A method for evaluating milk quality traits of Chinese Holstein cows

Abstract

The present invention discloses a method for evaluating the milk quality traits of Chinese Holstein cows. The present invention finds two SNPs sites in Chinese Holstein cows that are significantly correlated with the milk protein content, milk fat content and lactose content in the milk quality traits of dairy cows, and also finds that there is a chain reaction between the two SNPs sites. The present invention provides a simple and effective method for screening molecular markers closely related to the milk quality traits, and provides a basis for building a genetically stable high-quality dairy cow herd.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular breeding of dairy cows, and in particular to a method for evaluating milk quality traits of Chinese Holstein dairy cows. Background Art

[0002] my country is one of the world's largest milk consumers. As domestic dairy farming gradually becomes large-scale, standardized and modernized, milk production efficiency is also constantly improving. However, the quality and processing technology of domestic milk are still somewhat behind those of developed countries such as Europe. Dairy cow production performance measurement (Dairy Herd Improvement, DHI) is a measurement of dairy cow lactation performance and milk composition (fat rate, milk protein rate, dry matter, urea nitrogen, etc.). It is the core basic work for improving the production performance and genetic performance of dairy cows, and is also a leading technology for the transformation and upgrading of my country's dairy industry.

[0003] Single nucleotide polymorphism (SNP) is a genetic polymorphism marker with wide distribution, large number and high marker density. It is a molecular marker technology currently widely used in population genetics research due to its high genetic stability, good reproducibility of genetic analysis, high accuracy and easy and rapid high-throughput genotyping.

[0004] Interferon alpha-inducible protein 27-like protein 2A (IFI27 or ISG12a) was first discovered in estrogen-induced human breast cancer cells MCF 17, and can be induced by interferon alpha in a variety of human cell lines and expressed efficiently. Later, due to the presence of a conserved ISG12 motif, it was classified into the ISG12 gene family and renamed interferon-stimulated gene 12a (ISG12a). Because ISG12a expresses a protein of about 122 amino acids and has a 33% overlap with ZSG16, it is also classified into the FAM14 family. IFI27 (ISG12A) protein is localized in mitochondria, it mediates type I interferon (IFN)-induced cell apoptosis, and is currently widely concerned as a biomarker in the direction of immunotherapy, viral infection, etc., but research on the IFI27 gene as a molecular marker related to dairy cow milk quality traits has not yet been reported.

[0005] At present, studies have confirmed that the IFI27 (ISG12A) gene plays a role in adipocytes. Studies have shown that IFI27 (ISG12A) can regulate mitochondrial function and browning in adipocytes. The expression of IFI27 can be induced in adipose tissue by b3-adrenergic activation. Ifi27 gene knockout leads to reduced expression of key enzymes of the tricarboxylic acid cycle (TCA), electron transport chain subunits (ETC), and uncoupling protein 1 (Ucp1) in brown and beige adipocytes. In addition, the browning of subcutaneous white fat caused by b3-adrenergic agonists was also significantly prevented. There is no report that this gene is related to the lactation performance and milk composition of dairy cows. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a method for evaluating milk quality traits of Chinese Holstein cows.

[0007] The first object of the present invention is to provide a method for evaluating milk quality traits of Chinese Holstein cows.

[0008] The second object of the present invention is to provide a reagent for detecting the genotype of one or two of the molecular markers for use in evaluating milk quality traits of Chinese Holstein cows and / or preparing a kit for evaluating milk quality traits of Chinese Holstein cows.

[0009] The third object of the present invention is to provide a kit for evaluating the milk quality traits of Chinese Holstein cows.

[0010] The fourth object of the present invention is to provide the use of the method, the reagent, and / or the kit in molecular breeding of milk quality traits of Chinese Holstein cows.

[0011] In order to achieve the above object, the present invention is implemented by the following scheme:

[0012] A method for evaluating milk quality traits of Chinese Holstein cows, detecting one or two of the following molecular markers:

[0013] SNP site 1 is located at NC_037348.1:58736407, i.e., position 58736407 on chromosome 21 of the ARS-UCD1.2 version of the bovine genome;

[0014] SNP site 2 is located at NC_037348.1:58736566, which is position 58736566 on chromosome 21 of the bovine genome in the ARS-UCD1.2 version.

[0015] Preferably, the milk quality traits are milk protein, milk fat and / or lactose.

[0016] Preferably, the milk protein content in the milk quality trait of individual dairy cows whose genotype at SNP site 1 is GT is significantly higher than the milk protein content in the milk quality trait of individual dairy cows whose genotype is GG.

[0017] Preferably, the lactose content in the milk quality trait of individual dairy cows whose genotype at SNP site 1 is GG is significantly higher than that of individual dairy cows whose genotype is GT.

[0018] Preferably, the milk fat content in the milk quality trait of individual dairy cows with AG genotype at SNP locus 2 is Significant Higher than the milk fat content in the milk quality trait of individual dairy cows with GG genotype.

[0019] Preferably, the lactose content in the milk quality trait of individual dairy cows whose genotype at SNP site 2 is AG is higher than the lactose content in the milk quality trait of individual dairy cows whose genotype is GG.

[0020] Preferably, the milk fat content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 is GG and whose genotype at SNP site 2 is AG is significantly higher than the milk fat content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 is GG and whose genotype at SNP site 2 is GG.

[0021] Preferably, the milk protein content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 is GT and whose genotype at SNP site 2 is AG is significantly higher than the milk protein content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 is GG and whose genotype at SNP site 2 is GG.

[0022] More preferably, primers having nucleotide sequences as shown in SEQ ID NOs: 1 to 2 are used to detect one or both of the molecular markers.

[0023] More preferably, the DNA of the sample to be tested is amplified by PCR using primers with nucleotide sequences such as those shown in SEQ ID NOs: 1 to 2, and then sequenced.

[0024] More preferably, the PCR amplification system is: Green Taq Mix 10 μL, upstream primer (shown in SEQ ID NO: 1) 0.5 μL, downstream primer (shown in SEQ ID NO: 2) 0.5 μL, DNA template 1.0 μL, ddH2O 8 μL, and a total volume of 20 μL.

[0025] More preferably, the PCR amplification program is: 95°C for 5 min; 95°C for 30 s, 52°C for 45 s, 72°C for 90 s, 40 cycles; 72°C for 5 min.

[0026] The present invention also claims the use of a reagent for detecting the genotype of one or two of the following molecular markers in evaluating the milk quality traits of Chinese Holstein cows, and / or in preparing a kit for evaluating the milk quality traits of Chinese Holstein cows,

[0027] SNP site 1 is located at NC_037348.1:58736407, i.e., position 58736407 on chromosome 21 of the ARS-UCD1.2 version of the bovine genome;

[0028] SNP site 2 is located at NC_037348.1:58736566, which is position 58736566 on chromosome 21 of the bovine genome in the ARS-UCD1.2 version.

[0029] The present invention also claims a kit for evaluating the milk quality traits of Chinese Holstein cows, wherein the kit contains the reagent.

[0030] Preferably, the reagent is a primer.

[0031] More preferably, the nucleotide sequence of the primer is as shown in SEQ ID NO: 1 to 2.

[0032] The upstream sequence F is: 5'-GATTTCCTAATTCTGGCACCT-3' (SEQ ID NO: 1);

[0033] The downstream sequence R is: 5'-GGAAATGGACCTGAATTGAAGA-3' (SEQ ID NO: 2).

[0034] More preferably, the DNA of the sample to be tested is amplified by PCR using primers with nucleotide sequences such as those shown in SEQ ID NOs: 1 to 2, and then sequenced.

[0035] More preferably, the PCR amplification system is: Green Taq Mix 10 μL, upstream primer (shown in SEQ ID NO: 1) 0.5 μL, downstream primer (shown in SEQ ID NO: 2) 0.5 μL, DNA template 1.0 μL, ddH2O 8 μL, and a total volume of 20 μL.

[0036] More preferably, the PCR amplification program is: 95°C for 5 min; 95°C for 30 s, 52°C for 45 s, 72°C for 90 s, 40 cycles; 72°C for 5 min.

[0037] The results are interpreted as follows:

[0038] The sequencing results were compared with the ARS-UCD1.2 version of the cattle genome to determine the genotypes of the relevant loci.

[0039] SNP site 1 (I1-763 G>T) has two genotypes, GG and GT, located at NC_037348.1:58736407, i.e., position 58736407 of chromosome 21 of the ARS-UCD1.2 version of the bovine genome. The milk protein content of individual milk quality traits of dairy cows with GT genotype is higher than that of individual milk quality traits of dairy cows with GG genotype, and the lactose content of individual milk quality traits of dairy cows with GG genotype is higher than that of individual milk quality traits of dairy cows with GT genotype.

[0040] SNP site 2 (E2-77 A>G) has three genotypes: AA, AG, and GG. It is located at NC_037348.1:58736566, i.e., position 58736566 of chromosome 21 of the ARS-UCD1.2 version of the bovine genome. The milk fat content of individual dairy cows with AG genotype is Significant The milk fat content in the milk quality traits of individual dairy cows with the GG genotype is higher than that of individual dairy cows with the AG genotype. The lactose content in the milk quality traits of individual dairy cows with the AG genotype is higher than that of individual dairy cows with the GG genotype.

[0041] The milk fat content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 (I1-763 G>T) is GG and whose genotype at SNP site 2 (E2-77 A>G) is AG is significantly higher than that in the milk fat content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 (I1-763 G>T) is GG and whose genotype at SNP site 2 (E2-77 A>G) is GG.

[0042] The milk protein content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 (I1-763 G>T) is GT and whose genotype at SNP site 2 (E2-77 A>G) is AG is significantly higher than that of individual dairy cows whose genotype at SNP site 1 (I1-763 G>T) is GG and whose genotype at SNP site 2 (E2-77 A>G) is GG.

[0043] The use of the method, the reagent, and / or the kit in molecular breeding of milk quality traits of Chinese Holstein cows also falls within the protection scope of the present invention.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The present invention finds two SNPs sites in Chinese Holstein cows that are significantly correlated with milk protein content, milk fat content and lactose content in the milk quality traits of dairy cows, and also finds that there is a chain reaction between the two SNPs sites. The present invention provides a simple and effective method for screening molecular markers closely related to milk quality traits, and provides a basis for building a genetically stable high-quality dairy cow herd. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The results of PCR amplification of IFI27 gene in Chinese Holstein cows; M: DL-2 000 Marker; 1-5: PCR amplification products.

[0047] Figure 2 It is the sequencing map of two genotypes of SNP site 1 (I1-763 G>T) of IFI27 gene; Figure Ⅰ is an individual with genotype GG; Figure II It is an individual with genotype GT.

[0048] Figure 3 It is the sequencing map of three genotypes of SNP site 2 (E2-77 A>G) of IFI27 gene; Figure Ⅰ is an individual with genotype AA; Figure II is an individual with genotype AG; Figure III It is an individual with genotype AA.

[0049] Figure 4 This is the SNPs locus linkage reaction of the IFI271 gene in the Chinese Holstein cow population. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0051] Example 1 Amplification of IFI27 gene and screening of SNPs

[0052] 1. Experimental Methods

[0053] 126 Chinese Holstein cows that were grown in the same environment and in lactation were selected, and blood samples were collected by jugular vein blood sampling, and the blood samples were stored at -80°C. Blood genomic DNA was extracted using a blood DNA extraction kit (Beijing Tiangen), and the DNA was stored at -20°C.

[0054] 2. Experimental Results

[0055] 1. Primer design

[0056] A pair of primers was designed based on the bovine IFI27 gene sequence published in Ensembl (ENSBTAG00000003152) and synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0057] Wherein, the upstream sequence F is: 5'-GATTTCCTAATTCTGGCACCT-3' (SEQ ID NO: 1);

[0058] The downstream sequence R is: 5'-GGAAATGGACCTGAATTGAAGA-3' (SEQ ID NO: 2).

[0059] 2. PCR amplification and sequencing

[0060] PCR amplification was performed using blood genomic DNA from 126 Chinese Holstein cows as templates. The PCR system is shown in Table 1. The PCR amplification procedure is shown in Table 2. The PCR product was detected by 1.5% agarose electrophoresis under the conditions of 120V, 400mA, and 15min. After confirming that the band length was correct, single and without smear bands, and no specific bands, it was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0061] Table 1 PCR reaction system:

[0062]

[0063] Table 2 PCR reaction program:

[0064]

[0065] 3. Results Analysis

[0066] Seq Man software was used to observe overlapping peaks in the sequencing results.

[0067] 3. Experimental Results

[0068] PCR amplification electrophoresis results are as follows Figure 1 The length of the amplified target gene sequence was 869 bp. The results showed that the length of the electrophoresis band was consistent with expectations, and it was a single bright band without primer dimers and non-specific bands.

[0069] The sequencing results of the PCR amplification products revealed two overlapping peaks. The genotyping results of each site are as follows Figure 2 and Figure 3 As shown:

[0070] SNP site 1 (I1-763 G>T, Figure 2) There are two genotypes, GG and GT, located at NC_037348.1:58736407, which is the 58736407th position of chromosome 21 of the ARS-UCD1.2 version of the bovine genome.

[0071] SNP site 2 (E2-77 A>G, Figure 3 ) There are three genotypes: AA, AG and GG, located at NC_037348.1:58736566, which is the 58736566th position of chromosome 21 of the bovine genome of the ARS-UCD1.2 version.

[0072] Example 2 Population genetic characteristics of IFI27 gene SNPs sites

[0073] 1. Experimental Methods

[0074] Popgen32 was used to count the gene frequency, genotype frequency, effective number of alleles (Ne), and genetic heterozygosity (H) of the 126 Chinese Holstein cows in Example 1; the polymorphic information content (PIC) was calculated using PIC software; and χ 2 SPSS 23.0 software was used to perform a significance test on the differences between SNPs and dairy cow milk quality traits (P<0.05 indicated a significant difference, and the trait value was expressed as “mean ± standard error”).

[0075] 2. Experimental Results

[0076] The results of the population genetic characteristics analysis of SNPs are shown in Table 3.

[0077] Table 3 Results of population genetic characteristics analysis of IFI27 gene SNPs

[0078]

[0079] Note: 2 The value did not reach the significant level (P>0.05), χ 2 0.05 (df ​​= 2) = 5.99, χ 2 0.01 (df=2)=9.21.

[0080] The gene frequencies of alleles G and T at SNP site 1 (I1-763 G>T) were 0.9087 and 0.0913, respectively, and the dominant allele was G. The genotype frequencies of genotypes GG and GT were 0.8175 and 0.1825, respectively, and the dominant genotype was GG. The gene heterozygosity (H) and polymorphism information content (PIC) were both less than 0.25, indicating that this site was low polymorphic in the IFI27 gene.

[0081] The gene frequencies of alleles A and G at SNP site 2 (E2-77 A>G) were 0.3333 and 0.6667, respectively, and the dominant allele was G. The genotype frequencies of genotypes AA, AG and GG were 0.1270, 0.4127 and 0.4603, respectively, and the dominant genotype was GG. The gene heterozygosity (H) and polymorphism information content (PIC) were both between 0.25 and 0.50, indicating that this site was moderately polymorphic in the IFI27 gene.

[0082] The chi-square test showed that the two SNPs loci were in Hardy-Weinberg equilibrium in the Chinese Holstein dairy cow population (P>0.05). Example 3 Correlation analysis between IFI27 gene SNPs loci and dairy cow milk quality traits

[0083] 1. Experimental Methods

[0084] The milk quality traits of the milk produced by the 126 dairy cows in Example 1 were detected by a milk component analyzer, mainly including indicators such as milk yield, milk fat, milk protein, lactose, dry matter, somatic cells, urea nitrogen, and corrected milk. SPSS 23.0 software was used to perform a significant difference test between SNPs sites and dairy cow milk quality traits (P<0.05 indicated a significant difference, and the trait value was expressed as "mean ± standard error"), and the association between different genotypes of the IFI27 gene SNPs sites and dairy cow milk quality traits was analyzed.

[0085] 2. Experimental Results

[0086] The correlation analysis between IFI27 gene SNPs and milk fat traits of Chinese Holstein cows is shown in Table 4.

[0087] Table 4 Correlation analysis between IFI27 gene SNPs and milk quality traits in dairy cows

[0088]

[0089] Note: Different lowercase letters in the same index indicate significant differences (P<0.05), and no letters indicate no significant differences (P>0.05)

[0090] SNP site 1 (I1-763 G>T) was significantly correlated with the milk protein content and lactose content in the milk quality traits of dairy cows (P<0.05). The milk protein content in the milk quality traits of dairy cows with GT genotype was higher than that of dairy cows with GG genotype, and the lactose content in the milk quality traits of dairy cows with GG genotype was higher than that of dairy cows with GT genotype.

[0091] SNP locus 2 (E2-77 A>G) was significantly correlated with milk fat content and milk protein content in dairy cows' milk quality traits (P<0.05). Significant The milk fat content in the milk quality traits of individual dairy cows with the GG genotype is higher than the milk protein content in the milk quality traits of individual dairy cows with the AG genotype is higher than the milk protein content in the milk quality traits of individual dairy cows with the GG genotype.

[0092] Example 4 Correlation analysis between IFI27 gene SNPs and cow milk quality traits

[0093] 1. Experimental Methods

[0094] Haplo View 4.2 software was used to perform linkage analysis on the SNPs loci of the 126 Chinese Holstein cows in Example 1; SPSS 23.0 software was used to perform a significance test on the difference between SNPs haplotypes and dairy cow milk quality traits (P<0.05 indicated a significant difference, and the trait value was expressed as "mean ± standard error"), and the association between the IFI27 gene SNPs haplotype and dairy cow milk quality traits was analyzed.

[0095] 1. Experimental Results

[0096] The analysis found that there was a strong linkage relationship between SNP site 1 (I1-763 G>T) and SNP site 2 (E2-77 A>G), such as Figure 4 As shown. SNP site 1 (I1-763 G>T) and SNP site 2 (E2-77 A>G) form a haplotype domain, with a total of 5 different haplotypes with biological statistical significance (number of individuals n≥3), namely H1H1 (I1-763G>T genotype is GG type and E2-77 A>G genotype is GG type), H1H2 (I1-763 G>T genotype is GG type and E2-77 A>G genotype is AG type), H1H3 (I1-763 G>T genotype is GT type and E2-77 A>G genotype is AG type), H2H2 (I1-763 G>T genotype is GG type and E2-77 A>G genotype is AA type), H2H3 (I1-763G>T genotype is GT type and E2-77 A>G genotype is AA type).

[0097] The results of correlation analysis between IFI27 gene SNPs haplotypes and milk fat traits of Chinese Holstein cows are shown in Table 5.

[0098] Table 5 Correlation analysis between IFI27 gene SNPs haplotypes and milk fat traits in Chinese Holstein cows

[0099]

[0100] Note: Different lowercase letters in the same index indicate significant differences (P<0.05), and no letters indicate no significant differences (P>0.05)

[0101] Among them, the milk fat content in the milk quality traits of individual dairy cows with H1H2 (I1-763 G>T genotype is GG type and E2-77 A>G genotype is AG type) haplotype was significantly higher than that with H1H1 (I1-763 G>T genotype is GG type and E2-77 A>G genotype is GG type) haplotype (P<0.05), and the milk protein content in the milk quality traits of individual dairy cows with H1H3 (I1-763 G>T genotype is GT type and E2-77 A>G genotype is AG type) haplotype was significantly higher than that with H1H1 (I1-763 G>T genotype is GG type and E2-77A>G genotype is GG type) haplotype (P<0.05).

[0102] Example 5 A kit for detecting and / or evaluating cow milk quality traits

[0103] 1. Composition

[0104] Primers, ddH2O and Taq PCR Mix with nucleotide sequences as shown in SEQ ID NO: 1-2.

[0105] 2. Usage

[0106] Extract DNA from the sample to be tested and perform PCR testing.

[0107] The PCR amplification system was: Green Taq Mix 10 μL, upstream primer (shown in SEQ ID NO: 1) 0.5 μL, downstream primer (shown in SEQ ID NO: 2) 0.5 μL, DNA template 1.0 μL, ddH2O 8 μL, and a total volume of 20 μL.

[0108] The PCR amplification program was: 95°C for 5 min; 95°C for 30 s, 52°C for 45 s, 72°C for 90 s, 40 cycles; 72°C for 5 min.

[0109] The PCR products were detected by 1.5% agarose gel electrophoresis under the conditions of 120V, 400mA, 15min. After confirming that the band length was correct, single and without smear bands, and no specific bands, sequencing was performed.

[0110] 3. Interpretation of results

[0111] The sequencing results were compared with the ARS-UCD1.2 version of the cattle genome to determine the genotypes of the relevant loci.

[0112] SNP site 1 (I1-763 G>T) has two genotypes, GG and GT, located at NC_037348.1:58736407, i.e., position 58736407 of chromosome 21 of the ARS-UCD1.2 version of the bovine genome. The milk protein content of individual milk quality traits of dairy cows with GT genotype is higher than that of individual milk quality traits of dairy cows with GG genotype, and the lactose content of individual milk quality traits of dairy cows with GG genotype is higher than that of individual milk quality traits of dairy cows with GT genotype.

[0113] SNP site 2 (E2-77 A>G) has three genotypes: AA, AG, and GG. It is located at NC_037348.1:58736566, i.e., position 58736566 of chromosome 21 of the ARS-UCD1.2 version of the bovine genome. The milk fat content of individual dairy cows with AG genotype is Significant The milk fat content in the milk quality traits of individual dairy cows with the GG genotype is higher than that of individual dairy cows with the AG genotype. The lactose content in the milk quality traits of individual dairy cows with the AG genotype is higher than that of individual dairy cows with the GG genotype.

[0114] The milk fat content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 (I1-763 G>T) is GG and whose genotype at SNP site 2 (E2-77 A>G) is AG is significantly higher than that in the milk fat content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 (I1-763 G>T) is GG and whose genotype at SNP site 2 (E2-77 A>G) is GG.

[0115] The milk protein content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 (I1-763 G>T) is GT and whose genotype at SNP site 2 (E2-77 A>G) is AG is significantly higher than that of individual dairy cows whose genotype at SNP site 1 (I1-763 G>T) is GG and whose genotype at SNP site 2 (E2-77 A>G) is GG.

[0116] Example 6 A method for detecting and / or evaluating cow milk quality traits

[0117] 1. Methods

[0118] Same as "2. Usage" in Example 5.

[0119] 2. Interpretation of results

[0120] Same as "III. Result Interpretation" in Example 5.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for evaluating milk quality traits of Chinese Holstein cows, characterized in that: Detects one or both of the following molecular markers: SNP site 1 is located at NC_037348.1:58736407, i.e., position 58736407 on chromosome 21 of the ARS-UCD2.0 version of the bovine genome; SNP site 2 is located at NC_037348.1:58736566, i.e., position 58736566 on chromosome 21 of the bovine genome in the ARS-UCD2.0 version; The milk protein content in the milk quality trait of individual dairy cows with GT genotype at SNP site 1 was significantly higher than that of individual dairy cows with GG genotype. The lactose content in the milk quality traits of individual dairy cows with the GG genotype at SNP site 1 was significantly higher than that of individual dairy cows with the GT genotype. Milk fat content in individual milk quality traits of dairy cows with AG genotype at SNP locus 2 Significant The milk fat content in the milk quality trait of individual dairy cows with GG genotype is higher than that of dairy cows with GG genotype; The lactose content in the milk quality traits of individual cows with AG genotype at SNP locus 2 was higher than that of individual cows with GG genotype. The milk fat content in the milk quality trait of individual dairy cows with SNP site 1 genotype of GG and SNP site 2 genotype of AG was significantly higher than that of individual dairy cows with SNP site 1 genotype of GG and SNP site 2 genotype of GG. The milk protein content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 is GT and whose genotype at SNP site 2 is AG is significantly higher than that in the milk protein content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 is GG and whose genotype at SNP site 2 is GG.

2. Use of a reagent for detecting the genotype of one or two of the following molecular markers in evaluating the milk quality traits of Chinese Holstein cows, and / or in preparing a kit for evaluating the milk quality traits of Chinese Holstein cows, characterized in that: SNP site 1 is located at NC_037348.1:58736407, i.e., position 58736407 on chromosome 21 of the ARS-UCD2.0 version of the bovine genome; SNP site 2 is located at NC_037348.1:58736566, i.e., position 58736566 on chromosome 21 of the bovine genome in the ARS-UCD2.0 version; The milk protein content in the milk quality trait of individual dairy cows with GT genotype at SNP site 1 was significantly higher than that of individual dairy cows with GG genotype. The lactose content in the milk quality traits of individual dairy cows with the GG genotype at SNP site 1 was significantly higher than that of individual dairy cows with the GT genotype. Milk fat content in individual milk quality traits of dairy cows with AG genotype at SNP locus 2 Significant The milk fat content in the milk quality trait of individual dairy cows with GG genotype is higher than that of dairy cows with GG genotype; The lactose content in the milk quality traits of individual cows with AG genotype at SNP locus 2 was higher than that of individual cows with GG genotype. The milk fat content in the milk quality trait of individual dairy cows with SNP site 1 genotype of GG and SNP site 2 genotype of AG was significantly higher than that of individual dairy cows with SNP site 1 genotype of GG and SNP site 2 genotype of GG. The milk protein content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 is GT and whose genotype at SNP site 2 is AG is significantly higher than that in the milk protein content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 is GG and whose genotype at SNP site 2 is GG.

3. The application according to claim 2, characterized in that: The reagent is a primer with a nucleotide sequence as shown in SEQ ID NO: 1 to 2.

4. A kit for evaluating the milk quality traits of Chinese Holstein cows, characterized in that: The kit contains the reagent described in claim 3.

5. Application of the method according to claim 1 in molecular breeding of milk quality traits of Chinese Holstein cows, characterized in that: The milk quality traits are one or more of milk protein content, milk fat content and milk protein content.

6. Application of a reagent in molecular breeding of milk quality traits of Chinese Holstein cows, characterized in that: The milk quality traits are one or more of milk protein content, milk fat content and milk protein content; The reagent detects the genotype of one or two of the following molecular markers: SNP site 1 is located at NC_037348.1:58736407, i.e., position 58736407 on chromosome 21 of the ARS-UCD2.0 version of the bovine genome; SNP site 2 is located at NC_037348.1:58736566, i.e., position 58736566 on chromosome 21 of the bovine genome in the ARS-UCD2.0 version; The milk protein content in the milk quality trait of individual dairy cows with GT genotype at SNP site 1 was significantly higher than that of individual dairy cows with GG genotype. The lactose content in the milk quality traits of individual dairy cows with the GG genotype at SNP site 1 was significantly higher than that of individual dairy cows with the GT genotype. Milk fat content in individual milk quality traits of dairy cows with AG genotype at SNP locus 2 Significant The milk fat content in the milk quality trait of individual dairy cows with GG genotype is higher than that of dairy cows with GG genotype; The lactose content in the milk quality traits of individual cows with AG genotype at SNP locus 2 was higher than that of individual cows with GG genotype. The milk fat content in the milk quality trait of individual dairy cows with SNP site 1 genotype of GG and SNP site 2 genotype of AG was significantly higher than that of individual dairy cows with SNP site 1 genotype of GG and SNP site 2 genotype of GG. The milk protein content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 is GT and whose genotype at SNP site 2 is AG is significantly higher than that in the milk protein content in the milk quality traits of individual dairy cows whose genotype at SNP site 1 is GG and whose genotype at SNP site 2 is GG.

7. Use of the kit according to claim 4 in molecular breeding of milk quality traits of Chinese Holstein cows, characterized in that: The milk quality traits are one or more of milk protein content, milk fat content and milk protein content.

Images