Method for detecting milk production traits of dairy cows by using apom gene snps molecular markers and application thereof

Abstract

The application discloses a method for detecting milk production traits of dairy cows by using APOM gene SNPs molecular markers and application. The application provides application of a substance for detecting genotypes of four SNP sites, i.e. 23:g.27644015C>G and / or 23:g.27643343G>T and / or 23:g.27642597A>G and / or 23:g.27640015G>C and the like, in identifying or assisting in identifying milk production traits of dairy cows. Each site is related to 305-day milk yield, milk fat amount, milk fat rate, milk protein amount or milk protein rate, and can be used for breeding of dairy cows.

Description

Technical Field

[0001] This invention relates to a molecular marker and its application, and particularly to a method and application for detecting milk production traits in dairy cows using APOM gene SNPs molecular markers, belonging to the field of biotechnology. Background Technology

[0002] Apolipoproteins (APOs) are the protein fractions of plasma lipoproteins, involved in the metabolism and conversion of cholesterol and triglycerides. APOs are a crucial component of lipoproteins, binding and transporting lipids to various tissues for metabolism and utilization. Currently, more than 20 APO subtypes, including APOM, have been identified. Mutations in APO-related genes can result in proteins with different allelic genotypes and phenotypes, and their functions can be determined based on gene polymorphism.

[0003] The apolipoprotein M (APOM) gene is located on bovine chromosome 23, has 7 exons, a full-length gene of 2106 bp, and 2 transcripts with full-length mRNAs of 781 bp and 761 bp, encoding 223 and 18 amino acids respectively (bovine whole genome sequence ARS-UCD1.2). The protein encoded by this gene is an apolipoprotein, belonging to the lipoprotein family. It participates in the metabolism of fat-soluble vitamins, vitamin A-like metabolism and transport, and lipid signaling pathways. It can bind to fatty acids such as myristic acid, hexadecanoic acid, and stearic acid, and participate in lipoprotein transport. It is mainly found in high-density lipoprotein (HDL), with smaller amounts in low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL). Furthermore, APOM can maintain and protect β-cell function by reducing abnormal cholesterol accumulation in the pancreas and is regulated by insulin, playing a crucial role in stabilizing glucose metabolism levels in the body.

[0004] APOM has been shown to be the primary bioactive carrier of sphingosine-1-phosphate (S1P). APOM-S1P exerts its biological functions by activating S1P receptors on the cell membrane. Studies have shown that high-density lipoprotein (HDL) has anti-inflammatory, antioxidant, and anti-apoptotic effects. As a bioactive lipid, S1P can be carried by HDL. Extracellular S1P can bind to APOM and be transported to its corresponding S1P receptor, thereby activating downstream signaling pathways to perform complex biological functions, including regulating apoptosis and participating in the body's immune and glucose metabolism. Therefore, APOM acts as a physiological carrier of S1P, performing the function of transporting S1P, and the binding of the two effectively alleviates the extracellular degradation of S1P.

[0005] Semi-conservative DNA replication is a crucial pathway for biological evolution and propagation. Double-stranded DNA can denature and unwind into single strands under the action of various enzymes. With the participation of DNA polymerase, it replicates into two identical copies according to the base pairing principle. Experiments have shown that DNA can also denature and unwind at high temperatures, and then renature back into double strands when the temperature is lowered. Therefore, by controlling DNA denaturation and renaturation through temperature changes, and by adding designed primers, DNA polymerase, and dNTPs, specific genes can be replicated in vitro. Polymerase chain reaction (PCR) is a specific in vitro DNA amplification technique. Currently, it has become one of the most commonly used and important molecular biology techniques. PCR products can be sequenced after agarose gel electrophoresis for gene polymorphism identification; the detection method is simple and easy to perform. Summary of the Invention

[0006] The purpose of this invention is to provide a molecular marker associated with milk production traits in dairy cows and its application, wherein the SNP sites 23:g.27644015C>G and / or 23:g.27643343G>T and / or 23:g.27642597A>G and / or 23:g.27640015G>C are significantly or highly significantly associated with milk production traits (P<0.0001~0.0417).

[0007] On the one hand, the present invention provides the application of a substance for detecting the genotype of at least one of the following four SNP loci in identifying or assisting in the identification of milk production traits in dairy cows;

[0008] The four SNP sites are SNP site 23: g.27644015C>G, SNP site 23: g.27643343G>T, SNP site 23: g.27642597A>G and SNP site 23: g.27640015G>C.

[0009] On the other hand, the present invention provides the application of at least one of the following four SNP sites as a detection target in the identification or auxiliary identification of dairy cow milk production traits.

[0010] The four SNP sites are SNP site 23: g.27644015C>G, SNP site 23: g.27643343G>T, SNP site 23: g.27642597A>G and SNP site 23: g.27640015G>C.

[0011] In the above text, the milk production traits refer to milk yield and / or milk fat content and / or milk fat percentage and / or milk protein content and / or milk protein percentage.

[0012] In the above text, SNP site 23:g.27644015C>G was obtained by sequencing the product of PCR amplification using bovine genomic DNA as a template and primers 3F and 3R. This site is the 202nd position from the 5' end of the PCR product (sequence 21), which is the 27644015th position on chromosome 23 of the bovine genome ARS-UCD1.2 version reference sequence (Ensembl Gene ID: ENSBTAG00000008833, Transcript ID: ENSBTAT00000075540.1). The base at this site can be C or G; the genotype at this site is CC, GG, or CG.

[0013] SNP locus 23: g.27643343G>T was obtained by sequencing the product of PCR amplification using bovine genomic DNA as a template and primers 4F and 4R. This locus is the 184th position from the 5' end of the PCR product (sequence 22), which is the 27643343rd position on chromosome 23 of the bovine genome ARS-UCD1.2 version reference sequence. The base at this locus can be A or C; the genotype at this locus is AA, CC, or AC.

[0014] SNP locus 23: g.27642597A>G was obtained by sequencing the product of PCR amplification using bovine genomic DNA as a template and primers 5F and 5R. This locus is the 260th position from the 5' end of the PCR product (sequence 23), which is the 27642597th position on chromosome 23 of the bovine genome ARS-UCD1.2 version reference sequence. The base at this locus can be A or G; the genotype at this locus is AA, GG, or AG.

[0015] SNP locus 23: g.27640015G>C was obtained by sequencing the product of PCR amplification using bovine genomic DNA as a template and 10F and 10R primers. This locus is the 168th position from the 5' end of the PCR product (sequence 24), which is the 27640015th position on chromosome 23 of the bovine genome ARS-UCD1.2 version reference sequence. The base at this locus can be G or C; the genotype at this locus is GG, CC, or GC.

[0016] In the above text, the substance used to detect the genotype of at least one of the following four SNP sites may include primer pairs corresponding to amplify any one of the four SNP sites, specifically: primer pair 3 (composed of 3F and 3R) for amplifying SNP site 23: g.27644015C>G, primer pair 4 (composed of 4F and 4R) for amplifying SNP site 23: g.27643343G>T, primer pair 5 (composed of 5F and 5R) for amplifying SNP site 23: g.27642597A>G, and primer pair 10 (composed of 10F and 10R) for amplifying SNP site 23: g.27641305C>T.

[0017] The substances used to detect the genotype of at least one of the following four SNP loci may also include instruments and reagents related to MALDI-TOF-MS detection technology.

[0018] Thirdly, the present invention provides a method for identifying or assisting in the identification of milk production traits in dairy cows, which is any one of the following 1)-4):

[0019] 1) Includes the following steps: Detecting the genotype of SNP site 23:g.27644015C>G in the APOM gene of the tested dairy cows; the genotype of SNP site 23:g.27644015C>G is CC or GG (only homozygous types need to be considered);

[0020] The milk fat content and / or milk fat percentage of the tested dairy cows with the SNP locus 23:g.27644015C>G genotype CC were superior to or slightly superior to the tested dairy cows with the SNP locus 23:g.27644015C>G genotype GG.

[0021] 2) Includes the following steps: Detecting the genotype of SNP site 23:g.27643343G>T in the APOM gene of the tested dairy cows; the genotype of SNP site 23:g.27643343G>T is AA or CC;

[0022] The milk yield, milk fat content, milk protein content, and / or milk protein percentage of the tested dairy cows with the SNP locus 23:g.27643343G>T genotype AA were superior to or slightly superior to those of the tested dairy cows with the SNP locus 23:g.27643343G>T genotype CC.

[0023] 3) Includes the following steps: Detecting the genotype of SNP site 23:g.27642597A>G in the APOM gene of the tested dairy cows; the genotype of SNP site 23:g.27642597A>G is AA or GG;

[0024] The milk fat content, milk fat percentage, milk protein content, and / or milk protein percentage of the tested dairy cows with the SNP locus 23:g.27642597A>G genotype GG were superior to or slightly superior to those of the tested dairy cows with the SNP locus 23:g.27642597A>G genotype AA.

[0025] 4) Includes the following steps: Detecting the genotype of SNP site 23:g.27640015G>C in the APOM gene of the tested dairy cows; the genotype of SNP site 23:g.27640015G>C is GG or CC;

[0026] The milk fat content and / or milk fat percentage of the tested dairy cows with the SNP locus 23:g.27640015G>C genotype GG were superior to or slightly superior to those of the tested dairy cows with the SNP locus 23:g.27640015G>C genotype CC.

[0027] Fourthly, the present invention provides the application of the method described in the third aspect above in dairy cow screening or dairy cow breeding.

[0028] In the above applications, any of the following types of dairy cows were selected for milk production or breeding;

[0029] The tested dairy cows with the SNP locus 23:g.27644015C>G genotype CC were selected for breeding dairy cows with excellent milk fat content and / or milk fat percentage.

[0030] The purpose is to select dairy cows with the AA genotype at the SNP locus 23:g.27643343G>T; the purpose is to select dairy cows with excellent milk yield, milk fat content, milk protein content and / or milk protein percentage.

[0031] The purpose of selecting dairy cows with the genotype GG at the SNP locus 23:g.27642597A>G was to breed dairy cows with superior milk fat content, milk fat percentage, milk protein content, and / or milk protein percentage.

[0032] The tested dairy cows with the SNP locus 23:g.27640015G>C and the genotype GG were selected for breeding. The purpose was to breed dairy cows with excellent milk fat content and / or milk fat percentage.

[0033] Fifthly, the present invention also provides any one or a combination of primer pair 3, primer pair 4, primer pair 5 and primer pair 10;

[0034] The primer pair consists of primer pair 3, primer pair 4, primer pair 5 and primer pair 10;

[0035] Primer pair 3 is a primer pair consisting of primer 3F and primer 3R;

[0036] Primer pair 4 is a primer pair consisting of primer 4F and primer 4R;

[0037] Primer pair 5 is a primer pair consisting of primer 5F and primer 5R;

[0038] The primer pair 10 is a primer pair consisting of primer 10F and primer 10R;

[0039] The primer 3F is a single-stranded DNA molecule as shown in sequence 5 of the sequence listing, or a nucleotide with one or more nucleotides deleted, added, or changed from sequence 5, and with the same function as sequence 5;

[0040] The primer 3R is a single-stranded DNA molecule as shown in sequence 6 of the sequence listing, or a nucleotide in which one or more nucleotides of sequence 6 are deleted, added, or changed, and which has the same function as sequence 6.

[0041] The primer 4F is a single-stranded DNA molecule as shown in sequence 7 of the sequence listing, or a nucleotide in which one or more nucleotides of sequence 7 are deleted, added, or changed, and which has the same function as sequence 7.

[0042] The primer 4R is a single-stranded DNA molecule as shown in sequence 8 of the sequence listing, or a nucleotide in which one or more nucleotides of sequence 8 are deleted, added, or changed, and which has the same function as sequence 8.

[0043] The primer 5F is a single-stranded DNA molecule as shown in sequence 9 of the sequence listing, or a nucleotide with one or more nucleotides deleted, added, or changed from sequence 9, and with the same function as sequence 9;

[0044] The primer 5R is a single-stranded DNA molecule as shown in sequence 10 in the sequence listing, or a nucleotide in which one or more nucleotides are deleted, added, or changed from sequence 10, and which has the same function as sequence 10.

[0045] The primer 10F is a single-stranded DNA molecule as shown in sequence 19 in the sequence listing, or a nucleotide that has one or more nucleotides deleted, added, or changed from sequence 19, and has the same function as sequence 19.

[0046] The primer 10R is a single-stranded DNA molecule as shown in sequence 20 in the sequence listing, or a nucleotide that has one or more nucleotides deleted, added, or altered from sequence 20, and has the same function as sequence 20.

[0047] In a sixth aspect, the present invention also provides for the application of any one or combination of primer pairs described in the fifth aspect, as follows (a)-(f):

[0048] (a) Identifying or assisting in the identification of milk production traits in dairy cows;

[0049] (b) Cow selection;

[0050] (c) Dairy cow breeding;

[0051] (d) Prepare kits for identifying or assisting in the identification of milk production traits in dairy cows;

[0052] (e) Preparation of a kit for screening dairy cows;

[0053] (f) Preparation of a kit for dairy cow breeding.

[0054] The milk production, milk fat, and milk protein levels mentioned above are all statistics from the first lactation period (the lactation period after the first delivery).

[0055] The experiments of this invention demonstrate that the following molecular markers associated with milk production traits in dairy cows were discovered: 23:g.27644015C>G and / or 23:g.27643343G>T and / or 23:g.27642597A>G and / or 23:g.27640015G>C. These four molecular markers showed a significant or highly significant association with milk production traits (P<0.0001–0.0417). The molecular markers disclosed in this invention can be used to assist in the identification of dairy cow populations with excellent milk production traits (milk yield, milk fat content, milk fat percentage, milk protein content, and milk protein percentage), and have the following advantages: simplicity, speed, sensitivity, reliable, stable, and accurate results, suitable for the needs of large-scale laboratory testing. Attached Figure Description

[0056] Figure 1 The mutation locations are 23:g.27644015C>G, 23:g.27643343G>T, 23:g.27642597A>G and 23:g.27640015G>C. Detailed Implementation

[0057] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0059] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0060] The blood samples from Chinese Holstein cows were obtained from the Hebei Provincial Animal Husbandry Breeding Station.

[0061] The milk yield in the following examples refers to the milk yield of an individual over 305 days, specifically the total milk yield from the first day of calving to the 305th day. When the actual number of milking days is less than 305 days, the actual milk fat content is used as the milk fat content for 305 days; when the actual number of milking days exceeds 305 days, the milk fat content after the 306th day is not included.

[0062] The milk fat content in the following examples refers to the milk fat content over 305 days. The milk fat content over 305 days = milk fat percentage × milk production over 305 days. The milk fat percentage is obtained by monthly DHI (Dairy Herd Improvement) measurements. The average milk fat percentage for the lactation period can be calculated by plotting a lactation curve using more than 3 DHI data within the same lactation period.

[0063] In the following examples, the milk protein content refers to the milk protein content over 305 days. The milk protein content over 305 days = milk protein percentage × milk production over 305 days. The milk protein percentage is obtained by monthly DHI (Dairy Herd Improvement) measurements. The average milk protein percentage for the lactation period can be calculated by plotting a lactation curve using more than 3 DHI data within the same lactation period.

[0064] The above figures for milk production, milk fat content, and milk protein content are all based on statistics from the first lactation period (the lactation period after the first delivery).

[0065] Example 1, Related Basic Research

[0066] Our research group used liver tissues from three Chinese Holstein cattle at different lactation stages (dry period, early lactation, and peak lactation) as experimental materials. We used next-generation sequencing technology for transcriptome sequencing (RNA-sequencing, RNA-seq) and small RNA sequencing (small RNA-seq). We found that the expression level of the APOM gene was significantly increased in the early lactation stage (P<0.05). Combined with functional enrichment analysis and pathway analysis, we found that the APOM gene is a candidate gene affecting milk production traits.

[0067] Example 2: Gene Polymorphism Detection

[0068] I. A total of 111 female Holstein bulls from 80 Holstein bull families in Hebei Province were selected as the experimental population for gene polymorphism detection. These 111 Chinese Holstein bulls were randomly divided into 5 groups: the first group contained 23 individuals, and the remaining four groups contained 22 individuals each. The concentration of genomic DNA in their blood was accurately measured using a nucleic acid analyzer. This DNA was diluted to a concentration of 50 ng / μL and mixed in equal volumes to form 5 DNA pools, which were then used as templates for PCR amplification.

[0069] II. Based on the bovine APOM gene sequence (Ensembl Gene ID: ENSBTAG00000008833, Transcript ID: ENSBTAT00000075540.1), 10 pairs of primers were designed as shown in Table 1.

[0070] Table 1 shows the primer sequences for APOM gene PCR amplification.

[0071]

[0072]

[0073] In the table above, the sequences in column 3 from top to bottom are Sequence 1 to Sequence 20.

[0074] 3. Using the pooled DNA obtained in step 1 as a template, PCR amplification was performed using the primer pairs shown in Table 1 to obtain the PCR amplification products. The PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.

[0075] Table 2 shows the PCR reaction system.

[0076]

[0077] In Table 2, the forward primer F and the reverse primer R represent 1F and 1R, 2F and 2R, respectively.

[0078] Table 3 shows the PCR reaction conditions.

[0079]

[0080] IV. Sequencing the PCR amplification products

[0081] Sequencing of the PCR amplification products revealed four SNP markers in the experimental population: two SNP markers in the flanking sequence 2000 bp upstream of the APOM gene, one SNP marker in exon 2, and one SNP marker in the flanking sequence 2000 bp downstream. These four SNP markers are shown in Table 4, and their mutation locations are as follows: Figure 1 As shown.

[0082] Table 4 shows the four SNPs found in the APOM gene.

[0083]

[0084]

[0085] Among them, SNP site 23:g.27643343G>T is the name of SNP. SNP naming is generally based on the pattern when the SNP is first discovered. In the amplification product of this invention, 23:g.27643343G>T and the sequence when the SNP was discovered are the reverse complementary strands of the same double-stranded DNA. The polymorphic form of 23:g.27643343G>T site in the amplification product of this invention is A / C, and the genotypes are AA, AC and CC.

[0086] Of the four SNPs in the APOM gene mentioned above,

[0087] SNP locus 23:g.27644015C>G was obtained by sequencing the product of PCR amplification using bovine genomic DNA as a template and primers 3F and 3R. This locus is the 202nd position from the 5' end of the PCR product (sequence 21), which is the 27644015th position on chromosome 23 of the bovine genome ARS-UCD1.2 version reference sequence (Ensembl Gene ID: ENSBTAG00000008833, Transcript ID: ENSBTAT00000075540.1). The base at this locus can be C or G; the genotype at this locus is CC, GG, or CG.

[0088] SNP locus 23: g.27643343G>T was obtained by sequencing the product of PCR amplification using bovine genomic DNA as a template and primers 4F and 4R. This locus is the 184th position from the 5' end of the PCR product (sequence 22), which is the 27643343rd position on chromosome 23 of the bovine genome ARS-UCD1.2 version reference sequence. The base at this locus can be A or C; the genotype at this locus is AA, CC, or AC.

[0089] SNP locus 23: g.27642597A>G was obtained by sequencing the product of PCR amplification using bovine genomic DNA as a template and primers 5F and 5R. This locus is the 260th position from the 5' end of the PCR product (sequence 23), which is the 27642597th position on chromosome 23 of the bovine genome ARS-UCD1.2 version reference sequence. The base at this locus can be A or G; the genotype at this locus is AA, GG, or AG.

[0090] SNP locus 23: g.27640015G>C was obtained by sequencing the product of PCR amplification using bovine genomic DNA as a template and 10F and 10R primers. This locus is the 168th position from the 5' end of the PCR product (sequence 24), which is the 27640015th position on chromosome 23 of the bovine genome ARS-UCD1.2 version reference sequence. The base at this locus can be G or C; the genotype at this locus is GG, CC, or GC.

[0091] Example 3: Association Analysis

[0092] I. Obtaining the test group

[0093] The test population consisted of 1,123 Chinese Holstein cows.

[0094] II. Genotyping

[0095] Genotyping of 1123 Chinese Holstein cattle was performed based on the four SNPs of the APOM gene in Example 2, as detailed below:

[0096] Genomic DNA was extracted from blood samples of 1123 Chinese Holstein cows. The genomic samples were sent to Bomiao Biotechnology (Beijing) Co., Ltd. for genotyping of different SNP sites using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results are shown in Table 5.

[0097] III. Obtaining Milk Production Traits

[0098] Milk production traits were obtained from each cow in the test group.

[0099] Milk production traits include the following five indicators: milk yield, milk fat content, milk fat percentage, milk protein content, and milk protein percentage.

[0100] Each individual's record includes, in order: cow individual number, father number, mother number, grandfather number, grandmother number, maternal grandfather number, maternal grandmother number, date of birth, lactation period, calving date, milk yield, milk fat content, and milk protein content.

[0101] IV. Association Analysis Model between Single SNP Loci and Traits

[0102] Association analysis was performed on five indicators of milk production traits and genotypes using the MIXED procedure in SAS 9.2 software. An animal model was used for the association analysis; the specific model is as follows:

[0103] Y = μ + hys + b × M + G + a + e

[0104] Where Y represents the phenotypic value of each trait per cow (milk yield, milk fat content, milk fat percentage, milk protein content, or milk protein percentage); μ is the overall mean (i.e., the average phenotypic value of 1123 cows); hys represents the farm-year-season effect, where farm (1–2: 2 farms); calving year (1–7: 2013–2019); calving season (1: April–May; 2: June–August; 3: September–November; 4: December–March); M represents the calving age effect; b is the regression coefficient of the covariate M; G represents the genotype or haplotype combination effect; a represents the individual random additive genetic effect; and e is the random residual. Bonferroni correction was performed through multiple tests, and the significance level was equal to the original p-value divided by the number of genotype or haplotype combinations.

[0105] The association analysis results of the seven SNP sites of the APOM gene with milk production traits are shown in Table 5.

[0106] Table 5 shows the association analysis between the four SNP loci of the APOM gene and the milk production trait (least square mean ± standard error).

[0107]

[0108]

[0109] Note: * P<0.05 indicates a significant difference; ** P<0.01 indicates that the difference is highly significant. A,b Different superscripts in the same column of data indicate significant differences. A,B Different superscripts in the same column of data indicate highly significant differences.

[0110] As shown in the table above, all four SNPs were significantly or highly significantly associated with the traits of milk fat content and milk protein percentage (P < 0.0001–0.0417), three SNPs (23:g.27644015C>G, 23:g.27642597A>G, and 23:g.27640015G>C) were significantly or highly significantly associated with the trait of milk fat percentage (P = 0.002–0.0341), two SNPs (23:g.27643343G>T and 23:g.27642597A>G) were highly significantly associated with the trait of milk protein content (P < 0.0001, P = 0.0018), and 23:g.27643343G>T was highly significantly associated with the trait of milk yield (P = 0.0017).

[0111] Therefore, the milk production trait of the dairy cow can be determined by identifying the genotypes of the following four SNP loci:

[0112] The milk fat content / milk fat percentage of the tested dairy cows with the SNP locus 23:g.27644015C>G and the genotype CC was higher than that of the tested dairy cows with the SNP locus 23:g.27644015C>G and the genotype GG.

[0113] The milk yield / milk fat content / milk protein content / milk protein percentage of the tested dairy cows with SNP locus 23:g.27643343G>T genotype AA was higher than that of the tested dairy cows with SNP locus 23:g.27643343G>T genotype CC.

[0114] The milk fat content / milk fat percentage / milk protein content / milk protein percentage of the tested cows with SNP locus 23:g.27642597A>G and genotype GG were higher than those of the tested cows with SNP locus 23:g.27642597A>G and genotype AA.

[0115] The milk fat content / milk fat percentage of the tested dairy cows with the SNP locus 23:g.27640015G>C and the genotype GG was higher than that of the tested dairy cows with the SNP locus 23:g.27640015G>C and the genotype CC.

[0116] The genotypes of each SNP locus can be determined by amplification with the corresponding primers followed by sequencing.

[0117] V. Analysis of Genetic Effects

[0118] The significance of SNP additive effects, dominant effects, and substitution effects was tested using SAS 9.2 software.

[0119] The basic calculation formula is as follows:

[0120] a = (AA - BB) / 2, d = AB - (AA + BB) / 2, α = a + d(qp); 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 mean values ​​of the milk production trait for the corresponding genotypes; p is the frequency of allele A, and q is the frequency of allele B.

[0121] The results of tests for the additive effect, dominant effect, and allele substitution effect of the APOM gene are shown in Table 6.

[0122] The allele frequencies for each site are as follows:

[0123] Site 23: g.27644015C>G: p(C)=0.1555, q(G)=0.8445;

[0124] Locus 23: g.27643343G>T: p(C)=0.1194, q(A)=0.8806;

[0125] Locus 23: g.27642597A>G: p(A)=0.2179, q(G)=0.7821;

[0126] Site 23: g.27640015G>C: p(G)=0.1554, q(C)=0.8446.

[0127] Table 6 shows the results of tests for the additive, dominant, and substitution effects of APOM gene alleles.

[0128]

[0129] In the table above, additive effects are the heritable portions, dominant effects represent heterozygous effects, and substitution effects are the effects of allele substitution due to mutations, representing homozygous effects. This invention should place greater emphasis on additive and substitution effects; if both are significant, it indicates a significant homozygous effect that is heritable.

[0130] Note: * P<0.05 indicates a significant difference; ** P<0.01 indicates that the difference is highly significant.

[0131] The additive, dominant, and allele substitution effects of the four SNP loci on the five milk production traits were significant or highly significant (P < 0.05, P < 0.01). Taking the 23:g.27643343G>T locus as an example, the additive, dominant, and allele substitution effects on milk yield, milk fat content, and milk protein content, as well as the additive effect on milk protein percentage, were significant or highly significant (P < 0.05, P < 0.01). That is, each substitution of the C allele for the A allele would lead to an increase of 634.22 kg in milk yield, 23.609 kg in milk fat content, and 20.831 kg in milk protein content (P < 0.01).

[0132] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of substances used to detect the genotype of at least one of the following four SNP loci in the identification or auxiliary identification of milk production traits in dairy cows; The four SNP sites are SNP site 23: g.27644015C>G, SNP site 23: g.27643343G>T, SNP site 23: g.27642597A>G and SNP site 23: g.27640015G>C; The locations of all four SNPs were described using the bovine genome from ARS-UCD 1.2 as a reference genome. The dairy cows mentioned are Chinese Holstein cattle.

2. Application of at least one of the following four SNP loci as a detection target in identifying or assisting in the identification of milk production traits in dairy cows; Alternatively, at least one of the following four SNP sites can be used as a detection target in the development, identification, or auxiliary identification of dairy cow milk production trait products; The four SNP sites are SNP site 23: g.27644015C>G, SNP site 23: g.27643343G>T, SNP site 23: g.27642597A>G and SNP site 23: g.27640015G>C; The locations of all four SNPs were described using the bovine genome from ARS-UCD 1.2 as a reference genome. The dairy cows mentioned are Chinese Holstein cattle.

3. The application as described in claim 1 or 2, characterized in that: The milk production traits are milk yield and / or milk fat content and / or milk fat percentage and / or milk protein content and / or milk protein percentage.

4. A method for identifying or assisting in the identification of milk production traits in dairy cows, comprising any one of the following 1)-4): 1) Includes the following steps: testing the test cows APOM The genotype of SNP site 23:g.27644015C>G in the gene; the genotype of SNP site 23:g.27644015C>G is CC or GG; The milk fat content and / or milk fat percentage of the tested dairy cows with the SNP locus 23:g.27644015C>G genotype CC were superior to or slightly superior to the tested dairy cows with the SNP locus 23:g.27644015C>G genotype GG. 2) Includes the following steps: testing the test cows APOM The genotype of SNP site 23:g.27643343G>T in the gene; the genotype of SNP site 23:g.27643343G>T is AA or CC; The milk yield, milk fat content, milk protein content, and / or milk protein percentage of the tested dairy cows with the SNP locus 23:g.27643343G>T genotype AA were superior to or slightly superior to those of the tested dairy cows with the SNP locus 23:g.27643343G>T genotype CC. 3) Includes the following steps: testing the test cows APOM The genotype of SNP site 23:g.27642597A>G in the gene; the genotype of SNP site 23:g.27642597A>G is AA or GG; The milk fat content, milk fat percentage, milk protein content, and / or milk protein percentage of the tested dairy cows with the SNP locus 23:g.27642597A>G genotype GG were superior to or slightly superior to those of the tested dairy cows with the SNP locus 23:g.27642597A>G genotype AA. 4) Includes the following steps: testing the test cows APOM The genotype of SNP site 23:g.27640015G>C in the gene; the genotype of SNP site 23:g.27640015G>C is GG or CC; The milk fat content and / or milk fat percentage of the tested dairy cows with the SNP locus 23:g.27640015G>C genotype GG were superior to or slightly superior to the tested dairy cows with the SNP locus 23:g.27640015G>C genotype CC. The locations of all four SNPs were described using the bovine genome from ARS-UCD 1.2 as a reference genome. The dairy cows mentioned are Chinese Holstein cattle.

5. The application of the method according to claim 4 in screening for milk production traits in dairy cows or in breeding for milk production traits in dairy cows; The dairy cows mentioned are Chinese Holstein cattle.

6. The application according to claim 5, characterized in that: In the application, any of the following dairy cows in claim 4 are selected for milk production or breeding; The tested dairy cows with the SNP locus 23:g.27644015C>G genotype CC; The tested dairy cows had the AA genotype at SNP locus 23:g.27643343G>T. The tested dairy cows with the genotype GG at SNP locus 23:g.27642597A>G; The tested dairy cows had the genotype GG at the SNP locus 23:g.27640015G>C.

7. The application of any one or a combination of primer pairs 3, 4, 5, and 10 is any one of the following (a)-(f): The primer pair consists of primer pair 3, primer pair 4, primer pair 5 and primer pair 10; Primer pair 3 is a primer pair consisting of primer 3F and primer 3R; Primer pair 4 is a primer pair consisting of primer 4F and primer 4R; Primer pair 5 is a primer pair consisting of primer 5F and primer 5R; The primer pair 10 is a primer pair consisting of primer 10F and primer 10R; Primer 3F is the single-stranded DNA molecule shown in sequence 5 of the sequence listing; primer 3R is the single-stranded DNA molecule shown in sequence 6 of the sequence listing; Primer 4F is the single-stranded DNA molecule shown in sequence 7 of the sequence listing; primer 4R is the single-stranded DNA molecule shown in sequence 8 of the sequence listing; Primer 5F is the single-stranded DNA molecule shown in sequence 9 of the sequence listing; primer 5R is the single-stranded DNA molecule shown in sequence 10 of the sequence listing; Primer 10F is a single-stranded DNA molecule as shown in sequence 19 of the sequence listing; primer 10R is a single-stranded DNA molecule as shown in sequence 20 of the sequence listing; (a) To identify or assist in the identification of milk production traits in dairy cows; (b) Screening for milk production traits in dairy cows; (c) Breeding for milk production traits in dairy cows; (d) Prepare kits for identifying or assisting in the identification of milk production traits in dairy cows; (e) Preparation of a kit for screening milk production traits in dairy cows; (f) Preparation of a kit for breeding dairy cows with milk production traits; The dairy cows mentioned are Chinese Holstein cattle.

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