1030 th positions of SEQ ID No. 1) and 3R (reverse complementary to 1702-1721 th positions of SEQ ID No. 1), and then the PCR amplification product was recovered and sequenced.

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

The PCR amplification products of each subject were 711bp, where 452 was g.246757088G > T, namely SNP2 (corresponding to position 1462 from the 5' end of SEQ ID No.1 in the sequence Listing).

TABLE 7

TABLE 8

Genotyping based on g.24665770a > G.

1. Blood of a subject is taken, and genomic DNA is extracted.

2. The PCR amplification was performed using the genomic DNA as a template, using a primer set composed of 11F (as shown in SEQ ID No.1 at 11191-11209) and 11R (reverse complementary to SEQ ID No.1 at 11623-11642), and then the PCR amplification product was recovered and sequenced.

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

The PCR amplified products of each subject were 452bp, where position 210 was g.24665770A > G, namely SNP3 (corresponding to position 11400 from the 5' end of SEQ ID No.1 of the sequence Listing).

TABLE 9

Table 10

(III) detecting milk production characteristics

And (5) each cow in the tested population is subjected to milk production character detection respectively.

The milk production characteristics comprise the following five indexes: milk yield, milk fat amount, milk fat percentage, milk protein amount and milk protein percentage.

Each individual record comprises, in order, a bovine individual number, a father number, a mother number, a grandfather number, a grandmother number, an grandfather number, an grandmother number, a date of birth, a lactation period, a calving date, a milk yield, a milk fat yield, and a milk protein yield.

(IV), correlation analysis model of single SNP locus and character

Genotypes and milk production phenotype of SNP1 locus (i.e., CYP7A1 gene g.24676852G > A), SNP2 locus (i.e., CYP7A1 gene g.2467570G > T) and SNP3 locus (i.e., CYP7A1 gene g.24665770A > G) are shown in tables 11, 12 and 13.

Table 11 1123 genotypes of partial milk production trait phenotypes and 3 SNP loci of Hestent cows in China

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Table 12 1123 descriptive statistics of 5 milk production trait values for the chinese Holstein Niu Muniu population

Traits (3)	Average value of	Standard deviation of	Minimum value	Maximum value	Coefficient of variation
						Milk yield (kg)	8140.1308	1467.7241	12536.4375	3721.4801	0.1803
Milk fat quantity (kg)	304.0847	72.2922	562.4322	98.5694	0.2377
						Milk fat percentage (%)	3.7679	0.7615	6.9700	2.0000	0.2021
Milk protein quantity (kg)	257.0603	46.0290	398.1886	114.1625	0.1791
						Milk protein yield (%)	3.1691	0.2591	4.0100	2.3400	0.0817

TABLE 13 allele frequencies and genotype frequencies of 3 SNP loci of CYP7A1 gene

The result shows that 3 genotypes (SNP 1 genotypes for short) exist at the SNP1 locus, namely CC, TT or CT, wherein genotype CC is homozygous with SNP1 as C, genotype TT is homozygous with SNP1 as T, and genotype CT is heterozygous with SNP1 as C and T; the SNP2 locus has 3 genotypes (SNP 2 genotypes for short), namely CC, AA or CA, the genotype CC is homozygous with SNP2 as C, the genotype AA is homozygous with SNP2 as A, and the genotype CA is heterozygous with SNP2 as C and A; the SNP3 locus has 3 genotypes (SNP 3 genotypes for short), namely CC, TT or CT, the genotype CC is homozygous with SNP1 as C, the genotype TT is homozygous with SNP1 as T, and the genotype CT is heterozygous with SNP1 as C and T.

Five indicators of milk production traits and genotypes were analyzed in association using the MIXED procedure in SAS 9.2 software. The correlation analysis adopts an animal model, and the concrete model is as follows:

Y＝μ+bys+b×M+G+a+e

wherein Y: observed values of milk production traits (milk yield, milk fat percentage, milk protein amount or milk protein rate); mu: overall mean; hys: a field year season effect; b: regression coefficients of covariates M; m: a calving month-old effect; g: genotype effect; a: individual random additive genetic effects; e: random residual effect.

SNP1 locus in 1123 head population (i.e., CYP7A1 gene _g .24676224G>A) The typing is successful 1120, and the analysis results related to the milk production characteristics are shown in Table 14.

TABLE 14 CYP7A1 Gene g.24676852G > A milk production trait association analysis (least squares mean.+ -. Standard error)

Genotype of the type	Milk yield (kg)	Milk fat quantity (kg)	Milk fat percentage (%)	Milk protein quantity (kg)	Milk protein yield (%)
						TT(295)	6820.32±249.65b	250.95±10.107	3.701±0.1	207.34±4.86Bb	3.116±0.036ab
CT(549)	6875.08±249.1ab	251.79±10.085	3.695±0.1	209.82±4.849AaB	3.12±0.036a
						CC(276)	6983±251.72a	254.12±10.19	3.655±0.101	211.15±4.899Aab	3.098±0.036b
P value	0.0236*	0.394	0.105	0.0037**	0.0133*

Note that: * P (P)<0.05, indicating significant differences; * P<0.01, indicating that the difference was very significant. a, a step of, ^b the same column of data has different superscripts to indicate that the difference is obvious; ^A,B the same column of data has different superscripts to indicate that the differences are very significant.

As can be seen from table 14, SNP1 (g.246768224 g > a) was significantly associated with milk yield, milk protein amount and milk protein rate (p=0.0236-0.0037), for milk yield, milk protein amount and milk protein rate traits, the dominant allele is C:

The milk yield of the CC-genotype cows is higher than that of the TT-genotype cows, the milk yield of the CC-genotype cows has no obvious difference from that of the CT-genotype cows, and the milk yield of the TT-genotype cows has no obvious difference from that of the CT-genotype cows.

The milk protein amount of the CC genotype cow is higher than that of the TT genotype cow and the milk protein amount of the CT genotype cow is higher than that of the TT genotype cow, and the milk protein amount of the CT genotype cow is not obviously different from that of the CC genotype cow.

The milk protein rate of CC genotype cows is higher than that of CT genotype cows, the milk protein rate of CC genotype cows has no obvious difference from TT genotype cows, and the milk protein rate of TT genotype cows has no obvious difference from CT genotype cows.

The SNP2 locus (namely CYP7A1 gene g.246757088G > T) in 1123 head groups is successfully typed 1118 heads, and the analysis result related to the milk production characteristics is shown in Table 15.

TABLE 15 CYP7A1 Gene g.246757088G > T and milk production trait correlation analysis (least squares mean.+ -. Standard error)

Genotype of the type	Milk yield (kg)	Milk fat quantity (kg)	Milk fat percentage (%)	Milk protein quantity (kg)	Milk protein yield (%)
						CC(296)	6789.5±250.7B	260.78±10.175	3.808±0.1A	209.02±4.92Bb	3.13±0.036a
CA(545)	6864.1±250.02AB	260.31±10.144	3.777±0.1AB	211.71±4.899AaB	3.126±0.036ab
						AA(277)	6971.05±252.68A	262.33±10.254	3.731±0.101B	213.46±4.954Aab	3.107±0.036b
P value	0.0105*	0.629	0.0058**	0.0005**	0.0202*

Note that: * P (P)<0.05, indicating significant differences; * P<0.01, indicating that the difference was very significant. a, a step of, ^b the same column of data has different superscripts to indicate that the difference is obvious; ^A,B the same column of data has different superscripts to indicate that the differences are very significant.

As can be seen from table 15, SNP2 (g.246757088 g > T) is significantly correlated with milk yield, milk fat amount, milk protein amount and milk protein rate (p= 0.0202-0.0005), for milk yield, milk fat amount, milk protein amount and milk protein rate traits, the dominant allele is a:

the milk yield of the AA-type cows is higher than that of the CC-type cows, the milk yield of the CA-type cows is not obviously different from that of the AA-type cows, and the milk yield of the CA-type cows is not obviously different from that of the CC-type cows.

The milk fat percentage of the CC genotype cows is higher than that of the AA genotype cows, the milk fat percentage of the CC genotype cows has no significant difference from the CA genotype cows and the milk fat percentage of the AA genotype cows has no significant difference from the CA genotype cows.

The milk protein amount of the AA genotype cow is higher than that of the CC genotype cow and the milk protein amount of the CA genotype cow is higher than that of the CC genotype cow, and the milk protein amount of the CA genotype cow is not obviously different from that of the AA genotype cow.

The milk protein rate of the CC genotype cows is higher than that of the AA genotype cows, the milk protein rate of the CA genotype cows has no significant difference from the CC genotype cows and the milk protein rate of the CA genotype cows has no significant difference from the AA genotype cows.

The SNP3 locus (namely CYP7A1 gene g.24665770A > G) in 1123 head group is successfully typed 1116 heads, and the analysis result related to the milk production property is shown in Table 16.

Table 16 CYP7A1 gene g.24665770A > G and milk production property association analysis (least square mean value + -standard error)

Genotype of the type	Milk yield (kg)	Milk fat quantity (kg)	Milk fat percentage (%)	Milk protein quantity (kg)	Milk protein yield (%)
						TT(670)	6805.66±250.15B	256.34±10.169B	3.748±0.1B	210.93±4.95B	3.121±0.036
CT(382)	6965.54±252.75A	272.64±10.267A	3.883±0.101A	216.34±4.988A	3.123±0.036
						CC(64)	6966.21±264.15AB	273.09±10.719A	3.864±0.106A	218.71±5.194A	3.152±0.038
P value	0.0018**	<0.0001**	<0.0001**	<0.0001**	0.077

Note that: ^* P<0.05, indicating significant differences; ^** P<0.01, indicating that the difference was very significant. ^a,b The same column of data has different superscripts to indicate that the difference is obvious; ^A,B the same column of data has different superscripts to indicate that the differences are very significant.

As can be seen from table 16, SNP3 (g.24665770 a > G) correlated very significantly with milk yield, milk fat amount, milk fat percentage and milk protein amount (p=0.0018 to < 0.0001), for milk yield, milk fat amount, milk fat percentage and milk protein amount traits, the dominant allele was C.

Milk yield of CT genotype cows is higher than that of TT genotype cows.

The milk fat quantity, the milk fat percentage and the milk protein quantity of the CC genotype cow are all higher than those of the TT genotype cow, the milk fat quantity, the milk fat percentage and the milk protein quantity of the CT genotype cow are all higher than those of the TT genotype cow, and the milk fat quantity, the milk fat percentage and the milk protein quantity of the CC genotype cow are not obviously different from those of the CT genotype cow.

(V) genetic Effect analysis

And (3) performing significance test on SNP additive effect, dominant effect and substitution effect by using SAS 9.2 software.

The basic calculation formula is as follows:

a= (AA-BB)/2, d = AB- (aa+bb)/2, α = a+d (q-p); a is an additive effect, d is a dominant effect, and α is an allele replacement effect; AA. AB and BB are the least square average value of the milk production characteristics of the corresponding genotypes; p is the frequency of allele A and q is the frequency of allele B.

The results of the additive effect, dominant effect and allele replacement effect assays are shown in Table 17.

TABLE 17 test results of the additive, dominant and substitution effects of the CYP7A1 gene alleles

Note that: * P <0.05, indicating significant differences; * P <0.01, indicating that the difference is very significant.

From the results, the additive effect, dominant effect and allele substitution effect of SNP1 (g.24676858G > A) on milk yield, milk protein amount and milk protein rate respectively reach remarkable or extremely remarkable effect, namely, each T allele substitutes for C allele to reduce the milk yield by 81.622kg (P < 0.01), the milk protein amount by 1.898kg (P < 0.01) and the milk protein rate by 0.009% (P < 0.05). The additive effect and allele replacement effect of SNP2 (g.246757088 g > T) on milk yield, milk fat percentage, milk protein amount and milk protein rate were all significant, i.e. replacement of the a allele by each C allele resulted in a decrease in milk yield of 90.947kg (P < 0.01), an increase in milk fat percentage of 0.039% (P < 0.01), a decrease in milk protein amount of 2.216kg (P < 0.01) and an increase in milk protein rate of 0.011% (P < 0.01). The additive effect and allele replacement effect of SNP3 (g.24665770A > G) on milk protein amount and milk protein rate are both remarkable. That is, each T allele replaced the C allele resulting in a 3.057kg decrease in milk protein (P < 0.05) and a 0.023% decrease in milk protein rate (P < 0.05).

Analysis of haplotype

And constructing haplotypes by using Haploview 4.2 software, estimating haplotype frequency and linkage degree analysis, and carrying out association analysis with the characters. Using the MIXED procedure in SAS 9.2 software, the model is as follows:

Y＝μ+hys+b×M+G+a+e

y: observed values of milk production traits (milk yield, milk fat amount, milk fat percentage, milk protein amount, and milk protein rate); mu: overall mean; hys: a field year season effect; b: regression coefficients of covariates M; m: a calving month-old effect; g: haplotype combined effects; a: individual random additive genetic effects; e: random residual effect.

The haplotype analysis results are shown in Table 18.

Table 18 CYP7A1 haplotype consisting of 3 SNPs

In the 1123 study population, 3 SNPs (SNP 1, SNP2, and SNP 3) of the CYP7A1 gene together form a single haplotype block and form three haplotype combinations, namely haplotype H1, haplotype H2, and haplotype H3. Haplotype H1 (TAC) is a combination of one chromosome with SNP1 of T, SNP as A and SNP3 of C. Haplotype H2 (TCT) is a combination of one chromosome with SNP1 of T, SNP as C and SNP3 as T. Haplotype H3 (CCT) is a combination of SNP1 of C, SNP of C and SNP3 of T on one chromosome. Haplotype H1 frequency was 49%, haplotype H2 frequency was 28.3%, and haplotype H3 frequency was 22.1%. The 3 SNPs were subjected to linkage disequilibrium estimation, and the results showed that the 3 SNPs were in a completely linked state (r ² =1), as shown in fig. 2.

Haplotypes represent the linkage of the two SNPs on the same chromosome. Bovine was 2-fold, and the haplotype combinations of the two chromosomes of individuals in the population of 1123 heads tested were H1, H1H2, H2, respectively.

The genotype of the cow corresponding to H1H1 is TTAACC, and the genotype TTAACC is three SNP combined genotypes of which the genotype of SNP1 is TT, the genotype of SNP2 is AA and the genotype of SNP3 is CC. The genotype of the cow corresponding to H1H2 is TTCACT, and the genotype TTCACT is three SNP combined genotypes of which the genotype of SNP1 is TT, the genotype of SNP2 is CA and the genotype of SNP3 is CT. The genotype of the cow corresponding to H2H2 is TTCCTT, the genotype TTCCTT is three SNP combined genotypes of which the genotype of SNP1 is TT, the genotype of SNP2 is CC and the genotype of SNP3 is TT.

The results of analysis of the association of the CYP7A1 gene haplotype combinations with milk production traits are shown in Table 19 (haplotype combinations H3H3 with a frequency of less than 0.05 were deleted, and genotype CTCTTs of H2H3 combinations were not present in the present population but were deleted because the frequency of H2H3 was > 0.05). The haplotype combination of 3 SNPs of the CYP7A1 gene correlated with milk yield, milk fat mass, milk fat percentage, milk protein mass and milk protein rate to very significant levels (p=0.0088 < 0.0001), where haplotype H1 is the dominant haplotype for all 5 milk production traits.

TABLE 19CYP7A1 Gene haplotype combinations and milk production trait correlation analysis results (least squares mean.+ -. Standard error)

The milk yield of the cows of haplotype combination H1H1 (i.e., genotype TTAACC) is higher than that of the cows of haplotype combination H1H2 (i.e., genotype TTCACT) and the cows of haplotype combination H2H2 (i.e., genotype TTCCTT).

The milk fat content of the cows of haplotype combination H1H1 (i.e., genotype TTAACC) is higher than that of the cows of haplotype combination H1H2 (i.e., genotype TTCACT) and the cows of haplotype combination H2H2 (i.e., genotype TTCCTT).

The milk fat percentage of the cows of haplotype combination H2H2 (i.e., genotype TTCCTT) is higher than that of the cows of haplotype combination H1H2 (i.e., genotype TTCACT) and the cows of haplotype combination H1H1 (i.e., genotype TTAACC).

The milk protein amount of the cow of haplotype combination H1H1 (i.e., genotype TTAACC) is higher than that of the cow of haplotype combination H2H2 (i.e., genotype TTCCTT) and the milk protein amount of the cow of haplotype combination H1H2 (i.e., genotype TTCACT) is higher than that of the cow of haplotype combination H2H2 (i.e., genotype TTCCTT).

Haplotype analysis tends to express more information than single marker analysis, and some site interaction changes inside the gene can have a great effect on the phenotype, so that the haplotype analysis is obviously superior to conventional single marker analysis in complex trait association analysis.

The molecular marker disclosed by the invention can be applied to auxiliary identification of dairy cow groups with excellent milk production characteristics (milk yield in 305 days, milk fat amount, milk fat rate, milk protein amount and milk protein rate), and has the following advantages: simple, quick, sensitive, reliable, stable and accurate result, and is suitable for large-scale detection of laboratory population.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

SEQ ID No.1

ttcctgctgtttcttccccatctcctctttgtcgctctctgactgatctcaagaaatgggctgctctggggtctggtgttggattctattcttttcttgggcttaattgtctccctggacttcctgtggctcagatgataaagaatctgcctgcaatgcaggggacctgggtttgatccctgggtcaggaagatcccctggaggagggaatggcagcccactccagtattgttacccggaaaattccayggacgggggaacctggcaggctacagtccatggggttgcaaagagtctgacatgactgagcgactcacaactttcaatttcacattgtctcccaaggtgatatgatatagtttctgggctctaaacatcattcaaacactgatgacttgtgattcctatttccacctttgactcttctcccttgtttcaaactcaagttcaattgttaacctgacaacacagcagtctgtgtagctgaagacactgttttgatctgccgccctccaaaatctctttcccttctctcaagtgtcctgtatgtgactgagctcttgcctgtcttttgtcttacatgttaaccaagcttcccctcattcactctgctccaatgtatcctacatcctttctggttcttagatagaccaacctcatccttaactcaggacttctgcatttgctctttcttcttgaatttctcgtccttctagttttgtcttggctaatcccttgatctcagaggagcaagtcaccacgcctacagttgctctcatgccctgatagctctctgcactttgccttattttatgacttatattttattatctgtctctcgacgaaaatagtgccttcattaaagcagggaatcaggtcttattaatacctgtagtcagaatacctttaaagagttcttggtacagaatagacgttcaatcactacgtaagtgtgcaaagaaataaagtttgggaggatatacacaccaaacagctaacagttctgagtggggagatgtggcagaggtagattatgagttgactttccctttctactaagatatgtttcagttagtcttgaattatttgaaattaagctatactcctttaacaatcagaaaaattaataaacttattttcaattagaaaaaagtggttgataaagttttgatgctttaagataaactgtgttgtggggaacttgcctggcggtctagtggttaagacttcaccttccagtacagggggtgtgggttcaatccctggtcagggagataagatcctacatgccaaaaagctagaacataaaacagaaggaatattgtcataaatttaataaagacttaaaacactagactgtgtttagctggaggataaatttttgtgggaaactttctctcttgagggtgataggtaagtaacatgttactatatatagtaaaatctctctgcttttgtaaamgcactgaaacgtgaagcagcagaaacatttcactcagttctctttcctttgaactttacagctctctctcttgtaaatcatttaaattgatttctttttaaaggtaacaagatcaaatcattagcttaatgataacttgtacttacttttctttgccttattatattttacatctctatttgatatgttcatcttgaacaggtttatttgttcttttcataagagtcctaaatttgtaatactggctgtcattcccaggtctgaatgttaagtcaacatatgtttgagtgaccttcaacttaccaagtatggcaggcctctgattgttttggaacctcttctgatacttgtggacttagttcaaggccagttattttattttatttttttacctaatagggatggttaattgtttgctttgatcactcaagtgaagcatcttaccatgtataaaaagtctagctggagtctgttgttacatccttacctttctcatcacagacgcttctcctcagaggtttcacattagatttgcaaaatgatgagcctctctttgatttgggggattgttatagcagtgtgttgttgtttatacctacttggaatgagaagaaggtaagttttatctttaaattgcatttttgttcattcatttaaagttatttaactttcattgttataccatagagttggttttctgtacttacaaatgctggcattttcttacttaatatttttaaattaaaaaaaattttaagtaacttttttattatatctcacaagagatataatggcaatgtttcctttttagcattgaaatatattagtcttaatgtaaaaatggacaaacttgttaaacatcactgacagaaactttgtaagatctgtgtgtgtgcgtatgtctgtgtgtccacgtgtgcatataaagtgtggggtagtaaggacagatgtggtcagtgggatccacagttctatctcgtagattaaatagatgctctgactaaataaatgttctctttttagctagttcttgtgcactgtatacactcaggctgagctggacaagagcatagagcctgcagtgatcatcctgttattctttttttttttaagctttattttatgtattcattcagttttagcttcacttggactttgttgctgcaagggctttccctagttgtggtgagcgggggctactctttgctgtagtgctcaggcttctcactgcagtgcttcctcttgtcgtggagcacgggctctaggtgctcaaactgcgcgggcttcaggagttgggatgtgtggactcggtggttgtgactcgctggcgccagaccacaggctcagtagttgtggcacatggacttagttgctcctcggtaggtcagatcttcccagaccagggatcaaacccatgtccccttaacatcagggttaagaatctattattattactaatgataatcaatcatggtgaattgggaaactgctaaacacatgagacatcacttatttgtgtgcatcatttacaattggttgaagaaatacaataaagtcagatatatccaccataattatttaacttctgattggttcaaagagcatttgggggagggcatataaatattctcaactatgtcagtctccaaaacagaattacccactgcatatcttcagctatgtaggttgctatgggttgtccaccttttaacaggttttttttttttttccttttcagtattttaattcaggcttagtaatttctatttttaattaaactgcttacctgcaaactacaaaaataaaaatcattattttatgctatgttattctacaaagagggagagtggtttctgttggaaaataaatgtttattttattaaatgaaaacatttaaaatagcatgaacattaaattataatgctagattataagaaagagcattaaattataatcaataaccatgtcaatacctatcttattcaatttcattctataatgtgctaatttctcagattcccctcttcaactatcattacagaataatgtttaaactatgaagatttttaaaacttcaattctgcaatcctttctgtcattcagtatgctacctttagagaagacaataaaataaaacattttgagatactcaggcaaacaatgcctagaaacaaatataaacactacaatgtaatttcatttctacactgagagcaaaaatcttattggccttggttgatccttttatcctcatgattgggaagaatgagcagcatatattagacatttagtaagggcttctcgggtggcgcaggggtaaaggatccccctgccaatgcaggagatgcaagaaatgcgggtttgattccctgggtcaggaagatcccctggagatggaaacggcaacccactctagtattcttgcctgggaaacttcatggacagaggagcctggcggactacagtccatggagttgcaaaacagttggacatgattgagcatactgtacagaaacacacgtgtgcacacatacatatacgtgtttgctgaatggatgcatggatggatggatttatctatcatttttaccaacatatcatgttactaagttaagatgactcattattttagtaaaggagcagggccccaccaaccaaaaaaaatcaataaactatagagtttgaaatttgacatcagtggtagtaaacttaaaataaacaaagaactatgtacagttttccatatttaccatcttgtttcttacaggcagatgggcgaaccccctctggagaatgggctgattccgtatctgggttgtgctctgcaatttggtgccaatcctctcgagttcctcagggcaaatcaaaggaaacatggtcacgttttcacctgcagactgatgggaaactatgtccacttcatcacaaatcccttgtcatatcataaagtactgtgccatggaaaatactttgattggaaaaaatttcactttactgcttctgcaaaggtaaccagatttgcatttacataacattagacttgcttattttctaccttgtctatgtcaatctatttaaccattctgatgtacttcaattgaaagacagagatgttgaagagagctacccttacatgaagattcttagaaacacaataaaccaaattggacataatctcttggtactaaccatttaaaaagttgaaataggtatagatttgtcattttctaaccagtttacgagggtgaaataaaaactaaatgttttactggagtagacggtactgtataagacagattaaaaactcactcatgtcaataatttttacacaccacccaaagtgcacgtgggaacttaacgttcaatagttgttttaccacactcatgccacacaattgtttaattttgttaaggaattgtccatatattttggcatcactttaaaaggctcttctacagagaaaaatgaatcaatattaactgagtttctgcaaagaaatattagacctggtctctgctcctaagtaacttaaaatct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Claims (10)

1. The application is characterized in that: the application is any one of the following P1-P5:

the P1 is the application of a substance for detecting the polymorphism or genotype of three SNP such as SNP1, SNP2 and SNP3 in the identification or auxiliary identification of the milk production character of the dairy cow;

The P2 is the application of a substance for detecting haplotypes in identification or auxiliary identification of milk production traits of cows, wherein the haplotypes are polymorphism combinations of the SNP1, the SNP2 and the SNP3 on one chromosome of the cows;

the P3 is the application of a substance for detecting the polymorphism or genotype of the SNP1 in the identification or auxiliary identification of the milk production traits of the dairy cows;

the P4 is the application of a substance for detecting the polymorphism or genotype of the SNP2 in the identification or auxiliary identification of the milk production traits of the dairy cows;

the P5 is the application of a substance for detecting the polymorphism or genotype of the SNP3 in the identification or auxiliary identification of the milk production traits of the dairy cows;

SNP1 is one SNP of a dairy cow genome, is 249 th nucleotide of SEQ ID No.1 in a sequence table, and is C or T; SNP2 is one SNP of a dairy cow genome, is 1462 nucleotide of SEQ ID No.1 in a sequence table, and is C or A; SNP3 is one SNP of the genome of the dairy cow, is 11400 nucleotide of SEQ ID No.1 in the sequence table, and is C or T.

2. The application is characterized in that: the application is any one of Q1-Q5:

the Q1 is the application of a substance for detecting the polymorphism or genotype of three SNP such as SNP1, SNP2 and SNP3 in preparing and identifying or assisting in identifying dairy cow milk production character products;

The Q2 is the application of a substance for detecting haplotype in preparing and identifying or assisting in identifying dairy cow milk production character products; the haplotype is a polymorphism combination of three SNP1, SNP2 and SNP3 on one chromosome of the dairy cow;

the Q3 is the application of a substance for detecting the polymorphism or genotype of the SNP1 in preparing and identifying or assisting in identifying dairy cow milk production character products;

the Q4 is the application of a substance for detecting the polymorphism or genotype of the SNP2 in preparing and identifying or assisting in identifying dairy cow milk production character products;

the Q5 is the application of a substance for detecting the polymorphism or genotype of the SNP3 in preparing and identifying or assisting in identifying dairy cow milk production character products;

SNP1 is one SNP of a dairy cow genome, is 249 th nucleotide of SEQ ID No.1 in a sequence table, and is C or T; SNP2 is one SNP of a dairy cow genome, is 1462 nucleotide of SEQ ID No.1 in a sequence table, and is C or A; SNP3 is one SNP of the genome of the dairy cow, is 11400 nucleotide of SEQ ID No.1 in the sequence table, and is C or T.

3. The application is characterized in that: the application is any one of E1-E5:

the E1 is the application of a substance for detecting polymorphism or genotype of three SNP (single nucleotide polymorphism) 1, SNP2 and SNP3 in cow breeding or preparing cow breeding products;

The E2 is the application of a substance for detecting haplotype in cow breeding or preparing cow breeding products; the haplotype is a polymorphism combination of three SNP1, SNP2 and SNP3 on one chromosome of the dairy cow;

the E3 is the application of a substance for detecting the polymorphism or genotype of the SNP1 in cow breeding or preparing cow breeding products;

the E4 is the application of a substance for detecting the polymorphism or genotype of the SNP2 in cow breeding or preparing cow breeding products;

the E5 is the application of a substance for detecting the polymorphism or genotype of the SNP3 in cow breeding or preparing cow breeding products;

SNP1 is one SNP of a dairy cow genome, is 249 th nucleotide of SEQ ID No.1 in a sequence table, and is C or T; SNP2 is one SNP of a dairy cow genome, is 1462 nucleotide of SEQ ID No.1 in a sequence table, and is C or A; SNP3 is one SNP of the genome of the dairy cow, is 11400 nucleotide of SEQ ID No.1 in the sequence table, and is C or T.

4. A product characterized in that said product comprises the substance for detecting the polymorphism or genotype of three SNPs, namely, cow genome SNP1, SNP2 and SNP3, as defined in claim 1, or the substance for detecting haplotype, as defined in claim 1, or the substance for detecting the polymorphism or genotype of cow genome SNP1, as defined in claim 1, or the substance for detecting the polymorphism or genotype of cow genome SNP2, as defined in claim 1, or the substance for detecting the polymorphism or genotype of cow genome SNP3, as defined in claim 1, and can be any one of the following G1) to G3):

G1 A product for detecting single nucleotide polymorphism or genotype related to milk production traits of the dairy cows;

g2 Identification or auxiliary identification of products of milk production traits of the dairy cows;

g3 A product for breeding dairy cows.

5. A method for identifying or assisting in identifying milk production traits of dairy cows, wherein the method is a method A or a method B;

the method A is a method for identifying or assisting in identifying milk production traits of dairy cows, and comprises the steps of detecting genotypes of three SNP1, SNP2 and SNP3 in the dairy cows to be tested, and identifying or assisting in identifying the milk production traits of the dairy cows according to the genotypes of the three SNP of the dairy cows to be tested: the dairy cows with genotype of the three SNP are the dairy cows with genotype of TTAACC, and the dairy cows with milk production characters higher than or candidates higher than the genotype of the three SNP are the dairy cows with genotype of TTCACT or genotype of TTCCTT; genotype ttaac is three SNP combined genotypes with SNP1 genotype TT, SNP2 genotype AA and SNP3 genotype CC; genotype TTCACT is three SNP combined genotypes with SNP1 genotype TT, SNP2 genotype CA and SNP3 genotype CT; genotype TTCCTT is three SNP combined genotypes with SNP1 genotype TT, SNP2 genotype CC and SNP3 genotype TT;

the method B is a method for identifying or assisting in identifying milk production traits of dairy cows, and comprises detecting haplotypes in claim 1 in dairy cows to be tested, and identifying or assisting in identifying the milk production traits of the dairy cows according to the haplotypes of the dairy cows to be tested: the milk production character of the homozygous genotype dairy cows corresponding to the haplotype H1 is higher than or the candidate is higher than that of the homozygous genotype dairy cows corresponding to the haplotype H2; the haplotype H1 is that the SNP1 is T, the SNP2 is A and the SNP3 is C; the haplotype H2 is that the SNP1 is T, the SNP2 is C and the SNP3 is T.

6. The method for identifying or assisting in identifying the milk production characteristics of the dairy cows is characterized by comprising the following steps of: the method comprises detecting the genotype of the SNP1 or the SNP2 or the SNP3 in the dairy cow to be tested, and identifying or assisting in identifying dairy cow milk production traits according to the genotype of the SNP1 or the SNP2 or the SNP3 of the dairy cow to be tested:

the milk production character of the dairy cows with the genotype of CC of SNP1 is higher than or the candidate is higher than that of the dairy cows with the genotype of TT or CT; the CC is homozygous with SNP1 as C; the TT is homozygous with SNP1 as T; the CT is heterozygous with SNP1 of C and T;

the milk producing character of the dairy cows with the genotype of AA of SNP2 is higher or the candidate is higher than that of the dairy cows with the genotype of CC or CA; the AA is homozygous of SNP 2A; the CC is homozygous with SNP3 as C; the CA is heterozygous with SNP2 of C and A;

the milk production character of the dairy cows with the genotype of CC of SNP3 is higher than or the candidate is higher than that of the dairy cows with the genotype of TT or CT; the CC is homozygous with SNP3 as C; the TT is homozygous with SNP3 as T; the CT is heterozygous in which SNP3 is C and T.

7. Use of the method of claim 5 or 6 in dairy cow breeding.

8. The use according to any one of claims 1-3 and 7, the product according to claim 4, the method according to any one of claims 5-6, characterized in that: the breeding of the dairy cows is to cultivate dairy cows with high milk production characters or to breed dairy cow varieties with high milk production characters.

9. The use according to any one of claims 1-3 or 7, the product according to claim 4, the method according to any one of claims 5-6 or 8, characterized in that: the substance for detecting the polymorphism or genotype of three SNPs, namely SNP1, SNP2 and SNP3, or the substance for detecting the haplotype, or the substance for detecting the polymorphism or genotype of SNP3 is D1), D2) or D3) as follows:

d1 A PCR primer for amplifying the cow genome DNA fragments including SNP1, SNP2 and/or SNP3 locus;

d2 A PCR reagent comprising D1) the PCR primer;

d3 A kit containing D1) the PCR primer or D2) the PCR reagent.

10. The use or product or method according to claim 9, characterized in that: the PCR primers are 1F/1R, 3F/3R and/or 11F/11R:

1F/1R, a primer group consisting of single-stranded DNA shown in positions 4-23 of SEQ ID No.1 and single-stranded DNA reversely complementary to positions 598-617 of SEQ ID No.1 in the sequence table;

3F/3R, a primer group consisting of single-stranded DNA shown in 1011 th to 1030 th positions of SEQ ID No.1 and single-stranded DNA reversely complementary to 1702 th to 1721 th positions of SEQ ID No.1 in the sequence table;

11F/11R, a primer set consisting of single-stranded DNA shown in SEQ ID No.1 at 11191-11209 of the sequence Listing and single-stranded DNA reversely complementary to SEQ ID No.1 at 11623-11642.

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