CN117144020A

CN117144020A - SNP molecular marker of gene IDH2 related to dairy cow milk production traits and application thereof

Info

Publication number: CN117144020A
Application number: CN202311182283.1A
Authority: CN
Inventors: 孙东晓; 韩博; 宋雨; 许令娜
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2023-09-13
Filing date: 2023-09-13
Publication date: 2023-12-01

Abstract

The invention discloses an SNP molecular marker of a gene IDH2 related to milk production traits of cows and application thereof. The invention provides application of a substance for detecting polymorphism or genotype of at least one SNP locus in the following 4 SNP loci in identifying or assisting in identifying milk production traits of dairy cows; the 4 SNP loci are SNP locus g.21496168A > G, SNP locus g.21494708C > G, SNP locus g.21482140C > T and SNP locus g.214793970C > T. According to the invention, through genetic variation analysis of IDH2 genes in dairy cow association groups, 4 SNP, SNP1, SNP2, SNP3 and SNP4 are found to be respectively positioned in gene IDH2 genes related to milk production traits in dairy cow genomes, and can be used for auxiliary selection breeding of dairy cow molecular markers and breeding of dairy cow varieties with high milk production traits.

Description

SNP molecular marker of gene IDH2 related to dairy cow milk production traits and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and relates to an SNP molecular marker of a gene IDH2 related to milk production traits of dairy cows and application thereof.

Background

Isocitrate dehydrogenase 2 (Isocitrate Dehydrogenase (NADP (+)) 2, mitocondral, IDH2, encodes an enzyme that plays an important role in the decarboxylation of isocitrate to alpha-ketoglutarate, located on chromosome 21 of cattle, consisting of 11 exons, the full length of the gene 16318bp. There are three isoforms of IDH, depending on their localization, cytosolic NADP+ -dependent Isocitrate Dehydrogenase (IDHL), mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDH 2) and mitochondrial NAD+ -dependent isocitrate dehydrogenase (IDH 3). Of these three isoforms, the IDH2 gene encodes a protein that catalyzes the oxidative decarboxylation of isocitrate in the krebs cycle to α -ketoglutarate and NADPH, a molecule necessary for the utilization of anabolic pathways, such as the fatty acid extension process. Regarding NADPH production, IDH2 plays a key role in mitochondrial homeostasis, because mitochondria lack glucose-6-phosphate dehydrogenase (another key enzyme involved in the pentose phosphate pathway to produce NADPH).

IDH2 is nadp+ dependent, localizes to the mitochondrial matrix and is believed to play a key role in the metabolism of glucose, fatty acids, glutamine and mitochondrial homeostasis (maintaining redox balance). Oxidation of polyunsaturated fatty acids occurring in mitochondria may utilize NADPH provided by IDH 2. It has been found that amp-activated protein kinase (AMPK) promotes brown adipocyte differentiation, whereas AMPK alpha 1 may regulate IDH2 expression and thus affect alpha-ketoglutarate formation. Many studies have shown that IDH2 Knockout (KO) mice cause disease susceptibility ulcerative colitis, cardiac hypertrophy and others.

The polymerase chain reaction (Polymerase Chain Reaction, PCR) is a nucleic acid rapid amplification technique which simulates the natural DNA replication process in vitro, and is characterized by greatly increasing the trace amount of DNA, and has been invented by Mullis et al in the United states in 1983. PCR is to use DNA to become single chain at high temperature and primer and single chain at low temperature to combine based on the complementary pairing principle, and then regulate the temperature to the optimal reaction temperature of DNA polymerase to synthesize complementary chain along the direction from phosphate to five-carbon sugar (5 '-3'). Currently, this technology has become one of the most common and also important molecular biology techniques. The PCR product can be sequenced after agarose gel electrophoresis to identify the gene polymorphism, and the detection method is simple and easy to implement.

Disclosure of Invention

The invention aims to solve the technical problems of identifying or assisting in identifying milk production traits of cows or breeding cows.

In order to solve the technical problems, the invention provides the following technical scheme:

in a first aspect, the present invention provides the use of a substance for detecting a polymorphism or genotype of at least one of the following 4 SNP sites for the identification or assisted identification of milk production traits in dairy cows;

the 4 SNP loci are SNP locus g.21496168A > G, SNP locus g.21494708C > G, SNP locus g.21482140C > T and SNP locus g.214793970C > T.

In a second aspect, the invention provides an application of at least one SNP locus of the following 4 SNP loci as a detection target in identifying or assisting in identifying milk production traits of dairy cows;

or, the application of at least one SNP locus in the following 4 SNP loci serving as a detection target in developing and identifying or assisting in identifying dairy cow milk production trait products;

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

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

1) The method comprises the following steps: detecting the genotype of SNP locus g.214961688A > G in the IDH2 gene of the dairy cow to be tested; the genotype of the SNP locus g.21496168A > G is AA or AG or GG;

the milk yield and/or milk fat and/or milk protein of the tested dairy cows with the G genotype GG or AG of the SNP locus g.21496168A > are superior or assisted to those of the tested dairy cows with the G genotype AA of the SNP locus g.21496168A >;

2) The method comprises the following steps: detecting the genotype of SNP locus g.21494708C > G in the IDH2 gene of the dairy cow to be tested; the genotype of the SNP locus g.21494708C > G is CC or GG or CG;

the milk yield and/or milk fat and/or milk protein of the tested dairy cow with the genotype CC or CG of the SNP locus g.21494708C > is superior or assisted to that of the tested dairy cow with the genotype GG of the SNP locus g.21494708C >;

3) The method comprises the following steps: detecting the genotype of SNP locus g.21482140C > T in the IDH2 gene of the dairy cow to be tested; the genotype of the SNP locus g.21482140C > T is CC or TT or CT;

the milk yield and/or milk fat quantity and/or milk protein quantity of the tested dairy cows with the genotype of CC or CT of the SNP locus g.21482140C > are superior or assisted to those of the tested dairy cows with the genotype of TT of the SNP locus g.21482140C > respectively;

4) The method comprises the following steps: detecting the genotype of SNP locus g.21479397C > T in the IDH2 gene of the tested dairy cow; the genotype of the SNP locus g.21479397C > T is CC or TT or CT;

The milk yield and/or milk fat and/or milk protein of the tested dairy cows with the genotype of CC or CT of the SNP locus g.214793970C > are better than or assisted to be better than those of the tested dairy cows with the genotype of TT of the SNP locus g.214793970C > respectively;

5) The method comprises the following steps: detecting genotypes of four SNP loci of SNP locus g.21496168A > G, SNP locus g.21494708C > G, SNP locus g.21482140C > T and SNP locus g.214793970C > T in the IDH2 gene of the dairy cow to be tested, and identifying or assisting in identifying dairy cow milk production traits according to haplotype combination formed by the genotypes of the four SNP loci:

the haplotype combination is H1H1, H1H2, H1H3, H2H2 or H2H3;

milk yield of the milk cows of the haplotype combination H1H2 is higher than that of the milk cows of the haplotype combination H1H1 or the milk cows of the candidate milk cows are higher than that of the milk cows of the haplotype combination H1H 1;

milk cows of haplotype combination H2H3 have higher milk protein levels than or a candidate higher milk protein level than haplotype combination H1H1, haplotype combination H1H2, haplotype combination H1H3 or haplotype combination H2H 2;

the genotype of the haplotype combination H1H2 is CCCCGAG, which is 4 SNP combination genotypes of which the genotype of SNP1 is AG, the genotype of SNP2 is CG, the genotype of SNP3 is CC and the genotype of SNP4 is CC;

the genotype of the haplotype combination H1H1 is CCCCGGAA, which is 4 SNP combination genotypes of which the genotype of SNP1 is AA, the genotype of SNP2 is GG, the genotype of SNP3 is CC and the genotype of SNP4 is CC;

The genotype of the haplotype combination H2H3 is CTCTCTCCGG, which is 4 SNP combination genotypes of SNP1 genotype GG, SNP2 genotype CC, SNP3 genotype CT and SNP4 genotype CT;

the genotype of haplotype combination H1H3 is CTCTCTCGAG, which is 4 SNP combination genotypes of AG SNP1 genotype, CG SNP2 genotype, CT SNP3 genotype and CT SNP4 genotype.

In a fourth aspect, the invention provides the use of the method of the third aspect in cow screening or cow breeding.

In the above application, selecting the tested cows of any of the third aspects for milk production or breeding;

the SNP locus g.21496168A > G genotype is GG of the tested dairy cow;

the SNP locus g.21494708C > G genotype is the tested dairy cow of CC;

the SNP locus g.21482140C > T genotype is CC of the dairy cow to be tested;

the SNP locus g.21479397C > T genotype is CC of a tested dairy cow;

a test cow of the haplotype combination H1H 2;

and the haplotype combination H2H3 is used for testing cows.

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

identifying genotype or haplotype combination of each SNP locus according to the steps in the method of the third aspect, and selecting any one of the following tested cows for milk production or breeding;

The SNP locus g.21496168A > G genotype is GG of the tested dairy cow;

the SNP locus g.21494708C > G genotype is the tested dairy cow of CC;

the SNP locus g.21482140C > T genotype is CC of the dairy cow to be tested;

the SNP locus g.21479397C > T genotype is CC of a tested dairy cow;

a test cow of the haplotype combination H1H 2;

and the haplotype combination H2H3 is used for testing cows.

In a sixth aspect, the present invention provides any one of or a combination of primer pair 1, primer pair 3, primer pair 9 and primer pair 10;

the primer combination consists of the primer pair 1, the primer pair 3, the primer pair 9 and the primer pair 10;

the primer pair 1 is a primer pair consisting of a primer 1F and a primer 1R;

the primer pair 3 is a primer pair consisting of a primer 3F and a primer 3R;

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

the primer pair 10 is a primer pair consisting of a primer 10F and a primer 10R;

the primer 1F is a single-stranded DNA molecule shown as SEQ ID No.3 or a nucleotide which deletes, adds or alters one or more nucleotides in the SEQ ID No.3 and has the same function as the nucleotide in the SEQ ID No. 3;

the primer 1R is a single-stranded DNA molecule shown as SEQ ID No.4 or a nucleotide which deletes, adds or alters one or more nucleotides in the SEQ ID No.4 and has the same function as the SEQ ID No. 4;

The primer 3F is a single-stranded DNA molecule shown as SEQ ID No.5 or a nucleotide which deletes, adds or alters one or more nucleotides of the SEQ ID No.5 and has the same function as the SEQ ID No. 5;

the primer 3R is a single-stranded DNA molecule shown as SEQ ID No.6 or a nucleotide which deletes, adds or alters one or more nucleotides in the SEQ ID No.6 and has the same function as the SEQ ID No. 6;

the primer 9F is a single-stranded DNA molecule shown as SEQ ID No.7 or a nucleotide which deletes, adds or alters one or more nucleotides of the SEQ ID No.7 and has the same function as the SEQ ID No. 7;

the primer 9R is a single-stranded DNA molecule shown as SEQ ID No.8 or a nucleotide which deletes, adds or alters one or more nucleotides in the SEQ ID No.8 and has the same function as the SEQ ID No. 8;

the primer 10F is a single-stranded DNA molecule shown as SEQ ID No.9 or a nucleotide which deletes, adds or alters one or more nucleotides of the SEQ ID No.9 and has the same function as the SEQ ID No. 9;

the primer 10R is a single-stranded DNA molecule shown as SEQ ID No.10 or a nucleotide which deletes or adds or alters one or a plurality of nucleotides of the SEQ ID No.10 and has the same function as the SEQ ID No. 10.

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

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

(b) Screening dairy cows;

(c) Breeding dairy cows;

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

(e) Preparing a milk cow screening kit;

(f) Preparing a kit for breeding dairy cows.

SNP1 is one SNP of a dairy cow genome, is 358 th nucleotide of SEQ ID No.1 in a sequence table, and is T or C; SNP2 is one SNP of a dairy cow genome, is 1818 th nucleotide of SEQ ID No.1 in a sequence table, and is G or C; SNP3 is one SNP of a dairy cow genome, is 2435 nucleotide of SEQ ID No.2 in a sequence table, and is G or A; SNP4 is one SNP of the genome of the dairy cow, is 5178 nucleotide of SEQ ID No.2 in the sequence table, and is G or A.

The four single nucleotide polymorphism sites of the SNP1, the SNP2, the SNP3 and the SNP4 are positioned in an IDH2 gene on a 21 st chromosome of a dairy cow genome, the IDH2 gene is positioned at a 21478221-21494539 st chromosome of the dairy cow, and the nucleotide sequence of the IDH2 gene is sequentially composed of SEQ ID No.1 and SEQ ID No.2 in relation to the dairy cow milk production property.

The dairy cow milk production trait may be a combination of any one or more of milk yield, milk fat amount, and milk protein amount.

The genotype (i.e., allele) of the SNP1 can be genotype AA, genotype AG or genotype GG, wherein genotype AA is homozygous for SNP1 as A, genotype GG is homozygous for SNP1 as G, and genotype AG is heterozygous for SNP1 as A and G; the genotype (i.e., allele) of the SNP2 can be genotype CC, genotype CG or genotype GG, wherein the genotype CC is homozygous for the SNP2 with C, the genotype GG is homozygous for the SNP2 with G, and the genotype CG is heterozygous for the SNP2 with C and G; the genotype (i.e., allele) of the SNP3 can be genotype CC, genotype TT or genotype CT, wherein the genotype CC is homozygous for the SNP3 with C, the genotype TT is homozygous for the SNP3 with T, and the genotype CT is heterozygous for the SNP3 with C and T; the genotype (i.e., allele) of SNP4 can be genotype CC, genotype TT or genotype CT, genotype CC is homozygous for SNP4 for C, genotype TT is homozygous for SNP4 for T, genotype CT is heterozygous for SNP4 for C and T.

The haplotypes can be specifically haplotype H1, haplotype H2 and haplotype H3; the haplotype H1 (CCGA) is that the SNP1 is a, the SNP2 is G, the SNP3 is C, and the SNP4 is C; the haplotype H2 (CCCG) is that the SNP1 is G, the SNP2 is C, the SNP3 is C, and the SNP4 is C; the haplotype H3 (TTCG) was the SNP1 as G, the SNP2 as C, the SNP3 as T and the SNP4 as T.

The genotypes of the four SNPs SNP1, SNP2, SNP3, and SNP4 may be genotype cccggaa, genotype ccccgag, genotype ctctctgag, genotype cccccgg, and/or genotype TTCCTT. Genotype CCCCCGGAA is four SNP combined genotypes of SNP1 genotype AA, SNP2 genotype GG, SNP3 genotype CC and SNP4 genotype CC; genotype CCCCCCGAG is four SNP combined genotypes of which the SNP1 genotype is AG, the SNP2 genotype is CG, the SNP3 genotype is CC and the SNP4 genotype is CC; genotype ctctctctcgag is four SNP combination genotypes of SNP1 genotype AG, SNP2 genotype CG, SNP3 genotype CT, and SNP4 genotype CT. Cows with genotypes of four SNPs, SNP1, SNP2, SNP3 and SNP4, being genotype cccccggg, have milk production traits higher or candidate higher than cows with genotypes of four SNPs, SNP1, SNP2, SNP3 and SNP4, being genotype cccggaa. Cows with genotypes of four SNPs, SNP1, SNP2, SNP3 and SNP4, being genotype cccccggg, have milk yields and/or milk proteins higher or candidate higher than those of four SNPs, SNP1, SNP2, SNP3 and SNP4, being genotype cccggaa.

The breeding of the dairy cows is to cultivate the dairy cow variety with high milk yield.

The milk-producing traits described above may be, in particular, milk yield, milk fat and milk protein.

In the above application and method, the substance for detecting polymorphism or genotype of four SNPs, SNP1, SNP2, SNP3 and SNP4, or the substance for detecting haplotype, or the substance for detecting polymorphism or genotype of SNP1, or the substance for detecting polymorphism or genotype of SNP2, or the substance for detecting polymorphism or genotype of SNP3, or the substance for detecting polymorphism or genotype of SNP4, may be a substance for determining nucleotide types of SNP1, SNP2, SNP3 and/or SNP4 sites in the dairy genome by at least one of the following methods: DNA sequencing, restriction enzyme fragment length polymorphism, single-stranded conformational polymorphism, denaturing high performance liquid chromatography and SNP chips. The SNP chip comprises a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele specific primer extension reaction, a chip based on one-step method reaction, a chip based on primer connection reaction, a chip based on restriction enzyme reaction, a chip based on protein DNA binding reaction and a chip based on fluorescent molecule DNA binding reaction.

In the above application or method, the substance for detecting polymorphism or genotype of four SNPs of SNP1, SNP2, SNP3 and SNP4, or the substance for detecting haplotype, or the substance for detecting polymorphism or genotype of SNP3 may be the following D1), D2) or D3):

D1 PCR primers containing amplified bovine genomic DNA fragments including SNP1, SNP2, SNP3 and/or SNP4 sites;

d2 A PCR reagent comprising D1) the PCR primer;

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

The PCR primers are 1F/1R, 3F/3R and/or 11F/11R:

1F/1R, a primer group consisting of single-stranded DNA shown in positions 10-29 of SEQ ID No.1 and single-stranded DNA reversely complementary to positions 815-834 of SEQ ID No.1 in the sequence table;

3F/3R, a primer group consisting of single-stranded DNA shown in the 1393-1412 th sites of SEQ ID No.1 and single-stranded DNA reversely complementary to the 1896-1913 th sites of SEQ ID No.1 in the sequence table;

9F/9R, a primer set consisting of single-stranded DNA shown in SEQ ID No.2 at positions 2154-2173 and single-stranded DNA reversely complementary to SEQ ID No.2 at positions 2541-2558.

10F/10R, a primer set consisting of single-stranded DNA shown in the 4674 th to 4693 th positions of SEQ ID No.2 and single-stranded DNA reversely complementary to the 5418 th to 5437 th positions of SEQ ID No.2 in the sequence Listing.

In the above applications and methods, the PCR primers may or may not be labeled with a label. The label refers to any atom or molecule that can be used to provide a detectable effect and that can be attached to a nucleic acid. Markers include, but are not limited to, dyes; radiolabels, e.g. ³² P is as follows; binding moieties such as biotin (biotin); hapten such as Digoxin (DIG); a luminescent, phosphorescent or fluorescent moiety; and fluorescent dyes alone or in combination with a portion of the emission spectrum that can be suppressed or shifted by Fluorescence Resonance Energy Transfer (FRET). The label may provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, or the like. The label may be a charged moiety (positive or negative) or alternatively may be charge neutral. The label may comprise or be a combination of nucleic acid or protein sequences, provided that the sequence comprising the label is detectable. In some embodiments, the nucleic acid is directly detected without a label (e.g., directly reading the sequence).

In the above applications and methods, the product may be a reagent or a kit or a system, which may include a combination of a reagent or a kit, an instrument and analysis software, such as a product consisting of a PCR primer, a PARMS master mix reagent, an enzyme-labeled instrument and an on-line software SNP decoder (http:// www.snpway.com/SNP decoder01 /), a combination of a PCR primer, a PARMS master mix reagent, an on-line software SNP decoder and a fluorescent quantitative PCR instrument. The product may include the above-described substances for detecting polymorphisms or genotypes of SNP1, SNP2 and/or SNP3 sites in the genome of a dairy cow.

According to the invention, through genetic variation analysis of IDH2 genes in a dairy cow associated population, 4 SNP, SNP1, SNP2, SNP3 and SNP4 are found to be respectively positioned in gene IDH2 genes related to milk production traits in a dairy cow genome, namely 358 th, 1462 th, 14386 th and 17129 th of a sequence table SEQ ID No. 1. In the embodiment of the invention, the dominant allele of the SNP1 locus is G, the dominant allele of the SNP2 locus is C, the dominant allele of the SNP3 locus is C, and the dominant allele of the SNP4 locus is C, which indicates that the SNP1 locus, the SNP2 locus, the SNP3 locus and/or the SNP4 locus can be used for auxiliary selection breeding of dairy cow molecular markers and breeding of high-yield dairy cow varieties. These 4 SNP combinations total three haplotypes: haplotype H1 (CCGA), haplotype H2 (CCCG) and haplotype H3 (TTCG). Experiments prove that the milk production characters of the homozygous genotype dairy cows corresponding to the haplotype H2 (CCCG) are obviously higher than those of the homozygous genotype dairy cows corresponding to other haplotypes. The molecular marker of haplotype H2 (CCCG) can be used for early prediction and screening of milk production traits of cows, and can also be used for auxiliary selection breeding of the molecular markers of the cows and breeding of cow varieties with high milk production traits. In addition, in the embodiment of the invention, the dairy cows with genotypes of four SNP1, SNP2, SNP3 and SNP4 are higher or candidate higher than dairy cows with genotypes of four SNP1, SNP2, SNP3 and SNP4 are genotype CCCCCGG or genotype CCCCGGAA.

Drawings

FIG. 1 is an estimate of linkage disequilibrium for SNP1 (g.21496168A > G), SNP2 (g.21494708C > G), SNP3 (g.21482140C > T), and SNP4 (g.214793977C > T).

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The chinese Holstein cows in the examples below were from stock development limited in Peking head agriculture.

The data in the examples are all data for lactation 1. Lactation 1 refers to the lactation after the first delivery.

The milk yield in the examples is the individual's milk yield on day 305, which refers to the total milk yield from day one to day 305 of calving of the cow. When the actual milking days are less than 305 days, taking the actual milk yield as the milk yield of 305 days; when the actual milking days exceeded 305 days, the milk yield after 306 days was not counted. The milk yield is measured by monthly DHI (DHI, dairy Herd Improvement), and the milk yield in the lactation period of 305 days can be obtained by drawing a milk yield lactation curve according to the DHI data of more than 3 times in the same lactation period.

The milk fat amount in the examples refers to a milk fat amount of 305 days, a milk fat amount of 305 days=milk fat ratio×milk yield of 305 days. The milk fat percentage is determined by monthly DHI (DHI, dairy Herd Improvement), and the average milk fat percentage in the lactation period can be calculated by drawing a milk fat percentage lactation curve according to the DHI data of more than 3 times in the same lactation period.

The milk protein amount in the examples refers to 305 days milk protein amount, 305 days milk protein amount=milk protein rate×305 days milk yield. The milk protein rate is measured by monthly DHI (DHI, dairy Herd Improvement), and the average milk protein rate in the lactation period can be obtained by drawing a milk protein rate lactation curve through more than 3 DHI data in the same lactation period.

Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged.

Example 1 discovery of molecular markers

1. Related basic research

The subject group of the inventor carries out proteome analysis on liver tissues of different lactation stages (milk drying stage, lactation early stage and lactation peak stage) of Chinese Holstein cattle based on an iTRAQ technology, wherein 3252 proteins (FDR < 0.01) are totally identified, and 905 differentially expressed proteins (P <0.05 and FCI > 1.2) are totally identified. IDH2 expression was found to be significantly up-regulated during the initial and peak lactation period (P < 0.01), catalyzing oxidative decarboxylation of isocitrate to α -ketoglutarate and NADPH in the krebs cycle, NADPH being a necessary molecule for the utilization of anabolic pathways, such as fatty acid elongation processes.

2. Gene polymorphism detection

1. 45 Chinese Holstein bulls were selected as the test population for gene polymorphism detection. The frozen sperm DNA of 45 bulls is randomly mixed into 2 DNA pools, each pool consists of 22-23 frozen sperm DNA genome samples, the concentration of the DNA is accurately measured by a nucleic acid quality detector, and the DNA is diluted to 50 ng/. Mu.L and used for PCR amplification.

2. 20 pairs of primers as shown in Table 1 were designed based on the bovine IDH2 gene sequence (Ensembl ID is ENSBTAG00000014093, the nucleotide sequence of which consists of SEQ ID No.1 and SEQ ID No.2 in order from the 5' end).

Table 1 shows the sequence information of the primers for PCR amplification of IDH2 gene

3. And (3) taking the pool DNA obtained in the step (1) as a template, and respectively adopting each primer pair to carry out PCR amplification to obtain PCR amplification products. The PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.

Table 2 shows a PCR reaction system

Table 3 shows the PCR conditions

4. The PCR amplification product was sequenced. As a result, it was found that 2 SNP markers (referred to as SNP1 and SNP2, respectively) were present in the flanking sequences of 2000bp upstream of the IDH2 gene of the cow population, and 2 SNP (referred to as SNP3 and SNP 4) markers were present in the introns. The 4 SNP markers are shown in Table 4.

Table 4 shows 4 SNPs found from IDH2 gene

Gene position	Name of the name	SNPs	Physical location	Polymorphic forms
					5' regulatory region	SNP1	g.21496168A>G	Chr21:21496168bp	T/C
5' regulatory region	SNP2	g.21494708C>G	Chr21:21494708bp	G/C
					Introns	SNP3	g.21482140C>T	Chr21:21482140bp	G/A
Introns	SNP4	g.21479397C>T	Chr21:21479397bp	G/A

SNP1 corresponds to g.21496168A > G, is obtained by sequencing a product obtained by PCR amplification by using a primer pair consisting of 1F and 1R (the PCR amplification product is shown as 10 th-834 th site of SEQ ID No. 1), and has C or T as nucleotide, and corresponds to 358 th site of the SEQ ID No.1 from the 5' end in a sequence table. SNP2 corresponds to g.21494708C > G, is obtained by sequencing a product obtained by PCR amplification by using a primer pair consisting of 3F and 3R (the PCR amplification product is shown as 1393-1913 of SEQ ID No. 1), and the nucleotide is C or G, and corresponds to 1818 from the 5' end of SEQ ID No.1 in a sequence table. SNP3 corresponds to g.21482140C > T and is obtained by sequencing a product obtained by PCR amplification by using a primer pair consisting of 9F and 9R (the PCR amplification product is shown as 2154-2558 of SEQ ID No. 2), the nucleotide is G or A, and the nucleotide corresponds to 2435 from the 5' end of SEQ ID No.2 in the sequence table. SNP4 corresponds to g.21479397C > T and is obtained by sequencing a product obtained by PCR amplification by using a primer pair consisting of 10F and 10R (the PCR amplification product is shown as 4674-5437 bits of SEQ ID No. 2), and the nucleotide is G or A, and corresponds to 5178 bits from the 5' -end of SEQ ID No.2 in a sequence table. Y of SEQ ID No.1 in the sequence Listing represents T or C, S represents C or G; r of SEQ ID No.2 in the sequence Listing represents A or G.

g.21496168A > G, g.21494218C > G, g.21482140C > T and g.214793970C > T are the designations of SNP1, SNP2, SNP3, SNP4, respectively, the designations of SNP generally being according to the rules when the SNP was first discovered. The DNA is of a double-stranded structure and follows the base complementary pairing principle, and the sequence of SEQ ID No.1 and the sequence of the SNP1, SNP3 and SNP4 in the invention are the reverse complementary strand of the same double-stranded DNA. Thus, in the present invention, the polymorphic form of g.21496168A > G is C or T, the polymorphic form of g.21494708C > G is G or C, the polymorphic form of g.21482140C > T is G or A, and the polymorphic form of g.214793977C > T is G or A.

3. Correlation analysis

(one) obtaining a test population

The test population consisted of 926 Chinese Holstein cow.

(II) genotyping

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

I. Genotyping based on g.21496168a > G.

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

2. The PCR amplification is carried out by using the genome DNA as a template and a primer pair consisting of 1F (shown as 10 th to 29 th positions of SEQ ID No. 1) and 1R (reversely complementary to 815 th to 834 th positions of SEQ ID No. 1), and then the PCR amplification product is recovered and sequenced.

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

The PCR amplified products of each subject were 825bp, with position 349 being g.21496168A > G, SNP1 (corresponding to position 358 from the 5' end of SEQ ID No.1 in the sequence Listing).

Table 5 shows the reaction system for PCR amplification

Table 6 shows the reaction conditions for PCR amplification

II. Genotyping based on g.21494708c > G.

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

2. PCR amplification was performed using the genomic DNA as a template, using a primer set composed of 3F (as shown at positions 1393-1412 of SEQ ID No. 1) and 3R (reverse complementary to positions 1896-1913 of SEQ ID No. 1), and then the PCR amplification product was recovered and sequenced.

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

The PCR amplified products of each subject were 521bp, where position 426 was g.246757088G > T, namely SNP2 (corresponding to position 1818 from the 5' end of SEQ ID No.1 of the sequence Listing).

Table 7 shows the reaction system for PCR amplification

Table 8 shows the reaction conditions for PCR amplification

III, genotyping based on g.21482140C > T.

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

2. Using genomic DNA as a template, PCR amplification was performed using a primer set composed of 9F (as shown at positions 2154 to 2173 of SEQ ID No. 2) and 9R (reverse complementary to positions 2541 to 2558 of SEQ ID No. 2), and then the PCR amplified product was recovered and sequenced.

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

The PCR amplified products of each subject were 405bp, with position 282 being g.21482140C > T, namely SNP3 (corresponding to position 2435 from the 5' end of SEQ ID No.2 of the sequence Listing).

Table 9 shows the reaction system for PCR amplification

Table 10 shows the reaction conditions for PCR amplification

Genotyping based on g.21479397c > T.

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

2. Using genomic DNA as a template, PCR amplification was performed using a primer set consisting of 10F (as shown at 4674-4693 of SEQ ID No. 2) and 10R (reverse complement to 5418-5437 of SEQ ID No. 2), and then the PCR amplified product was recovered and sequenced.

The PCR amplification products of each subject were 764bp, where position 505 was g.21479397C > T, namely SNP3 (corresponding to position 5178 of SEQ ID No.2 from the 5' end of the sequence Listing).

(III) detecting milk production characteristics

And (5) each cow in the tested population is subjected to milk production character detection respectively.

The milk production characteristics comprise the following five indexes: milk yield, milk fat amount, milk fat percentage, milk protein amount and milk protein percentage.

Each individual record comprises, in order, a bovine individual number, a father number, a mother number, a grandfather number, a grandmother number, an grandfather number, an grandmother number, a date of birth, a lactation period, a calving date, a milk yield, a milk fat amount, a milk protein amount, a milk fat rate, and a milk protein rate.

(IV), correlation analysis model of single SNP locus and character

Genotypes and milk production phenotype of SNP1 locus (i.e., IDH2 gene g.21496168A > G), SNP2 locus (i.e., IDH2 gene g.21494708C > G), SNP3 locus (i.e., IDH2 gene g.21482140C > T) and SNP4 locus (i.e., IDH2 gene g.21479397C > T) are shown in Table 11, table 12, table 13 and Table 14.

Table 11 shows the phenotype of the partial milk production of the 926 Heston cow and the genotypes of the 4 SNP loci

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Table 12 is descriptive statistics of 5 milk production trait values for the 926 head Holstein Niu Muniu population

Traits (3)	Average value of	Standard deviation of	Minimum value	Maximum value	Coefficient of variation
						Milk yield (kg)	10412.35	1488.86	6057.96	14505.68	0.14
Milk fat quantity (kg)	352.9	61.37	184.8	537.97	0.17
						Milk fat percentage (%)	3.4	0.42	2.15	4.56	0.12
Milk protein quantity (kg)	315.97	48.37	157.73	457.53	0.15
						Milk protein yield (%)	3.04	0.19	2.35	3.51	0.06

Table 13 shows allele frequencies and genotype frequencies of 4 SNP loci of IDH2 gene

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The result shows that the SNP1 locus has 3 genotypes (SNP 1 genotypes for short), namely AA, AG or GG, genotype AA is homozygous for SNP 1A, genotype GG is homozygous for SNP 1G, genotype AG is heterozygous for SNP 1A and G; the SNP2 locus has 3 genotypes (SNP 2 genotypes for short), namely CC, GG or CG, the genotype CC is homozygous with SNP2 as C, the genotype GG is homozygous with SNP2 as G, the genotype CG is heterozygous with SNP2 as C and G; the SNP3 locus has 3 genotypes (SNP 3 genotypes for short), namely CC, CT or TT, the genotype CC is homozygous with SNP3 as C, the genotype TT is homozygous with SNP3 as T, and the genotype CT is heterozygous with SNP3 as C and T; the SNP4 locus has 3 genotypes (SNP 4 genotypes for short), namely CC, CT or TT, the genotype CC is homozygous with SNP4 as C, the genotype TT is homozygous with SNP4 as T, and the genotype CT is heterozygous with SNP4 as C and T.

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

Y＝μ+bys+b×M+G+a+e

wherein Y: observed values of milk production traits (milk yield, milk fat percentage, milk protein amount or milk protein rate); mu: overall mean; hys: a field year season effect; b: regression coefficients of covariates M; m: a calving month-old effect; g: genotype effect; a: individual random additive genetic effects; e: random residual effect.

The results of the SNP1 locus (i.e., IDH2 gene g.214961688A > G) and milk production trait association analysis in the 926 head population are shown in Table 14.

Table 14 shows the correlation analysis (least squares mean.+ -. Standard error) of IDH2 gene g.214961688A > G with milk production traits

Note that: ^* p is less than 0.05, which indicates that the difference is significant; ^** p < 0.01 indicates that the difference is extremely significant. ^a，b The same column of data has different superscripts to indicate that the difference is obvious; ^A，B the same column of data has different superscripts to indicate that the differences are very significant.

As can be seen from table 14, SNP1 (g.21496168 a > G) was significantly correlated with milk yield, milk fat and milk protein amounts (p=0.0004-0.0268), for milk yield, milk fat and milk protein amount traits, dominant allele is G:

the milk yield of the GG or AG genotype cows is higher than that of the AA genotype cows, and the milk yield of the GG genotype cows has no obvious difference with that of the AG genotype cows.

The milk fat content of the GG or AG genotype cows is higher than that of the AA genotype cows, and the milk fat content of the GG genotype cows is not significantly different from that of the AG genotype cows.

The milk protein amount of the GG or AG genotype cow is higher than that of the AA genotype cow, and the milk protein amount of the GG genotype cow is not obviously different from that of the AG genotype cow.

The analysis results of the association of SNP2 locus (i.e., IDH2 gene g.246757088G > T) and milk production characteristics in 926 populations are shown in Table 15.

Table 15 shows the correlation analysis (least squares mean.+ -. Standard error) of IDH2 gene g.246757088G > T and milk production characteristics

Note that: ^* P<0.05, indicating significant differences; ^** P<0.01, indicating that the difference was very significant. ^a,b The same column of data has different superscripts to indicate that the difference is obvious; ^A,B the same column of data has different superscripts to indicate that the differences are very significant.

As can be seen from table 15, SNP2 (g.246757088 g > T) was significantly correlated with milk yield, milk fat and milk protein amounts (p=0.0005-0.0261), for milk yield, milk fat and milk protein amount traits, the dominant allele is C:

the milk yield of CC or CG genotype cows is higher than that of GG genotype cows, and the milk yield of CC genotype cows has no obvious difference from that of CG genotype cows.

The milk fat content of CC or CG genotype cows is higher than that of GG genotype cows, and the milk fat content of CC genotype cows has no obvious difference from that of CG genotype cows.

The milk protein amount of CC or CG genotype cows is higher than that of GG genotype cows, and the milk protein amount of CC genotype cows has no obvious difference with that of CG genotype cows.

The analysis results of the association of SNP3 locus (i.e., IDH2 gene g.21482140C > T) and milk production characteristics in 926 populations are shown in Table 16.

Table 16 shows the correlation analysis (least squares mean.+ -. Standard error) of IDH2 gene g.21482140C > T and milk production traits

As seen from table 16, SNP3 (g.24665770 a > G) correlated very significantly with milk yield, milk fat and milk protein amounts (p=0.0004 to 0.0102), for milk yield, milk fat and milk protein amount traits, the dominant allele was C.

The milk yield of CC or CT genotype cows is higher than that of TT genotype cows, and the milk yield of CC genotype cows has no obvious difference from that of CT genotype cows.

The milk fat content of CC or CT genotype cows is higher than that of TT genotype cows, and the milk fat content of CC genotype cows has no obvious difference from that of CT genotype cows.

The milk protein amount of CC or CT genotype cows is higher than that of TT genotype cows, and the milk protein amount of CC genotype cows has no obvious difference from that of CT genotype cows.

The analysis results of the association of SNP4 locus (i.e., IDH2 gene g.21479397C > T) and milk production characteristics in 926 populations are shown in Table 17.

Table 17 shows the correlation analysis (least squares mean.+ -. Standard error) of IDH2 gene g.214793970C > T and milk production characteristics

Note that: ^* P<0.05, indicating significant differences; ^** P<0.01, indicating that the difference was very significant. ^a,b The same column of data has different superscripts to indicate that the difference is obvious; ^A,B identical toThe different superscripts of the column data indicate that the differences are very significant.

As seen from table 17, SNP4 (g.214793977C > T) correlated very significantly with milk yield, milk fat and milk protein amounts (p=0.0004 to 0.0054), for milk yield, milk fat and milk protein amount traits, the dominant allele was C.

The milk yield of CC or CT genotype cows is higher than that of TT genotype cows, and the milk yield of CC genotype cows has no obvious difference from CT genotype cows.

The milk protein amount of CC or CT genotype cows is higher than that of TT genotype cows, and the milk protein amount of CC genotype cows is not extremely obviously different from that of CT genotype cows.

(V) genetic Effect analysis

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

The basic calculation formula is as follows:

a= (AA-BB)/2, d = AB- (aa+bb)/2, α = a+d (q-p); a is an additive effect, d is a dominant effect, and α is an allele replacement effect; AA. AB and BB are the least square average value of the milk production characteristics of the corresponding genotypes; p is the frequency of allele A and q is the frequency of allele B.

The results of the additive effect, dominant effect and allele replacement effect assays are shown in Table 18.

Table 18 shows the results of the IDH2 gene allele additive, dominant and substitution tests

Note that: ^* p is less than 0.05, which indicates that the difference is significant; ^** p < 0.01, representing the difference poleIs remarkable.

From the results, the additive effect, dominant effect and allele replacement effect of SNP1 (g.21496168A > G) on milk yield and milk protein amount respectively reach remarkable or extremely remarkable, namely, each A allele replaces G allele to reduce the milk yield by 89.1011kg (P < 0.05), and the milk protein amount by 3.4797kg (P < 0.01). SNP2 (g.21494708C > G) has a pronounced additive effect on milk yield, milk protein yield, dominant effect and allele substitution effect, i.e. substitution of G allele by each C allele results in an increase in milk yield of 95.9159kg (P < 0.01) and milk protein yield of 3.5865kg (P < 0.01). SNP3 (g.21482140C > T) has a pronounced additive effect, dominant effect and allele substitution effect on milk yield, milk fat and milk protein. That is, each C allele replaced the T allele resulting in an increase in milk yield of 728.14kg (P < 0.01), an increase in milk fat mass of 19.5855kg (P < 0.01) and an increase in milk protein mass of 15.2512kg (P < 0.01). SNP4 (g.21479397C > T) has a pronounced additive, dominant and allele substitution effect on milk yield, milk fat and milk protein. That is, each substitution of the C allele for the T allele resulted in an increase in milk yield of 729.21kg (P < 0.01), an increase in milk fat mass of 30.5641kg (P < 0.01) and an increase in milk protein mass of 15.29kg (P < 0.05)

Analysis of haplotype

And constructing haplotypes by using Haploview 4.2 software, estimating haplotype frequency and linkage degree analysis, and carrying out association analysis with the characters. Using the MIXED procedure in SAS 9.4 software, the model is as follows:

Y＝μ+bys+b×M+G+a+e

y: observed values of milk production traits (milk yield, milk fat amount, milk fat percentage, milk protein amount, and milk protein rate); mu: overall mean; hys: a field year season effect; b: regression coefficients of covariates M; m: a calving month-old effect; g: haplotype combined effects; a: individual random additive genetic effects; e: random residual effect.

The haplotype analysis results are shown in Table 19.

Table 19 shows haplotypes composed of 4 SNPs of IDH2 gene

Haplotype type	Haplotype combinations	Frequency of
			H1	CCGA	0.502
H2	CCCG	0.344
			H3	TTCG	0.148

In the above-mentioned 926 study population, 4 SNPs (SNP 1, SNP2, SNP3, and SNP 4) of the IDH2 gene together form one haplotype block, and three haplotype combinations, i.e., haplotype H1, haplotype H2, and haplotype H3, are formed. Haplotype H1 (CCGA) is a combination of one chromosome with SNP1 of A, SNP of G, SNP of C and SNP4 of C. Haplotype H2 (CCCG) is a combination of one chromosome with SNP1 of G, SNP of C, SNP of C and SNP4 of C. Haplotype H3 (TTCG) is a combination of one chromosome with SNP1 of G, SNP of C, SNP of T and SNP4 of T. Haplotype H1 frequency was 50.2%, haplotype H2 frequency was 34.4% and haplotype H3 frequency was 14.8%. The 4 SNPs were subjected to linkage disequilibrium estimation, and the results showed that the 4 SNPs were in a completely linked state (r ² =1), as shown in fig. 1.

Haplotypes represent the linkage of the two SNPs on the same chromosome. Cattle are 2-fold, and haplotype combinations of two chromosomes of individuals in the population of 926 heads tested are H1H1, H1H2, H1H3, H2H2, H2H3, respectively.

The genotype of the cow corresponding to H1H1 is CCCCGGAA, the genotype of CCCCCCGGAA is SNP1 genotype AA, SNP2 genotype GG, SNP3 genotype CC and SNP4 genotype CC. The genotype of the cow corresponding to H1H2 is CCCCGAG, and the genotype CCCCCCGAG is four SNP combined genotypes of which the genotype of SNP1 is AG, the genotype of SNP2 is CC, the genotype of SNP3 is CC and the genotype of SNP4 is CC. The genotype of the cow corresponding to H1H3 is ctctctgags, which are four SNP combination genotypes of SNP1 genotype AG, SNP2 genotype CG, SNP3 genotype CT, and SNP4 genotype CT. The genotype of the cow corresponding to H2H2 is CCCCCGG, and the genotype of CCCCCGG is four SNP combined genotypes of which the SNP1 genotype is GG, the SNP2 genotype is CC, the SNP3 genotype is CC and the SNP4 genotype is CC. The genotype of the cow corresponding to H2H3 is CTCTCTCCGG, and the genotype CTCTCCGG is four SNP combined genotypes of which the SNP1 genotype is GG, the SNP2 genotype is CC, the SNP3 genotype is CT and the SNP4 genotype is CT.

The results of the analysis of the association of the IDH2 gene haplotype combinations with milk production traits are shown in Table 20 (haplotype combinations with a frequency of less than 0.05 were deleted). The haplotype combination of 4 SNPs of the IDH2 gene correlated with milk yield and milk protein yield to very significant levels (p=0.0009-0.0021), where haplotype H2 is the dominant haplotype for milk yield and milk protein yield traits.

Table 20 shows the results of IDH2 gene haplotype combinations and milk production profile correlation analysis (least squares mean.+ -. Standard error)

The milk yield of the cows of haplotype combination H1H2 (i.e., genotype CCCCCGGAG) was higher than that of the cows of haplotype combination H1H1 (i.e., genotype CCCCGGAA).

The milk protein amount of the cow of the haplotype combination H2H3 (i.e. genotype CTCTCCGG) is extremely higher than that of the cow of the haplotype combination H1H1 (i.e. genotype CCCCGGAA), the cow of the haplotype combination H1H2 (i.e. genotype CCCCGAG) and the cow of the haplotype combination H1H3 (i.e. genotype CTCTCTCGGG) is extremely higher than that of the cow of the haplotype combination H2H2 (i.e. genotype CCCCCGG).

Haplotype analysis tends to express more information than single marker analysis, and some site interaction changes inside the gene can have a great effect on the phenotype, so that the haplotype analysis is obviously superior to conventional single marker analysis in complex trait association analysis.

The molecular marker disclosed by the application can be applied to auxiliary identification of dairy cow groups with excellent milk production characteristics (milk yield in 305 days, milk fat amount, milk fat rate, milk protein amount and milk protein rate), and has the following advantages: simple, quick, sensitive, reliable, stable and accurate result, and is suitable for large-scale detection of laboratory population.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

1. Use of a substance for detecting a polymorphism or genotype of at least one of the following 4 SNP sites in the identification or assisted identification of milk production traits of dairy cows;

2. The application of at least one SNP locus in the following 4 SNP loci as a detection target in identifying or assisting in identifying milk production traits of dairy cows;

3. Use according to claim 1 or 2, characterized in that: the milk production property is milk yield and/or milk fat amount and/or milk protein amount.

4. A method for identifying or assisting in identifying milk production traits of a cow, which is any one of the following 1) -5):

The haplotype combination is H1H1, H1H2, H1H3, H2H2 or H2H3;

5. Use of the method of claim 4 in cow screening or cow breeding.

6. The use according to claim 5, characterized in that:

in the application, selecting any one of the tested cows of claim 4 for milk production or breeding;

the SNP locus g.21496168A > G genotype is GG of the tested dairy cow;

the SNP locus g.21494708C > G genotype is the tested dairy cow of CC;

the SNP locus g.21482140C > T genotype is CC of the dairy cow to be tested;

the SNP locus g.21479397C > T genotype is CC of a tested dairy cow;

a test cow of the haplotype combination H1H 2;

and the haplotype combination H2H3 is used for testing cows.

7. A method of breeding dairy cows comprising the steps of:

the method of claim 4, wherein the step of identifying the genotype or haplotype combination of each SNP locus comprises selecting any one of the following test cows for milk production or breeding;

the SNP locus g.21496168A > G genotype is GG of the tested dairy cow;

the SNP locus g.21494708C > G genotype is the tested dairy cow of CC;

the SNP locus g.21482140C > T genotype is CC of the dairy cow to be tested;

the SNP locus g.21479397C > T genotype is CC of a tested dairy cow;

a test cow of the haplotype combination H1H 2;

and the haplotype combination H2H3 is used for testing cows.

8. Any one of primer pair 1, primer pair 3, primer pair 9 and primer pair 10 or a primer combination;

the primer pair 1 is a primer pair consisting of a primer 1F and a primer 1R;

the primer pair 3 is a primer pair consisting of a primer 3F and a primer 3R;

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

9. The use of any one of the primer pairs or primer combinations of claim 8, which is any one of the following (a) - (f):

(b) Screening dairy cows;

(c) Breeding dairy cows;

(e) Preparing a milk cow screening kit;

(f) Preparing a kit for breeding dairy cows.