CN117512125A - A SNP molecular marker of HADHB, a gene related to dairy cow milk production traits, and its application - Google Patents

Abstract

The invention discloses a SNP molecular marker of a gene HADHB related to dairy cow milk production traits and application thereof. The invention provides application of a substance for detecting polymorphism or genotype of at least one SNP locus in 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. Experiments prove that 3 SNP, SNP1, SNP2 and SNP3 are respectively positioned in genes HADHB related to milk production traits in a dairy cow genome, the dominant allele of the SNP1 locus is C, the dominant allele of the SNP2 locus is C, and the dominant allele of the SNP3 locus is T, so that the SNP1 locus, the SNP2 locus and/or the SNP3 locus can be used for auxiliary selection breeding of dairy cow molecular markers and breeding of dairy cow varieties with high milk production traits.

Description

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

Technical Field

The invention belongs to the field of biotechnology and relates to a SNP molecular marker of a gene HADHB related to milk production traits of dairy cows and an application thereof.

Background Art

Beta Hydroxyacyl-CoAdehydrogenase trifunctional multienzyme complex subunit beta (HADHB), This gene encodes the alpha subunit of the mitochondrial trifunctional protein that 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, with the alpha subunit catalyzing both 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activities. Mutations in this gene result in trifunctional protein deficiency or LCHAD deficiency. The genes for the alpha and beta subunits of the mitochondrial trifunctional protein are located adjacent to each other in a head-to-head orientation in the human genome.

The HADHB gene encodes the β subunit of a mitochondrial trifunctional protein that is involved in catalyzing the last three steps of mitochondrial β-oxidation of long-chain fatty acids and plays an important role in controlling and regulating β-oxidation. In chickens, this gene plays an important role in liver lipid metabolism. It has been reported that this gene is expressed in the liver of rats and dogs, inducing peroxisomal and mitochondrial β-oxidation activity.

Polymerase Chain Reaction (PCR) is a rapid nucleic acid amplification technology that simulates the natural DNA replication process in vitro. Its biggest 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 uses the fact that DNA denatures into single strands at high temperatures in vitro. At low temperatures, primers and single strands combine according to the principle of complementary base pairing. The temperature is then adjusted to the optimal reaction temperature of DNA polymerase, and DNA polymerase synthesizes complementary chains along the direction from phosphate to pentose (5'-3'). At present, this technology has become one of the most commonly used and important molecular biology techniques. The PCR product can be sequenced after agarose gel electrophoresis to identify genetic polymorphisms, and the detection method is simple and easy.

Summary of the invention

The purpose of the present invention is to provide a SNP molecular marker of a gene HADHB related to milk production traits of dairy cows and an application thereof.

In a first aspect, the present invention provides a use of a substance for detecting the polymorphism or genotype of at least one of the following three SNP sites in identifying or assisting in identifying the milk production trait of a dairy cow;

The three SNP sites are SNP site g.73256269T>C, SNP site g.73256227A>C and SNP site g.73242290C>T.

In a second aspect, the present invention provides the use of at least one of the following three SNP sites as a detection target in identifying or assisting in identifying the milk production traits of dairy cows;

Or, use of at least one of the following three SNP sites as a detection target in developing and identifying or assisting in identifying a product for dairy cow milk production traits;

The three SNP sites are SNP site g.73256269T>C, SNP site g.73256227A>C and SNP site g.73242290C>T.

In the application described above, the milk production trait is milk yield, milk fat content and/or milk protein content.

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):

1) comprising the following steps: detecting the genotype of the SNP site g.73256269T>C in the HADHB gene of the tested dairy cow; the genotype of the SNP site g.73256269T>C is CC or CT or TT;

The milk yield, milk fat content and/or milk protein content of the test cows whose genotype of the SNP site g.73256269T>C is CC are better than or auxiliary better than the test cows whose genotype of the SNP site g.73256269T>C is TT or CT;

2) comprising the following steps: detecting the genotype of the SNP site g.73256227A>C in the HADHB gene of the tested dairy cow; the genotype of the SNP site g.73256227A>C is AA or AC or CC;

The milk production, milk fat content and/or milk protein content of the test cows whose SNP site g.73256227A>C genotype is CC are better than or auxiliary better than the test cows whose SNP site g.73256227A>C genotype is AA or AC;

3) comprising the following steps: detecting the genotype of the SNP site g.73242290C>T in the HADHB gene of the tested dairy cow; the genotype of the SNP site g.73242290C>T is CC or TT or CT;

The milk production, milk fat content and/or milk protein content of the test cows whose genotype of the SNP site g.73242290C>T is TT are better than or auxiliary better than the test cows whose genotype of the SNP site g.73242290C>T is CC or CT.

In a fourth aspect, the present invention provides application of the method described in the third aspect in dairy cow screening or dairy cow breeding.

In the above application, any of the following test cows in the third aspect is selected for milk production or breeding;

The SNP site g.73256269T>C genotype of the tested dairy cow is CC;

The SNP site g.73256227A>C genotype of the tested dairy cow is CC;

The genotype of the SNP site g.73242290C>T in the tested dairy cow is TT.

In a fifth aspect, the present invention provides a method for breeding dairy cows, comprising the following steps:

According to the steps in the method of the third aspect, the genotype of each SNP site is identified, and any of the following test cows are selected for milk production or breeding;

The SNP site g.73256269T>C genotype of the tested dairy cow is CC;

The SNP site g.73256227A>C genotype of the tested dairy cow is CC;

The genotype of the SNP site g.73242290C>T in the tested dairy cow is TT.

In a sixth aspect, the present invention provides any one or a combination of primers in primer pair 6 and primer pair 9;

The primer combination consists of the primer pair 6 and the primer pair 9;

The primer pair 6 is a primer pair consisting of primer 6F and primer 6R;

The primer pair 9 is a primer pair consisting of primer 9F and primer 9R;

The primer 6F is a single-stranded DNA molecule shown in SEQ ID No. 4 or a nucleotide with the same function as SEQ ID No. 4, with one or more nucleotides deleted, added or changed from SEQ ID No. 4;

The primer 6R is a single-stranded DNA molecule shown in SEQ ID No.5 or a nucleotide with the same function as SEQ ID No.5, with one or more nucleotides deleted, added or changed from SEQ ID No.5;

The primer 9F is a single-stranded DNA molecule shown in SEQ ID No.6 or a nucleotide with the same function as SEQ ID No.6 by deleting, adding or changing one or several nucleotides in SEQ ID No.6;

The primer 9R is a single-stranded DNA molecule shown in SEQ ID No.7 or a nucleotide with the same function as SEQ ID No.7, with one or more nucleotides deleted, added or changed from SEQ ID No.7.

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

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

(b) Dairy cow screening;

(c) dairy cattle breeding;

(d) preparing a test kit for identifying or assisting in identifying the milk production traits of dairy cows;

(e) preparing a kit for screening dairy cows;

(f) Preparing a kit for dairy cow breeding.

The dairy cow breeding mentioned above is to cultivate dairy cow breeds with high milk production traits.

In the above text,

The SNP1 is a SNP in the cow genome, which is the 2179th nucleotide of SEQ ID No.1 in the sequence list, which is T or C; the SNP2 is a SNP in the cow genome, which is the 2221st nucleotide of SEQ ID No.1 in the sequence list, which is A or C; the SNP3 is a SNP in the cow genome, which is the 5002nd nucleotide of SEQ ID No.2 in the sequence list, which is C or T.

The 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 trait of dairy cows. The nucleotide sequence of the gene consists of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 in sequence.

The genotype (i.e., allele) of the SNP1 may be genotype CC, genotype TT or genotype CT, genotype CC is the homozygous type of SNP1 with C, genotype TT is the homozygous type of SNP1 with T, and genotype CT is the heterozygous type of SNP1 with C and T; the genotype (i.e., allele) of the SNP2 may be genotype AA, genotype CC or genotype AC, genotype AA is the homozygous type of SNP2 with A, genotype CC is the homozygous type of SNP2 with C, and genotype AC is the heterozygous type of SNP2 with A and C; the genotype (i.e., allele) of the SNP3 may be genotype CC, genotype TT or genotype CT, genotype CC is the homozygous type of SNP1 with C, genotype TT is the homozygous type of SNP1 with T, and genotype CT is the heterozygous type of SNP1 with C and T.

The high milk production trait mentioned above may specifically be high milk yield, high milk fat content and/or high milk protein content.

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 be used to determine the nucleotide types of the SNP1, SNP2 and/or SNP3 sites in the above 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.

In the above application or method, the substance for detecting the polymorphism or genotype of the three SNPs SNP1, SNP2 and/or SNP3 may be as follows:

D1) containing PCR primers for amplifying a dairy cow genomic DNA fragment including the SNP1, SNP2 and/or SNP3 sites;

D2) a PCR reagent containing the PCR primers described in D1);

D3) A kit containing the PCR primers described in D1) or the PCR reagents described in D2).

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

In the above-mentioned application or method, the PCR primer may be labeled or not labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be attached to a nucleic acid. Markers 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 fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The marker can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, weight determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or, alternatively, can be charge neutral. The marker can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence containing the marker is detectable. In some embodiments, the nucleic acid is directly detected (e.g., directly reading the sequence) without a marker.

In the above application or method, the product may be a reagent or a kit or a system, and the system may include a combination product of a reagent or a kit, an instrument and an analysis software, such as a product consisting of PCR primers, PARMS master mix reagents, an ELISA reader and an online software SNP decoder (http://www.snpway.com/snpdecoder01/), and a combination product consisting of PCR primers, PARMS master mix reagents, an online software SNP decoder and a fluorescence quantitative PCR instrument. The product may include the above-mentioned substance for detecting the polymorphism or genotype of the SNP1, SNP2 and/or SNP3 sites in the cow genome.

The experiment of the present invention proves that the present invention analyzes the genetic variation of the HADHB gene in the dairy cow associated population and finds that three SNPs, SNP1, SNP2 and SNP3, are respectively located in the gene HADHB related to the milk production trait in the dairy cow genome, namely, the 2179th position of SEQ ID No.1, the 2221st position of SEQ ID No.1 and the 5002nd position of SEQ ID No.2 in the sequence list. In the embodiment of the present invention, 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, the SNP2 site and/or the SNP3 site can be used for molecular marker-assisted selection breeding of dairy cows and the breeding of high milk production trait dairy cow varieties.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

The Chinese Holstein cows in the following examples were from the Hebei Provincial Animal Husbandry Breeding Station.

The data in the embodiments are all data of lactation period 2. Lactation period 2 refers to the lactation period after the second parturition.

The milk production in the embodiment is the individual 305-day milk production, which refers to the total milk production from the first day of calving 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 306 days is not included. The milk production is obtained by measuring the monthly DHI (DHI, Dairy Herd Improvement). The milk production lactation curve is drawn by using the DHI data of more than 3 times in the same lactation period to calculate the milk production of 305 days in the lactation period.

The milk fat amount in the embodiment 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 obtained by measuring the monthly DHI (DHI, Dairy Herd Improvement). The average milk fat rate of the lactation period can be obtained by drawing a milk fat rate lactation curve based on more than 3 DHI data in the same lactation period.

The milk protein amount in the embodiment refers to the 305-day milk protein amount, and the 305-day milk protein amount = milk protein rate × 305-day milk production. The milk protein rate is obtained by measuring the monthly DHI (DHI, Dairy Herd Improvement). The average milk protein rate of the lactation period can be obtained by drawing a milk protein rate lactation curve based on more than three DHI data in the same lactation period.

Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

Example 1. Discovery of molecular markers

1. Related basic research

The inventor's research group used liver tissues from three Chinese Holstein cows at different lactation stages (dry period, early lactation, and peak lactation) as experimental materials, and used the second-generation sequencing technology to perform transcriptome sequencing (RNA-sequencing, RNA-seq) and small RNA sequencing (small RNA sequencing, small RNA-seq). It was found that compared with the dry period, the expression of HADHB in the early and peak lactation periods was significantly upregulated (P<0.01), which can promote the increase of bile acid synthesis, thereby improving lipid absorption for milk synthesis.

2. Gene polymorphism detection

1. A total of 655 Chinese Holstein cows in Hebei Province were selected as the test population for gene polymorphism detection. Genomic DNA was extracted from blood samples, and the concentration of DNA was accurately measured using a nucleic acid quality detector. The DNA was diluted to a concentration of 50 ng/μL and mixed into 5 pools of DNA in equal amounts for PCR amplification as a template.

2. According to the bovine HADHB gene sequence (Ensembl ID is ENSBTAG00000005287, whose nucleotide sequence is composed 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.

Table 1 shows the primer sequence information for PCR amplification of HADHB gene

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

Table 2 shows the PCR reaction system

Table 3 shows the PCR reaction conditions.

4. Sequencing of the PCR amplified product. The results showed that the HADHB gene of the cow population had two SNP markers (respectively called SNP1 and SNP2) in the upstream 2000bp flanking sequence and one SNP marker (respectively called SNP3) in the 3'UTR. The three SNP markers are shown in Table 4.

Table 4 shows the three SNPs found in the HADHB gene

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

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

g.73256269T>C, g.73256227A>C and g.73242290C>T are the names of SNP1, SNP2 and SNP3 respectively. The naming of SNP is generally named according to the rule when the SNP is first discovered. DNA is a double-stranded structure and follows the principle of base complementary pairing. The SEQ ID No.1 in the present invention and the sequence when SNP1, SNP2 and SNP3 are found are the reverse complementary chains of the same double-stranded DNA. Therefore, the polymorphic form of g.73256269T>C in the present invention is T or C, the polymorphic form of g.73256227A>C is A or C, and the polymorphic form of g.73242290C>T is C or T.

3. Correlation Analysis

(I) Obtaining the test population

The test population consisted of 655 Chinese Holstein cows.

(II) Genotyping

Each individual in the test group was genotyped separately.

I. Genotyping based on g.73256269T>C.

1. Take blood from the test individual and extract genomic DNA.

2. Using genomic DNA as a template, PCR amplification was performed using a primer pair consisting 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), and then the PCR amplification product was recovered and sequenced.

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

The PCR amplification products of each test individual were 820 bp, of which the 183rd position was g.73256269T>C, i.e. SNP1 (corresponding to the 2179th position from the 5' end of SEQ ID No.1 in the sequence table).

Table 5 is the reaction system for PCR amplification

Table 6 shows the reaction conditions for PCR amplification

II. Genotyping based on g.73256227A>C.

1. Take blood from the test individual and extract genomic DNA.

2. Using genomic DNA as a template, PCR amplification was performed using a primer pair consisting 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), and then the PCR amplification product was recovered and sequenced.

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

The PCR amplification products of each test individual were 820 bp, of which the 225th position was g.73256227A>C, i.e. SNP2 (corresponding to the 2221st position from the 5' end of SEQ ID No.1 in the sequence table).

Table 7 is the reaction system for PCR amplification

Table 8 shows the reaction conditions for PCR amplification

III. Genotyping based on g.73242290C>T.

1. Take blood from the test individual and extract genomic DNA.

2. Using genomic DNA as a template, PCR amplification was performed using a primer pair consisting 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), and then the PCR amplification product was recovered and sequenced.

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

The PCR amplification products of each test individual were 242 bp, of which the 210th position was g.73242290C>T, i.e. SNP3 (corresponding to the 5002nd position from the 5' end of SEQ ID No. 2 in the sequence table).

Table 9 is the reaction system for PCR amplification

Table 10 shows the reaction conditions for PCR amplification

(III) Testing milk production traits

Each cow in the test group was tested for milk production traits.

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

The records of each individual include the cow's individual number, father number, mother number, grandfather number, grandmother number, maternal grandfather number, maternal grandmother number, date of birth, lactation period, calving date, milk production, milk fat content and milk protein content.

(IV) Association analysis model between single SNP loci and traits

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

Table 11 shows the phenotypes of some milk production traits and the genotypes of three SNP loci of 655 Chinese Holstein cows

Table 12 shows the descriptive statistics of the phenotypic values of five milk production traits in a population of 655 Chinese Holstein cows

Characteristics average value Standard Deviation Minimum Maximum Milk production (kg) 10840.58 1895.22 4757 16512 Milk fat content (kg) 390.53 85.75 145 741 Milk fat percentage (%) 3.608 0.505 2.212 5.431 Milk protein amount (kg) 321.73 58.17 138 467 Milk protein rate (%) 2.971 0.191 2.199 3.538

Table 13 shows the allele frequency and genotype frequency of the three SNP sites of the HADHB gene

The results showed that there were three genotypes at the SNP1 site (referred to as SNP1 genotypes), namely CC, TT or CT. Genotype CC was the homozygous type of SNP1 with C, genotype TT was the homozygous type of SNP1 with T, and genotype CT was the heterozygous type of SNP1 with C and T. There were three genotypes at the SNP2 site (referred to as SNP2 genotypes), namely AA, CC or AC. Genotype AA was the homozygous type of SNP2 with A, genotype CC was the homozygous type of SNP2 with C, and genotype AC was the heterozygous type of SNP2 with A and C. There were three genotypes at the SNP3 site (referred to as SNP3 genotypes), namely CC, TT or CT. Genotype CC was the homozygous type of SNP3 with C, genotype TT was the homozygous type of SNP3 with T, and genotype CT was the heterozygous type of SNP3 with C and T.

The MIXED procedure in SAS 9.2 software was used to conduct association analysis on the five indicators of milk production traits and genotypes. The association analysis used an animal model, and the specific model is as follows:

Y＝μ+hys+b×M+G+a+e

Wherein, Y: observed value of milk production traits (milk yield, milk fat content, milk fat rate, milk protein content or milk protein rate); μ: overall mean; hys: field year and season effect; b: regression coefficient of covariate M; M: calving age effect; G: genotype effect; a: individual random additive genetic effect; e: random residual effect.

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

genotype Milk production (kg) Milk fat content (kg) Milk fat percentage (%) Milk protein amount (kg) Milk protein rate (%) 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

Note: ^* P<0.05 indicates significant difference; ^** P<0.01 indicates extremely significant difference. Data in the same column ^{a and b} with different superscripts indicate significant difference; Data in the same column ^{A and B} with different superscripts indicate extremely significant difference.

As shown in Table 14, SNP1 (g.73256269T>C) is significantly associated with milk yield, milk fat content and milk protein content (P<0.0001). For the traits of milk yield, milk fat content and milk protein content, the dominant allele is C:

The milk production of CC genotype cows was higher than that of TT or CT genotype cows, and the milk production of CT genotype cows was higher than that of TT genotype cows.

The milk fat content of CC genotype cows was higher than that of TT or CT genotype cows, and the milk fat content of CT genotype cows was higher than that of TT genotype cows.

The milk protein content of CC genotype cows was higher than that of TT or CT genotype cows, and there was no significant difference in milk protein content between CT genotype cows and TT genotype cows.

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

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

Note: ^* P<0.05 indicates significant difference; ^** P<0.01 indicates extremely significant difference. Data in the same column ^{a and b} with different superscripts indicate significant difference; Data in the same column ^{A and B} with different superscripts indicate extremely significant difference.

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 the traits of milk yield, milk fat content and milk protein content, the dominant allele is C:

The milk production of CC genotype cows was higher than that of AA or AC genotype cows, and the milk production of AC genotype cows was higher than that of AA genotype cows.

The milk fat content of CC genotype cows was higher than that of AA or AC genotype cows, and the milk fat content of AC genotype cows was higher than that of AA genotype.

The milk protein content of CC genotype cows was higher than that of AA or AC genotype cows, and there was no significant difference in milk protein content between AC genotype cows and AA genotype cows.

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

genotype Milk production (kg) Milk fat content (kg) Milk fat percentage (%) Milk protein amount (kg) Milk protein rate (%) 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

Note: ^* P<0.05 indicates significant difference; ^** P<0.01 indicates extremely significant difference. Data in the same column ^{a and b} with different superscripts indicate significant difference; Data in the same column ^{A and B} with different superscripts indicate extremely significant difference.

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

The milk production of cows with TT genotype was higher than that of cows with CC or CT genotypes, and there was no significant difference in milk production between cows with CT genotype and CC genotype.

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

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

(V) Analysis of genetic effects

SAS 9.2 software was used to perform significance tests on SNP additive effect, dominant effect and substitution effect.

The basic calculation formula is as follows:

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

The results of the tests for additive effect, dominant effect and allele substitution effect are shown in Table 17.

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

Note: ^* P<0.05 indicates a significant difference; ^** P<0.01 indicates an extremely significant difference.

The results show that the additive effect and allele substitution effect of SNP1 (g.73256269T>C) on milk yield and milk protein content are extremely significant, and the additive effect, dominant effect and allele substitution effect on milk fat content are extremely significant, that is, each C allele replacing the T allele will lead to an increase in milk production of 471.25kg (P < 0.01), an increase in milk fat content of 24.1742kg (P < 0.01), and an increase in milk protein content of 14.4302kg (P < 0.01). The additive effect, dominant effect and allele substitution effect of SNP2 (g.73256227A>C) on milk yield, milk fat content, milk fat rate, milk protein content and milk protein rate were significant or extremely significant, that is, each C allele replacing the A allele would lead to an increase of 538kg in milk production (P＜0.01), an increase of 36.6914kg in milk fat content (P＜0.01), an increase of 0.1472% in milk fat rate (P＜0.05), an increase of 17.5372kg in milk protein content (P＜0.01) and an increase of 0.00162kg in milk protein rate (P＜0.01). The additive effect and allele substitution effect of SNP3 (g.73242290C>T) on milk yield, milk fat content, milk protein content and milk protein rate were significant or extremely significant, that is, each T allele replacing the C allele would lead to an increase of 140.15 kg in milk yield (P < 0.05), an increase of 10.0302 kg in milk fat content (P < 0.01), an increase of 7.5665 kg in milk protein content (P < 0.01), and an increase of 0.02341 kg in milk protein rate (P < 0.05).

(VI) Haplotype analysis

Haploview 4.2 software was used to construct haplotypes, estimate haplotype frequencies and linkage analysis, and conduct association analysis with traits. Using the MIXED procedure in SAS 9.2 software, the model is as follows:

Y＝μ+hys+b×M+G+a+e

Y: observed values of milk production traits (milk yield, milk fat content, milk fat percentage, milk protein content and milk protein percentage); μ: overall mean; hys: field year and season effect; b: regression coefficient of covariate M; M: calving age effect; G: haplotype combination effect; a: individual random additive genetic effect; e: random residual effect.

The molecular markers disclosed in the present invention can be used to assist in identifying a group of dairy cows with excellent milk production traits (milk production at 305 days, milk fat content, milk fat percentage, milk protein content and milk protein percentage), and have the following advantages: simplicity, rapidity, sensitivity, reliable, stable and accurate results, and are suitable for the needs of large-scale group testing in laboratories.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims (9)

1. The application of substances used to detect the polymorphism or genotype of at least one SNP site among the following three SNP sites in identifying or assisting in identifying milk production traits of dairy cows; The three SNP sites are SNP site g.73256269T>C, SNP site g.73256227A>C and SNP site g.73242290C>T. 2. The application of at least one SNP site among the following three SNP sites as a detection target in identifying or assisting in identifying milk production traits of dairy cows; Or, the application of at least one SNP site among the following three SNP sites as a detection target in the development of products to identify or assist in the identification of milk production traits of dairy cows; The three SNP sites are SNP site g.73256269T>C, SNP site g.73256227A>C and SNP site g.73242290C>T. 3. The application according to claim 1 or 2, characterized in that the milk production traits are milk production, milk fat and/or milk protein. 4. A method for identifying or assisting in identifying milk production traits of dairy cows, which is any one of the following 1)-5): 1) includes the following steps: detecting the genotype of the SNP site g.73256269T>C in the HADHB gene of the test cow; the genotype of the SNP site g.73256269T>C is CC or CT or TT; The milk production, milk fat amount and/or milk protein amount of the test cows whose g.73256269T>C genotype is CC are better than or assist better than those of the SNP g.73256269T>C genotype. TT or CT test cows; 2) including the following steps: detecting the genotype of the SNP site g.73256227A>C in the HADHB gene of the test cow; the genotype of the SNP site g.73256227A>C is AA or AC or CC; The milk production, milk fat amount and/or milk protein amount of the test cows whose g.73256227A>C genotype is CC are better than or assist better than those of the SNP g.73256227A>C genotype. AA or AC test cows; 3) including the following steps: detecting the genotype of the SNP site g.73242290C>T in the HADHB gene of the test cow; the genotype of the SNP site g.73242290C>T is CC or TT or CT; The milk production, milk fat amount and/or milk protein amount of the test cows whose g.73242290C>T genotype is TT at the SNP site are better than or assist better than those with the SNP g.73242290C>T genotype. CC or CT test cows. 5. Application of the method of claim 4 in dairy cow screening or dairy cow breeding. 6. Application according to claim 5, characterized in that: In the application, any one of the following test cows in claim 4 is selected for milk production or breeding; The SNP site g.73256269T>C is a test cow with CC genotype; The SNP site g.73256227A>C is a test cow with CC genotype; The SNP site g.73242290C>T genotype is the test cow of TT. 7. A method of dairy cow breeding, including the following steps: According to the steps in the method of claim 4, the genotype or haplotype combination of each SNP site is identified, and any of the following test cows are selected for milk production or breeding; The SNP site g.73256269T>C is a test cow with CC genotype; The SNP site g.73256227A>C is a test cow with CC genotype; The SNP site g.73242290C>T genotype is the test cow of TT. 8. Any one or primer combination of primer pair 6 and primer pair 9; The primer combination consists of the primer pair 6 and the primer pair 9; The primer pair 6 is a primer pair composed of primer 6F and primer 6R; The primer pair 9 is a primer pair composed of primer 9F and primer 9R; The primer 6F is a single-stranded DNA molecule shown in SEQ ID No. 4 or a nucleoside that deletes, adds or changes one or several nucleotides in SEQ ID No. 4 and has the same function as SEQ ID No. 4. acid; The primer 6R is a single-stranded DNA molecule shown in SEQ ID No. 5 or a nucleoside that deletes, adds or changes one or several nucleotides in SEQ ID No. 5 and has the same function as SEQ ID No. 5. acid; The primer 9F is a single-stranded DNA molecule shown in SEQ ID No. 6 or a nucleoside that deletes, adds or changes one or several nucleotides in SEQ ID No. 6 and has the same function as SEQ ID No. 6 acid; The primer 9R is a single-stranded DNA molecule shown in SEQ ID No. 7 or a nucleoside that deletes, adds or changes one or several nucleotides in SEQ ID No. 7 and has the same function as SEQ ID No. 7 acid. 9. The application of any one of the primer pairs or primer combinations according to claim 8 is any one of the following (a)-(f): (a) Identify or assist in identifying the milk production traits of dairy cows; (b) Cow screening; (c) Dairy cattle breeding; (d) Prepare kits for identifying or assisting in identifying milk production traits of dairy cows; (e) Preparing a kit for dairy cow screening; (f) Preparing a kit for dairy cow breeding.