CN117402977A

CN117402977A - Method for detecting milk production traits of dairy cows based on SNP markers of ACOX2 genes and application of method

Info

Publication number: CN117402977A
Application number: CN202311192285.9A
Authority: CN
Inventors: 孙东晓; 韩博; 王哲; 许令娜
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2023-09-15
Filing date: 2023-09-15
Publication date: 2024-01-16

Abstract

The invention discloses a method for detecting milk production traits of dairy cows based on an ACOX2 gene SNP marker and application thereof. The technical scheme to be protected by the invention is the application of a substance for detecting the polymorphism or genotype of SNP1 or SNP2 in the identification or auxiliary identification of milk production traits of dairy cows. Experiments prove that the related molecular markers of SNP1 or SNP2 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

Method for detecting milk production traits of dairy cows based on SNP markers of ACOX2 genes and application of method

Technical Field

The invention relates to a method for detecting milk production traits of dairy cows based on an ACOX2 gene SNP marker and application thereof in the technical field of molecular biology.

Background

The ACOX2 gene is located on bovine chromosome 22, has 15 exons, has the total length of 30897bp, is a coding gene of acyl-CoA oxidase 2, and belongs to acyl-CoA oxidase families. The gene family contains 3 subtypes of ACOX1, ACOX2 and ACOX3, and the three subtypes are involved in peroxidase metabolism and sugar metabolism. The ACOX2 gene catalyzes the conversion of cholesterol into bile acid through a coenzyme A ester intermediate of oxidized bile acid, thereby affecting the content of fatty acid in tissues, and being an important regulatory gene in the fatty acid metabolic process. Studies show that the ACOX2 gene participates in the degradation of a D-type oxidation system and branched chain fatty acid, influences the beta-oxidation of fatty acid by acyl-CoA dehydrogenase, and further regulates and controls fatty acid metabolism.

Deficiency of the ACOX2 gene causes bile acid synthesis deficiency, and is common in adolescent populations. Studies have demonstrated that the lack of this gene will hinder the conversion of cholesterol to bile acids, accumulating toxic C27 bile acid intermediates, and thus reducing C24 bile acid synthesis. In the research on liver cancer, experiments show that ACOX2 over-expression has an inhibition effect on tumors, and the inhibition effect on tumors is proved to reduce proliferation and metastasis of liver cancer in vitro and in vivo through PPARα pathway. Furthermore, wang Xiao et al performed transcriptome differential expression analysis of the muscovy duck ileum, and found that the ACOX2 gene was significantly positively correlated with abdominal fat weight and abdominal fat rate.

Semi-retained replication of DNA is an important pathway for biological evolution and passage. Double-stranded DNA can be denatured and unwound into single strands under the action of various enzymes, and the same two-molecule copies are copied according to the base complementary pairing principle under the participation of DNA polymerase. In experiments, it was found that DNA can be denatured and melted even at high temperatures, and can be double-stranded again after the temperature is lowered. Therefore, the denaturation and renaturation of the DNA are controlled by temperature change, and the in vitro replication of the specific gene can be completed by adding the designed primer, DNA polymerase and dNTP. Polymerase Chain Reaction (PCR) technology is a specific DNA in vitro amplification technique. 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 component characters of cows or breeding cows.

In order to solve the above technical problems, the present invention provides an application, where the application is the following P1 or P2:

the P1 is the application of a substance for detecting the polymorphism or genotype of the SNP1 in the identification or auxiliary identification of milk constituent characters of cows;

the P2 is the application of a substance for detecting the polymorphism or genotype of the SNP2 in the identification or auxiliary identification of the milk constituent characters of the dairy cows;

SNP1 is one SNP of a dairy cow genome, is 561 nucleotide of SEQ ID No.1 in a sequence table, and is C or T; SNP2 is one SNP of the genome of the dairy cow, is 170 th nucleotide of SEQ ID No.2 in a sequence table, and is C or T.

The SNP1 and SNP2 single nucleotide polymorphism sites are respectively positioned in an upstream 5 'regulatory region and a downstream 3' regulatory region of an ACOX2 gene on a No. 22 chromosome of a dairy cow genome, the ACOX2 gene is positioned at 42,836,492-42,867,389 th position of the No. 22 chromosome of the dairy cow and is related to milk production traits of the dairy cow, and the nucleotide sequence of the SNP1 single nucleotide polymorphism sites is a DNA molecule shown as the 2 nd, 001-32 nd 898 th position of a sequence A in a sequence table.

In order to solve the technical problem, the invention also provides an application, wherein the application is Q1 or Q2:

the Q1 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 ingredient character products;

the Q2 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 ingredient character products;

In order to solve the technical problem, the invention also provides an application, wherein the application is E1 or E2:

the E1 is the application of a substance for detecting the polymorphism or genotype of the SNP1 in cow breeding or preparing cow breeding products;

the E2 is the application of a substance for detecting the polymorphism or genotype of the SNP2 in cow breeding or preparing cow breeding products;

The dairy cow milk ingredient may be any one and/or a combination of a plurality of milk yield, milk fat amount, milk fat percentage, milk protein amount and milk protein percentage.

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

In order to solve the technical problems, the invention also provides a product which contains the substance for detecting the SNP1 polymorphism or genotype of the genome of the dairy cow or the substance for detecting the SNP2 polymorphism or genotype of the genome of the dairy cow and can be any one of the following G1) to G3):

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

g2 Identification or auxiliary identification of milk component character 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 component traits of cows, which comprises detecting genotypes of the SNP1 of the cows to be tested, and identifying or assisting in identifying milk component traits of the cows according to the genotypes of the SNP1 of the cows to be tested: the milk component characters of the dairy cows with genotype of CC or TC of SNP1 are higher than or candidate higher than those of the dairy cows with genotype of TT; the CC is homozygous with SNP1 as C; the TT is homozygous with SNP1 as T; the TC is heterozygous for SNP1 of C and T.

In order to solve the technical problems, the invention also provides a method for identifying or assisting in identifying milk component traits of cows, which comprises detecting genotypes of the SNP2 of the cows to be tested, and identifying or assisting in identifying milk component traits of the cows according to the genotypes of the SNP2 of the cows to be tested: the milk component characters of the dairy cows with genotype of CC or TC of SNP2 are higher than or candidate higher than those of the dairy cows with genotype of TT; the CC is homozygous with SNP2 as C; the TT is homozygous with SNP2 as T; the TC is heterozygous for SNP2 of C and T.

The invention also provides a method for breeding dairy cows, which comprises the steps of selecting dairy cows with genotype of SNP1 of CC or TC as parents for breeding; SNP1 is one SNP of a dairy cow genome, is 561 nucleotide of SEQ ID No.1 in a sequence table, and is C or T; the CC is homozygous in which the SNP1 is C; the TC is heterozygous for SNP1 of C and T.

The invention also provides a method for breeding dairy cows, which comprises the steps of selecting dairy cows with genotype of SNP2 of CC or TC as parents for breeding; the SNP2 is one SNP of a dairy cow genome, is 170 th nucleotide of SEQ ID No.2 in a sequence table, and is C or T; the CC is homozygous in which the SNP2 is C; the TC is heterozygous for SNP2 of C and T.

The breeding of cows is to cultivate cow varieties with high milk content.

The milk components described above may specifically be milk component amounts, milk fat fractions, milk protein amounts and/or milk protein fractions.

In the above application and method, the substance for detecting SNP1 polymorphism or genotype, or the substance for detecting SNP2 polymorphism or genotype may be a substance for determining the nucleotide type of SNP1 and/or SNP2 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 SNP1 polymorphism or genotype, or the substance for detecting SNP2 polymorphism or genotype may be the following D1), D2) or D3):

d1 A PCR primer for amplifying the cow genome DNA fragments including the SNP1 and/or SNP2 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 primer is a primer group consisting of single-stranded DNA shown in the 1 st to 19 th positions of SEQ ID No.1 and single-stranded DNA reversely complementary to the 726 th to 744 th positions of SEQ ID No.1 in the sequence table, and/or a primer group consisting of single-stranded DNA shown in the 1 st to 20 th positions of SEQ ID No.2 in the sequence table and single-stranded DNA reversely complementary to the 381 th to 400 th positions of SEQ ID No. 2.

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 can include the above-described substances for detecting polymorphisms or genotypes of SNP1 and/or SNP2 sites in the genome of a dairy cow.

In the application, the product or the method, the dairy cow is Chinese Holstein cow.

In the embodiment of the invention, through genetic variation analysis of ACOX2 genes in a dairy cow association group, 2 SNP are found, wherein SNP1 and SNP2 are positioned in genes ACOX2 genes related to milk production traits in a dairy cow genome, namely 561 th and 170 th positions of sequence table SEQ ID No.1 and SEQ ID No. 2. In the embodiment of the invention, the dominant allele of the SNP1 locus is C, and the dominant allele of the SNP2 locus is C, which indicates that the SNP1 locus and/or the SNP2 locus can be used for auxiliary selection breeding of dairy cow molecular markers and breeding of dairy cow varieties with high milk yield traits.

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 stock development limited in Peking head agriculture.

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.

Lactation 1 refers to the lactation after the first delivery. Lactation 2 refers to the lactation after the second delivery.

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). The ACOX2 gene was found to be significantly differentially expressed during the initial and peak lactation phases (P < 0.01) compared to the dry phase.

2. Gene polymorphism detection

1. 45 Chinese Holstein bull in Beijing area are selected as test groups for detecting gene polymorphism. The 45 Chinese Holstein bulls are randomly divided into 2 groups (a group of 22 bulls and a group of 23 bulls), genomic DNA of the frozen bull semen 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 2 pools of DNA in equal quantity, and the pool of DNA is used as a template for PCR amplification.

2. 21 pairs of primers as shown in Table 1 were designed based on the sequence of the bovine ACOX2 gene and the sequences of its upstream 5 'regulatory region and downstream 3' regulatory region (Ensembl ID is ENSBTAG00000004178, transcript ID is ENSBTAT00000005472.5, as shown in sequence A, wherein the ACOX2 gene sequence is at positions 2,001-32,898 of sequence A).

TABLE 1 ACOX2 Gene PCR amplification primer sequence information

3. And (2) taking the mixed pool DNAs obtained in the step (1) as templates, 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 2 PCR reaction System

TABLE 3 PCR reaction conditions

4. The PCR amplification product was sequenced. As a result, it was found that 1 SNP marker (referred to as SNP1 and SNP2, respectively) was present in the flanking sequences of 2000bp upstream and downstream, respectively, of the ACOX2 gene of the bull population. The 2 SNP markers are shown in Table 4.

TABLE 4ACOX2 Gene found 2 SNPs

Gene position	Name of the name	SNPs	Physical location	Polymorphic forms
					5' regulatory region	SNP1	g.42838675C>T	Chr22:42838675bp	C/T
3' regulatory region	SNP2	g.42869152T>C	Chr22:42869152bp	T/C

SNP1 corresponds to g.4283875C > T, is obtained by sequencing a product obtained by PCR amplification by using a primer pair consisting of 6F and 6R (the PCR amplification product is shown as 3624-4367 (1-744) of a sequence A, the nucleotide is C or T, and the nucleotide is shown as 4184 (561) of the sequence A from the 5' end (561) of the sequence A in the corresponding sequence table, and is expressed as Y). SNP2 corresponds to 42869152T > C, is obtained by sequencing a product obtained by PCR amplification by using a primer pair consisting of 21F and 21R (the PCR amplification product is shown as 34492-34891 of a sequence A, namely 1-400 of SEQ ID No. 2), the nucleotide is T or C, and the nucleotide corresponds to 34661 th from the 5' end of the sequence A in the sequence table (namely 170 th position of SEQ ID No. 2) and is expressed by Y.

3. Correlation analysis

(one) obtaining a test population

The test population consisted of 922 chinese holstein cows.

(II) genotyping

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

I. Genotyping based on g.4283875c > T.

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

2. Using genomic DNA as a template, PCR amplification was performed using a primer pair consisting of 6F (as shown at positions 3624-3642 of SEQ ID No.1, i.e., positions 1-19) and 6R (reverse complementary to positions 4349-4367 of SEQ ID No.1, i.e., reverse complementary to positions 726-744 of SEQ ID No. 1), and then the PCR amplified product was 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 tested individual are 744bp (shown as SEQ ID No. 1), wherein the 561 th position is g.4283875C > T, namely SNP1 (4184 th position from the 5' end of the sequence A in the corresponding sequence table, namely 561 th position of SEQ ID No. 1).

TABLE 5

TABLE 6

II. Genotyping based on g.42869152T > C.

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

2. Using genomic DNA as a template, PCR amplification was performed using a primer pair consisting of 21F (shown as 34492-34511 of SEQ ID No.2, i.e., 1-20) and 21R (reverse complement to 34872-34891 of SEQ ID No.2, i.e., 381-400 of SEQ ID No. 2), and then the PCR amplified 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 amplified products of each tested individual are 400bp (shown as SEQ ID No. 2), wherein the 170 th position is g.42869152T > C, namely SNP2 (corresponding to the 34661 th position from the 5' end of the sequence A in the sequence table, namely the 170 th position of SEQ ID No. 2).

TABLE 7

TABLE 8

(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 the SNP1 locus (i.e., ACOX2 gene g.4283875C > T) and the SNP2 locus (i.e., ACOX2 gene g.42869152T > C) are shown in Table 9, table 10 and Table 11.

Table 9 922 genotype of milk production phenotype and 2 SNP loci of Henstein Niu Muniu (part)

Table 10 922 presents descriptive statistics of 5 milk production trait values for Chinese Holstein Niu Muniu populations

Traits (3)	Average value of	Standard deviation of	Minimum value	Maximum value	Coefficient of variation
						Milk yield (kg)	10421.71	1472.83	6057.96	14505.68	0.14
Milk fat quantity (kg)	353.05	60.78	184.80	518.18	0.17
						Milk fat percentage (%)	3.40	0.42	2.15	4.56	0.12
Milk protein quantity (kg)	316.30	47.68	178.36	457.53	0.15
						Milk protein yield (%)	3.04	0.19	2.35	3.51	0.06

TABLE 11 allele frequencies and genotype frequencies of 2 SNP loci of ACOX2 Gene

The SNP1 locus has 3 genotypes (SNP 1 genotype for short), namely CC, TT or TC, the genotype CC is homozygous with SNP1 as C, the genotype TT is homozygous with SNP1 as T, and the genotype TC is heterozygous with SNP1 as C and T; the SNP2 locus has 3 genotypes (SNP 2 genotypes for short), namely CC, TT or TC, the genotype CC is homozygous with SNP2 as C, the genotype TT is homozygous with SNP2 as T, and the genotype TC is heterozygous with SNP2 as T and C.

Five indicators of milk production traits and genotypes were analyzed in association using the MIXED procedure in SAS 9.4 software. The correlation analysis adopts an animal model, and the concrete model is as follows:

Y＝μ+hys+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.

The results of SNP1 locus (i.e., ACOX2 gene g.4283875C > T) and milk production trait association analysis are shown in Table 12. The number of cows producing milk in lactation 1 is 922 and the number of cows producing milk in lactation 2 is 681.

TABLE 12 correlation analysis of ACOX2 Gene g.4283875C > T and milk production Property (least squares mean.+ -. Standard error)

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 12, at the first lactation, SNP1 reached significant or very significant levels of correlation with milk yield, milk fat and milk protein (p= 0.0403-p=0.0006). At the second lactation, SNP1 reached very significant levels of correlation with milk yield, milk fat and milk protein (P < 0.0001).

For SNP1, i.e., ACOX2 gene g.4283875C > T, the milk yield of CC genotype cows is significantly higher than that of TT genotype cows, the milk yield of TC genotype cows is significantly higher than that of TT genotype cows, and the milk yield of CC genotype cows has no significant difference from that of TC genotype cows. The milk yield is that of lactation period 1.

For SNP1, i.e., ACOX2 gene g.4283875C > T, the milk fat content of CC genotype cows is higher than that of TT genotype cows, and the milk fat content of TC genotype cows is higher than that of TT genotype cows. The milk fat amount is the milk fat amount of lactation period 1.

For SNP1, i.e., ACOX2 gene g.4283875c > T, the milk protein amount of the CC genotype cows was significantly higher than the TT genotype cows and the milk protein amount of the TC genotype cows was significantly higher than the TT genotype cows, and the milk protein amount of the CC genotype cows was not significantly different from the TC genotype cows. The milk protein amount is the milk protein amount in lactation period 1.

For SNP1, i.e., ACOX2 gene g.4283875C > T, the milk yield of CC genotype cows is significantly higher than that of TT genotype cows, the milk yield of TC genotype cows is significantly higher than that of TT genotype cows, and the milk yield of CC genotype cows has no significant difference from that of TC genotype cows. The milk yield is that of lactation period 2.

For SNP1, i.e. ACOX2 gene g.4283875c > T, the milk fat mass of the CC genotype cows was significantly higher than the TT genotype cows, the milk fat mass of the TC genotype cows was significantly higher than the TT genotype cows, and the milk fat mass of the CC genotype cows was not significantly different from the TC genotype cows. The milk fat amount is the milk fat amount of lactation period 2.

For SNP1, i.e., ACOX2 gene g.4283875c > T, the milk protein amount of the CC genotype cows was significantly higher than the TT genotype cows, the milk protein amount of the TC genotype cows was significantly higher than the TT genotype cows, and the milk protein amount of the CC genotype cows was not significantly different from the TC genotype cows. The milk protein amount is milk protein amount in lactation period 2.

The analysis results of the association of SNP2 locus (namely ACOX2 gene g.42869152T > C) and milk production characteristics are shown in Table 13. The number of cows producing milk in lactation 1 is 922 and the number of cows producing milk in lactation 2 is 681.

TABLE 13 ACOX2 Gene g.42869152T > C and milk production trait correlation analysis (least squares mean.+ -. Standard error)

As can be seen from table 13, at the first lactation, SNP2 reached a significant correlation level with milk protein amount (p=0.0165). At the second lactation period, SNP site 2 reached very significant levels of correlation with milk yield, milk fat and milk protein (P < 0.0001), and SNP2 reached significant levels of milk fat percentage (p=0.0117).

For SNP2, i.e., ACOX2 gene g.42869152T > C, the milk protein amount of TT genotype cows is higher than that of TC genotype cows. The milk protein amount is the milk protein amount in lactation period 1.

For SNP2, i.e., ACOX2 gene g.42869152t > C, milk yield of the CC genotype cows is significantly higher than that of the TT genotype cows, and milk yield of the TC genotype cows is significantly higher than that of the TT genotype cows. The milk yield is that of lactation period 2.

For SNP2, i.e. ACOX2 gene g.42869152t > C, the milk fat mass of the CC genotype cows was significantly higher than the TT genotype cows, the milk fat mass of the TC genotype cows was significantly higher than the TT genotype cows, and the milk fat mass of the CC genotype cows was not significantly different from the TC genotype cows. The milk fat amount is the milk fat amount of lactation period 2.

For SNP2, i.e. ACOX2 gene g.42869152t > C, the milk fat percentage of the cow of the CC genotype is very significantly higher than that of the cow of the TC genotype, and the cow of the CC genotype is higher than that of the cow of the TT genotype. The milk fat ratio is that of lactation period 2.

For SNP2, i.e., ACOX2 gene g.42869152t > C, the milk protein amount of the CC genotype cows is significantly higher than the TT genotype cows and the milk protein amount of the TC genotype cows is significantly higher than the TT genotype cows. The milk protein amount is milk protein amount in lactation period 2.

(V) genetic Effect analysis

And (3) performing significance test on SNP additive effect, dominant effect and substitution effect by using SAS 9.4 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 14.

TABLE 14ACOX2 Gene allele additive, dominant, and substitution results

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

For the first lactation period data: the additive effect and the allele substitution effect of the SNP1 locus are mainly reflected on milk yield, milk fat quantity and milk protein quantity traits, and the additive effect and the allele substitution effect of the SNP2 locus are mainly reflected on milk yield and milk protein quantity. Taking the SNP1 (g.4283875C > T) locus as an example, the allele additive effect and the allele substitution effect on milk yield, the allele additive effect and the allele substitution effect on milk fat yield, the additive effect and the allele substitution effect on milk protein yield reach remarkable or extremely remarkable, namely, each C allele substituted T allele can lead to the increase of 190.49kg (P < 0.01) of milk yield, the increase of 6.28kg (P < 0.05) of milk fat yield and the increase of 6.475kg (P < 0.01) of milk protein yield.

For the second lactation data: the additive effect, the allelic dominant effect and the allelic substitution effect of 2 SNP loci are mainly expressed on milk yield, milk fat content and milk protein content traits, and by taking SNP2 (g.42869152T > C) loci as an example, the additive effect, the dominant effect and the allelic substitution effect of the alleles on milk yield, the additive effect, the dominant effect and the allelic substitution effect of the alleles and the allelic substitution effect of the milk fat content are extremely remarkable, namely, each C allele substitution T allele can lead to 406.18kg (P < 0.01) of milk fat content, 13.891kg (P < 0.01) of milk fat content, 12.364kg (P < 0.01) of milk protein content.

The molecular marker disclosed by the invention can be applied to auxiliary identification of dairy cow groups with excellent milk production characteristics (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

Sequence A (SEQ ID No.1 in italics, SEQ ID No.2 in bold)

Claims

1. The application is characterized in that: the application is the following P1 or P2:

2. The application is characterized in that: the application is Q1 or Q2:

3. The application is characterized in that: the application is E1 or E2:

4. The product is characterized in that: the product contains the substance for detecting the polymorphism or genotype of the genome SNP1 of the dairy cow as set forth in claim 1 or the substance for detecting the polymorphism or genotype of the genome SNP2 of the dairy cow as set forth in claim 1, and can be any one of the following G1) -G3):

g3 A product for breeding dairy cows.

5. The method for breeding the dairy cows is characterized by comprising the following steps of: the method comprises the steps of selecting cows with genotype CC or TC of SNP1 as parents for breeding; SNP1 is one SNP of a dairy cow genome, is 561 nucleotide of SEQ ID No.1 in a sequence table, and is C or T; the CC is homozygous in which the SNP1 is C; the TC is heterozygous for SNP1 of C and T.

6. The method for breeding the dairy cows is characterized by comprising the following steps of: the method comprises the steps of selecting cows with genotype of CC or TC of SNP2 as parents for breeding; the SNP2 is one SNP of a dairy cow genome, is 170 th nucleotide of SEQ ID No.2 in a sequence table, and is C or T; the CC is homozygous in which the SNP2 is C; the TC is heterozygous for SNP2 of C and T.

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

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

d2 A PCR reagent comprising D1) the PCR primer;

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

9. The use or product or method according to claim 8, wherein: the PCR primer is a primer group consisting of single-stranded DNA shown in the 1 st-19 th positions of SEQ ID No.1 and single-stranded DNA reversely complementary to the 726 th-744 th positions of SEQ ID No.1 in the sequence table, and/or a primer group consisting of single-stranded DNA shown in the 1 st-20 th positions of SEQ ID No.2 in the sequence table and single-stranded DNA reversely complementary to the 381 th-400 th positions of SEQ ID No. 2.

10. The use or product or method according to claim 8 or 9, characterized in that: the milk cow is Chinese Holstein cow.