SNP markers associated with milk production traits in Holstein cows in southern China and their application

Abstract

The present invention provides SNP markers related to the milk production trait of Holstein dairy cows in southern China and applications thereof, wherein the first SNP marker is located at 39037752bp on the reverse strand of chromosome 18 of the genome of Holstein dairy cows in southern China, the base is C or T, the 305d milk yield of CC genotype individuals of the first SNP marker is extremely significantly higher than that of CT genotype individuals and TT genotype individuals, and the total milk yield and peak milk yield are significantly higher than those of CT genotype individuals and TT genotype individuals; the second SNP marker is located at 39037665bp on the reverse strand of chromosome 18 of the genome of Holstein dairy cows in southern China, the base is G or A, and the total milk yield and peak milk yield of GG genotype individuals of the second SNP marker are significantly higher than those of GA genotype individuals. The two SNP sites provided by the present invention enrich the molecular marker genetic resource library for dairy cow breeding, and using the same for molecular marker-assisted selection is conducive to improving the milk yield of Holstein dairy cows in southern China, and will not affect the reproductive traits of Holstein dairy cows in southern China.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to two SNP markers related to the milk production traits of Holstein cows in southern China and applications thereof. Background Art

[0002] Compared with the northern region, the southern region of my country has geographical and climatic characteristics such as complex topography, high temperature and humidity, and relatively dense population. These characteristics restrict the development model of increasing the total milk production by expanding the scale of breeding in terms of capital, land, climate, and labor. In addition, the southern region of my country has not paid enough attention to the selection and breeding of dairy cows for a long time. The dairy cows as a whole have problems such as a small number of good breeds, low yields, complex population resources, and poor genetic quality. Therefore, improving dairy cow breeds and cultivating local high-yield dairy cow groups to increase milk production is a key way to help dairy companies increase their profits and promote the development of my country's dairy industry.

[0003] With the rapid development of molecular biology technology, molecular genetic markers and marker-assisted selection have been studied more and more widely in livestock and poultry breeding. At present, the most widely used molecular genetic markers are: Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphism DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Single Nucleotide Polymorphism (SNP), etc. Among them, SNP markers have the advantages of genetic stability, low mutation rate, and easy automatic detection. Therefore, the development of SNP genetic markers related to dairy cow milk production traits will effectively promote the breeding of high-yield dairy cow breeds (lines).

[0004] Snapshot technology is a new sequencing technology. This technology is based on the principle of fluorescent labeling single base extension. It uses primers to amplify the fragment where the target SNPs are located. The amplified product is purified and used as a template for single base extension. Sequencer, four fluorescently labeled ddNTPs and extension primers with the 5'-end close to the SNP site are used for PCR reaction. The primers are terminated after one base extension. After detection by the sequencer, the SNP site corresponding to the extension product is determined according to the moving position of the peak. The type of base incorporated can be known according to the color of the peak, thereby determining the genotype of the sample. This technology has the advantages of accurate typing, high throughput, fast detection speed, and is not limited by the polymorphic characteristics of the SNP site and the number of samples.

[0005] In the process of dairy cow breeding, by finding key genes related to milk production in dairy cows and screening SNP markers closely related to increasing milk production for breeding, early selection can be achieved and breeding accuracy can be improved, thereby accelerating the genetic breeding process, fundamentally improving the milk production performance of dairy cows, and promoting the sustainable and healthy development of my country's dairy industry. However, there are currently few SNP markers related to the milk production traits of Holstein cows in southern China, and the gene pool is scarce. Summary of the invention

[0006] The embodiment of the present invention provides two SNP markers related to the milk production traits of Holstein cows in southern China and applications thereof, so as to solve the problem of lack of SNP markers related to the milk production traits of Holstein cows in southern China in the related art.

[0007] In the first aspect, the present invention provides two SNP markers associated with the milk production traits of southern Chinese Holstein cows. The first SNP marker is located at 39037752bp on the reverse strand of chromosome 18 of the southern Chinese Holstein cow genome, and the base is C or T. The 305d milk yield of individuals with CC genotype of the first SNP marker is extremely significantly higher than that of individuals with CT genotype and TT genotype, and the total milk yield and peak milk yield are significantly higher than those of individuals with CT genotype and TT genotype; the second SNP marker is located at 39037665bp on the reverse strand of chromosome 18 of the southern Chinese Holstein cow genome, and the base is G or A. The total milk yield and peak milk yield of individuals with GG genotype of the second SNP marker are significantly higher than those of individuals with GA genotype.

[0008] In a second aspect, the present invention provides a primer pair for detecting the above-mentioned SNP marker:

[0009] Forward primer: 5'-CCATACTCAGCCACAGCACAGC-3', as shown in SEQ ID NO: 1;

[0010] Reverse primer: 5′-GGGGCGAAATGAAAACTTCAACT-3′, as shown in SEQ ID NO:2.

[0011] In a third aspect, the present invention provides a kit for detecting the above-mentioned SNP marker, the kit comprising the above-mentioned primer pair and at least one of the following single-base extension primers:

[0012] Single base extension primer for the first SNP marker:

[0013] 5'-TTTTTTTTTTTTTTGAAGGTTGTTCTCATTCAGTATGGG-3', as shown in SEQ ID NO: 3;

[0014] Single base extension primer for the second SNP marker:

[0015] 5'-TTTTTTTGTCGTCTTCCYTGTCGTGAAC-3', as shown in SEQ ID NO: 4, wherein Y in the nucleotide sequence is a degenerate base, representing C or T.

[0016] In a fourth aspect, the present invention provides uses of the above-mentioned SNP markers, the above-mentioned primer pairs, and the above-mentioned kit in assisting the identification of milk production traits of Holstein cows in southern China.

[0017] In a fifth aspect, the present invention provides a method for breeding southern China Holstein dairy cows, by detecting the above-mentioned SNP markers in the southern China Holstein dairy cows to be tested, and evaluating the milk production traits of the southern China Holstein dairy cows to be tested.

[0018] The method for breeding Holstein cows in southern China specifically includes the following steps:

[0019] Extracting genomic DNA from Holstein cows in southern China;

[0020] Using the genomic DNA of the Holstein cows from southern China as a template, the primer pairs were used to perform a multiplex PCR amplification reaction;

[0021] The single-base extension primers are used to perform a single-base extension reaction on the PCR amplification products, and the extension products are sequenced to determine the genotypes of the first SNP marker and the second SNP marker, and the varieties with superior milk production traits are selected according to the genotypes.

[0022] On the basis of the above technical scheme, one of the parents used for hybrid breeding meets at least one of the following conditions: (i) the first SNP marker contains allele C, (ii) the second SNP marker contains allele G. Thereby, the dominant allele spreads rapidly in the population, accelerating the process of cultivating offspring with superior milk production traits. Preferably, an individual that meets at least one of the following conditions is selected as an individual with superior milk production traits: (i) the first SNP marker is a CC genotype, (ii) the second SNP marker is a GG genotype. More preferably, an individual whose first SNP marker is a CC genotype and whose second SNP marker is a GG genotype is selected as an individual with superior milk production traits. Preferably, the Holstein cows in southern China are Holstein cows in Wuhan.

[0023] The beneficial effects brought about by the technical solution provided by the present invention include:

[0024] (1) The present invention provides two SNP markers that are significantly associated with the total milk yield and peak milk yield of Holstein cows in southern China. The rs41871650 site where the first SNP marker is located and the rs209034260 site where the second SNP marker is located are both located on the HP gene. At the same time, the rs41871650 site is significantly associated with the total milk yield and peak milk yield of Holstein cows at 305 days. poleThese two SNP markers enrich the molecular marker genetic resource library for dairy cattle breeding, and using them for molecular marker-assisted selection is conducive to improving the milk production of Holstein dairy cows in southern China, while not affecting the reproductive traits of Holstein dairy cows in southern China.

[0025] (2) The present invention combines rs41871650 and rs209034260 loci for marker-assisted selection, which can rapidly spread the dominant genotypes that improve the milk production traits (305d milk volume, total milk volume and peak milk) of dairy cows within the group, thereby fundamentally improving the lactation performance of the offspring group, greatly promoting the improvement of dairy cow production performance and the improvement of dairy cow genetic quality, and is conducive to cultivating a local high-yield dairy cow group. At the same time, the selection of the dominant genotype will not have an adverse effect on the reproductive level of the group, especially the age of first calving and the calving interval, which is conducive to the balanced breeding of important economic traits of Holstein dairy cows in southern China.

[0026] (3) The present invention greatly increases the accuracy of selection by combining the dominant genotypes of two SNP markers, which helps to achieve early selection and precision breeding of dairy cows. In addition, the rs41871650 locus and the rs209034260 locus are significantly associated with multiple milk production traits. By combining the 305d milk yield, total milk yield and peak milk traits, the selection efficiency of individuals with excellent milk production traits is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 to Figure 3 This is the result of rs41871650 site typing;

[0029] Figures 4 to 6 This is the rs209034260 site typing result diagram. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly and completely described in conjunction with specific embodiments below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0031] The present invention uses modern molecular biology technology and statistical methods to find two SNP markers that are closely related to increasing milk production. The two SNP markers are located on the haptoglobin (HP) gene. The HP gene is located in the 39037402-39043531bp interval on the reverse strand of chromosome 18 of dairy cows. It is an α2 globulin synthesized by the liver, accounting for about 1% of the total plasma protein, and the molecular weight reaches 1000-2000kDa. Since hemoglobin (Hb) has peroxidase activity, HP can protect body tissues from oxidative damage by binding to it; once HP binds to Hb, the iron ions in Hb cannot be used by iron-loving bacteria. Therefore, HP has a certain indirect antibacterial activity. At the same time, HP also has the effects of promoting angiogenesis, regulating lipid metabolism and immunomodulation. HP can be used as an effective marker molecule for the occurrence, severity and prognosis of diseases such as mastitis, endometritis, foot-and-mouth disease and pneumonia in dairy cows. In addition, this molecule can also indirectly reflect the changes in milk composition and milk quality, and serve as a candidate indicator for evaluating milk quality. However, there are currently no reports on the polymorphic sites of the HP gene and the effects of genetic variation on dairy cow milk production traits.

[0032] The information of the two SNP markers obtained by the present invention is shown in Table 1.

[0033] Table 1 Information of two SNP markers used for association analysis

[0034]

[0035] Note: The location of the SNP is marked in the form of "chromosome number: position on the chromosome".

[0036] Example 1: Acquisition and identification of two SNP markers closely related to increasing milk production

[0037] 1.1 Collection of blood samples and phenotypic data

[0038] A total of 785 Chinese southern Holstein cows from a large-scale dairy farm in Wuhan, Hubei Province were selected as experimental subjects. Blood was collected from the tail vein, anticoagulated with EDTA, and stored at -20°C. The milk production traits (lactation days, 305-day milk production, total milk yield, peak milk, peak day, milk fat rate, and milk protein rate), reproductive traits (age at first calving, calving interval), and health traits (somatic cell count) of these cows were monitored. A total of 777 complete data on first-parity cows, 386 data on second-parity cows, and 92 data on third-parity cows were obtained.

[0039] 1.2 Extraction and detection of total DNA

[0040] Whole genome DNA was extracted from blood samples of 785 Holstein cows in southern China using the TIA Namp Blood DNA Kit. 200 μL of buffer GB and 20 μL of proteinase K were added to 200 μL of whole blood and mixed thoroughly by inversion. After standing at 56°C, 350 μL of buffer BD was added and mixed thoroughly by inversion before passing through the adsorption column. 500 μL of buffer GDB and 600 μL of rinse solution PWB were added to the adsorption column and centrifuged. The residual rinse solution in the adsorption column was dried at room temperature. Finally, the adsorption column was transferred to a centrifuge tube, elution buffer TB was added dropwise, and the solution was collected after standing at room temperature and centrifuged. 1 μL of DNA sample was tested for purity and concentration by 1% agarose gel electrophoresis, and then diluted to a working concentration of 5-10 ng / μL.

[0041] 1.3 PCR reaction

[0042] According to the reference sequence provided by NCBI, PCR amplification primers were designed using Primer3.0 online software. The two SNP markers at the rs41871650 site and the rs209034260 site shared a pair of PCR amplification primers:

[0043] Forward primer: 5′-CCATACTCAGCCACAGCACAGC-3′;

[0044] Reverse primer: 5′-GGGGCGAAATGAAAACTTCAACT-3′.

[0045] The PCR reaction system (20 μL) contained 1× GC-I buffer (purchased from Takara), 3.0 mM Mg 2+ , 0.3mM dNTP, 1U hot start Taq enzyme (purchased from Qiagen), 1μL sample DNA and 0.5μL each of forward and reverse primers, with a primer concentration of 1.5μM. The reaction process is: (1) 95℃2min; (2) 94℃20s, 65℃40s (-0.5℃ / cycle), 72℃1.5min, three steps for a total of 11 cycles; (3) 94℃20s, 59℃30s, 72℃1.5min, three steps for a total of 24 cycles; (4) 72℃2min. The nucleotide sequence of the amplified product is shown in SEQ ID NO:5, with a length of 309bp:

[0046]

[0047]

[0048] 1.4 PCR product purification

[0049] 5U SAP enzyme and 2U Exonuclease I enzyme were added to 10μL PCR product, incubated at 37℃ for 1h, and then inactivated at 75℃ for 15min.

[0050] 1.5Snapshot single base extension reaction

[0051] The purified PCR products were subjected to single base extension reaction using ABI's Snapshot Multiplex kit.

[0052] The single base extension primer (SF) for rs41871650 is:

[0053] 5'-TTTTTTTTTTTTTTGAAGGTTGTTCTCATTCAGTATGGG-3';

[0054] The single base extension primer (SF) for rs209034260 is:

[0055] 5'-TTTTTTTGTCGTCTTCCYTGTCGTGAAC-3'.

[0056] Among them, Y is a degenerate base, representing C or T.

[0057] The extension reaction system (10 μL) included 5 μL Snapshot Multiplex kit (purchased from ABI), 2 μL purified multiple PCR product, 2 μL ultrapure water, and 1 μL extension primer mixture, where the primer concentration of rs41871650 was 1.6 μM and the primer concentration of rs209034260 was 0.8 μM. The reaction process was: (1) 96°C for 1 min; (2) 96°C for 10 s, 55°C for 5 s, and 60°C for 30 s, for a total of 28 cycles.

[0058] 1.6 Purification of extension products

[0059] 1U SAP enzyme was added to 10μL of extension product, incubated at 37℃ for 1h, and then inactivated at 75℃ for 15min.

[0060] 1.7 Sequencing of extension products

[0061] 0.5 μL of the purified extension product was mixed with 0.5 μL Liz 120SIZE STANDARD and 9 μL Hi-Di, and then denatured at 95°C for 5 minutes before loading on an ABI3730XL sequencer. The raw data was analyzed using Gene Mapper4.1 (Applied Biosystems, USA). The genotype frequencies and allele frequencies of the two loci were calculated using Excel, and the Hardy-Weinberg equilibrium of the two loci was analyzed using the chi-square test.

[0062] Example 2: Association analysis between SNP and production traits

[0063] The genotype frequency and allele frequency of each locus were calculated using Excel, and the Hardy-Weinberg equilibrium of each locus was analyzed using the chi-square test. The normality test of milk fat percentage, milk protein percentage and somatic cell count was performed using the univariate procedure of the SAS online program (https: / / welcome.oda.sas.com / ), the data were transformed using the BOX-COX method, and outliers were eliminated using the "mean ± 3 times standard deviation". The GLM procedure was used to test the effect of genotype on the corrected traits, and the test model was Y = μ + P + S + G + e. The test model for the age of first calving was Y = μ + G + e; where Y is the individual trait phenotypic value, μ is the population mean, P is the fixed effect of parity, S is the fixed effect of calving season, G is the fixed effect of genotype, and e is the random error effect. The DUNCAN method was used to perform multiple comparisons of the individual trait phenotypic values ​​of different genotypes.

[0064] 2.1 Gene and genotype frequencies

[0065] The DNA of cow blood was detected by 1% agarose gel electrophoresis, and the bands were bright and free of protein contamination. Using NanoDrop2000 nucleic acid concentration meter, more than 95% of the samples had a mass concentration greater than 10 ng / μL, and the concentration and purity could meet the requirements of Snapshot typing experiment.

[0066] After testing, the gene frequency and genotype frequency of the rs41871650 locus and rs209034260 locus are shown in Table 2. Both genes are in Hardy-Weinberg equilibrium (P>0.05).

[0067] Table 2 Genotype frequency and gene frequency of two SNP loci

[0068]

[0069] 2.2 Phenotypic data statistics

[0070] The statistical results of 10 milk production traits (lactation days, 305-day milk yield, total milk yield, peak milk, peak day, milk fat rate, milk protein rate, somatic cell count, age at first calving, and calving interval) of the Holstein dairy cow herd in southern China selected for the experiment are shown in Table 3. The overall production level of the experimental herd is high, but the differences between individuals are also relatively large, and there is a large space for breeding.

[0071] Table 3 Statistical results of milk production traits

[0072]

[0073] 2.3 Association analysis

[0074] Association analysis showed the effects of different genotypes at different loci of the HP gene on milk production traits. The results are shown in Table 4.

[0075] The effect of rs41871650 locus of HP gene on 305d milk yield, total milk yield, peak milk yield and age at first calving reached a significant level (P<0.05), and there was no significant correlation with other traits. The results of multiple comparisons showed that the 305d milk yield of individuals with CC genotype at rs41871650 locus of HP gene was extremely significantly higher than that of CT genotype and TT genotype (P<0.01), with an average increase of 4.54% (CT type) and 3.84% (TT type); the total milk yield of individuals with CC genotype was significantly higher than that of CT genotype and TT genotype (P<0.05), with an average increase of 7.32% (CT type) and 6.46% (TT type); the peak milk yield of individuals with CC genotype was significantly higher than that of CT genotype and TT genotype (P<0.05), with an average increase of 3.28% (CT type) and 2.98% (TT type). There was no significant difference in the age of first birth between individuals with CC genotype at rs41871650 and those with CT and TT genotypes (P>0.05), but there was a significant difference between individuals with CT and TT genotypes (P<0.05).

[0076] The rs209034260 locus of the HP gene reached a significant level for total milk volume and peak milk (P<0.05), and had no significant correlation with other traits. The results of multiple comparisons showed that the total milk volume of individuals with the GG genotype at the rs209034260 locus was extremely significantly higher than that of the GA genotype (P<0.05), with an average increase of about 3.66%; the peak milk volume of individuals with the GG genotype at the rs209034260 locus was significantly higher than that of the GA genotype (P<0.05), with an average increase of about 2.51%.

[0077] Table 4 Association analysis between HP gene SNP loci and milk production traits in dairy cows (LSM±sE)

[0078]

[0079] Note: Different lowercase letters indicate significant differences (P<0.05) between different genotypes of the same trait at the same locus, and different uppercase letters indicate extremely significant differences (P<0.01) between different genotypes of the same trait at the same locus, the same below.

[0080] The research results of the present invention show that there are SNP sites on the HP gene that can affect the milk production traits of dairy cows, and the rs41871650 site and the rs209034260 site on the HP gene can be used to perform marker-assisted selection on the milk production traits of dairy cow groups.

[0081] The milk yield of individuals with CC genotype at rs41871650 locus at 305 days was extremely significantly higher than that of individuals with CT genotype and TT genotype. The total milk yield and peak milk yield of individuals with CC genotype were significantly higher than those of individuals with CT genotype and TT genotype. At the same time, there was no significant difference in the first calving age of individuals with CC genotype and those with CT genotype and TT genotype. Peak milk refers to the highest milk yield on the day before 150 days after delivery. Peak milk is positively correlated with the milk yield of the entire parity. According to statistics, for every 1kg increase in peak milk, the milk yield of the entire parity can increase by 200-250kg. The first calving age is the number of days between the first calving date and the birth date of the dairy cow. It can simultaneously reflect the early or late sexual maturity of the individual cow and its ability to conceive, and is one of the important reproductive traits. Studies have confirmed that the first calving age has a significant effect on milk fat content, milk protein and production life, and the milk production traits are improved as the first calving age decreases. In general, the test results of the present invention show that the comprehensive milk production traits (305d milk volume, total milk volume and peak milk) of individuals with CC genotype at rs41871650 are better than those of individuals with TT genotype, and have no significant effect on the age of first calving and calving interval. The total milk volume and peak milk volume of individuals with GG genotype at rs209034260 are significantly higher than those of GA genotype, and there are no significant differences in other production traits between individuals with GG genotype and other genotypes.

[0082] In production practice, individuals with allele T in the first SNP marker and / or allele G in the second SNP marker can be selected as one of the parents, and the dominant alleles of the two loci can be rapidly spread in the population through hybrid breeding, thereby accelerating the breeding process of new varieties with superior milk production traits. Individuals with CC genotype at rs41871650 and GG genotype at rs209034260 can also be selected as the most dominant individuals to breed new varieties with superior milk production traits, without negatively affecting the reproductive traits of the individuals.

[0083] Applying the two SNP markers provided by the present invention to seed selection can achieve early seed selection and improve breeding accuracy, thereby accelerating the genetic breeding process, fundamentally improving the milk production performance of dairy cows, and promoting the sustainable and healthy development of my country's dairy cow industry.

[0084] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein. Sequence Listing <110> Wuhan Academy of Agricultural Sciences <120> SNP markers associated with milk production traits in Holstein cows in southern China and their application <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty one <212> DNA <213> Artificial Sequence <400> 1 cctactcagc cacagcacag c 21 <210> 2 <211> twenty three <212> DNA <213> Artificial Sequence <400> 2 ggggcgaaat gaaaacttca act 23 <210> 3 <211> 38 <212> DNA <213> Artificial Sequence <400> 3 ttttttttttttttgaaggt gttctcattc agtatggg 38 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <400> 4 tttttttgtc gtcttccytg tcgtgaac 28 <210> 5 <211> 309 <212> DNA <213> Artificial Sequence <400> 5 ccatactcag ccacagcaca gctcttgtca aagctcagga tcccggccgc ataccaggtg 60 tcgtcttcct tgtcgtgaac gacgaaggcg ctgccggcgt cgccatagca ggtgtcgtcc 120 tggtacttgg acaggccgac gcagaaggtg ttctcattca gtatgggttg cacccctaca 180 gggctcttag ctgtcttatt tttaggtgcg tcgacgccct catagtgttt cacacacttg 240 tcttggtcag ccacaggtag catgacatac ttcagatgct ccgtaaagtt gaagttttca 300 tttcgcccc 309

Claims

1. Use of a second SNP marker in assisting identification of milk production traits of Holstein cows in southern China, characterized in that: The second SNP marker is located at 39037665bp on the reverse strand of chromosome 18 in the genome of Holstein cows in southern China, numbered rs209034260, with a base of G or A. The total milk yield and peak milk of individuals with the GG genotype of the second SNP marker are significantly higher than those of individuals with the GA genotype.

2. Use of a kit for detecting a second SNP marker in assisting identification of milk production traits of Holstein cows in southern China, characterized in that: The kit for detecting the second SNP marker contains a primer pair for detecting the second SNP marker and a single base extension primer; The second SNP marker is located at 39037665bp on the reverse strand of chromosome 18 of the genome of southern Chinese Holstein cows, numbered as rs209034260, with a base of G or A. The total milk yield and peak milk yield of individuals with the GG genotype of the second SNP marker are significantly higher than those of individuals with the GA genotype. The primer pair comprises: Forward primer: 5'-CCATACTCAGCCACAGCACAGC-3', Reverse primer: 5′-GGGGCGAAATGAAAACTTCAACT-3′; The single base extension primer is: 5'-TTTTTTTGTCGTCTTCCYTGTCGTGAAC-3'; Among them, Y in the nucleotide sequence is a degenerate base, representing C or T.

3. A method for breeding Holstein cows in southern China, characterized in that: By detecting the second SNP marker on the Chinese southern Holstein cows to be tested, the milk production traits of the Chinese southern Holstein cows to be tested are evaluated, and individuals with dominant alleles and genotypes that can increase milk production are selected to carry out breeding work; the second SNP marker is located at 39037665bp on the reverse strand of chromosome 18 of the Chinese southern Holstein cow genome, numbered rs209034260, and the base is G or A. The total milk yield and peak milk of individuals with the GG genotype of the second SNP marker are significantly higher than those of individuals with the GA genotype.

4. The method for breeding Holstein cows in southern China according to claim 3, characterized in that: The following steps are involved: Extracting genomic DNA from Holstein cows in southern China; The genomic DNA of the Holstein cow in southern China to be tested is used as a template, and a multiplex PCR amplification reaction is performed using a primer pair; the primer pair includes: Forward primer: 5'-CCATACTCAGCCACAGCACAGC-3', Reverse primer: 5′-GGGGCGAAATGAAAACTTCAACT-3′; The single base extension primer of the second SNP marker is used to perform a single base extension reaction on the PCR amplification product, and the extension product is sequenced to determine the genotype of the second SNP marker, and individuals with the target genotype are selected to cultivate milk-producing superior varieties; the single base extension primer of the second SNP marker is: 5'-TTTTTTTGTCGTCTTCCYTGTCGTGAAC-3'; Among them, Y in the nucleotide sequence is a degenerate base, representing C or T.

5. The method for breeding Holstein cows in southern China according to claim 4, characterized in that: One of the parents used for hybrid breeding meets at least the following conditions: the second SNP marker contains allele G.

6. The method for breeding Holstein cows in southern China according to claim 5, characterized in that: Individuals with the CC genotype as the first SNP marker and the GG genotype as the second SNP marker were selected as dominant individuals for milk production traits; the first SNP marker was located at 39037752bp on the reverse strand of chromosome 18 in the genome of Holstein cows in southern China, numbered rs41871650, and the base was C or T. The milk yield of individuals with the CC genotype of the first SNP marker at 305 days was extremely significantly higher than that of individuals with the CT genotype and the TT genotype, and the total milk yield and peak milk yield were significantly higher than those of individuals with the CT genotype and the TT genotype.

7. The method for breeding Holstein cows in southern China according to any one of claims 3 to 6, characterized in that: The South China Holstein cows are Holstein cows from Wuhan area.

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