CN112746112B - SNP marker related to milk production peak days of Holstein cows in south China and application thereof - Google Patents

Abstract

The invention relates to an SNP marker related to the peak milk producing day of southern Holstein cows in China and application thereof, wherein the SNP marker is positioned at 19648602bp of No. 2 chromosome of genome of the southern Holstein cows in China, the basic group is G or C, and the peak milk producing day of a CC genotype individual of the SNP marker is obviously earlier than that of a GC genotype individual and a GG genotype individual. The SNP marker can detect the genotype of a cow at birth, is not limited by age and sex, and has high accuracy. In actual production, the CC genotype individual marked by the SNP is selected for breeding, so that the dominant allele and genotype which are beneficial to the dairy cows to produce milk in advance in the peak day can be rapidly diffused in the dairy cow group, the generation interval is shortened, the breeding cost is reduced, the breeding process of high-yield dairy cows is accelerated, and the sustainable development of the dairy cow industry in China is promoted.

Description

SNP marker related to milk production peak days of Holstein cows in south China and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to an SNP marker related to the peak milk production day of Holstein cows in south China and application thereof.

Background

The milk production traits are the most important production traits of the dairy cows, and comprise the indexes of total milk production, 305d milk production, peak milk production day and the like. The peak milk yield is the highest milk yield obtained by comparing the milk yields of the cow several times before the birth, and the peak milk yield indicates the days of the peak milk yield after the birth. Although the peak milk production day and the peak milk cannot directly display the milk yield, the occurrence time of the peak milk production day and the peak milk yield have great influence on the milk yield in the whole lactation period. Generally, the ideal milk production peak occurs 45-60 days after delivery, the milk production peak occurs too early (less than 30 days), the actual peak milk amount which the cow should reach is not realized, and the lactation potential is not fully exerted; the milk production peak day is too late (more than 70 days), which indicates that the cattle only have problems in nutrition in the perinatal period and have potential milk loss. Therefore, shortening the arrival time of the peak milk production day and improving the peak milk quantity have positive promotion effects on improving the economic benefit of cow breeding.

With the rapid development of molecular biology technology, molecular genetic markers and marker-assisted selection are widely researched and applied in livestock breeding, and a new way is opened up for fundamentally improving the genetic quality of dairy cows and accelerating the breeding process of dairy cows. The Snapshot technology is a typing technology based on the principle of fluorescence labeling single base extension, also called as micro sequencing technology, can realize the typing of SNPs with medium flux, and is widely used for SNPs identification and correlation analysis of human genetic diseases at present because of the characteristics of accurate typing, simultaneous detection of a plurality of sites, no limitation on polymorphism of SNP sites, small sample amount required for detection, high detection speed, high accuracy, relatively low cost and the like. Therefore, the Snapshot technology is used for screening the SNP genetic marker related to the dairy peak daily trait, so that a marker gene can be provided for molecular breeding of the dairy cow, and genetic materials of the molecular breeding of the dairy cow are enriched.

Disclosure of Invention

The SNP marker is a missense mutation site on the NRF2 gene, mutation at the site causes the conversion of amino acid glutamine (Gln) of encoded protein into histidine (His), possibly changes the activity of the protein and has certain influence on the production performance of the cow, and the SNP marker provides molecular materials for the cultivation of high-yield and high-quality cows, so that the early selection of the holstein cows in the southern China is realized, and the aims of improving the production performance of the cows, reducing the production cost and improving the production benefit are finally achieved.

The technical scheme provided by the invention is explained as follows:

in a first aspect, the invention provides an SNP marker related to the peak milk producing day of southern Holstein cows in China, the SNP marker is positioned at 19648602bp of chromosome 2 of the genome of the southern Holstein cows in China, the base is G or C, and the peak milk producing day of a CC genotype individual of the SNP marker is obviously earlier than that of a GC genotype individual and that of a GG genotype individual.

In a second aspect, the present invention provides a primer set for detecting the above-mentioned SNP marker, comprising:

an upstream primer: 5'-GCTTTTATAGCAGAGCCCAGTACCA-3', as shown in SEQ ID NO: 1;

a downstream primer: 5 '-TCTACRGGGAATGGGATATGGAGAG-3' as shown in SEQ ID NO. 2. Wherein R in the nucleotide sequence is degenerate basic group and represents G or A.

In a third aspect, the present invention provides a kit for detecting the SNP marker, the kit comprising the primer set, and further comprising a single-base extension primer: 5 '-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTGCAGCACGTGATTCCCW-3' as shown in SEQ ID NO. 3. Wherein, W in the nucleotide sequence is degenerate base, and represents A or T.

In a fourth aspect, the invention provides the use of the SNP marker, the primer pair and the kit in the auxiliary identification of the peak dairy day traits of southern Holstein cows in China.

In a fifth aspect, the invention provides a method for breeding Chinese southern Holstein cows, which comprises the steps of detecting the SNP marker of the Chinese southern Holstein cows to be detected, predicting the daily property of the peak milk production period of the Chinese southern Holstein cows to be detected, and selecting dominant allele and genotype individuals which can advance the peak milk production period for breeding.

The method for breeding the southern Holstein cows in China comprises the following steps:

extracting the genome DNA of the southern Holstein cow to be detected in China;

taking the genome DNA of the Chinese southern Holstein cow to be detected as a template, and carrying out multiple PCR amplification reaction by using the primer pair;

performing single base extension reaction on the PCR amplification product by using the single base extension primer, sequencing the extension product, and determining the genotype of the SNP marker; predicting the characteristics of the Holstein cows in south China to be detected in the peak milk producing days, and selecting the CC genotype individuals marked by the SNP as dominant varieties with early peak milk producing days.

On the basis of the technical scheme, the process of the multiplex PCR amplification reaction comprises the following steps: firstly, 95 ℃ for 2 min; ② sequentially carrying out 11 cycles of 94 ℃ 20s and 65 ℃ 40s, reducing the temperature by 0.5 ℃ and 72 ℃ for 1.5min in each cycle; thirdly, 24 cycles of 94 ℃ for 20s, 59 ℃ for 30s and 72 ℃ for 1.5min are carried out in sequence; fourthly, 2min at 72 ℃.

On the basis of the technical scheme, the single base extension reaction process comprises the following steps: 1min at 96 ℃; ② 96 ℃ for 10s, 55 ℃ for 5s and 60 ℃ for 30s, and the three steps are 28 cycles.

On the basis of the technical scheme, the Chinese southern Holstein cow is a Holstein cow in Wuhan region.

The technical scheme provided by the invention has the beneficial effects that:

1. the SNP marker rs43706509 site related to the peak milk producing day of Holstein cows in south China is located on the NRF2 gene, the detection of the SNP marker provides scientific basis for marker-assisted selection of the peak milk producing day characters of the Holstein cows in south China, the SNP site enriches a molecular marker genetic resource library for breeding cows, and the molecular marker-assisted selection of the Holstein cows in south China is utilized to facilitate shortening of arrival time of the peak milk producing day, maintenance of peak milk yield, and improvement of fetal milk yield of cow groups so as to improve the feeding benefit.

2. The SNP marker related to the peak milk production day of Holstein cows in south China can detect the genotype of the cows at birth, is not limited by age and sex, and has high accuracy. In actual production, by selecting the rs43706509 locus CC genotype individual in the early stage, the dominant allele and genotype which are beneficial to the dairy cows to produce milk in advance in the peak day can be rapidly diffused in the dairy cow group, the generation interval is shortened, the breeding cost is reduced, the breeding process of high-yield dairy cows is accelerated, and the sustainable development of the dairy cow industry in China is promoted.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIGS. 1 to 3 are graphs showing the rs43706509 locus typing results.

Detailed Description

To make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

It should be noted that the experimental methods used in the following examples are all conventional methods unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. R, W in the nucleotide sequences referred to in the examples below are all degenerate bases, R represents G or A, W represents A or T.

The invention finds an SNP marker which is obviously related to the dairy cow peak date through modern molecular biology technology and statistical method, and the SNP marker is positioned on the gene of the nuclear factor E2 related factor 2 (NRF 2 for short). NRF2 is a key nuclear transcription factor of 1 in the organism, belongs to a transcription factor leucine zipper transcription activator (CNC) family member, and is a key factor for protecting cells from various stress injuries. NRF2 can form a dimer with small nuclear Maf protein after entering the nucleus, so that adenine and uracil rich cis-acting elements (AREs) ARE identified and combined with antioxidant stress, and transcription of a series of enzymes related to substance metabolism and transport and II-phase antioxidant enzyme genes is started. Research shows that the streptococcus uberis infected mouse mammary epithelial cells can activate an NRF2 pathway to a certain extent, increase the level of NRF2 protein, promote the expression of superoxide dismutase (SOD) in a regulatory region and the transcription of genes such as heme oxygenase 1 (HO-1) and the like, and help an organism to actively resist partial infection. After the diethylenetriamine/nitric oxide polymer is used for inducing bovine mammary epithelial cells to generate oxidative stress, the expression level of NRF2 mRNA and protein is reduced. The melatonin can up-regulate the expression of NRF2 and HO-1 genes of bovine mammary epithelial cells and activate an NRF2 antioxidant pathway, thereby treating inflammation and oxidative stress injury. The reports show that NRF2 and the antioxidant signal path mediated by the NRF2 can play an important regulation and control role in the oxidative damage of the mammary gland of the dairy cow and the antioxidant stress response, and can be used as a candidate gene for developing a molecular breeding marker for the production traits of the dairy cow. However, the influence of the genetic variation of the NRF2 gene on the production traits of the dairy cows is not reported at present.

Example 1: acquisition and identification of SNP markers closely related to the Dairy Peak day 1.1 Experimental population and phenotypic data

785 Chinese Holstein cows in a certain large-scale dairy farm in Wuhan Hubei are taken as experimental objects, tail vein blood collection is carried out, EDTA anticoagulation is carried out, and the milk is preserved at the temperature of-20 ℃. The milk production traits (lactation days, milk yield of 305d, total milk yield, peak milk production day, milk fat rate and milk protein rate), reproductive traits (initial day age, calving interval) and health traits (somatic cell count) of the cows are monitored, and 777 complete first-born cow data, 386 second-born cow data and 92 third-born cow data are obtained.

1.2 extraction of blood genomic DNA

Blood DNA was extracted using a TIANAmp Blood DNAkit Blood genomic DNA extraction kit.

(1) 200 mu L of cow whole blood is taken and put into a 1.5mL of an EP tube without RNase, 200 mu L of a premixed solution of a buffer GB and 20 mu L of an RNaseK is added, the mixture is fully inverted and mixed evenly, the mixture is placed at 56 ℃ for 10min, the mixture is inverted and mixed for a plurality of times, and the solution is clear (if the solution is not clear completely, the lysis time is prolonged until the solution is clear).

(2) After standing at room temperature for 2-5min, 350. mu.L of buffer BD was added, and the mixture was thoroughly mixed by inversion, whereby a flocculent precipitate may appear.

(3) And (3) adding the solution and flocculent precipitate obtained in the step (2) into an adsorption column CG2 (the adsorption column CG2 is placed into a collecting pipe), centrifuging at 12000rpm (about 13400g) for 30s, pouring waste liquid in the collecting pipe, and placing an adsorption column CG2 into the collecting pipe.

(4) 500. mu.L of buffer GDB was added to the adsorption column CG2, centrifuged at 12000rpm (-13400 g) for 30s, the waste liquid in the collection tube was discarded, and the adsorption column CG2 was placed in the collection tube.

(5) Add 600. mu.L of rinsing liquid PWB (check whether absolute ethanol has been added before use) to adsorption column CG2, centrifuge at 12000rpm (-13400 g) for 30s, pour off the waste liquid in the collection tube, and place adsorption column CG2 in the collection tube.

(6) And (5) repeating the operation step.

(7) The adsorption column CG2 together with collection tube 12000rpm (-13400 g) was centrifuged for 2min, and the waste liquid was decanted. The adsorption column CG2 was left at room temperature until the residual rinse solution in the adsorption material was completely dried.

(8) Transferring the adsorption column CG2 into a 1.5mL centrifuge tube, suspending and dripping 50-200 μ L of elution buffer TB into the middle position of the adsorption membrane, standing at room temperature for 2min, centrifuging at 12000rpm (-13400 g) for 2min, and collecting the solution into the centrifuge tube.

1.3 obtaining mutation information of related candidate genes

Randomly selecting 16 cow DNA samples, carrying out conventional PCR amplification on CDS full length of NRF2, detecting the amplification effect by agarose gel electrophoresis, and sending to Wuhan division company of Biotechnology Limited in Beijing Optimalaceae for sequencing. Using MAFFT on-line software (https://www.ebi.ac.uk/Tools/msa/mafft/) Comparing the results obtained by sequencing, finding that NRF2 has 2 candidate single nucleotide polymorphism SNP markers (1 missense mutation and 1 synonymous mutation), and further analyzing the correlation between missense mutation sites and dairy cow production traits. The information on this SNP marker is shown in Table 1.

TABLE 1 SNP marker information for association analysis

Note: using "chromosome number located: location on chromosome form-labeling the location of an SNP with 1.4Snapshot Single Nucleotide Polymorphism (SNP) typing

After the DNA sample is subjected to quality inspection and concentration measurement, the sample is diluted to the working concentration of 5-10 ng/mu L, and Snapshot is adopted to carry out typing on related sites. The method mainly comprises the following steps:

(1) primer design

PCR amplification primers were designed using Primer3.0 online software based on the reference sequence provided by NCBI.

An upstream primer: 5'-GCTTTTATAGCAGAGCCCAGTACCA-3' the flow of the air in the air conditioner,

a downstream primer: 5 '-TCTACRGGGAATGGGATATGGAGAG-3'.

(2) Multiplex PCR amplification

The PCR reaction system (20. mu.L) contained 1 XGC-I buffer (purchased from Takara Co., Ltd.), 3.0mM Mg²⁺0.3mM dNTP, 1U Hot Start Taq enzyme (purchased from Qiagen), 1. mu.L sample DNA and 1. mu.L LPCR primer. The concentration of the primer pair at the rs43706509 site is 3. mu.M.

The reaction process is as follows: firstly, the temperature is 95 ℃ for 2 min; ② sequentially carrying out 11 cycles of 94 ℃ 20s and 65 ℃ 40s, reducing the temperature by 0.5 ℃ and 72 ℃ for 1.5min in each cycle; ③ 24 cycles of 94 ℃ for 20s, 59 ℃ for 30s and 72 ℃ for 1.5min are carried out in sequence; fourthly, 2min at 72 ℃. Length of amplified fragment: 560bp, as shown in SEQ ID NO. 4:

GCTTT TATAG CAGAG CCCAG TACCA GCAAC GGCAT GCCCT CCTCT GCTAC TTTAA GCCAG TCACT CTCTG AACTT CTAAA CGGGC CCATT GATCT CTCTG ATCTG TCACT TTGTA AAGCT TTCAA TCAAA ACCAC CCTGA AAGCA CAACA GCAGA ATTCA ATGAT TCTGA CTCTG GCATT TCACT GAACA CAACA AGTCC AAGCA TGGCA TCACC AGACC ACTCA GTGGA ATCTT CCATC TATGG AGACA CATTG CTTGG CTTCA GTGAT TCTGA AATGG AAGAG ATAGA TAGTA CCCCT GGAAA TGTCA AACAG AAGGG TCCCA AAACA CCGTC AGTGT GGCCT CCTGG GGACC CAGTC CAACC TTTGT CGTCA TCACA GGGGA ACAGC GCTGC AGCAC GTGAT TCCCA

(rs43706509)TGTG AAAAC GCACC AAAGA AAGAA GTACC TGTAA GTCCG GGTCA TCGAA AAACG CCATT CACAA AAGAC AAACA TTCAA GCCGC TTGGA GGCTC ACCTC ACAAG AGATG AGCTA CGGGC AAAAG CTCTC CATAT CCCAT TCCCT GTAGA。

(3) PCR product purification

To 10 μ of the LPCR product were added 5U of SAP enzyme (available from Promega) and 2U of Exonuclease I enzyme (available from Epicentre), incubated at 37 ℃ for 1h and then inactivated at 75 ℃ for 15 min.

(4) Single base extension reaction of SNaPshot

The purified PCR product was subjected to a single base extension reaction using Snapshot multiplex Kit of ABI.

The single base extension primer (SF) at rs43706509 site is as follows:

5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTGCAGCACGTGATTCCCW-3’；

the extension reaction system (10. mu.L) included 5. mu.L of Snapshot multiplex Kit (available from ABI), 2. mu.L of purified multiplex PCR product, 2. mu.L of ultrapure water, and 1. mu.L of extension primer mixture, and the concentration of extension primer was 2.4. mu.M. The reaction process is as follows: 1min at 96 ℃; ② 96 ℃ for 10s, 55 ℃ for 5s and 60 ℃ for 30s, and the three steps are 28 cycles.

(5) Purification of extension products

Add 1U SAP enzyme to 10. mu.L extension product, incubate for 1h at 37 ℃ and inactivate for 15min at 75 ℃.

(6) Sequencing of extension products

mu.L of the purified extension product was taken, mixed with 0.5. mu.L of Liz120 sizerandsard and 9. mu.L of Hi-Di, denatured at 95 ℃ for 5 minutes and applied to an ABI3730XL sequencer, and the raw data obtained was analyzed by GeneMapper4.1 (applied biosystems, USA).

Example 2: statistical analysis

Calculating the genotype frequency and the allele frequency of each site by using Excel, and analyzing the Hardy-Weinberg equilibrium condition of each site by using a Chi-square test. The data of each character are subjected to an orthomorphic test by using the univariate process of an SAS online program (https:// welome. oda. SAS. com /), the data are converted by using a BOX-COX method, and abnormal values are eliminated by using the 'average value +/-3 times standard deviation'. Testing the influence of the genotype on each corrected property by using a GLM process, wherein a testing model is Y ═ mu + P + S + G + e, and a testing model for the influence on the early-birth day age is Y ═ mu + G + e; wherein Y is an individual character phenotype value, mu is a population mean value, P is a fixed effect of the number of fetuses, S is a fixed effect of the calving season, G is a fixed effect of the genotype, and e is a random error effect. Multiple comparisons of individual trait phenotype values for different genotypes were performed using the DUNCAN method.

2.1 statistical analysis of production traits

For 10 production trait indexes of experimental herds: lactation days, 305d milk yield, total milk yield, peak milk production day, milk fat rate, milk protein rate, day age of primordial birth, calving interval are collated and analyzed in terms of somatic cell count. As can be seen from Table 2, the overall production level of the experimental population is high, but the difference between individuals is large, and a large breeding space exists.

TABLE 2 data of experimental cattle resource group production traits

2.2 genomic DNA detection and SNP typing

The DNA of the milk cow blood is detected by 1 percent agarose gel electrophoresis, and the strip is bright and has no protein pollution. The mass concentration of more than 95% of the sample is more than 10 ng/mu L by adopting a NanoDrop2000 nucleic acid concentration meter, and the concentration and the purity can meet the requirements of Snapshot typing experiments.

The allele frequency and genotype frequency at rs43706509 locus were analyzed by Excel and subjected to the hardy-weinberg equilibrium test (table 3). The genotyping result shows that the rs43706509 locus is in a Hardy-Weinberg unbalanced state (P <0.05), and may be related to the application of artificial insemination technology of experimental groups and non-random mating of cow groups.

TABLE 3 genotype frequencies and Gene frequencies at SNP sites

2.3 Association analysis of SNP sites and production traits

The influence of different genotypes of the rs43706509 locus of the NRF2 gene on production traits is analyzed by adopting SAS software (Table 4). The result shows that the rs43706509 site has a remarkable effect (P <0.05) on the peak days of milk production and has no remarkable effect (P >0.05) on other character indexes. Multiple comparison results show that: the milk production peak day of rs43706509 locus CC genotype individual is obviously lower than that of GC genotype and GG genotype individuals (P is less than 0.05). Wherein, the milk production peak day of the CC genotype individual is 22.43d and 20.03d earlier than the appearance time of the peak milk of the GC genotype individual and the GG genotype individual respectively, and the average peak milk volume among the genotypes is close.

TABLE 4 correlation analysis of SNP and production traits of NRF2 Gene (least squares means. + -. standard error)

Note: different lower case letters indicate that the difference is obvious (P <0.05) when compared with different genotypes of the same character at the same site, and different upper case letters indicate that the difference is extremely obvious (P <0.01) when compared with different genotypes of the same character at the same site, and the difference is the same below.

Research results show that the NRF2 gene can be used as an important candidate gene influencing the peak milk production day, and the rs43706509 site of the NRF2 gene can be used for marker-assisted selection of the peak milk production day traits of a dairy cow population. In the production practice, marker-assisted selection can be carried out on the rs43706509 locus, the arrival time of the milk production peak day is shortened, the peak milk quantity is maintained, the fetal secondary milk quantity of the milk cow group is increased, and the feeding benefit is increased.

The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

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Claims (9)

1. An application of SNP marker in auxiliary identification of milk production peak daily traits of southern Holstein cows in China is characterized in that: the SNP marker is located on 19648602bp of No. 2 chromosome of genome of southern Holstein cow in China, the base is G or C, and the CC genotype individual of the SNP marker obviously precedes the GC genotype individual and the GG genotype individual at the peak of milk production.

2. The application of a primer pair for detecting SNP markers in the auxiliary identification of the milk production peak daily traits of southern Holstein cows in China is characterized in that:

the SNP marker is located at 19648602bp of No. 2 chromosome of southern Holstein cow genome in China, and the base is G or C;

the primer pair comprises:

an upstream primer: 5'-GCTTTTATAGCAGAGCCCAGTACCA-3' the flow of the air in the air conditioner,

a downstream primer: 5 '-TCTACRGGGAATGGGATATGGAGAG-3';

wherein R in the nucleotide sequence is degenerate basic group and represents G or A.

3. The utility model provides an use of kit for detecting SNP mark in supplementary appraisal china southern lotus Stent milk cow peak daily property of bringing milk which characterized in that: the SNP marker is located at 19648602bp of No. 2 chromosome of southern Holstein cow genome in China, and the base is G or C;

the kit contains a primer pair, wherein the primer pair comprises:

an upstream primer: 5'-GCTTTTATAGCAGAGCCCAGTACCA-3' the flow of the air in the air conditioner,

a downstream primer: 5 '-TCTACRGGGAATGGGATATGGAGAG-3';

wherein R in the nucleotide sequence is degenerate basic group and represents G or A.

4. The use of the kit for detecting SNP markers according to claim 3, for the auxiliary identification of the peak-daily milk production traits of southern Holstein cows in China, characterized in that: the kit also contains a single base extension primer:

5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTGCAGCACGTGATTCCCW-3’；

wherein, W in the nucleotide sequence is degenerate base, and represents A or T.

5. A method for breeding Chinese southern Holstein cows is characterized in that: by detecting SNP markers of the Chinese southern Holstein cows to be detected, evaluating the characteristics of the Chinese southern Holstein cows to be detected in the peak milk production days, and selecting dominant alleles and genotype individuals which can advance the peak milk production days for breeding; the SNP marker is located on 19648602bp of No. 2 chromosome of southern Holstein cow genome in China, and the base is G or C.

6. The method of breeding a southern holstein cow in china according to claim 5, comprising the steps of:

extracting the genome DNA of the southern Holstein cow to be detected in China;

performing multiplex PCR amplification reaction by using the primer pair of claim 2 by using the genomic DNA of a southern Holstein cow to be detected in China as a template;

carrying out single-base extension reaction on a PCR amplification product by using the single-base extension primer of claim 4, sequencing the extension product, determining the genotype of the SNP marker, evaluating the daily property of the peak of milk production of the southern Holstein cow to be detected, and selecting a CC genotype individual of the SNP marker as a dominant individual with advanced peak of milk production.

7. The method of breeding a southern holstein cow in china according to claim 6, wherein: the process of the multiplex PCR amplification reaction is as follows: firstly, 95 ℃ for 2 min; ② sequentially carrying out 11 cycles of 94 ℃ 20s and 65 ℃ 40s, reducing the temperature by 0.5 ℃ and 72 ℃ for 1.5min in each cycle; ③ 24 cycles of 94 ℃ for 20s, 59 ℃ for 30s and 72 ℃ for 1.5min are carried out in sequence; fourthly, 2min at 72 ℃.

8. The method of breeding a southern holstein cow in china according to claim 6, wherein: the single base extension reaction process is as follows: 1min at 96 ℃; ② 96 ℃ for 10s, 55 ℃ for 5s and 60 ℃ for 30s, and the three steps are 28 cycles.

9. The method for breeding southern holstein cows in china according to any one of claims 5 to 8, which comprises the steps of: the southern Holstein cows in China are Holstein cows in Wuhan region.

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