SNP molecular marker of gene HADHB related to milk production traits in dairy cows and its application

Abstract

The present invention discloses an SNP molecular marker of gene HADHB related to milk production traits in dairy cows and its application. The present invention provides the application of a substance for detecting the polymorphism or genotype of at least one of the following 3 SNP sites in the identification or auxiliary identification of milk production traits in dairy cows; the 3 SNP sites are SNP site g.73256269T>C, SNP site g.73256227A>C, and SNP site g.73242290C>T. Experiments of the present invention have demonstrated that the 3 SNPs, SNP1, SNP2, and SNP3 are respectively located in the gene HADHB related to milk production traits in the dairy cow genome. The dominant allele of the SNP1 site is C, the dominant allele of the SNP2 site is C, and the dominant allele of the SNP3 site is T, indicating that the SNP1 site, SNP2 site, and / or SNP3 site can be used for molecular marker-assisted selection breeding of dairy cows and the breeding of dairy cow varieties with high milk production traits.

Description

Technical Field

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

[0002] Beta Hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB), this gene encodes the alpha subunit of mitochondrial trifunctional protein, which catalyzes the last three steps of mitochondrial beta-oxidation of long-chain fatty acids. The mitochondrial membrane-bound heterocomplex is composed of four alpha and four beta subunits, and the alpha subunit catalyzes 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activities. Mutations in this gene lead to trifunctional protein deficiency or LCHAD deficiency. The genes of the alpha and beta subunits of mitochondrial trifunctional protein are adjacent to each other in the human genome in a head-to-head orientation.

[0003] The HADHB gene encodes the beta subunit of mitochondrial trifunctional protein, which participates in catalyzing the last three steps of mitochondrial beta-oxidation of long-chain fatty acids and plays an important role in controlling and regulating beta-oxidation. In chickens, this gene plays an important role in liver lipid metabolism. It has been reported that this gene is expressed in the livers of rats and dogs, inducing peroxisomal and mitochondrial beta-oxidation activities.

[0004] Polymerase Chain Reaction (PCR) is a nucleic acid rapid amplification technique that simulates the natural DNA replication process in vitro. Its greatest feature is that it can greatly increase trace amounts of DNA. It was invented by Mullis et al. in the United States in 1983. PCR utilizes the fact that DNA denatures into single strands at high temperatures in vitro, primers bind to the single strands according to the principle of base complementary pairing at low temperatures, and then the temperature is adjusted to the optimal reaction temperature of DNA polymerase. DNA polymerase synthesizes complementary strands along the direction from phosphate to pentose sugar (5'-3'). Currently, this technology has become one of the most commonly used and important molecular biology techniques. After PCR products are electrophoresed on agarose gel and then sequenced, gene polymorphism identification can be carried out, and the detection method is simple and easy to perform. Summary of the Invention

[0005] The object of the present invention is to provide an SNP molecular marker of a gene HADHB related to milk production traits of dairy cows and its application.

[0006] In a first aspect, the present invention provides an application of a substance for detecting the polymorphism or genotype of at least one of the following 3 SNP loci in identifying or assisting in identifying the milk production traits of dairy cows;

[0007] The 3 SNP loci are SNP locus g.73256269T>C, SNP locus g.73256227A>C, and SNP locus g.73242290C>T.

[0008] In a second aspect, the present invention provides an application of at least one of the following 3 SNP loci as a detection target in identifying or assisting in identifying the milk production traits of dairy cows;

[0009] Or, an application of at least one of the following 3 SNP loci as a detection target in developing a product for identifying or assisting in identifying the milk production traits of dairy cows;

[0010] The 3 SNP loci are SNP locus g.73256269T>C, SNP locus g.73256227A>C, and SNP locus g.73242290C>T.

[0011] In the above-mentioned application, the milk production traits are milk yield, milk fat content, and / or milk protein content.

[0012] In a third aspect, the present invention provides a method for identifying or assisting in identifying the milk production traits of dairy cows, which is any one of the following 1)-5):

[0013] 1) It includes the following steps: detecting the genotype of SNP locus g.73256269T>C in the HADHB gene of the test dairy cow; the genotype of SNP locus g.73256269T>C is CC or CT or TT;

[0014] The milk yield, milk fat content, and / or milk protein content of the test dairy cow with the genotype of CC at SNP locus g.73256269T>C is better than or assisted better than that of the test dairy cow with the genotype of TT or CT at SNP locus g.73256269T>C;

[0015] 2) It includes the following steps: detecting the genotype of SNP locus g.73256227A>C in the HADHB gene of the test dairy cow; the genotype of SNP locus g.73256227A>C is AA or AC or CC;

[0016] The milk yield, milk fat content, and / or milk protein content of the test dairy cow with the genotype of CC at SNP locus g.73256227A>C is better than or assisted better than that of the test dairy cow with the genotype of AA or AC at SNP locus g.73256227A>C;

[0017] 3) It includes the following steps: detecting the genotype of the SNP locus g.73242290C>T in the HADHB gene of the test dairy cows; the genotype of the SNP locus g.73242290C>T is CC or TT or CT;

[0018] The milk yield, milk fat yield and / or milk protein yield of the test dairy cows with the genotype TT of the SNP locus g.73242290C>T are better than or assist in being better than those of the test dairy cows with the genotype CC or CT of the SNP locus g.73242290C>T.

[0019] Fourthly, the present invention provides the application of the method described in the third aspect in dairy cow screening or dairy cow breeding.

[0020] In the above-mentioned application, the test dairy cows of any one of the following in the third aspect are selected for milk production or breeding;

[0021] The test dairy cows with the genotype CC of the SNP locus g.73256269T>C;

[0022] The test dairy cows with the genotype CC of the SNP locus g.73256227A>C;

[0023] The test dairy cows with the genotype TT of the SNP locus g.73242290C>T.

[0024] Fifthly, the present invention provides a method for dairy cow breeding, including the following steps:

[0025] Identifying the genotypes of each SNP locus according to the steps in the method described in the third aspect, and selecting the test dairy cows of any one of the following for milk production or breeding;

[0026] The test dairy cows with the genotype CC of the SNP locus g.73256269T>C;

[0027] The test dairy cows with the genotype CC of the SNP locus g.73256227A>C;

[0028] The test dairy cows with the genotype TT of the SNP locus g.73242290C>T.

[0029] Sixthly, the present invention provides any one of primer pair 6 and primer pair 9 or a primer combination;

[0030] The primer combination is composed of the primer pair 6 and the primer pair 9;

[0031] The primer pair 6 is a primer pair composed of primer 6F and primer 6R;

[0032] The primer pair 9 is a primer pair composed of primer 9F and primer 9R;

[0033] The primer 6F is a single-stranded DNA molecule shown in SEQ ID No. 4, or a nucleotide sequence obtained by deleting, adding, or altering one or more nucleotides in SEQ ID No. 4, and having the same function as SEQ ID No. 4.

[0034] The primer 6R is a single-stranded DNA molecule shown in SEQ ID No. 5, or a nucleotide sequence obtained by deleting, adding, or altering one or more nucleotides in SEQ ID No. 5, and having the same function as SEQ ID No. 5.

[0035] The primer 9F is a single-stranded DNA molecule shown in SEQ ID No. 6, or a nucleotide sequence obtained by deleting, adding, or altering one or more nucleotides in SEQ ID No. 6, and having the same function as SEQ ID No. 6.

[0036] The primer 9R is a single-stranded DNA molecule shown in SEQ ID No. 7, or a nucleotide sequence obtained by deleting, adding, or altering one or more nucleotides in SEQ ID No. 7, and having the same function as SEQ ID No. 7.

[0037] In a seventh aspect, the present invention provides the use of any one or a combination of the primer pairs described in the sixth aspect, which is any one of the following (a)-(f):

[0038] (a) Identifying or assisting in identifying milk production traits of dairy cows;

[0039] (b) Screening dairy cows;

[0040] (c) Breeding dairy cows;

[0041] (d) Preparing a kit for identifying or assisting in identifying milk production traits of dairy cows;

[0042] (e) Preparing a kit for screening dairy cows;

[0043] (f) Preparing a kit for breeding dairy cows.

[0044] The above-mentioned dairy cow breeding refers to breeding dairy cow varieties with high milk production traits.

[0045] In the above text,

[0046] The SNP1 is an SNP in the dairy cow genome, which is the 2179th nucleotide of SEQ ID No.1 in the sequence listing, and it is T or C; the SNP2 is an SNP in the dairy cow genome, which is the 2221st nucleotide of SEQ ID No.1 in the sequence listing, and it is A or C; the SNP3 is an SNP in the dairy cow genome, which is the 5002nd nucleotide of SEQ ID No.2 in the sequence listing, and it is C or T.

[0047] The above three single nucleotide polymorphism sites, SNP1, SNP2 and SNP3, are located in the HADHB gene on chromosome 11 of the dairy cow genome. The HADHB gene is related to the milk production traits of dairy cows, and the nucleotide sequence of this gene consists of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 in sequence.

[0048] The genotype (i.e., allele) of the SNP1 can be genotype CC, genotype TT or genotype CT. Genotype CC is the homozygous type where SNP1 is C, genotype TT is the homozygous type where SNP1 is T, and genotype CT is the heterozygous type where SNP1 is C and T; the genotype (i.e., allele) of the SNP2 can be genotype AA, genotype CC or genotype AC. Genotype AA is the homozygous type where SNP2 is A, genotype CC is the homozygous type where SNP2 is C, and genotype AC is the heterozygous type where SNP2 is A and C; the genotype (i.e., allele) of the SNP3 can be genotype CC, genotype TT or genotype CT. Genotype CC is the homozygous type where SNP1 is C, genotype TT is the homozygous type where SNP1 is T, and genotype CT is the heterozygous type where SNP1 is C and T.

[0049] The above-mentioned high milk production traits can specifically be high milk yield, high milk fat content and / or high milk protein content.

[0050] In the above application or method, the substance for detecting the polymorphism or genotype of the three SNPs, SNP1, SNP2 and SNP3, or the substance for detecting the polymorphism or genotype of SNP1, or the substance for detecting the polymorphism or genotype of SNP2, or the substance for detecting the polymorphism or genotype of SNP3 can determine the nucleotide types at the SNP1, SNP2 and / or SNP3 sites in the above-mentioned dairy cow genome by at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography and SNP chip. Among them, the SNP chip includes a chip based on nucleic acid hybridization reaction, a chip based on single-base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction endonuclease reaction, a chip based on protein-DNA binding reaction, and a chip based on fluorescent molecule-DNA binding reaction.

[0051] In the above application or method, the substance for detecting the polymorphism or genotype of the three SNPs, i.e., SNP1, SNP2, and / or SNP3, may be any of the following D1), D2), or D3):

[0052] D1) A PCR primer containing a fragment of dairy cow genomic DNA that amplifies regions including the SNP1, SNP2, and / or SNP3 loci;

[0053] D2) A PCR reagent containing the PCR primer described in D1);

[0054] D3) A kit containing the PCR primer described in D1) or the PCR reagent described in D2).

[0055] The PCR primers described in D1) above are primer pair 6 and primer pair 9 in the sixth aspect above.

[0056] In the above application or method, the PCR primer may or may not be labeled with a labeling agent. The labeling agent refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Labeling agents include but are not limited to dyes; radioactive labels such as 32 P; binding moieties such as biotin; haptens such as digoxin (DIG); luminescent, phosphorescent, or fluorescent moieties; and individual fluorescent dyes or fluorescent dyes combined with moieties that can suppress or shift the emission spectrum through fluorescence resonance energy transfer (FRET). The label can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The label can be a charged moiety (positive or negative charge) or, alternatively, can be charge-neutral. The label can include nucleic acid or protein sequences or combinations thereof, as long as the sequence containing the label is detectable. In some embodiments, nucleic acids are directly detected without a label (e.g., directly reading the sequence).

[0057] In the above application or method, the product may be a reagent, a kit, or a system. The system may include a combined product of a reagent or a kit, an instrument, and analysis software, such as a product composed of a PCR primer, a PARMS master mix reagent, a microplate reader, and the online software SNP decoder (http: / / www.snpway.com / snpdecoder01 / ), or a combined product composed of a PCR primer, a PARMS master mix reagent, the online software SNP decoder, and a real-time fluorescence quantitative PCR instrument. The product may include the substances for detecting the polymorphism or genotype of the SNP1, SNP2, and / or SNP3 loci in the dairy cow genome described above.

[0058] Experimental verification of the present invention shows that through genetic variation analysis of the HADHB gene in dairy cattle related populations, three SNPs, namely SNP1, SNP2, and SNP3, are found to be located in the HADHB gene related to milk production traits in the dairy cattle genome, specifically at positions 2179 of SEQ ID No.1, 2221 of SEQ ID No.1, and 5002 of SEQ ID No.2. In the examples of the present invention, the dominant allele at the SNP1 locus is C, the dominant allele at the SNP2 locus is C, and the dominant allele at the SNP3 locus is T, indicating that the SNP1 locus, SNP2 locus, and / or SNP3 locus can be used for molecular marker-assisted selection breeding of dairy cattle and breeding of dairy cattle breeds with high milk production traits. Detailed implementation manners

[0059] The present invention will be further described in detail below in conjunction with specific implementation manners. The provided examples are only for clarifying the present invention and not for limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way.

[0060] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0061] The Chinese Holstein cows in the following examples are from the Hebei Provincial Animal Husbandry Improved Variety Workstation.

[0062] The data in the examples are all data of lactation period 2. Lactation period 2 refers to the lactation period after the second calving.

[0063] The milk production in the examples is the individual 305-day milk production, which refers to the total milk production from the first day of calving of the cow to the 305th day. When the actual milking days are less than 305 days, the actual milk production is used as the 305-day milk production; when the actual milking days exceed 305 days, the milk production after the 306th day is not included. The milk production is measured by DHI (DHI, Dairy Herd Improvement) every month, and the 305-day milk production of this lactation period can be calculated by drawing a milk production lactation curve through more than 3 DHI data in the same lactation period.

[0064] The milk fat amount in the examples refers to the 305-day milk fat amount, and the 305-day milk fat amount = milk fat rate × 305-day milk production. The milk fat rate is measured by DHI (DHI, Dairy Herd Improvement) every month, and the average milk fat rate of this lactation period can be calculated by drawing a milk fat rate lactation curve through more than 3 DHI data in the same lactation period.

[0065] The milk protein amount in the examples refers to the milk protein amount in 305 days. The milk protein amount in 305 days = milk protein rate × milk yield in 305 days. The milk protein rate is obtained by monthly DHI (DHI, Dairy Herd Improvement) measurement, and the average milk protein rate in the lactation period can be calculated by drawing a milk protein rate lactation curve based on DHI data more than 3 times within the same lactation period.

[0066] Unless otherwise specified, in the following quantitative tests of the examples, three repeated experiments are set, and the results are averaged.

[0067] Example 1. Discovery of Molecular Markers

[0068] I. Related Basic Research

[0069] The research group where the inventor belongs used the liver tissues of 3 Chinese Holstein cows at different lactation stages (dry period, early lactation period, peak lactation period) as test materials, and carried out transcriptome sequencing (RNA-sequencing, RNA-seq) and small RNA sequencing (small RNA sequencing, small RNA-seq) using the second-generation sequencing technology. It was found that compared with the dry period, the expression level of HADHB was significantly up-regulated in the early lactation period and the peak lactation period (P<0.01), which can promote the increase of bile acid synthesis, thereby improving lipid absorption for milk synthesis.

[0070] II. Gene Polymorphism Detection

[0071] 1. A total of 655 Chinese Holstein cows in the Hebei region were selected as the test population for gene polymorphism detection. The genomic DNA of blood samples was extracted, and the concentration of DNA was accurately measured using a nucleic acid quality detector, and these DNAs were all diluted to a concentration of 50 ng / μL and equally mixed into 5 pool DNAs as templates for PCR amplification.

[0072] 2. According to the bovine HADHB gene sequence (Ensembl ID is ENSBTAG00000005287, and its nucleotide sequence consists of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 in sequence), 27 pairs of primers as shown in Table 1 were designed.

[0073] Table 1 shows the primer sequence information for HADHB gene PCR amplification

[0074]

[0075]

[0076] 3. Using the pooled DNA obtained in step 1 as a template, perform PCR amplification with each primer pair to obtain PCR amplification products. The PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.

[0077] Table 2 is the PCR reaction system

[0078]

[0079] Table 3 is the PCR reaction conditions

[0080]

[0081] 4. Sequence the PCR amplification products. The results show that there are 2 SNP markers (designated as SNP1 and SNP2 respectively) in the upstream 2000bp flanking sequence of the HADHB gene in the cow population and 1 SNP (designated as SNP3) marker in the 3'UTR. The 3 SNP markers are shown in Table 4.

[0082] Table 4 is the 3 SNPs found in the HADHB gene

[0083] Gene Location Name SNPs Physical Location Polymorphic Form 5' Regulatory Region SNP1 g.73256269T>C Chr11:73256269bp T / C 5’UTR SNP2 g.73256227A>C Chr11:73256227bp A / C Intron between Exon 5 / 6 SNP3 g.73242290C>T Chr11:73242290bp C / T

[0084] Among them, SNP1 corresponds to g.73256269T>C, which was obtained by sequencing analysis of the product obtained by PCR amplification using the primer pair composed of 6F and 6R (this PCR amplification product is shown in positions 1997 - 2816 of SEQ ID No.1), its nucleotide is T or C, corresponding to the 2179th position from the 5' end in SEQ ID No.1 in the sequence listing. SNP2 corresponds to g.73256227A>C, which was obtained by sequencing analysis of the product obtained by PCR amplification using the primer pair composed of 6F and 6R (this PCR amplification product is shown in positions 1997 - 2816 of SEQ ID No.1), its nucleotide is A or C, corresponding to the 2221st position from the 5' end in SEQ ID No.1 in the sequence listing. SNP3 corresponds to g.73242290C>T, which was obtained by sequencing analysis of the product obtained by PCR amplification using the primer pair composed of 9F and 9R (this PCR amplification product is shown in positions 4793 - 5034 of SEQ ID No.2), its nucleotide is C or T, corresponding to the 5002nd position from the 5' end in SEQ ID No.2 in the sequence listing. Y in SEQ ID No.1 in the sequence listing represents T or C, and M represents C or A. Y in SEQ ID No.2 in the sequence listing represents T or C.

[0085] The designations of g.73256269T>C, g.73256227A>C, and g.73242290C>T are for SNP1, SNP2, and SNP3 respectively. The naming of SNPs is generally carried out according to the rules when they are first discovered. DNA has a double-stranded structure and follows the base complementary pairing principle. SEQ ID No.1 in the present invention and the sequences when SNP1, SNP2, and SNP3 were discovered are reverse complementary strands of the same double-stranded DNA. Therefore, the polymorphic forms of g.73256269T>C in the present invention are T or C, the polymorphic forms of g.73256227A>C are A or C, and the polymorphic forms of g.73242290C>T are C or T.

[0086] III. Association analysis

[0087] (I). Obtaining the test population

[0088] The test population consists of 655 Chinese Holstein cows.

[0089] (II). Conducting genotype typing

[0090] Each individual in the test population is subjected to genotype typing separately.

[0091] I. Genotype typing based on g.73256269T>C.

[0092] 1. Take the blood of the test individual and extract genomic DNA.

[0093] 2. Using genomic DNA as a template, perform PCR amplification with the primer pair composed of 6F (as shown in positions 1997 - 2015 of SEQ ID No.1) and 6R (reverse complementary to positions 2793 - 2816 of SEQ ID No.1), then recover the PCR amplification product and perform sequencing.

[0094] The reaction system for PCR amplification is shown in Table 5. The reaction conditions for PCR amplification are shown in Table 6.

[0095] The PCR amplification product of each test individual is 820bp, and the 183rd position is g.73256269T>C, that is, SNP1 (corresponding to the 2179th position from the 5' end of SEQ ID No.1 in the sequence listing).

[0096] Table 5 is the reaction system for PCR amplification

[0097]

[0098] Table 6 is the reaction conditions for PCR amplification

[0099]

[0100] II. Genotyping based on g.73256227A>C

[0101] 1. Take the blood of the test individual and extract genomic DNA.

[0102] 2. Using the genomic DNA as a template, perform PCR amplification with the primer pair composed of 6F (as shown in positions 1997 - 2015 of SEQ ID No.1) and 6R (reverse complementary to positions 2793 - 2816 of SEQ ID No.1), then recover the PCR amplification product and perform sequencing.

[0103] The reaction system for PCR amplification is shown in Table 7. The reaction conditions for PCR amplification are shown in Table 8.

[0104] The PCR amplification product of each test individual is 820bp, and the 225th position is g.73256227A>C, that is, SNP2 (corresponding to the 2221st position from the 5' end of SEQ ID No.1 in the sequence listing).

[0105] Table 7 is the reaction system for PCR amplification

[0106]

[0107] Table 8 is the reaction conditions for PCR amplification

[0108]

[0109] III. Genotyping based on g.73242290C>T

[0110] 1. Take the blood of the test individual and extract genomic DNA.

[0111] 2. Using the genomic DNA as a template, perform PCR amplification with the primer pair composed of 9F (as shown in positions 4793 - 4815 of SEQ ID No.2) and 9R (reverse complementary to positions 5015 - 5034 of SEQ ID No.2), then recover the PCR amplification product and perform sequencing.

[0112] The reaction system for PCR amplification is shown in Table 9. The reaction conditions for PCR amplification are shown in Table 10.

[0113] The PCR amplification product of each test individual is 242bp, and the 210th position is g.73242290C>T, that is, SNP3 (corresponding to the 5002nd position from the 5' end of SEQ ID No.2 in the sequence listing).

[0114] Table 9 is the reaction system for PCR amplification

[0115]

[0116] Table 10 shows the reaction conditions for PCR amplification

[0117]

[0118] (III). Detection of milk production traits

[0119] For each cow in the test population, milk production traits were detected separately

[0120] Milk production traits include the following five indicators: milk yield, milk fat yield, milk fat percentage, milk protein yield, and milk protein percentage

[0121] The records of each individual sequentially include the individual number of the cow, sire number, dam number, grandfather number, grandmother number, maternal grandfather number, maternal grandmother number, date of birth, lactation period, calving date, milk yield, milk fat yield, and milk protein yield

[0122] (IV). Association analysis model between a single SNP locus and traits

[0123] The genotypes of SNP1 locus (i.e., g.73256269T>C in the HADHB gene), SNP2 locus (i.e., g.73256227A>C in the HADHB gene), and SNP3 locus (i.e., g.73242290C>T in the HADHB gene) and the phenotypes of milk production traits are shown in Tables 11, 12, and 13

[0124] Table 11 shows the partial phenotypes of milk production traits and the genotypes of 3 SNP loci in 655 Chinese Holstein cows

[0125]

[0126]

[0127]

[0128] Table 12 shows the descriptive statistics of the phenotypic values of 5 milk production traits in a population of 655 Chinese Holstein cows

[0129] Trait Mean Standard Deviation Minimum Maximum Milk Yield (kg) 10840.58 1895.22 4757 16512 Milk Fat Yield (kg) 390.53 85.75 145 741 Milk Fat Percentage (%) 3.608 0.505 2.212 5.431 Milk Protein Yield (kg) 321.73 58.17 138 467 Milk Protein Percentage (%) 2.971 0.191 2.199 3.538

[0130] Table 13 shows the allele frequencies and genotype frequencies of 3 SNP loci in the HADHB gene

[0131]

[0132]

[0133] The results showed that there were three genotypes at the SNP1 locus (abbreviated as SNP1 genotype), namely CC, TT or CT. Genotype CC was the homozygous type with SNP1 being C, genotype TT was the homozygous type with SNP1 being T, and genotype CT was the heterozygous type with SNP1 being C and T. There were three genotypes at the SNP2 locus (abbreviated as SNP2 genotype), namely AA, CC or AC. Genotype AA was the homozygous type with SNP2 being A, genotype CC was the homozygous type with SNP2 being C, and genotype AC was the heterozygous type with SNP2 being A and C. There were three genotypes at the SNP3 locus (abbreviated as SNP3 genotype), namely CC, TT or CT. Genotype CC was the homozygous type with SNP3 being C, genotype TT was the homozygous type with SNP3 being T, and genotype CT was the heterozygous type with SNP3 being C and T.

[0134] The MIXED procedure in SAS 9.2 software was used to conduct association analysis between five indexes of milk production traits and genotypes. The association analysis adopted the animal model, and the specific model was as follows:

[0135] Y = μ + hys + b×M + G + a + e

[0136] Among them, Y: the observed value of milk production traits (milk yield, milk fat yield, milk fat percentage, milk protein yield or milk protein percentage); μ: the overall mean; hys: the effect of farm-year-season; b: the regression coefficient of the covariate M; M: the effect of calving month age; G: the genotype effect; a: the individual random additive genetic effect; e: the random residual effect.

[0137] The results of the association analysis between the SNP1 locus (i.e., g.73256269T>C of the HADHB gene) and milk production traits are shown in Table 14. Table 14 shows the association analysis between g.73256269T>C of the HADHB gene and milk production traits in the second lactation period (least squares mean ± standard error)

[0138] Genotype Milk Yield (kg) Milk Fat Yield (kg) Milk Fat Percentage (%) Milk Protein Yield (kg) Milk Protein Percentage (%) CC(397) 10873±66.5485A 387.93±2.953Aa 3.5777±0.02743 324.74±2.953A 2.9878±0.008911 CT(230) 10626±74.6693B 380.21±3.2572Ab 3.5953±0.0306 315.93±3.2572B 2.9774±0.01017 TT(28) 10091±162.53C 351.42±6.6827Bc 3.5045±0.06532 299.92±6.6827B 2.9838±0.02329 P value <0.0001** <0.0001** 0.3795 <0.0001** 0.585

[0139] Note: * P < 0.05 indicates a significant difference; ** P < 0.01 indicates a highly significant difference. a,b Different superscripts for the same column of data indicate a significant difference; A,B Different superscripts for the same column of data indicate a highly significant difference.

[0140] As obtained from Table 14, SNP1 (g.73256269T>C) was significantly associated with milk yield, milk fat yield and milk protein yield (P < 0.0001). For the traits of milk yield, milk fat yield and milk protein yield, the dominant allele was C;

[0141] The milk yield of CC genotype cows is higher than that of TT genotype or CT genotype cows, and the milk yield of CT genotype cows is higher than that of TT genotype cows.

[0142] The milk fat content of CC genotype cows is higher than that of TT genotype or CT genotype cows, and the milk fat content of CT genotype cows is higher than that of TT genotype cows.

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

[0144] The results of the association analysis between SNP2 locus (i.e., g.73256227A>C in HADHB gene) and milk production traits are shown in Table 15.

[0145] Table 15 shows the association analysis between g.73256227A>C in HADHB gene and milk production traits in the second lactation period (least square means ± standard error)

[0146]

[0147] Note: * P<0.05 indicates significant difference; ** P<0.01 indicates extremely significant difference. a,b Different superscripts in the same column of data indicate significant differences; A,B Different superscripts in the same column of data indicate extremely significant differences.

[0148] As shown in Table 15, SNP2 (g.73256227A>C) is significantly associated with milk yield and milk protein content (P = 0.0184 - 0.0015). For milk yield, milk fat content and milk protein content traits, the dominant allele is C:

[0149] The milk yield of CC genotype cows is higher than that of AA genotype or AC genotype cows, and the milk yield of AC genotype cows is higher than that of AA genotype cows.

[0150] The milk fat content of CC genotype cows is higher than that of AA genotype or AC genotype cows, and the milk fat content of AC genotype cows is higher than that of AA genotype cows.

[0151] The milk protein content of CC genotype cows is higher than that of AA genotype or AC genotype cows, and there is no significant difference in the milk protein content between AC genotype cows and AA genotype cows.

[0152] The results of the association analysis between SNP3 locus (i.e., g.73242290C>T in HADHB gene) and milk production traits are shown in Table 16. Table 16 shows the association analysis between g.73242290C>T in HADHB gene and milk production traits in the second lactation period (least square means ± standard error)

[0153] Genotype Milk Yield (kg) Milk Fat Yield (kg) Milk Fat Percentage (%) Milk Protein Yield (kg) Milk Protein Percentage (%) CC(96) 10476±99.0923B 368.81±4.1983Bc 3.5376±0.04017 308.42±4.1983B 2.9573±0.01389 CT(156) 10545±82.7589B 379.36±3.5761ABb 3.6164±0.03376 315.16±3.5761B 2.9901±0.01141 TT(403) 10884±66.0831A 387.96±2.9518Aa 3.574±0.02725 325.03±2.9518A 2.9875±0.008838 P value <0.0001** <0.0001** 0.1681 <0.0001** 0.0674

[0154] Note: * P < 0.05 indicates significant difference; ** P < 0.01 indicates extremely significant difference. a,b Different superscripts in the same column of data indicate significant difference; A,B Different superscripts in the same column of data indicate extremely significant difference.

[0155] As shown in Table 16, SNP3 (g.73242290C>T) was extremely significantly associated with milk yield, milk fat yield, milk fat percentage and milk protein yield (P<0.0001). For the traits of milk yield, milk fat yield and milk protein yield, the dominant allele was T.

[0156] The milk yield of cows with TT genotype was higher than that of cows with CC genotype or CT genotype, and there was no significant difference in milk yield between cows with CT genotype and CC genotype.

[0157] The milk fat yield of cows with TT genotype was higher than that of cows with CC genotype or CT genotype, and the milk fat yield of cows with CT genotype was higher than that of CC genotype.

[0158] The milk protein yield of cows with TT genotype was higher than that of cows with CC genotype or CT genotype, and there was no significant difference in milk protein yield between cows with CT genotype and CC genotype.

[0159] (V). Genetic effect analysis

[0160] SAS 9.2 software was used to conduct significance tests on SNP additive effect, dominant effect and substitution effect.

[0161] The basic calculation formulas are as follows:

[0162] a = (AA - BB) / 2, d = AB - (AA + BB) / 2, α = a + d(q - p); a is the additive effect, d is the dominant effect, and α is the allele substitution effect; AA, AB, and BB are the least squares means of milk production traits for the corresponding genotypes; p is the frequency of allele A, and q is the frequency of allele B.

[0163] The test results of additive effect, dominant effect and allele substitution effect are shown in Table 17.

[0164] Table 17 shows the test results of additive effect, dominant effect and substitution effect of alleles of HADHB gene

[0165]

[0166] Note: * P < 0.05 indicates significant difference; ** P < 0.01 indicates extremely significant difference.

[0167] The results showed that the additive effect and allelic substitution effect of SNP1 (g.73256269T>C) on milk yield and milk protein content reached extremely significant levels, and the additive effect, dominance effect, and allelic substitution effect on milk fat content reached extremely significant levels. That is, each C allele replacing the T allele would result in an increase in milk yield of 471.25 kg (P<0.01), an increase in milk fat content of 24.1742 kg (P<0.01), and an increase in milk protein content of 14.4302 kg (P<0.01). The additive effect, dominance effect, and allelic substitution effect of SNP2 (g.73256227A>C) on milk yield, milk fat content, milk fat percentage, milk protein content, and milk protein percentage reached significant or extremely significant levels respectively. That is, each C allele replacing the A allele would result in an increase in milk yield of 538 kg (P<0.01), an increase in milk fat content of 36.6914 kg (P<0.01), an increase in milk fat percentage of 0.1472% (P<0.05), an increase in milk protein content of 17.5372 kg (P<0.01), and an increase in milk protein percentage of 0.00162 kg (P<0.01). The additive effect and allelic substitution effect of SNP3 (g.73242290C>T) on milk yield, milk fat content, milk protein content, and milk protein percentage reached significant or extremely significant levels respectively. That is, each T allele replacing the C allele would result in an increase in milk yield of 140.15 kg (P<0.05), an increase in milk fat content of 10.0302 kg (P<0.01), an increase in milk protein content of 7.5665 kg (P<0.01), and an increase in milk protein percentage of 0.02341 kg (P<0.05).

[0168] (VI) Haplotype analysis

[0169] The Haploview 4.2 software was used to construct haplotypes, estimate haplotype frequencies and linkage degrees, and perform association analysis with traits. Using the MIXED procedure in SAS 9.2 software, the model was as follows:

[0170] Y = μ + hys + b×M + G + a + e

[0171] Y: Observed values of milk production traits (milk yield, milk fat content, milk fat percentage, milk protein content, and milk protein percentage); μ: Overall mean; hys: Farm-year-season effect; b: Regression coefficient of covariate M; M: Calving month effect; G: Haplotype combination effect; a: Individual random additive genetic effect; e: Random residual effect.

[0172] The molecular markers disclosed in the present invention can be applied to assist in identifying dairy cattle populations with excellent milk production traits (305-day milk yield, milk fat content, milk fat percentage, milk protein content, and milk protein percentage), and have the following advantages: simple, fast, sensitive, reliable, stable, and accurate results, and are suitable for the needs of large-scale laboratory detection.

[0173] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. Use of a substance for detecting the polymorphism or genotype of at least one of the following 3 SNP loci in identifying or assisting in identifying milk production traits of dairy cows; The 3 SNP loci are SNP locus g.73256269T>C, SNP locus g.73256227A>C, and SNP locus g.73242290C>T; The SNP locus g.73256269T>C is the 2178th nucleotide of Sequence Listing SEQ ID No.1, and the nucleotide is T or C; The SNP locus g.73256227A>C is the 2220th nucleotide of Sequence Listing SEQ ID No.1, and the nucleotide is A or C; The SNP locus g.73242290C>T is the 5002nd nucleotide of Sequence Listing SEQ ID No.2, and the nucleotide is C or T; The milk production traits are milk yield, milk fat yield, and / or milk protein yield; The dairy cows are Chinese Holstein cows.

2. Use of at least one of the following 3 SNP loci as a detection target in identifying or assisting in identifying milk production traits of dairy cows; Or, use of at least one of the following 3 SNP loci as a detection target in developing products for identifying or assisting in identifying milk production traits of dairy cows; The 3 SNP loci are SNP locus g.73256269T>C, SNP locus g.73256227A>C, and SNP locus g.73242290C>T; The SNP locus g.73256269T>C is the 2178th nucleotide of Sequence Listing SEQ ID No.1, and the nucleotide is T or C; The SNP locus g.73256227A>C is the 2220th nucleotide of Sequence Listing SEQ ID No.1, and the nucleotide is A or C; The SNP locus g.73242290C>T is the 5002nd nucleotide of Sequence Listing SEQ ID No.2, and the nucleotide is C or T; The milk production traits are milk yield, milk fat yield, and / or milk protein yield; The dairy cows are Chinese Holstein cows.

3. A method for identifying or assisting in identifying milk production traits of dairy cows, which is any one of the following 1)-3): 1) The following steps are included: detecting the genotype of the SNP locus g.73256269T>C in the gene of the tested dairy cow HADHB ; The SNP locus g.73256269T>C is the 2178th nucleotide of Sequence Listing SEQ ID No.1, and the nucleotide is T or C; The genotype of the SNP locus g.73256269T>C is CC or CT or TT; The milk yield, milk fat yield, and / or milk protein yield of the tested dairy cows with the genotype CC of the SNP locus g.73256269T>C is better than or assisted better than that of the tested dairy cows with the genotype TT or CT of the SNP locus g.73256269T>C; 2) It includes the following steps: detecting the genotype of the SNP site g.73256227A>C in the gene of the test dairy cow HADHB ; The SNP locus g.73256227A>C is the 2220th nucleotide of Sequence Listing SEQ ID No.1, and the nucleotide is A or C; The genotype of the SNP locus g.73256227A>C is AA or AC or CC; The milk yield, milk fat yield and / or milk protein yield of the tested dairy cows with the CC genotype at the SNP locus g.73256227A>C are superior to or assist in being superior to those of the tested dairy cows with the AA or AC genotype at the SNP locus g.73256227A>C; 3) The method includes the following steps: detecting the genotype of SNP locus g.73242290C>T in the gene of the tested dairy cow HADHB ; The SNP locus g.73242290C>T is the 5002nd nucleotide of Sequence Listing SEQ ID No.2, and the nucleotide is C or T; The genotype of the SNP locus g.73242290C>T is CC or TT or CT; The milk yield, milk fat yield and / or milk protein yield of the tested dairy cows with the TT genotype at the SNP locus g.73242290C>T are superior to or assist in being superior to those of the tested dairy cows with the CC or CT genotype at the SNP locus g.73242290C>T; The dairy cows are Chinese Holstein cows.

4. Use of the method according to claim 3 in dairy cow screening or dairy cow breeding; The dairy cow screening or dairy cow breeding is to screen or cultivate dairy cow breeds with high milk production traits; The milk production trait is milk yield, milk fat yield and / or milk protein yield; The dairy cows are Chinese Holstein cows.

5. According to the use according to claim 4, characterized in that: In the said use, the tested dairy cows of any one of the following in claim 3 are selected for milk production or breeding; The tested dairy cows with the CC genotype at the SNP locus g.73256269T>C; The tested dairy cows with the CC genotype at the SNP locus g.73256227A>C; The tested dairy cows with the TT genotype at the SNP locus g.73242290C>T.

6. A method for dairy cow breeding, comprising the following steps: Identify the genotype or haplotype combination of each SNP locus according to the steps in the method according to claim 3, and select the tested dairy cows of any one of the following for milk production or breeding; The tested dairy cows with the CC genotype at the SNP locus g.73256269T>C; The tested dairy cows with the CC genotype at the SNP locus g.73256227A>C; The tested dairy cows with the TT genotype at the SNP locus g.73242290C>T; The dairy cow breeding is to cultivate dairy cow breeds with high milk production traits; The milk production trait is milk yield, milk fat yield and / or milk protein yield; The dairy cows are Chinese Holstein cows.

7. Use of any one of the following primer pairs or primer combinations, which is any one of the following (a)-(f): (a) Identify or assist in identifying the milk production traits of dairy cows; the milk production trait is milk yield, milk fat yield and / or milk protein yield; the dairy cows are Chinese Holstein cows; (b) Dairy cow screening; the dairy cow screening is to screen dairy cow breeds with high milk production traits; the milk production trait is milk yield, milk fat yield and / or milk protein yield; the dairy cows are Chinese Holstein cows; (c) Dairy cow breeding; the dairy cow breeding is to cultivate dairy cow breeds with high milk production traits; the milk production trait is milk yield, milk fat yield and / or milk protein yield; the dairy cows are Chinese Holstein cows; (d) Preparation of a kit for identifying or assisting in the identification of milk production traits in dairy cows; the milk production traits being milk yield, milk fat content and / or milk protein content; the dairy cows being Chinese Holstein cows; (e) Preparation of a kit for screening dairy cows; the screening of dairy cows being for dairy cow breeds with high milk production traits; the milk production traits being milk yield, milk fat content and / or milk protein content; the dairy cows being Chinese Holstein cows; (f) Preparation of a kit for dairy cow breeding; the dairy cow breeding being for breeding dairy cow breeds with high milk production traits; the milk production traits being milk yield, milk fat content and / or milk protein content; the dairy cows being Chinese Holstein cows; Any one or primer combination of the following primer pairs is any one or primer combination of primer pair 6 and primer pair 9; The primer combination consists of primer pair 6 and primer pair 9; Primer pair 6 is a primer pair composed of primer 6F and primer 6R; Primer pair 9 is a primer pair composed of primer 9F and primer 9R; Primer 6F is a single-stranded DNA molecule shown in SEQ ID No. 4; primer 6R is a single-stranded DNA molecule shown in SEQ ID No. 5; Primer 9F is a single-stranded DNA molecule shown in SEQ ID No. 6; primer 9R is a single-stranded DNA molecule shown in SEQ ID No. 7.

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